MXPA06008853A - Methods of diagnosing and treating pre-eclampsia or eclampsia - Google Patents

Abstract

Disclosed herein are methods for treating pre-eclampsia or eclampsia using compounds that increase VEGF or P1GF levels or compounds that decrease sF1t-1 levels. Also disclosed here in are methods for monitoring the treatment of pre-eclampsia or eclampsia by detecting the levels of sF1t-1, VEGF, or P1GF. Also disclosed herein are methods for diagnosing pre-eclampsia and eclampsia by detecting the levels of sF1t-1, VEGF, and P1GF in a subject.

Description

METHODS OF DIAGNOSING AND TREATING PRE-ECLAMPSIA OR ECLAMPSIA Field of the Invention In general, this invention relates to the detection and treatment of subjects who have pre-eclampsia or eclampsia. Background of the Invention Pre-eclampsia is a syndrome of hypertension, edema and proteinuria that affects 5-10% of pregnancies and results in substantial fetal and maternal morbidity and mortality. Pre-eclampsia is responsible for at least 200,000 maternal deaths worldwide each year. Symptoms of pre-eclampsia typically appear after the 20th week of pregnancy and are usually detected by routine monitoring of the mother's blood pressure and urine. However, these control methods are ineffective in diagnosing the syndrome at an early stage, which could reduce the risk of the developing subject or fetus, if effective treatment is available. Currently there is no known cure for eclampsia. Pre-eclampsia can vary in severity from mild to a life threatening condition. A mild form of pre-eclampsia can be treated with bed rest and frequent monitoring. For moderate to severe cases, hospitalization is recommended and a medication is prescribed to treat blood pressure or anti-convulsant medications to prevent attacks. If the condition becomes a threatening condition for the life of the mother or baby, the pregnancy ends and the baby is born premature. The proper development of the fetus and the placenta is mediated by several growth factors. One of these growth factors is vascular endothelial growth factor (VEGF). VEGF is a specific mitogen of endothelial cells, an angiogenic inducer and a mediator of vascular permeability. It has also been shown that VEGF is important for the repair of glomerular capillaries. VEGF binds as a homodimer to one of two transmembrane homologous tyrosine kinase receptors, the fs-type tyrosine kinase receptor (Flt-1) and the kinase domain receptor (KDR), which are differentially expressed in endothelial cells obtained from of many different tissues. Flt-1 but not KDR, has a high expression in trophoblast cells that contribute to the formation of the placenta. Placental growth factor (PlGF) is a member of the VEGF family that is also involved in the development of the placenta. PlGF is expressed by cytotrophoblasts and syncytiotrophoblasts and is able to induce proliferation, migration and activation of endothelial cells. PlGF binds as a homodimer to the Flt-1 receptor, but not to the KDR receptor. Both P1GF and VEGF contribute to the mitogenic activity and to angiogenesis that are critical for the development of the placenta. Recently, a soluble form of the Flt-1 receptor (sFlt-1) was identified in a cultured medium of human umbilical vein endothelial cells and subsequently in vivo expression in placental tissue was demonstrated. sFlt-1 is a splice variant of the Flt-1 receptor that lacks the transmembrane and cytoplasmic domains. sFlt-1 binds to VEGF with high affinity, but does not stimulate the mitogenesis of endothelial cells. It is believed that sFlt-1 acts as a "physiological fossa" to negatively regulate the VEGF signaling pathway. Therefore, the regulation of sFlt-1 levels act by modulating VEGF and the VEGF signaling pathways. Careful regulation of the VEGF and PlGF signaling pathways is critical to maintaining proper proliferation, migration and angiogenesis by the trophoblast cells in the developing placenta. There is a need for methods to accurately diagnose subjects at risk or who have pre-eclampsia, particularly before the onset of more severe symptoms. A treatment is also needed. Compendium of the Invention A means has been discovered to diagnose and effectively treat pre-eclampsia and eclampsia before the development of symptoms. Using gene expression analysis, we have discovered that sFlt-1 levels are markedly elevated in placental tissue samples from pregnant women suffering from pre-eclampsia. It is known that sFlt-1 antagonizes VEGF and PlGF acting as a "physiological fossa" and, in pre-eclamptic or ecclastic women, sFlt-1 can reduce in the placenta the necessary amounts of these essential angiogenic and mitogenic factors. An excess of sFlt-1 can also produce eclampsia by breaking the endothelial cells that maintain the blood-brain barrier and / or the endothelial cells that line the choroid plexus of the brain, thus leading to cerebral edema and the attacks observed in eclampsia. In the present invention, compounds are administered which increase the levels of VEGF and PIGF to a subject to treat or prevent pre-eclampsia or eclampsia by counteracting the effects of the high level of sFlt-1. In addition, anti-bodies directed against sFlt-1 are used to competitively inhibit the binding of VEGF or PlGF to this sFlt-1, thereby increasing the levels of free VEGF and PlGF. Anti-sense and RNA interference nucleobases oligomers are also used to reduce sFlt-1 levels. Finally, the present invention provides the use and control of sFlt-1, VEGF and PIGF as screening tools for early diagnosis and treatment of pre-eclampsia or eclampsia, or a predisposition to it. It has also been discovered that levels of PIGF in the urine can be used as a diagnostic tool to detect pre-eclampsia or eclampsia, or a predisposition to them. The free form of PIGF has an average molecular weight of about 30 kDa and is small enough to filter through the kidney and release into the urine. PIGF, when formed in complex with sFlt-1, has a much higher molecular weight and therefore would not be released into the urine. When the levels of sFlt-1 are increased, sFlt-1 can form complexes with PIGF, thereby reducing the levels of free PIGF released into the urine. As a result, urinalysis of free PIGF levels can be used to diagnose pre-eclampsia or eclampsia or in a patient at risk of having them. Accordingly, in one aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia in a subject by administering to the subject a compound capable of binding sFlt-1, where administration is for a time and in an amount sufficient to treat or prevent pre-eclampsia or eclampsia in a subject. In a preferred embodiment, the compound is a purified sFlt-1 anti-body or a fragment thereof that is linked to an antigen. In a related aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia in a subject by administration to the subject of a compound (for example nicotine, theophylline, adenosine, nifedipine, minoxidil or magnesium sulfate) which increases the level of a growth factor capable of binding to sFlt-1, where the administration is carried out for a period of time and in an amount sufficient to Treat or prevent pre-eclampsia or eclampsia in a subject. In another related aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia in a subject by administering to the subject an anti-sense nucleobase oligomer complementary to at least a portion of a nucleic acid sequence. of sFlt-1 being administration sufficient to treat or prevent pre-eclampsia or eclampsia in a subject. In one embodiment, the antisense oligonucleotide oligomer has a length of 8 to 30 nucleotides. In another related aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia in a subject, by administering to the subject a double helix RNA (dsRNA) containing at least a portion of a nucleic acid sequence of sFlt- 1, the administration being sufficient to treat or prevent pre-eclampsia or eclampsia in the subject. In one embodiment, the double-stranded RNA is processed into small interfering RNA (siRNA) of 19 to 25 nucleotides in length. In various embodiments of the above aspects, the candidate compound is a growth factor such as vascular endothelial growth factor (VEGF), including all isoforms such as VEGF189, VEGF121 or VEGF165; placental growth factor (PIGF) including all isoforms; or fragments of them. In preferred embodiments, the candidate compound is an anti-body that binds sFlt-1. In other embodiments of the above aspects, the method also involves administering to a subject an anti-hypertensive compound. In other embodiments of the above aspects, the subject is a pregnant woman, a woman after childbirth or a non-human animal (e.g., a cow, a horse, a sheep, a pig, a goat, a dog or a cat) . In another aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia. The method involves administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a VEGF or PIGF polypeptide. In one embodiment, the composition contains a VEGF polypeptide. In another embodiment, the composition contains a PIGF polypeptide. In a related aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia. This method involves administration to a subject in need of such treatment of an effective amount of a pharmaceutical composition comprising a nucleic acid molecule encoding VEGF or PIGF. In one embodiment, the composition contains a VEGF nucleic acid molecule. In another embodiment, the composition contains a PIGF nucleic acid molecule. In another related aspect, the invention provides a method for treating or preventing pre-eclampsia or eclampsia in a subject. The method involves the step of administering to the subject a compound (eg, chemical compound, polypeptide, peptide, anti-body or a fragment thereof) that inhibits the binding of the growth factor to a sFlt-1 polypeptide, with sufficient administration to treat or prevent pre-eclampsia or eclampsia in a subject. In one embodiment, the compound binds to sFlt-1 and blocks growth factor binding. In various embodiments of the above aspects, the method also includes the step of administering to a subject an anti-hypertensive compound (e.g., adenosine, nifedipine, minoxidil and magnesium sulfate). In other embodiments of the above aspects, the subject is a pregnant woman, a woman after childbirth or a non-human animal (e.g., a cow, a horse, a sheep, a pig, a goat, a dog or a cat). In another aspect, the invention provides a method for diagnosing in a subject the condition or propensity to develop pre-eclampsia or eclampsia, which includes measuring the level of free PIGF in a sample of the subject's urine. This method can be used to determine the absolute levels of free PIGF that are below a threshold level and are diagnostic of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia. The normal urinary concentration of urinary PIGF is approximately 400-800 pg / l during the middle pregnancy. In preferred embodiments, a free PIGF level of less than 400 pg / ml, preferably less than 300, 200, 100, 50 or 10 pg / ml is diagnostic of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia. eclampsia or eclampsia. This method can be used to determine the relative levels of free PIGF compared to a reference sample where a decrease (eg, 10, 20, 25, 50, 75, 90% or more) at the level of free PIGF compared to a normal reference sample is diagnosis of pre-eclampsia or eclampsia or the propensity to develop eclampsia or pre-eclampsia. In this case, the normal reference sample may be a previous sample taken from the same subject or a sample taken from a matched subject (e.g., equipped for gestational age) who is pregnant but who does not have pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia. In additional embodiments, the reference sample is a standard, a level or a number derived from such a normal reference sample. The standard or reference level can also be a value derived from a normal subject that is equipped to the subject of the sample by at least one of the following criteria: gestational age of the fetus, age of the mother, blood pressure before pregnancy, blood pressure during pregnancy, body mass index (BMI) of the mother, weight of the fetus, previous diagnosis of pre-eclampsia or eclampsia, and family history of pre-eclampsia or eclampsia. In preferred embodiments, the measurement is performed using an immunological assay such as an ELISA assay, preferably a sandwich ELISA assay, or a fluorescence immunoassay.

In preferred embodiments, the method also includes the steps of (a) measuring the level of at least one sFlt-1 polypeptide, PIGF and VEGF in a subject sample, where the sample is a body fluid selected from the group that consists of urine, blood, serum, plasma or cerebro-spinal fluid, and (b) comparing the level of at least one of sFlt-1, PIGF and VEGF of the subject to the level of the same polypeptide in a reference sample, where a increase in the level of sFlt-1 or a reduction in the level of the VEGF or PIGF polypeptide of the subject sample compared to the reference sample is a diagnostic indicator of pre-eclampsia or eclampsia, or a propensity to develop pre-eclampsia or eclampsia. eclampsia or eclampsia. In preferred embodiments, sFlt-1 or sFlt-1 and PIGF are measured in a serum sample from a subject identified by a PIGF assay in urine as being at risk of developing pre-eclampsia or eclampsia. Desirably, this method further includes calculating the ratio between the levels of at least one of sFlt-1, VEGF, and PIGF of step (a) using a metric, where an alteration in the subject's sample relative to The metric in the reference sample diagnoses pre-eclampsia or eclampsia in a subject or a propensity to develop pre-eclampsia or eclampsia. Preferably, the metric is PAAI (as described above) and a PAAI value greater than 20 is a diagnostic indicator of pre-eclampsia or eclampsia. In preferred embodiments, sFlt-1 is free, ligated or total sFlt-1, and PIGF and VEGF are free PIGF and free VEGF. In various embodiments of the above aspects, the sample is a body fluid, such as serum or urine. In one embodiment, a level of sFlt-1 greater than 2 ng / ml is indicative of pre-eclampsia or eclampsia. In preferred embodiments of the above aspects, the level of sFlt-1 polypeptide measured is the level of bound or total free sFlt-1 polypeptide. In other preferred embodiments of the above aspects, the level of VEGF or PIGF is the level of VEGF or free PIGF. In another aspect, the invention provides a method for diagnosing in a subject the condition or propensity to develop pre-eclampsia or eclampsia. This method involves measuring the level of a nucleic acid molecule of sFlt-1, VEGF or PIGF in a sample of the subject and comparing this level with a reference sample, diagnosing an alteration in the levels of pre-eclampsia or eclampsia in the subject, or diagnosing a propensity to develop pre-eclampsia or eclampsia. In another aspect, the invention provides a method for diagnosing in a subject the condition or propensity to develop pre-eclampsia or eclampsia. This method involves the determination of the nucleic acid sequence of a sFlt-1, VEGF or PIGF gene in a subject and comparison with a reference sequence, diagnosing an alteration in the nucleic acid sequence of the subject that changes the level of the gene product in the subject a state of pre-eclampsia or eclampsia, or a propensity to develop pre-eclampsia or eclampsia. In one embodiment, the alteration is a polymorphism in the nucleic acid sequence. In various embodiments of the above aspects, the sample is a bodily fluid (eg, urine, amniotic fluid, serum, plasma or cephalo-rachidian fluid) of the subject in which sFlt-1, VEGR or PIGF can normally be detected. . In other embodiments, the sample is a tissue or a cell. Non-limiting examples include placental tissue or placental cells, endothelial cells and leukocytes (e.g., monocytes). In other embodiments of the above aspects, the subject is a non-pregnant woman, a pregnant woman or a woman after childbirth. In other embodiments of the above aspects, the subject is a non-human animal (e.g., a cow, a horse, a sheep, a pig, a goat, a dog or a cat). In other embodiments of the above aspects, at least one of the measured levels is the level of sFlt-1 (free, bound or total). In other embodiments of the above aspects, when the level of VEGF is measured, the level of sFlt-1 or PIGF is also measured. In various embodiments of the above aspects, an increase in the level of sFlt-1 nucleic acid or polypeptide with respect to a reference is a diagnostic indicator of pre-eclampsia or eclampsia. In other embodiments of the above aspects, a reduction in the level of free VEGF polypeptide or VEGF nucleic acid with respect to a reference is a diagnostic indicator of pre-eclampsia or eclampsia. In other embodiments of the above aspects, a reduction in the level of free PIGF polypeptide or PIGF nucleic acid with respect to a reference is a diagnostic indicator of pre-eclampsia or eclampsia. In additional embodiments of the above aspects, the levels are measured on two or more occasions and a change in the levels between the measurements is a diagnostic indicator of pre-eclampsia or eclampsia. In a preferred embodiment, the level of sFlt-1 increases from the first measurement to the next measurement. In another preferred embodiment, the VEGF or PIGF level decreases from the first measurement to the next measurement. In another aspect, the invention provides a diagnostic kit for the diagnosis of pre-eclampsia or eclampsia in a subject comprising a nucleic acid sequence, or a fragment thereof, selected from the group consisting of a nucleic acid molecule of sFlt-1, VEGF and PIGF, or a sequence complementary thereto, or any combination thereof. In a preferred embodiment, the kit comprises at least two probes for the detection of a nucleic acid molecule of sFlt-1, VEGF or PIGF. In a related aspect, the invention provides a kit for the diagnosis of pre-eclampsia or eclampsia in a subject comprising a means for detecting a sFlt-1, VEGF or PIGF polypeptide, and any combination thereof. In one embodiment, the detection means is selected from the group consisting of an immunological assay, an enzymatic assay and a colorimetric assay. In other embodiments of the above aspects, the kit diagnoses a propensity to develop pre-eclampsia or eclampsia in a pregnant or a pregnant woman. In preferred embodiments of the above aspects, the kit detects sFlt-1 or PIGF. In other preferred embodiments of the above aspects, when the kit detects VEGF, then sFlt-1 or PIGF is also detected. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia, the method including contacting a cell expressing a nucleic acid molecule of sFlt-1, VEGF or PIGF with a candidate compound, and comparing the level of expression of the nucleic acid molecule in the cell that has come into contact with the candidate compound with the level of expression in a control cell that has not come into contact with the candidate compound, identifying an alteration in the Expression of the nucleic acid molecule of sFlt-1, VEGF or PlGF that the candidate compound is a compound that improves pre-eclampsia or eclampsia. In another aspect, the invention provides a composition comprising a purified anti-body or an antigen-binding fragment thereof that specifically binds sFlt-1. In a preferred embodiment, the anti-body prevents the binding of a growth factor to sFlt-1. In another embodiment, the anti-body is a monoclonal anti-body. In other preferred embodiments, the anti-body or an antigen-binding fragment thereof is a human or humanized anti-body. In other embodiments, the anti-body lacks a Fe portion. In other embodiments, the anti-body is a structure F (ab ') 2 and Fa-t >; or a Fv structure. In other embodiments, the anti-body or antigen-binding fragment thereof is present in a pharmaceutically acceptable carrier. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia. This method includes contacting a cell expressing a sFlt-1, VEGF or PIGF polypeptide with a candidate compound and comparing the level of expression of the polypeptide in the cell that has been in contact with the candidate compound with the level of expression of the polypeptide. in a control cell that has not been in contact with the candidate compound, identifying an alteration in the expression of the sFlt-1 VEG or PIGF polypeptide that the candidate compound is a compound that improves pre-eclampsia or eclampsia. In one embodiment, the alteration in expression is assayed using an immunological assay, an enzymatic assay or an immunoassay. In one embodiment, the alteration in expression is a reduction in the level of sFlt-1. In another embodiment, the alteration in expression is an increase in the level of VEGF or PIGF. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia. The method includes contacting a cell expressing a sFlt-1, VEGF, or PIGF polypeptide with a candidate compound, and comparing the biological activity of the polypeptide in the cell that has been in contact with the candidate compound with the level of biological activity in a control cell that has not been in contact with the candidate compound, identifying an increase in the biological activity of the sFlt-1, VEGF or PIGF polypeptide that the candidate compound is a compound that improves pre-eclampsia or eclampsia. In one embodiment, the increase in biological activity is assayed using an immunological assay, an enzymatic assay or an immunoassay. In one embodiment, the alteration in expression is a reduction in the activity of sFlt-1. In another embodiment, the alteration in expression is an increase in the activity of VEGF or PIGF. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia. The method involves the detection of the binding between a sFlt-1 polypeptide and a growth factor in the presence of a candidate compound, identifying a reduction in binding with respect to the binding between the sFlt-1 polypeptide and the growth factor in the absence of the candidate compound that the candidate compound is a compound that improves pre-eclampsia or eclam-psia. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PIGF. In another aspect, the invention provides a method for identifying a polypeptide or fragment thereof, which prevents binding between a sFlt-1 polypeptide and a growth factor. The method involves the detection of the binding between a sFlt-1 polypeptide and a growth factor in the presence of the candidate polypeptide, identifying a reduction in binding, with respect to the binding between the sFlt-1 polypeptide and the growth factor in the absence of the candidate polypeptide, that the candidate polypeptide is a polypeptide that prevents binding between a sFlt-1 polypeptide and a growth factor. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PlGF. In one embodiment, the alteration is a reduction in the level of sFlt-1. In other embodiments, the alteration is an increase in the level of VEGF or PIGF. In other embodiments, the alteration is in transcription or translation. In another embodiment, when the method identifies a candidate compound that increases VEGF expression, the candidate compound also increases the expression of PIGF or reduces the expression of sFlt-1. In another aspect, the invention provides a pharmaceutical composition that includes a VEGF or PIGF polypeptide or a portion thereof, formulated in a pharmaceutically acceptable carrier. In a related aspect, the invention provides a pharmaceutical composition comprising a PIGF nucleic acid molecule, or a portion thereof formulated in a pharmaceutically acceptable carrier. In one embodiment, the composition also contains a VEGF nucleic acid molecule, or a portion thereof. In another aspect, the invention provides a composition comprising a purified anti-body or an antigen-binding fragment thereof that specifically binds sFlt-1.

In a preferred embodiment, the anti-body prevents the binding of a growth factor to sFlt-1. In another embodiment, the anti-body is a monoclonal anti-body. In other preferred embodiments, the anti-body or an antigen-binding fragment thereof is a human or humanized anti-body. In other embodiments, the anti-body lacks a Fe portion. In other embodiments, the anti-body is a F (ab ') 2 and Fab structure or an Fv structure. In other embodiments, the anti-body or antigen-binding fragment thereof is present in a pharmaceutically acceptable carrier. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia.This method includes contacting a cell expressing a sFlt-1, VEGF or PIGF polypeptide with a candidate compound and comparing the level of expression of the polypeptide in the cell that has been in contact with the candidate compound with the level of expression of the polypeptide in a control cell that has not been in contact with the candidate compound, identifying an alteration in the expression of the sFlt polypeptide -1 VEG or PIGF that the candidate compound is a compound that improves pre-eclampsia or eclampsia In one embodiment, the alteration in expression is assayed using an immunological assay, an enzyme assay or an immunoassay. embodiment, the alteration in expression is a reduction in the level of sFlt-1 In another embodiment, the alteration in expression is an increase in the VEGF or PIGF vel In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia. The method includes contacting a cell expressing a sFlt-1, VEGF, or PIGF polypeptide with a candidate compound, and comparing the biological activity of the polypeptide in the cell that has been in contact with the candidate compound with the level of biological activity in a control cell that has not been in contact with the candidate compound, identifying an increase in the biological activity of the sFlt-1, VEGF or PIGF polypeptide that the candidate compound is a compound that improves pre-eclampsia or eclampsia. In one embodiment, the increase in biological activity is assayed using an immunological assay, an enzymatic assay or an immunoassay. In one embodiment, the alteration in expression is a reduction in the activity of sFlt-1. In another embodiment, the alteration in expression is an increase in the activity of VEGF or PIGF. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia. The method involves the detection of the binding between a sFlt-1 polypeptide and a growth factor in the presence of a candidate compound, identifying a reduction in binding with respect to the binding between the sFlt-1 polypeptide and the growth factor in the absence of the candidate compound that the candidate compound is a compound that improves pre-eclampsia or eclampsia. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PIGF. In another aspect, the invention provides a method for identifying a polypeptide or fragment thereof, which prevents binding between a sFlt-1 polypeptide and a growth factor. The method involves the detection of the binding between a sFlt-1 polypeptide and a growth factor in the presence of the candidate polypeptide, identifying a reduction in binding, with respect to the binding between the sFlt-1 polypeptide and the growth factor in the absence of the candidate polypeptide, that the candidate polypeptide is a polypeptide that prevents binding between a sFlt-1 polypeptide and a growth factor. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PIGF. In another aspect, the invention provides a method for identifying a compound that improves pre-eclampsia or eclampsia, which comprises detecting the binding of a sFlt-1 polypeptide and a candidate compound, wherein the compound that binds the sFlt-1 polypeptide improves the eclampsia or eclampsia. In a related aspect, the invention provides a compound identified according to the previous aspect, wherein the compound is a polypeptide that specifically binds to an sFlt-1 polypeptide and prevents the binding of the sFlt-1 polypeptide to VEGF or PIGF. In a preferred embodiment, the polypeptide is an anti-body, in another preferred embodiment, the polypeptide is a fragment of sFlt-1, VEGF or PIGF. In preferred embodiments of the above aspects, the compound that improves pre-eclampsia or eclampsia reduces the expression levels or the biological activity of sFlt-1. In preferred embodiments of the above aspects, the compound that improves pre-eclampsia or eclampsia increases the expression levels or the biological activity of VEGF or PIGF. For the purposes of the present invention, the following abbreviations and terms are defined below. By "alteration" is meant a change (increase or decrease) in the expression levels of a gene or a polypeptide as detected by methods known in the art such as those described above. As used herein, an increase or reduction includes a change of 10% of the expression levels, preferably a change of 25%, more preferably a change of 40% and even more preferably a change of 50% or greater in the levels of expression. "Alteration" may also indicate a change (increase or decrease) in the biological activity of any of the polypeptides of the invention (e.g., sFlt-1, VEGF or PIGF). Examples of biological activity of PIGF or VEGF include receptor binding as measured by immunoassays, ligand binding assays or Scatchard plot analysis, and induction of cell proliferation or migration as measured by BrdU labeling, cell count experiments or quantitative assays for DNA synthesis such as the incorporation of 3H-thymidine. Examples of biological activity for sFlt-1 include binding to PIGF and VEGF measured by immunoassays, ligand binding assays or Scatchard plot analysis. Other examples of biological activity for each of the polypeptides are described in this document. As used herein, an increase or reduction includes a change of 10% in biological activity, preferably a change of 25%, more preferably a change of 40% and even more preferably a change of 50% or greater in activity biological By "oligomer of anti-sense nucleobases" is meant an oligomer of nucleobases, regardless of length, that is complementary to the coding strand of mRNA of a sFlt-1 gene. By "oligomer of nucleobases" is meant a compound that includes a chain of at least eight nucleobases, preferably at least twelve and even more preferably minus sixteen bases, linked together by linking groups. Included in this definition are natural and unnatural oligonucleotides, both modified and unmodified, as well as oligonucleotide mimetics such as protein nucleic acids, blocked nucleic acids and arabinonucleic acids. Numerous nucleobases and linking groups, including those described in patent applications US 20030114412 and 20030114407, incorporated herein by reference, can be employed in the nucleobase oligomers of the invention. The oligomer of nucleobases can also be directed to translation start and stop sites. Preferably, the anti-sense nucleobase oligomer comprises from about 8 to 30 nucleotides. The anti-sense nucleobase oligomer may also contain at least 40, 60, 85, 120 or more consecutive nucleotides that are complementary to sFlt-1 mRNA or DNA, and they can be as long as the mRNA or the full-length gene. By "compound" is meant any small molecule chemical, anti-body, nucleic acid molecule or polypeptide, or fragments thereof. By "chimeric anti-body" is meant a polypeptide comprising at least the antigen binding portion of an anti-body molecule linked to at least part of another protein (typically an immunoglobulin constant domain). By "double-stranded RNA (dsRNA)" is meant a ribonucleic acid molecule composed of both a sense chain and an anti-sense chain. The dsRNAs are typically used to mediate interference with RNA. By "expression" is meant the detection of a gene or polypeptide by conventional methods known in the art. For example, expression of a polypeptide is often detected by Western blotting, expression of DNA is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by Northern blotting. , PCR or RNase protection assays. By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, or 60% of the entire length of the reference nucleic acid or polypeptide molecule. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. By "homologue" is meant any gene or sequence of proteins bearing at least 30% homology, more preferably 40% homology, 50%, 60%, 70%, 80%, and most preferably 90 homology. % or greater with a known sequence of a gene or protein over the entire length of the comparison sequence. A "homologous" protein may also have at least one biological activity of the comparison protein. In the case of the polypeptides, the length of the comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids and even more preferably 35 amino acids or greater. In the case of nucleic acids, the length of the comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides and even more preferably at least 110 nucleotides. "Homology" may also refer to a substantial analogy between an epitope used to generate anti-bodies and the protein or fragment thereof to which the anti-bodies are directed. In this case, the homology refers to a sufficient similarity to induce the production of anti-bodies that can specifically recognize the protein in question. By "humanized anti-body" is meant a variant of an immunoglobulin amino acid sequence or fragment thereof that is capable of binding to a predetermined antigen. Usually the anti-body will contain both the light chain and at least the variable domain of a heavy chain. The antibody can also include the CH1, joint, CH2, CH3 or CH4 regions of the heavy chain. The humanized anti-body comprises a flanking region (FR) having substantially the amino acid sequence of a human immunoglobulin and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human immunoglobulin (the sequences of "import"). Generally, a humanized anti-body has one or more amino acid residues introduced from a non-human source. In general, the humanized anti-body will comprise substantially all of at least one, and typically two variable domains (Fab, Fab ', F (ab') 2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized anti-body will optimally comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. By "complementarity determining region (CDR)" are meant the three hypervariable sequences in the variable regions within each of the light and heavy immunoglobulin chains. By "flanking region (FR)" are meant amino acid sequences located on either side of the three hypervariable sequences (CDR) of the light and heavy immunoglobulin chains. The FR and CDR regions of the humanized anti-body do not need to correspond accurately to the parental sequences, for example, the import CDR or the consensus FR can be mutagenized by substitution, insertion or deletion of at least one residue, so that the The rest of the CDR or FR in that site does not correspond to the consensus or the anti-body of import. However, such mutations will not be extensive. Normally, at least 75%, preferably 90% and even more preferably at least 95% of the humanized anti-body residues will correspond to those of the parental FR and CDR sequences. By "hybridizing" is meant forming base pairs with a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of astringency. (See, for example, Wahl and Berger (1987) Methods Enzymol .152: 399; Ki mel, Methods Enzymol. 152: 507, 1987). For example, the concentration of astringent salt will normally be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate and even more preferably less than about 250 M NaCl and trisodium citrate 25 mM. Low stringency hybridization can be obtained in the absence of an organic solvent, for example, formamide, whereas high stringency hybridization can be obtained in the presence of formamide at least 35%, and more preferably at least about 50% formamide. Stringent temperature conditions will typically include temperatures of at least about 30 ° C, more preferably of at least about 37 ° C and even more preferably of at least about 42 ° C. Additional variable parameters, such as hybridization time, detergent concentration, for example, sodium dodecyl sulfate (SDS) and the inclusion or exclusion of supporting DNA, are well known to those skilled in the art. When necessary, the different levels of astringency are achieved by combining these different conditions. In a preferred embodiment, hybridization will be performed at 30 ° C in 750 mM NaCl, 75 mM trisodium citrate and 1% SDS. In a more preferred embodiment, hybridization will be carried out at 37 ° C in 500 M NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide and denatured salmon sperm DNA (DNases) at 100 mg / ml . In a more preferred embodiment, the hybridization will be carried out at 42 ° C in 250 mM NaCl, 25 M trisodium citrate, 1% SDS, 50% formamide and 200 mg / ml DNAs. For the specialists in the matter, useful variations of these conditions will be evident. For most applications, the washing steps that follow hybridization will also vary in astringency. The conditions of wash stringency can be defined by the salt concentration and by the temperature. As indicated above, wash stringency can be increased by reducing the salt concentration or increasing the temperature. For example, an astringent salt concentration for the washing steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and even more preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions for the washing steps will normally include a temperature of at least about 25 ° C, more preferably of at least about 42 ° C and even more preferably of at least about 68 ° C. In a preferred embodiment, the washing steps will be carried out at 25 ° C in 30 mM NaCl, 3 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing steps will be carried out at 42 ° C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing steps will be carried out at 68 ° C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. Other variations of these conditions will be evident to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Nati, Acad. Sci., United States 72: 3961, 1975); Ausubel and collaborators (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By "intrauterine growth retardation (IUGR)" is meant a syndrome that results in a birth weight that is less than 10 percent of the fetal weight foreseen for the gestational age of the fetus. The current World Health Organization criteria for a low birth weight is a weight less than 2,500 g (5 lbs, 8 oz) or below the 10 percent percentile for gestational age according to the tables of United States weight at birth for gestational age by race, parity, and sex of the child (Zhang and Bowes, Obstet, Gynecol, 86: 200-208, 1995). These babies with low birth weight are also called "small for gestational age (SGA)". Pre-eclampsia is a condition known to be associated with IURG or SGA. "Measurement system" means a measurement. A measurement system may be used, for example, to compare the levels of a polypeptide or nucleic acid molecule of interest. Examples of suitable measurement systems include, but are not limited to, mathematical formulas or algorithms, such as relationships. The measurement system to be used is the one that best discriminates between sFlt-1, VEGF or PIGF levels in a subject who has pre-eclampsia or eclampsia and a normal control subject. Depending on the measurement system used, the diagnosis indicator of eclampsia or pre-eclampsia may be significantly above or below a reference value (eg, of a control subject who does not have pre-eclampsia or eclampsia) . The level of sFlt-1 is measured by measuring the amount of free, bound (ie, bound to growth factor) or total (bound + free) sFlt-1. The levels of VEGF or PIGF are determined by measuring the amount of free PIGF or free VEGF (ie, not bound to sFlt-1). An illustrative measurement system is [sFlt-1 / (VEGF + PIGF)], also called the anti-angiogenic index of pre-eclampsia (PAAI). By "anti-angiogenesis index of pre-eclampsia (PAAI)" is meant the ratio of sFlt-1 / VEGF + PIGF used as an indicator of anti-angiogenic activity. A PAAI greater than 20 is considered indicative of pre-eclampsia or at risk of pre-eclampsia. By "operably linked" it is meant that a gene and one or more regulatory sequences are connected in such a way that expression of the gene is allowed when the appropriate molecules (eg, transcription activating proteins) bind to the regulatory sequences. By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the treated animal while maintaining the therapeutic properties of the compound with which it is administered. An illustrative pharmaceutically acceptable carrier is saline physiological solution. Other physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (20th edition), A. Gennaro, editor, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania, United States. By "placental growth factor (PlGF)" is meant a mammalian growth factor that is homologous to the protein defined by accession number of GenBank P49763 and having biological activity of PlGF. PIGF is a glycosylated homodimer that belongs to the VEGF family and can be found in two different isoforms by means of alternative splicing mechanisms. PIGF is expressed by cyto- and syncytiotrophoblasts of the placenta and the biological activities of PIGF include induction of proliferation, migration and activation of endothelial cells, particularly trophoblast cells. By "pre-eclampsia" is meant the multisystem disorder that is characterized by hypertension with proteinuria or edema, or both, glomerular dysfunction, cerebral edema, hepatic edema or coagulation abnormalities due to pregnancy or the influence of a recent pregnancy. Pre-eclampsia usually occurs after the twentieth week of gestation. Pre-eclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (BP) > 140 mm Hg and a diastolic BP > 90 mm Hg after 20 weeks of gestation (measures usually twice, separated by a period of 4-168 hours), (2) new onset proteinuria (1 + by test strips in urinalysis,> 300 mg of protein in urine collected for 24 hours, or a single random urine sample that has a protein / creatinine ratio >; 0.3), and (3) resolution of hypertension and proteinuria at 12 weeks after delivery. Severe pre-eclampsia is generally defined as (1) a diastolic BP > 110 mm Hg (usually measured twice, separated by a period of 4-168 hours) or (2) proteinuria characterized by a measure of 3.5 g or more protein in urine collected over 24 hours or two random urine samples with a level of protein of at least 3+ measured by test strips. In pre-eclampsia, hypertension and proteinuria usually appear with an interval of seven days.

In severe pre-eclampsia, severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevation of liver enzymes and low platelet levels) or eclampsia may appear simultaneously or only one symptom at a time. Occasionally, severe pre-eclampsia can lead to the development of attacks. This severe form of syndrome is called "eclampsia." Eclampsia may also include dysfunction or injury to various organs or tissues such as the liver (eg, hepatocellular injury, periportal necrosis) and the central nervous system (eg, cerebral edema and cerebral hemorrhage). It is believed that the etiology of the attacks is secondary to the development of cerebral edema and focal spasm of small blood vessels of the kidney. By "protein" or "polypeptide" or "polypeptide fragment" is meant any chain of more than two amino acids, independently of post-translational modification (eg, glycosylation or phosphorylation), which constitutes all or part of a polypeptide or natural peptide, or which constitutes a non-natural polypeptide or peptide. By "reduced or inhibited" is meant the ability to produce a total reduction preferably of 20% or more, more preferably 50% or more and even more preferably 65% or more of the level of protein or nucleic acid, detected by the assays mentioned above (see "expression") compared to samples not treated with oligomers of anti-sense nucleobabs or dsRNA used for RNA interference. By "small interfering RNA (siRNA)" is meant an isolated dsRNA molecule, preferably with a length greater than 10 nucleotides, more preferably longer than 15 nucleotides, and even more preferably with a length greater than 19 nucleotides that is used to identify the target gene or mRNA to be degraded. A range of 19-25 nucleotides is the most preferred size for siRNAs. The siRNAs can also include short hairpin RNAs in which the two strands of a siRNA duplex are included within a single RNA molecule. The siRNA includes any form of dsRNA (proteolytically cleaved products of a larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA and recombinantly produced RNA) as well as altered RNA that differs from natural RNA by addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include the addition of a non-nucleotide material, such as the ends of the RNA of 21 to 23 nt or internally (in one or more nucleotides of the RNA). In a preferred embodiment, the RNA molecules contain a 3 'hydroxyl group. The nucleotides in the RNA molecules of the present invention may also comprise non-conventional nucleotides, including unnatural nucleotides or deoxy-ribonucleotides. Collectively, all these altered RNAs are called RNA analogues. The siRNAs of the present invention need only be sufficiently similar to natural RNA to have the ability to mediate RNA interference (RNAi). As used herein, "RNAi" refers to the ATP-directed directed cleavage and degradation of a specific mRNA molecule by introducing small interfering RNA or dsRNA into a cell or an organism. As used herein, "mediated RNAi" refers to the ability to distinguish or identify the RNA to be degraded. By "soluble Flt-1 (sFlt-1)" (also known as sVEGF-Rl) is meant the soluble form of the Flt-1 receptor, which is homologous to the protein defined by GenBank accession number U01134, and which has biological activity of sFlt-1. The biological activity of a sFlt-1 polypeptide can be assayed using any conventional method, for example, by testing the binding of sFlt-1 to VEGF. sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-1 can bind to VEGF and PIGF with high affinity, but it can not induce proliferation or angiogenesis and is therefore functionally different from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical endothelial cells and subsequently demonstrated to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any member or isofor of the sFlt-1 family. By "specifically binds" is meant a compound or anti-body which recognizes and binds to a polypeptide of the invention, but which does not substantially recognize or bind to other molecules in a sample, eg, a biological sample, which includes naturally a polypeptide of the invention. In one example, an anti-body that binds specifically to sFlt-1 does not bind to Flt-1. By "subject" is meant a mammal, including, but not limited to, a human being or a non-human mammal such as a cow, horse, dog, sheep or cat. Included in this definition are pregnant mammals, post-partum and non-pregnant. By "substantially identical" is meant an amino acid sequence that differs only by conservative amino acid substitutions, for example, substitution of one amino acid by another of the same class (eg, glycine by valine, lysine by arginine, etc.) or by one or more non-conservative substitutions, deletions or insertions located at positions of the amino acid sequence that do not destroy the function of the protein. Preferably, the amino acid sequence has a homology of at least 70%, more preferably at least about 80% and even more preferably at least about 90%, with another amino acid sequence. The methods to determine the identity are available in computer programs that can be acquired by the public. Methods of computer programs for determining identity between two sequences include, but are not limited to, the CGC software package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215: 403 (1990) The well-known Smith Waterman Algorithm can also be used to determine identity.The BLAST program is available to the public at NCB1 and other sources (BLAST Manual, Altschul, et al., NCB1 NLM NIH, Bethesda, Maryland 20894, United States; BLAST 2.0 at http: // ww. ncbi. nlm. nih gov / blast /). These software programs compare similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. By "symptoms of pre-eclampsia" is meant any of the following: (1) a systolic blood pressure (BP) > 140 mm Hg and a diastolic BP > 90 mm Hg after 20 weeks of gestation, (2) new start proteinuria (1 + per dipstick in urinalysis,> 300 mg of protein in urine collected for 24 hours, or a protein / creatinine ratio in random urine > 0.3) and (3) resolution of hypertension and proteinuria at 12 weeks after delivery. Symptoms of pre-eclampsia may also include renal dysfunction and endotheliosis or globular hypertrophy. By "symptoms of eclampsia" is meant the development of any of the following symptoms due to pregnancy or the influence of a recent pregnancy: seizures, coma, thrombocytopenia, hepatic edema, pulmonary edema and cerebral edema. By "therapeutic amount" is meant an amount that when administered to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a qualitative or quantitative reduction in the symptoms of pre-eclampsia or eclampsia as described herein. A "therapeutic amount" can also mean an amount that when administered to a patient suffering from pre-eclampsia or eclampsia is sufficient to produce a reduction in the expression levels of sFlt-1 or an increase in the expression levels of VEGF or PIGF as measured by the assays described in this document. By "treatment" is meant the administration of a compound or a pharmaceutical composition for prophylactic and / or therapeutic purposes. To "treat a disease" or use for "therapeutic treatment" refers to the administration of a treatment to a subject already suffering from a disease to improve the condition of the subject. Preferably, the subject has been diagnosed with pre-eclampsia or eclampsia based on the identification of any of the characteristic symptoms described below or through the use of the diagnostic methods described herein. To "prevent a disease" refers to a prophylactic treatment of a subject who is not yet ill, but who is susceptible or at risk of developing a particular disease in another way. Preferably, it is determined that a subject is at risk of developing pre-eclampsia or eclampsia using the diagnostic methods described herein. Thus, in the claims and embodiments, the treatment is administration to a mammal for therapeutic or prophylactic purposes. By "trophoblast" is meant the layer of mesoectodermal cells that cover the blastocyst that erodes the uterine mucosa and through which the embryo receives nutrition from the mother; the cells contribute to the formation of the placenta. By "vascular endothelial growth factor (VEGF) "is meant a mammalian growth factor that is homologous to the growth factor defined in the US patents ,332,671; 5,240,848; 5,194,596; and Charnock-Jones and collaborators . { Biol. Reproduction, 48: 1120-1128, 1993) and has biological activity of VEGF. VEGF exists as a glycosylated homodimer and includes at least four differently linked isoforms. The biological activity of native VEGF includes the promotion of the selective growth of vascular endothelial cells or umbilical vein endothelial cells and the induction of angiogenesis. As used herein VEGF includes any member or isoform of the VEGF family (eg, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF165, or VEGF 121). Preferably, VEGF is the isoform VEGF121 or VEGF165 (Tischer et al, J. "Biol. Chem. 266, 11947-11954, 1991; Neufed et al. Cancer Metastasis 15: 153-158, 1996), which is described in US Pat. 6,447,768; 5,219,739; and 5,194,596, incorporated herein by reference, Also included are mutant forms of VEGF such as VEGF with selectivity of KDR and VEGF with Flt selectivity described in Gille et al. (J. Biol. Chem. 276: 3222-3230, 2001) Although human VEGF is preferred, the invention is not limited to human forms and may include other animal forms of VEGF (eg, mouse, dog or chicken). DNA, normally derived from a plasmid or bacteriophage, into which fragments of DNA can be inserted or cloned.A recombinant vector will contain one or more unique restriction sites, and may be able to replicate autonomously in a defined host or organism of vehic ulo in such a way that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, after transfection in a recipient cell, an RNA is expressed. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims. Brief Description of the Drawings Figure 1 shows mRNA and protein expression of sFlt-1 in pre-eclampsia. Figure 1A shows the expression of placental sFlt-1 mRNA from three patients with pre-eclampsia (PI, P2, P3) and three full-term pregnancies at normal pressure (N1, N2, N3) as determined by transfer analysis Northern The major band (7.5 kb) is the full-length Flt-1 mRNA and the smaller and more abundant band (3.4 kb) is the sFlt-1 mRNA alternatively bound. GAPDH is included as a control and the arrowhead indicates 28S RNA. Patients Pl and P2 had severe pre-eclampsia while patient P3 had mild pre-eclampsia. Figure IB is a graph showing the levels of sFlt-1 in the serum of patients with mild pre-eclampsia (mild PE), patients with severe pre-eclampsia (severe PE), and normotensive pregnant women at term (normal). The levels of sFlt-1 were measured by an ELISA performed for sFlt-1 using a commercially available kit (R & D Systems, Minneapolis, Minneapolis, United States). Additional controls included patients with preterm deliveries for other reasons (pre-term) to rule out specific changes in gestational age. The number of patients tested is shown in parenthesis on the X axis. Samples were collected before delivery (t = 0) and 48 hours after delivery (t = 48). Figure 1C is a graph showing the ratios of anti-angiogenesis indices (PAAI = sFlt-l / (VEGF + PIGF) at the time of delivery (t = 0), determined by ELISA for all patients described in the Figure IB Figure 2A-2F are photomicrographs showing the anti-angiogenic effect of excess sFlt-1 in pre-eclampsia Endothelial tube assays were performed using serum from four normal pregnant control women and from four patients with pre-eclampsia. eclampsia: An experiment representative of a normal control and a patient with pre-eclampsia is shown Figures 2A, 2B and 2C show assays performed using serum from a normal patient, while Figures 2D, 2E and 2F show assays performed using serum of a patient with pre-eclampsia In Figure 2A, t = 0, (10% of a normal pregnant woman's term); in Figure 2B, t = 48 (10% serum from a normal pregnant woman 48 hours after delivery); in Figure 2 C, t = 0 + exogenous sFlt-1 (10 ng / ml); in Figure 2D, t = 0 (10% serum of a pre-ecclastic woman before delivery); in Figure 2E, t = 48 (10% serum from a pre-eclastic woman 48 hours after delivery), and in Figure 2F, t = 0 + exogenous VEGF (10 ng / ml) + PIGF (10 ng / ml). ml). The tube test was quantified and the average tube length +/- SEM is displayed in pixel at the bottom of each panel. Figures 3A and 3B are graphs demonstrating that the inhibition of VEGF and PIGF induced vasodilation of renal micro-vessels by sFlt-1. Figure 3A demonstrates that the increase in rat renal arteriolar relaxation responses to sFlt-1 (S), VEGF (V), PIGF (P) was measured at three different doses. V + and P + represent vasodilator responses of the individual reagents in the presence of sFlt-1 at 100 ng / ml. All experiments were performed on 6 different dissected rat kidney micro-vessels and the data are shown as mean +/- SEM. The * represents the statistical significance with p < 0.01 compared to single reagents alone. Figure 3B shows the increase in relaxation responses at physiological doses: VEGF 100 pg / ml (V), PIGF 500 pg / ml (P), sFlt-1 10 ng / ml (S), VEGF (100 pg / ml ) + PIGF 500 pg / ml (V + P) or VEGF (100 pg / ml) + PlGF 500 pg / ml + sFlt-1 10 ng / ml (V + P + S). All experiments were performed on 6 different dissected rat kidney micro-vessels and the data are shown as mean +/- SEM. The * represents the statistical significance with p < 0.05 compared to V + P. Figures 4A and 4B show the induction by sFlt-1 of glomerular endotheliosis. Figure 4A is a photomicrograph showing haematoxylin and eosin staining (H & E) in a capillary occlusion in animals treated with sFlt-1 with enlarged glomeruli and swollen cytoplasm compared to controls. In animals treated with sFlt-1 after periodic acid Schiff staining (PAS), "glomerular endotheliosis" with cytoplasm with bubbles is shown. All optical microscopy photographs were taken at 60 magnifications, with the original magnification. Figure 4B is an electron micrograph of glomeruli treated with sFlt-1 confirming cytoplasmic swelling of endocapillary cells. Immunofluorescence (IF) for fibrin photographs was taken at 40 magnifications and EM photographs were taken at 2,400 magnifications, originally magnified. All figures were reproduced with the same magnification.

Figures 5A-5C show the sFlt-1 levels measured before and after the onset of pre-eclampsia by gestational age. Figure 5A is a graph showing average serum concentrations in pg / ml for normotensive controls (lighter line with white triangles), cases before pre-eclampsia (filled circles) and "end-point" samples of cases after of pre-eclampsia (filled squares) at 4-5 weeks of gestational age before the onset of labor. The parentheses indicate the typical error of the mean. The asterisks indicate significant differences with respect to the control samples within the same window of gestational age after the logarithmic transformation: * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 5B is a graph showing the mean serum concentrations of sFlt-1 in pg / ml for cases before and after the onset of pre-eclampsia at intervals of weeks before pre-eclampsia. PE indicates the arithmetic mean of 43 end-point samples (obtained during or after the onset of pre-eclampsia). The mean gestational age (days) is indicated in parentheses below each time interval. The horizontal line indicates the level in the samples of the end point. Vertical lines mark the period < 5 weeks before pre-eclampsia. Figure 5C is a graph showing mean serum concentrations of sFlt-1 in pg / ml per gestational age window for normotensive controls and cases before pre-eclampsia, after excluding samples obtained in the 5 weeks after beginning of pre-eclampsia. There are no significant differences. Figures 6A-6C show PIGF levels before and after pre-eclampsia by gestational age. Figure 6A is a graph showing the PIGF levels in all samples obtained before delivery. The parentheses indicate the typical error of the mean. The asterisks indicate significant differences with respect to the control samples within the same interval after the logarithmic transformation: ** p < 0.01, *** p < 0.001. Figure 6B is a graph showing the mean serum concentrations of PIGF in pg / ml for cases before and after the onset of pre-eclampsia within weeks intervals before pre-eclampsia. PE indicates the arithmetic mean of 43 end-point samples (obtained during or after the onset of pre-eclampsia). The mean gestational age (days) is indicated in parentheses below each time interval. The horizontal line indicates the level in the samples of the end point. Vertical lines mark the period < 5 weeks before pre-eclampsia. Figure 6C is a graph showing mean serum concentrations of PlGF in pg / ml per window of gestational age for normotensive controls and cases of onset of pre-eclampsia. Figures 7A and 7B show the levels of sFlt-1 and PIGF by state and severity of pre-eclampsia. Figure 7A is a graph showing the average arithmetic serum concentrations of sFlt-1 (black bars) and PIGF (white bars) at 23-32 weeks of gestation in controls and cases (before the start of treatment). clinical disease) with mild pre-eclampsia, severe pre-eclampsia, pre-eclampsia with onset < 37 weeks, pre-eclampsia with a small baby for gestational age (SGA), and pre-eclampsia with onset < 34 weeks The sample numbers are recorded below each pair of columns. Adjustment for gestational age and body mass index resulted in minor changes without affecting the level of meaning. Figure 7B is a graph showing the mean arithmetic serum concentrations of sFlt-1 (black bars) and PIGF (white bars) at 33-41 weeks of gestation in controls and cases (before the onset of clinical disease) with mild pre-eclampsia, severe pre-eclampsia, pre-eclampsia with onset < 37 weeks and pre-eclampsia with a SGA baby. The sample numbers are recorded below each pair of columns. Adjustment for gestational age and body mass index resulted in minor changes without affecting the level of meaning. Figure 8 is a self-radiogram showing the expression of flt, sFlt-1, and variants or related fragments in PBMCs isolated from normal patients and with pre-eclampsia. Protein lysates were analyzed by Western blotting using an anti-body that recognizes the N-terminus of the Flt-1 protein. Figures 9A to 9D are graphs showing the concentration of urinary PIGF by intervals of gestational age. Figure 9A is a graph showing the average concentrations of PIGF before and after the onset of clinical pre-eclampsia according to gestational age. Bars I represent standard errors. Figure 9B is a graph showing the average PIGF expressed as pg per mg of creatinine before and after the onset of clinical pre-eclampsia. Bars I represent standard errors. Figure 9C is a graph showing the average concentrations of PIGF before and after the onset of clinical pre-eclampsia, using only random urine samples. Bars I represent standard errors. Figure 10 is a graph showing the mean concentrations of PIGF according to the condition and severity of pre-eclampsia, before and after normalization by creatinine. The concentrations of PIGF and pg per mg of creatinine are shown at 21-32 weeks of gestation in controls and in women who later had clinical pre-eclampsia (PE) according to whether they had pre-eclampsia tenuous, severe pre-eclampsia, pre-eclampsia with onset at less than 37 weeks gestation, pre-eclampsia and a small product for gestational age (SGA), or pre-eclampsia with an appearance less than 34 weeks gestation. The samples of women in whom pre-eclampsia developed were obtained before the onset of the clinical disease. The given values P are for comparison with the samples of the controls. Bars I represent standard errors. Figure 11 is a graph showing a longitudinal trace of placental growth factor concentrations within individual women by gestational age. Figures 12A and 12B are graphs showing traces of urinary PIGF concentrations and ratios of sFlt-1 to PIGF in serum at 21-32 weeks per day of gestation. The values were obtained from paired samples of urine and serum obtained from 20 weeks before the development of pre-eclampsia at less than 37 weeks of gestation and from 69 normo-tensive controls. Figure 12A shows the concentrations of urinary PIGF. Figure 12B shows serum ratios of sFlt-1 to PIGF. Figure 13 is a graph showing the mean urinary concentrations of the placental growth factor (PIGF) in normo-tensive women with products that were not born small for gestational age (SGA), normo-tensive women with SGA products, women with gestational hypertension, and women in whom pre-eclampsia developed before 37 weeks of gestation. The concentrations of urinary PIGF in pg / ml and in pg per mg of creatinine are shown at 21-32 weeks of gestation in normo-tensive women whose products were not born small for gestational age (NT-SGA), normo- Tensives with SGA products (NT + SGA), women who later developed gestational hypertension (GH), and women who subsequently developed pre-eclampsia before 37 weeks of gestation (PE <37 weeks). Samples of women in whom gestational hypertension or pre-eclampsia developed were obtained before the onset of clinical disease. Mean gestational age at sample collection was similar in all groups. N indicates the number of samples. The given values P are for the comparisons with the samples of the controls (NT-SGA). Bars I represent standard errors. Detailed Description It has been found that sFlt-1 levels are elevated in blood serum samples taken from pre-eclamptic women. sFlt-1 binds to VEGF and PIGF with high affinity and blocks the mitogenic and angiogenic activity of these growth factors. In this way, sFlt-1 is an excellent diagnostic marker for pre-eclampsia and VEGF and PIGF can be used to treat pre-eclampsia. In addition, therapeutic agents have been discovered which interfere with the binding of purified sFlt-1 to VEGF or PIGF, or agents that increase levels of biologically active VEGF or PIGF, and which can be used to treat or prevent pre-eclampsia or eclampsia in a subject. Such agents include, but are not limited to, anti-bodies against sFlt-1, oligonucleotides for anti-sense or RNAi that reduce the levels of sFlt-1, compounds that increase the levels of VEGF or PIGF, and small molecules that bind to sFlt. -1 and block the binding site to the growth factor. The invention also characterizes methods for measuring levels of growth factors; The methods can be used as diagnostic tools for early detection of pre-eclampsia or an increased risk of developing pre-eclampsia or eclampsia. Although the detailed description presented herein refers specifically to sFlt-1, VEGF or PIGF it will be apparent to a person skilled in the art that the detailed description can also be applied to sFlt-1, VEGF or members of the PIGF family, isoforms and / or variants, and to growth factors that have been shown to bind sFlt-1. The following examples are to illustrate the invention and should not be considered as limiting. Example 1. Increase of sFlt-1 and Protein mRNA Levels in Pregnant Women with Pre-Eclampsia In an attempt to identify new secreted factors that play a pathological role in pre-eclampsia, a profile of gene expression has been made of placental tissue from women with and without pre-eclampsia using Affymetrix U95A micro-array chips. It has been found that the gene for sFlt-1 was positively regulated in women with pre-eclampsia. To confirm the positive regulation of sFlt-1 in pre-eclampsia, Northern blots were performed to analyze placental sFlt-1 mRNA levels (Figure 1A) and ELISA assays to measure serum protein levels of sFlt-1 ( Figure IB) in pregnant women pre-eclamptic compared to normotensive pregnant women. Pre-eclampsia was defined as (1) a systolic blood pressure (BP) > 140 mm Hg and a diastolic BP > 90 mm Hg after 20 weeks of gestation, (2) new start proteinuria (1 + by urine test strips,> 300 mg of protein in urine collected for 24 hours, or a protein / creatinine ratio in urine random >; 0.3), and (3) resolution of hypertension and proteinuria at twelve weeks after delivery. Patients with hypertension, proteinuria or underlying kidney disease were excluded. Patients were divided into mild and severe pre-eclampsia based on the presence or absence of nephritic interval proteinuria (> 3 g of protein in urine collected for 24 hours or a protein / creatinine ratio in urine greater than 3.0). The mean protein / creatinine ratios in urine in the group of mild pre-eclampsia were 0.94 +/- 0.2 and in the group of severe pre-eclampsia they were 7.8 +/- 2.1. The mean gestational ages of the various groups were as follows: normal 38.8 +/- 0.2 weeks, mild pre-eclampsia 34 +/- 1.2 weeks, severe pre-eclampsia 31.3 +/- 0.6 weeks, and pre-term 29.5 +/- 2.0 weeks Placental samples were obtained immediately after delivery. Four random samples were taken from each placenta, placed in RNA stabilization solution (Ambion, Austin, Texas, United States) and stored at -70 ° C. RNA isolation was performed using a Qiagen RNAeasy Maxi kit (Qiagen, Valencia, California, United States). An increase was detected both in placental sFlt-1 mRNA and in sFlt-1 protein in maternal serum in pregnant pre-eclamptic women compared to normotensive pregnant women. The mean serum level of sFlt-1 was almost four times higher in patients with severe pre-eclampsia compared to normal control pregnant women. To exclude the possibility that this effect was due to the previous gestational age of pre-ecclastic cases, sFlt-1 levels were also measured in normotensive women with similar gestational age and with preterm delivery for other reasons (gestational ages 23-36 weeks) and no significant differences were found in this group compared to normotensive term pregnancies. The probes used for the Northern blots were obtained by PCR and included a 500 bp fragment in the human Flt-1 cDNA coding region of pUClld, and a GAPDH cDNA that was used as the normalization control. In normal pregnancy there is a balance between pro- and anti-angiogenic factors secreted by the placenta that is necessary for an adequate placental development. It was hypothesized that in pre-eclampsia, the increase in the production of sFlt-1 and the reduction in the production of VEGF and PIGF displaces the balance in favor or against angiogenesis. To treat the net anti-angiogenic activity, the serum levels of VEGF and PIGF were measured and it was found that the serum levels of PIGF and VEGF were lower in patients with pre-eclampsia compared to the normal control patients (mean PlGF, 235.3 +/- 45.3 pg / ml versus 464 +/- 116.6 pg / ml) as described (Tidwell et al., Am. J. Obstet, Gynecol., 184: 1267-1272, 2001). When sFlt-1 was incorporated, the levels of VEGF and PIGF in an anti-angiogenic index, or PAAI, as an indicator of net anti-angiogenic activity, it was discovered that pre-ecliptic patients could be clearly separated from normal patients and that PAAI seemed to correlate with the severity of pre-eclampsia (Figure 1C). This PAAI can be used as a diagnostic tool for the detection of pre-eclampsia in pregnant women. Example 2. The Serum of Women with Pre-Eclampsia Inhibits Angiogenesis in an In Viral Endothelial Tube Assay It was hypothesized that the excess of circulating sFlt-1 in patients with pre-eclampsia produces an endothelial dysfunction and leads to an anti-endothelial state. angiogenic To solve this, an endothelial tube assay was used as an in vitro model of angiogenesis. Reduced growth factor Matrigel (7 mg / ml, Collaborative Biomedical Products, Bedford, Massachusetts, United States) was placed in wells (100 ml / well) of a pre-cooled 48-well cell culture plate and incubated at 37 ° C. ° C for 25-30 minutes to allow polymerization. Human umbilical vein endothelial cells (30,000 + 300 ml of endothelial basal medium without serum, Clonetics, Walkersvi-lle, Maryland, United States) were treated in passages 3-5 with 10% patient serum, cultured in the wells coated with Matrigel and incubated at 37 ° C for 12-16 hours. The tube formation was then evaluated by means of an inverted phase contrast microscope at 4x magnification (Nikon Corporation, Tokyo, Japan) and analyzed quantitatively (tube area and total length) using the simple PCI image analysis software. The conditions of the tube formation test were adjusted such that normal human umbilical vein endothelial cells would only form tubes in the presence of endogenous growth factors such as VEGF. Under these conditions, it was found that although serum from normotensive women induced endothelial cells to form regular tube-like structures, serum from women with pre-eclampsia inhibited tube formation (Figure 2). Notably, at 48 hours after delivery this anti-angiogenic effect had disappeared, which suggests that the inhibition of the tubes detected with the serum of patients with pre-eclampsia was probably due to a circulating factor released by the placenta. When sFlt-1 was added to normotensive serum at doses similar to those found in patients with pre-eclampsia, tube formation did not occur, mimicking the effects observed with the serum of pre-eclamptic women. When exogenous VEGF and PlGF were added to the assay using pre-eclastic serum, tube formation was restored (Figure 2). For these tests, recombinant human VEGF, human PIGF and human Flt-lFc were used. These results suggested that the anti-angiogenic properties of pre-ecliptic serum were due to antagonism of VEGF and PIGF by endogenous sFlt-1. These results also suggested that the addition of purified VEGF and / or PlGF can reverse or mitigate the pre-eclastic state and can be used therapeutically. Example 3. sFlt-1 Inhibits VEGF-Induced Vessel-Dilatation and PIGF from Kidney Micro-Vessels The causative role of sFlt-1 in vasoconstriction was determined using an experiment of microvascular vascular reactivity in vi tro. Micro-vascular reactivity experiments were performed as previously described using rat renal micro-vessels (Sato et al., J. Surg. Res., 90: 138-143, 2000). Kidney artery microvessels (70-170 mm internal diameter) were dissected from rat kidneys using a 10X magnification dissection microscope (Olympus Optical, Tokyo, Japan). The micro-vessels were placed in an isolated micro-vessel chamber, cannulated with double glass micro-pipettes measuring 30-60 mm in diameter and fixed with a 10-0 nylon mono-filament suture (? thicon, Somerville, New Jersey). A buffer solution of oxygenated Krebs (95% oxygen and 5% carbon dioxide) was circulated continuously at 37 ° C through the vessel chamber and a reservoir containing a total of 100 ml of the solution. The vessels were pressurized to 40 mm Hg in a non-flow state using a burette manometer filled with Krebs buffer solution. With an inverted microscope (40 x to 200 x; Olympus CK2, Olympus Optical) connected to a video camera, the image of the glass is projected on a black and white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, Vermont, United States) was used to measure the lumen's internal diameter. The measurements were recorded with a register (Graphtec, Irvine, California, United States). The glasses were allowed to bathe in the micro-vessel chamber for at least 30 minutes before any intervention. In all the experimental groups, relaxation responses of the renal micro-vessels were examined after pre-contraction of the micro-vessels with U46619 (thromboxane agonist) at 40-60% of their initial diameter at a distension pressure of 40 mm Hg. Once the steady-state tone was reached, responses to various reagents such as VEGF, PlGF and sFlt-1 were examined. For these tests, recombinant rat VEGF, mouse PlGF and mouse Flt-lFc were used. All drugs were applied extra-luminally. Measurements were made when the response had stabilized (typically 2-3 minutes after administering the drug). In each vessel, one to four interventions were performed. The vessels were washed with a Krebs buffer solution and allowed to equilibrate in Krebs buffer solution without drug for 20-30 minutes between interventions. It was found that sFlt-1 alone did not produce significant vasoconstriction, however it blocked the increase in dose response in vasodilation induced by VEGF or PIGF (Figure 3A). In addition, it was discovered that VEGF and PIGF, at the physiological levels observed in pregnancy, induced a significant dose-dependent arteriolar relaxation, and this effect was blocked by the addition of 10 ng / ml of sFlt-1, a concentration observed in women with severe pre-eclampsia (Figure 3B). This result suggested that circulating sFlt-1 in patients with pre-eclampsia may oppose vasorelaxation, thus contributing to hypertension. These results confirm the conclusion that sFlt-1 is responsible for many of the clinical and pathological symptoms of pre-eclampsia, including hypertension. The inhibition of sFlt-1, through the use of targeted anti-bodies, for example, could reverse the effects of the protein in pre-eclamptic women and such inhibitors of sFlt-1 could potentially be used as a therapeutic agent. Example 4. Effects of sFlt-1 on an Animal Model of Pre-Eclampsia Based on the above results, it was hypothesized that the addition of exogenous sFlt-1 would produce hypertension and proteinuria in an animal model. It has been shown that adenoviruses expressing sFlt-1 produce sustained systemic levels of sFlt-1 associated with significant anti-tumor activity (Kuo et al., Proc. Nati, Acad. Sci. USA, 98: 4605-4610, 2001 ). This recombinant adenovirus encoding murine sFlt-1 was injected into the tail vein of pregnant Sprague-Dawley rats on day 8-9 of pregnancy. As controls, adenoviruses encoding murine Fe and sFlkl-Fc (Flkl electro-domain fusion protein from mouse VEGF receptor and Fe protein) were used in equivalent doses. It has been shown that Flkl binds to VEGF, but not to PIGF. Therefore, sFlk-lFc was chosen as a control to help discriminate between the anti-VEGF activity and the anti-PlGF activity of sFlt-1. 1 x 109 pfu of Ad Fe, Ad sFlt-1, or Ad sFlk-lFc were injected into pregnant and non-pregnant Sprague-Dawley rats by injections into the tail vein. These adenoviruses have been previously described (Kuo et al., Supra) and were generated at the Harvard Vector Core Laboratory. In pregnant rats, adenoviruses were injected on day 8-9 of pregnancy (in the first part of the second trimester) and blood pressure was measured on day 16-17 of pregnancy (in the first part of the third trimester). In nonpregnant animals, BP was measured on day 8 after adenovirus injection. BP were measured in the rats after anesthesia with sodium pentobarbital (60 mg / kg, i.p.). The carotid artery was isolated and cannulated with a 3-Fr high-fidelity micro-tip catheter connected to a pressure transducer (Millar Instruments, Houston, Texas, United States). The Millar Mikro-Tip catheter was advanced into the artery to record blood pressure. Blood pressure and heart rate were recorded in a register (model 56-1X 40-006158, Gould Instrument Systems, Cleveland, Ohio, United States) and averaged over a period of 10 minutes. Blood, tissue and urine samples were then collected before euthanasia. Albumin in urine was measured by a conventional test strip and quantified by a competitive enzyme bound immunoassay (ELISA) as described elsewhere in this document (Cohen et al., Kidney Intl., 45: 1673- 1679, 1994). Urine creatinine was measured by a picric acid colorimetric kit (Sigma, St. Louis, Missouri, United States). Intra-arterial blood pressures were measured in the first part of the third trimester of pregnancy to mimic the natural pathology of pre-eclampsia. These experiments were also performed on non-pregnant female Sprague-Dawley rats to determine whether the effects of sFlt-1 are direct or indirect through their effects on the placenta. It was confirmed by means of Western blot analysis that the systemic levels of sFlt-1 at day of blood pressure measurement were in the range of 25-350 ng / ml in the various animals treated with sFlt-1 on the day of the measurement of BP. Table 1 shows the blood pressure and proteinuria in the different experimental groups.

Table 1. Blood pressure and proteinuria in N MAP rats (mm U Hg ratio) alb: cr Fe (P) 5 75.6 ± 62 ± 21 11.1 sFlt-1 4 109.0 ± 6923 ± 658 * (P) 19.3 * sFlk-lFc 4 72.8 ± 50 ± 32 (P) 14.7 Fe (NP) 5 89.3 ± 5.7 138 ± 78 sFlt-1 6 117.9 ± 12947 ± 2776 * (NP) 12.9 * sFlk-lFc 4 137.3 ± 2269 ± 669 * (NP) 2.3 * Pregnant (P) and non-pregnant (NP) rats were administered adenoviruses expressing Fe (control), sFlt-1 or sFlk-lFc protein. Median arterial blood pressure (MAP = diastolic + 1/3 pulse pressure in mm Hg) ± SEM and the albumin: Cr ratio in urine (mg albumin per gram of creatinine) ± SEM were measured eight days later, corresponding to of the first part of the third trimester in pregnant rats. N = the number of animals in each experimental group. The * represents statistical significance with p < 0.01 compared to the control group (Fe). Pregnant rats treated with sFlt-1 had significant hypertension and a significant albuminuria of nephrotic range compared to Fe controls. Nonpregnant rats given sFlt-1 also developed hypertension and proteinuria. Notably, nonpregnant rats treated with sFlk-Fc developed hypertension and proteinuria, while pregnant rats treated with sFlk-Fc did not. Therefore, in pregnancy, VEGF antagonism alone is insufficient to produce pre-eclampsia, possibly due to the presence of high levels of PIGF. In the non-pregnant state, in which PIGF is virtually absent, VEGF antagonism alone is insufficient to break the pro / anti-angiogenic balance and produce renal pathologies similar to those associated with pre-eclampsia. Various staining techniques were used to examine the renal injury that was observed in all rats treated with sFlt-1 (Figure 4). Kidneys collected from the rats were fixed in Bouin's solution, cut into sections and stained with H & E and PAS dyes. For electron microscopy, renal tissue was fixed in glutaraldehyde, was included in a mixture of araldite-epon and ultrafine renal sections (1 μm) were cut, stained with toluene blue and evaluated using Zeiss EM 10 at various magnifications . Immuno-fluorescence for fibrin deposits within the glomeruli was performed using anti-fibrin polyclonal anti-body (ICN, Switzerland). Global and diffuse glomerular endotheliosis was the renal lesion universally observed in rats treated with sFlt-1. A glomerular increase was detected with occlusion of the capillary loops due to swelling and hypertrophy of the endocapillary cells. In the glomerular epithelial cells, numerous apparent protein resorption droplets were observed. No segmental glomerulosclerosis was observed. "Asymmetric double contours" and focal fibrin deposition were seen within the glomeruli. This finding of fibrin deposition in the absence of significant mesangial interposition is similar to that which has been described as typical of the pre-partum phase of the human disease (Kincaid-Smith, Am. J. Kidney Dis., 17: 144 -148, 1991). The immuno-fluorescence for fibrin showed foci of fibrin deposition within the glomeruli of the animals treated with sFlt-1, but not of the animals treated with Fe. The nonpregnant rats treated with sFlkl developed the same lesion. In fact, when sFlkl was used at the same levels as sFlt-1, kidney injury was more severe in nonpregnant rats, since there are fewer circulating pro-angiogenic molecules to antagonize sFlt-1. These results suggested that elevated levels of sFlt-1 may be responsible for the glomerular endotheliosis associated with pre-eclampsia, but this effect was independent of the placenta, since glomerular changes were detected in non-pregnant rats as well as in pregnant rats. These results also suggested that antagonism of VEGF and PIGF is important in the pathology of pre-eclampsia since hypertension and proteinuria occurred in non-pregnant mice treated with sFlk-1 but not in pregnant mice treated with sFlk-1 when the levels of PIGF are high. The animal model created in this document could be used as an experimental model to test new therapeutical compounds. Using this animal model, the efficacy of potential therapeutic compounds as well as pharmacology and toxicity can be studied. Example 5. Effects of sFlt-1 on an Animal Model of Pre-Eclampsia Based on the above results, it was hypothesized that the addition of exogenous sFlt-1 would produce hypertension and proteinuria in an animal model. It has been shown that adenoviruses expressing sFlt-1 produce sustained systemic levels of sFlt-1 associated with significant anti-tumor activity (Kuo et al., Proc. Nati, Acad. Sci. USA, 98: 4605-4610, 2001 ). This recombinant adenovirus encoding murine sFlt-1 was injected into the tail vein of pregnant Sprague-Dawley rats on day 8-9 of pregnancy. As controls, adenoviruses encoding murine Fe and sFlkl-Fc (Flkl electro-domain fusion protein from mouse VEGF receptor and Fe protein) were used in equivalent doses. It has been shown that Flkl binds to VEGF, but not to PlGF. Therefore, sFlk-lFc was chosen as a control to help discriminate between the anti-VEGF activity and the anti-PlGF activity of sFlt-1. 1 x 109 pfu of Ad Fe, Ad sFlt-1, or Ad sFlk-lFc were injected into pregnant and non-pregnant Sprague-Dawley rats by injections into the tail vein. These adenoviruses have been previously described (Kuo et al., Supra) and were generated at the Harvard Vector Core Laboratory. In pregnant rats, adenoviruses were injected on day 8-9 of pregnancy (in the first part of the second trimester) and blood pressure was measured on day 16-17 of pregnancy (in the first part of the third trimester). In nonpregnant animals, BP was measured on day 8 after adenovirus injection. BP were measured in the rats after anesthesia with sodium pentobarbital (60 mg / kg, i.p.). The carotid artery was isolated and cannulated with a 3-Fr high-fidelity micro-tip catheter connected to a pressure transducer (Millar Instruments, Houston, Texas, United States). The Millar Mikro-Tip catheter was advanced into the artery to record blood pressure. Blood pressure and heart rate were recorded in a register (model 56-1X 40-006158, Gould Instrument Systems, Cleveland, Ohio, United States) and averaged over a period of 10 minutes. Blood, tissue and urine samples were then collected before euthanasia. Albumin in urine was measured by a conventional test strip and quantified by a competitive enzyme bound immunoassay (ELISA) as described elsewhere in this document (Cohen et al., Kidney Intl., 45: 1673- 1679, 1994). Urine creatinine was measured by a picric acid colorimetric kit (Sigma, St. Louis, Missouri, United States). Intra-arterial blood pressures were measured in the first part of the third trimester of pregnancy to mimic the natural pathology of pre-eclampsia. These experiments were also performed on non-pregnant female Sprague-Dawley rats to determine whether the effects of sFlt-1 are direct or indirect through their effects on the placenta. It was confirmed by means of Western blot analysis that the systemic levels of sFlt-1 at day of blood pressure measurement were in the range of 25-350 ng / ml in the various animals treated with sFlt-1 on the day of the measurement of BP. Table 1 shows the blood pressure and proteinuria in the different experimental groups. Table 1. Blood pressure and proteinuria in N MAP rats (mm U Hg ratio) alb: cr Fe (P) 5 75.6 ± 62 ± 21 11.1 sFlt-1 4 109.0 ± 6923 ± 658 * (P) 19.3 * sFlk-lFc 4 72.8 ± 50 ± 32 (P) 14.7 Fe (NP) 5 89.3 ± 5.7 138 ± 78 sFlt-1 6 117.9 ± 12947 ± 2776 * (NP) 12.9 * sFlk-lFc 4 137.3 ± 2269 ± 669 * (NP) 2.3 * Pregnant (P) and non-pregnant (NP) rats were administered adenoviruses expressing Fe (control), sFlt-1 or sFlk-lFc protein. Mean arterial blood pressure (MAP = diastolic + 1/3 pulse pressure in mm Hg) + SEM and the ratio of albumin: Cr in urine (mg of albumin per gram of creatinine) ± SEM were measured eight days later, corresponding to the first part of the third trimester in pregnant rats. N = the number of animals in each experimental group. The * represents statistical significance with p < 0.01 compared to the control group (Fe). Pregnant rats treated with sFlt-1 had significant hypertension and a significant albuminuria of nephrotic range compared to Fe controls. Nonpregnant rats given sFlt-1 also developed hypertension and proteinuria. Notably, nonpregnant rats treated with sFlk-Fc developed hypertension and proteinuria, while pregnant rats treated with sFlk-Fc did not. Therefore, in pregnancy, VEGF antagonism alone is insufficient to produce pre-eclampsia, possibly due to the presence of high levels of PIGF. In the non-pregnant state, in which PlGF is practically absent, antagonism of VEGF alone is insufficient to break the pro / anti-angiogenic balance and produce renal pathologies similar to those associated with pre-eclampsia. Various staining techniques were used to examine the renal injury that was observed in all rats treated with sFlt-1 (Figure 4). Kidneys collected from the rats were fixed in Bouin's solution, cut into sections and stained with H & E and PAS dyes. For the electronic microscopy, the renal tissue was fixed in glutaraldehyde, it was included in a mixture of araldite-epon and ultra thin renal sections (1 μm) were cut, stained with toluene blue and evaluated using Zeiss EM 10 a various increases. Immuno-fluorescence for fibrin deposits within the glomeruli was performed using anti-fibrin polyclonal anti-body (ICN, Switzerland). Global and diffuse glomerular endotheliosis was the renal lesion universally observed in rats treated with sFlt-1. A glomerular increase was detected with occlusion of the capillary loops due to swelling and hypertrophy of the endocapillary cells. In the glomerular epithelial cells, numerous apparent protein resorption droplets were observed. No segmental glomerulosclerosis was observed. "Asymmetric double contours" and focal fibrin deposition were seen within the glomeruli. This finding of fibrin deposition in the absence of significant mesangial interposition is similar to that which has been described as typical of the pre-partum phase of human disease (Kincaid-Smith, Am. J.) Kidney Dis., 17: 144-148, 1991.) Immuno-fluorescence for fibrin showed foci of fibrin deposition within the glomeruli of the animals treated with sFlt-1, but not of the animals treated with Fe. The nonpregnant rats treated with sFlkl developed In fact, when sFlkl was used at the same levels as sFlt-1, the renal lesion was more severe in nonpregnant rats, since there are fewer circulating pro-angiogenic molecules to antagonize sFlt-1. that high levels of sFlt-1 may be responsible for glomerular endotheliosis associated with pre-eclampsia, but this effect was independent of the placenta, since glomerular changes were detected in non-pregnant rats as well as in rats These results also suggested that antagonism of VEGF and PIGF is important in the pathology of pre-eclampsia since hypertension and proteinuria occurred in non-pregnant mice treated with sFlk-1 but not in pregnant mice treated with sFlk- 1 when the PIGF levels are high. The animal model created in this document could be used as an experimental model to test new therapeutic compounds. Using this animal model, the efficacy of potential therapeutic compounds as well as pharmacology and toxicity can be studied. Example 6. Effects of sFlt-1 in an Animal Model of Eclampsia Pregnant rats in the first part of their second trimester of pregnancy are injected with exogenous sFlt-1. The rats are then monitored and tested during the first part of their third trimester for the development of eclampsia. The tests used for the detection of eclampsia can include MRI of the brains of the rats for the development of edema, EEG of the rat brain for the development of attacks and histology of the brains of the rat to determine if endothelial injury has occurred along the hematoenic barrier and the choroid plexus using specific endothelial markers. The animal model created in this document can be used as an experimental model to test new therapeutic compounds. Using this animal model, the efficacy of potential therapeutic compounds as well as pharmacodynamics and toxicity can be studied.

Example 7: The Ratio of PIGF / Creatinine in Urine Serves as Diagnosis of Pre-Eclampsia Urine samples were obtained from 10 women at 16 weeks of gestation (five normal, four with mild pre-eclampsia and one with severe pre-eclampsia) . These samples were provided by Dr. Ravi Thadhani of the Massachusetts General Hospital. The mean ratios of free PIGF / creatinine in urine (pg of PIGF per mg of creatinine) for normal pregnant women were 78 +/- 10.7 and for the four women with mild pre-eclampsia were 33 +/- 5.0 and for The patient with severe pre-eclampsia was 17. In this way, an alteration in the relationship between PIGF and creatinine in urine is useful as a diagnostic indicator of pre-eclampsia in a patient. Example 8: Levels of Protein sFlt-1 and PIGF as a Diagnostic Indicator of Pre-Eclampsia and Eclampsia in Women For this study archived samples of the calcium assay for the prevention of pre-eclampsia were used to analyze the gestational models of sFlt-1 , Free PIGF and free VEGF circulating in normotensive and pre-ecplastic pregnancies. The calcium trial for the prevention of pre-eclampsia, or CPEP, was a double-blind randomized clinical trial conducted during 1992-1995 to evaluate the effects of the daily supplement of 2 g of elemental calcium or placebo on the incidence and severity of pre-eclampsia. -eclampsia (Levine et al., N. Engl. J. Med. 377: 69-76, 1997; Levine et al., Control Clin. Triais 17: 442-469, 1996). Healthy nulliparous women with simple pregnancies between 13 and 21 weeks of gestation were included in 5 participating US medical centers and followed up to 24 hours after delivery using a common protocol and identical data collection forms. At the time of inclusion, all CPEP participants had a blood pressure < 135/85 mm Hg, and none had renal dysfunction or proteinuria. Gestational age was determined by ultrasound examination. Serum samples were obtained from the participants before inclusion in the trial (13-21 weeks), at 26-29 weeks, at 36 weeks if they were still pregnant, and when hypertension or proteinuria was detected. The "endpoint samples" were samples obtained during or after the onset of symptoms and signs of pre-eclampsia, but before delivery as described elsewhere in this document (Levine et al., 1996, supra). The blood samples obtained from the CPEP trial were obtained thanks to the collaboration of Dr. Richard Levine at the NIH. Participants Subjects were selected with complete information on the results, from which serum samples had been obtained at < 22 weeks and they had a man born alive. Of the 4,589 CPEP participants, 253 who lost track, 21 whose pregnancy ended before 20 weeks, 13 absences of maternal or perinatal outcome data, 4 no history of smoking, 9 with hypertension not verified by the groups were excluded. of revision, and another 32 with children stillborn, leaving 4,257 women with adequate information and live births. Among these, 2,156 had male babies. After excluding a woman whose baby had a chromosomal abnormality, 381 with gestational hypertension and 43 without an initial serum sample, 1,731 women remained. Of these, 175 developed pre-eclampsia and 1,556 remained normotensive throughout the pregnancy. As the calcium supplement had no effect on the risk and severity of pre-eclampsia and was not related to the concentrations of pro- and anti-angiogenic molecules, cases and controls were chosen without taking into account the treatment with CPEP. For each case of pre-eclampsia, a normotensive control was selected, equivalent in the inclusion site, gestational age in the collection of the first serum sample (within a week) and freezer storage time at -70 ° C. (in 12 months). 120 equivalent pairs ("cases" and "controls") were randomly selected for the analysis of the 657 serum samples obtained before delivery (Table 2, presented below) The mean gestational age at collection of the first serum sample was of 112.8 and 113.6 days in cases and controls respectively, the average duration of storage in the freezer was 9.35 and 9.39 years.

TABLE 2: Characteristics of cases and controls in the inclusion in the CPEP and of their newborn babies Characteristics Cases Controls (n = 120) (n = 120) Age (years) 20.8 + 4.5 20.2 ± 3.6 Height (cm) 161.0 ± 6.7 163.0 ± 6.9 * Weight (kg) 71.0 ± 19.4 66.8 ± 17.1 mass index 27.3 + 6.8 25.1 ± 6.1 ** body blood pressure 109.5 ± 8.8 105.7 + 9.0 ** systolic (mm Hg) Blood pressure 62.0 ± 7.9 59.4 ± 7.4 ** diastolic (mm Hg) Loss of an e- 23 (19.2) 25 (20.8 previous razo [n (%)] Current smoker 9 (7.5) 13 (10.8 [n (%)] Health insurance 8 (6.7) 13 (10.8 private [n (%)] Married [n (%)] 25 (20.8) 24 (20.0 Race / ethnicity 24 (20.0) 35 (29.2 White, non-Hispanic [n (%)] White Hispanic 21 (17.5) 14 (11.7) [n (%)] Afro-American 69 (57.5) 68 (56.7) [n (%)] Other, unknown 6 (5.0) 3 (2.5) Ln (%)] Weight in the nation- 3100 ± 796 3255 + 595 lie ( g) Childbirth &<37 weeks 29 (24.2) 9 (7.5) ** [n (%)] Small for 18 (15.0) 4 (3.3) ** gestational age (< tenth percentile) [n (%)] Mean + standard deviation unless indicated * p < 0.05 ** p < 0.01 For this study, hypertension was defined as a diastolic blood pressure of at least 90 mm Hg on two separate occasions for a period of 4-168 hours. Severe hypertension was defined as a diastolic blood pressure of at least 110 mm Hg on two separate occasions for a period of 4-168 hours, or an occasion if the woman had received anti-hypertensive therapy. Proteinuria was defined as 300 mg or more of protein in a 24-hour urine collection, two random urine samples separated by a period of 4-168 hours containing at least 1+ protein by means of a dipstick, a single urine sample with a protein / creatinine ratio of at least 0.35, or a single random urine sample containing at least 2+ protein per test strip. Severe proteinuria was diagnosed by a 24-hour urine collection sample containing at least 3.5 g of protein or by two random urine samples with at least 3+ protein by means of a dipstick. Pre-eclampsia was defined as hypertension and proteinuria that occurred less than 7 days apart; Severe pre-eclampsia was defined as pre-eclampsia with severe hypertension, severe proteinuria, HELLP syndrome (hemolysis, elevated liver enzymes and low platelet concentration) or eclampsia. The onset of pre-eclampsia was the time of detection of the first elevation of blood pressure or proteinuria in the urine sample that led to the diagnosis of pre-eclampsia. Small size for gestational age (SGA) was defined as a birth weight lower than the percentile of 10% for gestational age according to the United States tables of birth weight for gestational age by race, parity and sex of the baby (Zhang and Bowes 1995, supra). Procedures in cough The trials were conducted at the Beth Israel Deaconess Medical Center by laboratory personnel who were unaware of the patients' diagnosis and other relevant clinical information. The samples were randomly ordered for analysis. Enzyme-linked immunosorbent assays (ELISA) were performed for human sFlt-1, free PIGF and free VEGF according to the manufacturer's instructions, using kits purchased from R &D Systems (Minneapolis, Minneapolis, United States). Aliquots of serum samples that had been stored at -70 ° C were diluted at room temperature, diluted with BSA / Tris-buffered saline and incubated for 2 hours in a 96-well plate pre-coated with anti-body. of capture directed against sFlt-1, PIGF or VEGF. The wells were then washed three times, incubated 20 minutes with a substrate solution containing hydrogen peroxide and tetramethylbenzidine and the reaction was quenched with 2N sulfuric acid. The optical density was determined at 450 nm (correction of wavelength at 550 nm). All the tests were carried out in duplicate. Protein concentrations were calculated using a standard curve derived from known concentrations of the respective recombinant proteins. If the difference between the duplicates exceeded 25%, the trial was repeated and the initial results were discarded. The assays had sensitivities of 5, 7 and 5 pg / ml for sFlt-1, PIGF and VEGF, respectively, with inter- and intra-assay coefficients of variation of 7.6% and 3.3% for sFlt-1, of 11.2% and 5.4%. % for PIGF, and 7.3% and 5.4% for VEGF. Statistical Analysis In the analysis of the maternal or child characteristics to compare the categorical or continuous variables, chi square tests and t tests were used respectively. Although the values of the arithmetic mean of the concentrations are given in the text and in the figures, the statistical test was performed after a logarithmic transformation, unless otherwise indicated. The adjustment was made using logistic reversion in logarithmically transformed concentrations. Results Of the 120 cases, 80 developed pre-eclampsia and 40 developed severe pre-eclampsia, including 3 with the HELLP syndrome and 3 with eclampsia. The patients in the case group were lower than the control patients, had a higher body mass index and had a higher initial blood pressure (Table 2). In addition, higher proportions of patients in the case group had pregnancies complicated by premature delivery of small children for gestational age (SGA). Case patients contributed an average of 2.9 serum samples to the study; the controls with 2.6 samples. First, it was confirmed that the levels of sFlt-1, PIGF and VEGF at the time of active disease were altered in patients with pre-eclampsia compared with controls with a similar gestational age in this group of CPEP study. Samples taken at the time of established clinical pre-eclampsia (end-point sample) had dramatically increased sFlt-1 levels, reduced PIGF levels and reduced VEGF levels compared to controls with gestational ages (4,382 vs. 1,643 pg / ml sFlt-1, p <0.0001, 137 vs. 669 pg / ml PIGF, p <0.0001, and 6.41 vs. 13.86 pg / ml VEGF, p = 0.06) for cases and controls, respectively, in 23 couples of similar gestational age) similar to the previously published reports (Maynard et al, J ".Clin.Invest. 111: 649-658, 2003.) To evaluate the gestational model of the levels of sFlt-1, PIGF and VEGF, Circulating concentrations of sFlt-1, PIGF and VEGF were measured from serum samples obtained from case patients and control patients within various windows of gestational age.The gestational model of the protein sFlt-1 for 120 women pre- eclámpticas and 120 control women are shown in Figure 5A. The levels of Flt-1 in control patients remained constant until 33-36 weeks, at which time they rose approximately 145 pg / ml per week until delivery. Among case patients before the chemical symptoms, sFlt-1 appeared to begin to rise at 21-24 weeks, with a stepwise elevation and a statistically significant difference of controls at 29-31 weeks (Figure 5A). In general, the differences between the case and control patients measured before the onset of the chemical symptoms were 17% (p <0.05) at mid-gestation. The endpoint samples were significantly elevated compared to the samples taken before the disease. To evaluate the mechanisms of sFlt-1 elevation before the onset of clinical disease, we represented concentrations of sFlt-1 in all pre-eclamptic women in weeks before the onset of pre-eclampsia (Figure 5B). Mean concentrations of sFlt-1 in patient samples of cases were represented by complete weeks before the onset of pre-eclampsia. Beginning 5 weeks before preeclampsia, sFlt-1 concentrations rose substantially up to one week before the onset of the disease, at which time they reached the concentrations observed in the endpoint samples. Increases in sFlt-1 at 4, 3, 2 and 1 week before pre-eclampsia occurred with few changes in mean gestational age and can not be explained by an increase in the latter part of the third trimester as gestational age advances . From 8-6 to 5 weeks before pre-eclampsia, the level of sFlt-1 increased to 962 pg / ml, while the mean gestational age increased 31 days. Approximately one third of this increase in sFlt-1 can not be attributed to the progress of pregnancy. When the level of sFlt-1 was represented by gestational age in controls and in cases after removing the samples obtained < 5 weeks before the onset of pre-eclampsia, no substantial differences were observed (Figure 5C). These data suggest that the highest concentration of sFlt-1 in patients in cases before the onset of pre-eclampsia is due to acute elevations in sFlt-1 in the 5 weeks prior to the onset of clinical disease. The gestational model of the PIGF protein was then represented in the same group of patients as shown in Figure 6A. The control PIGF protein concentrations were elevated during the first two quarters, reached a maximum at 29-32 weeks and were reduced during the last part of gestation. Among patients in the cases, or group of cases, before pre-eclampsia, concentrations of the PlGF protein followed a similar gestational model, but were significantly lower than the controls from week 13 to week 16. In general, the differences in PIGF between patients in the case group and the controls measured before the onset of clinical symptoms were 35% (p <; 0.0001) in the middle of gestation. The levels of PlGF in cases before the onset of pre-eclampsia is represented by weeks before pre-eclampsia (Figure 6B) and by gestational age after extracting samples < 5 weeks before pre-eclampsia (Figure 6C). One week before the onset of pre-eclampsia, concentrations approached those observed after the onset of pre-eclampsia (Figure 6B). Compared with the controls, the PIGF levels of patients in the case group were moderately reduced from delivery, with no further substantial reductions at 5 and 3 weeks before delivery. The concentrations of control patients remained elevated from 17-15 to 3 weeks before delivery, and then decreased dramatically. The graph showing the PlGF levels excluding the samples obtained < 5 weeks before pre-eclampsia indicates a smaller reduction in cases with respect to controls at 29-32 weeks of gestation and none in samples obtained from case patients at 33-36 weeks (Figure 6C ). This suggests that the reduction of PIGF concentrations in the weeks prior to the disease was responsible for the dramatically low levels of PlGF detected at the beginning of the disease (or the endpoint samples shown in Figure 6A). VEGF concentrations throughout pregnancy were very low and similar in controls and in cases before pre-eclampsia, with the exception of a significant reduction in case patients at 37-41 weeks. The mean concentrations of VEGF at 23-32 weeks in the cases excluding the samples obtained 5 weeks before pre-eclampsia did not differ significantly from the controls (11.6 vs 12.8 pg / ml), while the concentrations in the cases included the samples < 5 weeks before delivery if they did (5.1 vs. 12.8 pg / ml, p <0.01). At 33-41 weeks the VEGF concentrations of cases > 5 o < 5 weeks before pre-eclampsia were higher and lower than the controls respectively (11.2 pg / ml and 8.3 versus 9.7 pg / ml), although these differences were not significant. Figure 7 represents sFlt-1 and PIGF at 23-32 weeks (Figure 7A) and 33-41 weeks (Figure 7B) by state of pre-eclampsia and severity. The graphs show that increases in sFlt-1 and reductions in PIGF before the onset of pre-eclampsia were associated with the severity of the disease, the time of onset and the presence of an SGA child. At 23-32 weeks, the levels of sFlt-1 and PlGF in the patients of cases with an SGA child before the onset of pre-eclampsia were significantly higher or lower, respectively, than the corresponding concentrations in the control patients with a SGA child. In addition, compared to control patients with preterm delivery, patients in cases with preterm delivery had a higher sFlt-1 level and a significantly lower level of PlGF. It was then determined whether circulating concentrations of PIGF and / or sFlt-1 could be used during the first trimester to identify women at risk of developing pre-eclampsia. At 8-20 weeks, after adjusting for gestational age, body mass index and sFlt-1, patients in cases with PIGF in the lowest quartile of distribution of control values had an almost 12-fold increase in eclampsia a < 34 weeks (odds ratio [OR] 11.7, p <0.05) compared to cases with PlGF in the upper three quartiles (Table 3). The risk of pre-eclampsia to < 34 weeks in the lower quartile, compared with the upper quartile increased almost 16 times (OR 15.8, p <0.01).

TABLE 3: Reasons for Inequality (OR) for Pre-eclampsia of Early Start by Control PIGF Distribution Quartiles at 8-20 Weeks Start of PE < 34 weeks Start of PE < 37 weeks PIGF Cases Controls OR Adj. * Cases Controls OR Adj * (pg / ml) (N) (N) (95% Cl) (N) (N) (95% Cl) Q4 > 267.5 2 30 1.0 Referent 4 30 1.0 Referent Q3 > 128.6- 30 0.7 (0.1-8.9) 30 1.3 (0.3- 267.5 5.8) Q2 > 70.1- 30 2.3 (0. 30 2.6 (0.6- 128.6 19.3) 12.1) Ql < 70.1 30 15.8 (1.5- 17 30 22.3 (3.7- 172.8) ** 135.6) *** * Inequality ratios adjusted for gestational age, body mass index, log sFlt-1 ** p < 0.01 *** p < 0.001 95% CI = 95% confidence limits These results demonstrate that sFlt-1 levels begin to rise dramatically approximately 5 weeks before the onset of pre-eclampsia symptoms. Parallel with the increase in sFlt-1, free PIGF levels and levels of Free VEGFs are reduced, suggesting that the reduction in PIGF and VEGF may be due at least partially to antagonism by sFlt-1 and not due to a reduction in placental production of PIGF and VEGF. Three subgroups of pre-eclampsia - severe pre-eclampsia, early onset of the disease and SGA children - had higher concentrations of sFlt-1 and lower concentrations of PlGF at 23-32 weeks and at 33-41 weeks than controls or women with mild pre-eclampsia. A small but significant reduction in the level of free PlGF that started early in the second trimester among women destined to develop pre-eclampsia has also been demonstrated. These results demonstrate that a reduction in PIGF levels can be a useful agent for predicting early onset pre-eclampsia. In the present invention, the gestational model of sFlt-1 in normal pregnancy is described for the first time, observing relatively stable levels throughout gestation followed by a constant increase starting at 33-36 weeks. This elevation corresponds to the last gestational reduction in PIGF observed in normal pregnancy by other authors (Torry et al., J. Soc. Gynecol, Invest. 10: 178-188, 1998, Taylor et al., Am. ". O set. Gynecol., 188: 177-182, 2003) and in the results described in this document.The temporal association, together with the knowledge that sFlt-1 interferes with the ELISA measurement of PlGF (Maynard et al., Supra) suggests that the reduction The levels of free PIGF during the latter part of pregnancy may be due to the elevation of sFlt-1 levels During the first and second trimesters, when placental growth is needed to be able to respond to increasing fetal demands, concentrations of PIGF are elevated and sFlt-1 concentrations are low, creating a relatively pro-angiogenic state Later in pregnancy, when it may be necessary to tune and stop placental vascular growth, a rise in anti-angiogenic sFlt-1 and a resulting reduction in PIGF. In women with pre-eclampsia, the elevation of sFlt-1 begins earlier in pregnancy, approximately 5 weeks before the onset of symptoms, to approximately 29-32 weeks of gestation on average. Thus, in the case of pre-eclampsia, anti-angiogenic "brakes" can be applied too early and too forcefully, resulting in an exaggeration of a normal physiological process that stops placental growth. It seems clear that the pathological placental changes that characterize pre-eclampsia occur early in pregnancy (10-14 weeks), before the dramatic elevation in sFlt-1. The resulting placental ischemia itself may potentiate the production of sFlt-1, ultimately triggering a burst in sFlt-1. In addition to the large differences observed in the 5 weeks prior to the development of clinical symptoms, women destined to develop pre-eclampsia had small but statistically significant reductions in free PIGF levels already at 13-16 weeks of gestation. This reduction in PIGF was generally not accompanied by a reciprocal increase in sFlt-1 levels. Nevertheless, there was a trend towards slightly higher sFlt-1 levels during the first trimester although it was not statistically significant (for example, in the window 17-20 weeks, the mean levels of sFlt-1 in the cases were 865.77 pg / ml versus to 795.25 in the controls). This reduction in PIGF levels early in pregnancy may reflect reduced placental PIGF production in pregnancies compromised by conditions such as pre-eclampsia or SGA. Importantly, in patients with pre-eclampsia complicated by SGA, a statistically significant increase was found both in the elevation of sFlt-1 and in the reduction of PIGF before the presentation of the disease. It is also possible that there is no change in the placental production of PIGF in pre-eclamptic patients and that the elevation of local sFlt-1 levels in the placenta may contribute to the reduction of circulating free PIGF levels. This is confirmed by the finding that the placental PIGF, measured by immunohistochemistry, is not altered in pre-eclampsia (Zhou et al, Am J. Pathol, 160: 1405-1423, 2002). In summary, we have shown that sFlt-1 begins elevation in pre-eclampsia at least 5 weeks before the onset of clinical disease that is accompanied by levels of free PIGF and free circulating VEGF. The reduction of PIGF during the first trimester can serve to predict pre-eclampsia and the elevation of sFlt-1 can serve to predict the proximity of the clinical disease. These data together with the work in animals described above demonstrates that sFlt-1 alone induces symptoms similar to those of pre-eclampsia in rodents suggest a probable etiologic role of sFlt-1 in the pathogenesis of pre-eclampsia. Our limited data on SGA children and preterm birth in controls, compared to patients in the case group, suggests that the greatest alterations in protein levels observed in pre-ecplastic pregnancies with a SGA child are more substantial than the difference due only to the restriction of uterine growth or premature delivery in the absence of pre-eclampsia. Example 8. Effects of sFlt-1 on an Animal Model of Eclampsia Pregnant rats in the first part of their second trimester of pregnancy are injected with exogenous sFlt-1. The rats are then monitored and tested during the first part of their third trimester for the development of eclampsia. The tests used for the detection of eclampsia can include MRI of the brains of the rats for the development of edema, EEG of the brain of the rat for the development of attacks and histology of the brains of the rat to determine if endothelial injury has occurred along the blood-brain barrier and the choroid plexus using specific endothelial markers. The animal model created in this document can be used as an experimental model to test new therapeutics. Using this animal model, the efficacy of potential therapeutic compounds as well as pharmacology and toxicity can be studied. Example 9: The Ratio of PIGF / Creatinine in Urine Serves as Diagnosis of Pre-Eclampsia Urine samples were obtained from 10 women at 16 weeks of gestation (five normal, four with mild pre-eclampsia and one with severe pre-eclampsia) . These samples were provided by Dr. Ravi Thadhani of the Massachusetts General Hospital. The mean ratios of free PIGF / creatinine in urine (pg of PIGF per mg of creatinine) for normal pregnant women were 78 +/- 10.7 and for the four women with mild pre-eclampsia were 33 +/- 5.0 and for The patient with severe pre-eclampsia was 17. In this way, an alteration in the relationship between PlGF and creatinine in urine is useful as a diagnostic indicator of pre-eclampsia in a patient. Example 10: Levels of Protein sFlt-1 and PIGF as a Diagnostic Indicator of Pre-Eclampsia and Eclampsia in Women For this study archived samples of the calcium assay for the prevention of pre-eclampsia were used to analyze the gestational models of sFlt-1, Free PIGF and free VEGF circulating in normotensive and pre-ecplastic pregnancies. The calcium trial for the prevention of pre-eclampsia, or CPEP, was a double-blind randomized clinical trial conducted during 1992-1995 to evaluate the effects of the daily supplement of 2 g of elemental calcium or placebo on the incidence and severity of pre-eclampsia. -eclampsia (Levine et al., N. Engl. J. Med. 377: 69-76, 1997; Levine et al., Control Clin. Triais 17: 442-469, 1996). Healthy nulliparous women with simple pregnancies between 13 and 21 weeks of gestation were included in 5 participating US medical centers and followed up to 24 hours after delivery using a common protocol and identical data collection forms. At the time of inclusion, all CPEP participants had a blood pressure < 135/85 mm Hg, and none had renal dysfunction or proteinuria. Gestational age was determined by ultrasound examination. Serum samples were obtained from the participants before inclusion in the trial (13-21 weeks), at 26-29 weeks, at 36 weeks if they were still pregnant, and when hypertension or proteinuria was detected. The "endpoint samples" were samples obtained during or after the onset of symptoms and signs of pre-eclampsia, but before delivery as described elsewhere in this document (Levine et al., 1996, supra). The blood samples obtained from the CPEP trial were obtained thanks to the collaboration of Dr. Richard Levine at the NIH.

Parti cipants Subjects were selected with complete information on the results, from which serum samples were obtained at < 22 weeks and they had a man born alive. Of the 4,589 CPEP participants, 253 who lost track, 21 whose pregnancy ended before 20 weeks, 13 absences of maternal or perinatal outcome data, 4 no history of smoking, 9 with hypertension not verified by the groups were excluded. of revision, and another 32 with children stillborn, leaving 4,257 women with adequate information and live births. Among these, 2,156 had male babies. After excluding a woman whose baby had a chromosomal abnormality, 381 with gestational hypertension and 43 without an initial serum sample, 1,731 women remained. Of these, 175 developed pre-eclampsia and 1,556 remained normotensive throughout the pregnancy. As the calcium supplement had no effect on the risk and severity of pre-eclampsia and was not related to the concentrations of pro- and anti-angiogenic molecules, cases and controls were chosen without taking into account the treatment with CPEP. For each case of pre-eclampsia, a normotensive control was selected, equivalent in terms of inclusion site, gestational age in the collection of the first serum sample (within a week) and storage time in a freezer at -70. ° C (in 12 months). 120 equivalent pairs ("cases" and "controls") were randomly selected for the analysis of the 657 serum samples obtained before delivery (Table 2, presented below) The mean gestational age at collection of the first serum sample was of 112.8 and 113.6 days in cases and controls respectively, the average duration of storage in the freezer was 9.35 and 9.39 years.

TABLE 2: Characteristics of cases and controls in the inclusion in the CPEP and of their newborn babies Characteristics Cases Controls (n = 120) (n = 120) Age (years) 20.8 ± 4.5 20.2 ± 3.6 Height (cm) 161.0 ± 6.7 163.0 ± 6. 9 * Weight (kg) 71.0 ± 19.4 66.8 ± 17.1 mass index 27.3 ± 6.8 25.1 ± 6.1 ** body blood pressure 109.5 + 8.8 105.7 ± 9.0 ** systolic (mm Hg) Blood pressure 62.0 ± 7.9 59.4 + 7.4 ** diastolic (mm Hg) Loss of one package- 23 (19.2) 25 (20.8) previous ration [n (%)] Current smoker 9 (7.5) 13 (10.8) [n (%)] Health insurance 8 (6.7) 13 (10.8) private [n (%)] Married [n (%)] 25 (20.8) 24 (20.0) Race / Ethnicity 24 (20.0) 35 (29.2) White, not Hispanic [n (%)] White Hispanic 21 (17.5) 14 ( 11.7) [n (%)] Afro-American 69 (57.5) 68 (56.7) [n (%)] Other, unknown 6 (5.0) 3 (2.5) [n (%)] Weight in the year 3100 + 796 3255 + 595 term (g) Childbirth < 37 weeks 29 (24.2) 9 (7.5) ** [n (%)] Small for 18 (15.0) 4 (3.3) ** gestational age (< tenth percentile) [n (%)] Mean ± standard deviation unless indicated * p < 0.05 ** p < 0.01 For this study, hypertension was defined as a diastolic blood pressure of at least 90 mm Hg on two separate occasions for a period of 4-168 hours. Severe hypertension was defined as a diastolic blood pressure of at least 110 mm Hg on two separate occasions for a period of 4-168 hours, or an occasion if the woman had received anti-hypertensive therapy. Proteinuria was defined as 300 mg or more of protein in a 24-hour urine collection, two random urine samples separated by a period of 4-168 hours containing at least 1+ protein by means of a dipstick, a single urine sample with a protein / creatinine ratio of at least 0.35, or a single random urine sample containing at least 2+ protein per test strip. Severe proteinuria was diagnosed by a 24-hour urine collection sample containing at least 3.5 g of protein or by two random urine samples with at least 3+ protein by means of a dipstick. Pre-eclampsia was defined as hypertension and proteinuria that occurred less than 7 days apart; Severe pre-eclampsia was defined as pre-eclampsia with severe hypertension, severe proteinuria, HELLP syndrome (hemolysis, elevation of liver enzymes and low platelet concentration) or eclampsia. The onset of pre-eclampsia was the moment of detection of the first elevation of blood pressure or proteinuria in the urine sample that led to the diagnosis of pre-eclampsia. Small size for gestational age (SGA) was defined as a birth weight lower than the percentile of 10% for gestational age according to the United States tables of birth weight for gestational age by race, parity and sex of the baby (Zhang and Bowes 1995, supra). Procedures The trials were conducted at the Beth Israel Deaconess Medical Center by the laboratory personnel who did not know the diagnosis of the patients and other relevant clinical information. The samples were randomly ordered for analysis. Immuno-absorbent assays with bound enzyme (ELISA) were performed for human sFlt-1, free PlGF and free VEGF according to the manufacturer's instructions, using kits purchased from R &D Systems (Minneapolis, Minneapolis, United States). Aliquots of serum samples that had been stored at -70 ° C were thawed at room temperature, diluted with BSA / Tris-buffered saline and incubated for 2 hours in a 96-well plate pre-coated with anti-body capture. directed against sFlt-1, PIGF or VEGF. The wells were then washed three times, incubated 20 minutes with a substrate solution containing hydrogen peroxide and tetramethylbenzidine and the reaction was quenched with 2N sulfuric acid. The optical density was determined at 450 nm (correction of wavelength at 550 nm). All the tests were carried out in duplicate. Protein concentrations were calculated using a standard curve derived from known concentrations of the respective recombinant proteins. If the difference between the duplicates exceeded 25%, the trial was repeated and the initial results were discarded. The assays had sensitivities of 5, 7 and 5 pg / ml for sFlt-1, PIGF and VEGF, respectively, with inter- and intra-assay coefficients of variation of 7.6% and 3.3% for sFlt-1, of 11.2% and 5.4%. % for PIGF, and 7.3% and 5.4% for VEGF. Statistical Analysis In the analysis of the maternal or child characteristics to compare the categorical or continuous variables, chi square tests and t tests were used respectively. Although the values of the arithmetic mean of the concentrations are given in the text and in the figures, the statistical test was performed after a logarithmic transformation, unless otherwise indicated. The adjustment was made using logistic reversion in logarithmically transformed concentrations. RESULTS Of the 120 cases, 80 developed pre-eclampsia and 40 developed severe pre-eclampsia, including 3 with the HELLP syndrome and 3 with eclampsia. The patients in the case group were lower than the control patients, had a higher body mass index and had a higher initial blood pressure (Table 2). In addition, higher proportions of patients in the case group had pregnancies complicated by premature delivery of small children for gestational age (SGA). Case patients contributed an average of 2.9 serum samples to the study; the controls with 2.6 samples. First, it was confirmed that the levels of sFlt-1, PIGF and VEGF at the time of active disease were altered in patients with pre-eclampsia compared with controls with a similar gestational age in this group of CPEP study. Samples taken at the time of established clinical pre-eclampsia (end-point sample) had dramatically increased sFlt-1 levels, reduced PIGF levels and reduced VEGF levels compared to controls with gestational ages (4,382 vs. 1,643 pg / ml sFlt-1, p <0.0001, 137 vs. 669 pg / ml PIGF, p <0.0001, and 6.41 vs. 13.86 pg / ml VEGF, p = 0.06) for cases and controls, respectively, in 23 couples of similar gestational age) similar to previously published reports (Maynard et al., J. Clin. Invest. 111: 649-658, 2003). To evaluate the gestational model of the levels of sFlt-1, PIGF and VEGF, the circulating concentrations of sFlt-1, PIGF and VEGF were measured from serum samples obtained from case patients and control patients within various windows of gestational age . The gestational model of the sFlt-1 protein for 120 pre-eclamptic women and 120 control women is shown in Figure 5A. The levels of Flt-1 in the control patients remained constant until 33-36 weeks, at which time they rose approximately 145 pg / ml per week until delivery. Among case patients before the chemical symptoms, sFlt-1 appeared to begin to rise at 21-24 weeks, with a stepwise elevation and a statistically significant difference of controls at 29-31 weeks (Figure 5A). In general, the differences between case and control patients measured before the onset of chemical symptoms were 17% (p <; 0.05) mid-gestation. The endpoint samples were significantly elevated compared to the samples taken before the disease. To evaluate the mechanisms of sFlt-1 elevation before the onset of clinical disease, we represented concentrations of sFlt-1 in all pre-eclamptic women in weeks before the onset of pre-eclampsia (Figure 5B). Mean concentrations of sFlt-1 in patient samples from cases were represented by complete weeks before the onset of pre-eclampsia. Beginning 5 weeks before pre-eclampsia, the concentrations of sFlt-1 rose substantially up to one week before the onset of the disease, at which point they reached the concentrations observed in the endpoint samples. Increases in sFlt-1 at 4, 3, 2 and 1 week before pre-eclampsia occurred with few changes in mean gestational age and can not be explained by an increase in the latter part of the third trimester as gestational age advances . From 8-6 to 5 weeks before pre-eclampsia, the level of sFlt-1 increased to 962 pg / ml, while the mean gestational age increased 31 days. Approximately one third of this increase in sFlt-1 can not be attributed to the progress of pregnancy. When the level of sFlt-1 was represented by gestational age in controls and in cases after removing the samples obtained < 5 weeks before the onset of pre-eclampsia, no substantial differences were observed (Figure 5C). These data suggest that the highest concentration of sFlt-1 in patients in cases before the onset of pre-eclampsia is due to acute elevations in sFlt-1 in the 5 weeks prior to the onset of clinical disease. The gestational model of the PIGF protein was then represented in the same group of patients as shown in Figure 6A. The control PIGF protein concentrations were elevated during the first two quarters, reached a maximum at 29-32 weeks and were reduced during the last part of gestation. Among patients in the cases, or group of cases, before pre-eclampsia, PIGF protein concentrations followed a similar gestational model, but were significantly lower than controls from week 13 to week 16. In general, differences in PlGF between patients in the case group and controls measured before the onset of clinical symptoms were 35% (p <0.0001) in the middle of pregnancy. PIGF levels in cases before the onset of pre-eclampsia are represented by weeks before pre-eclampsia (Figure 6B) and by gestational age after extracting samples < 5 weeks before pre-eclampsia (Figure 6C). One week before the onset of pre-eclampsia, concentrations approached those observed after the onset of pre-eclampsia (Figure 6B). Compared with the controls, the PIGF levels of patients in the case group were moderately reduced from delivery, with no further substantial reductions at 5 and 3 weeks before delivery. The concentrations of control patients remained elevated from 17-15 to 3 weeks before delivery, and then decreased dramatically. The graph showing the PIGF levels excluding the samples obtained < 5 weeks before pre-eclampsia indicates a smaller reduction in cases with respect to controls at 29-32 weeks of gestation and none in samples obtained from case patients at 33-36 weeks (Figure 6C ). This suggests that the reduction of PIGF concentrations in the weeks prior to the disease was responsible for the dramatically low levels of PIGF detected at the beginning of the disease (or the endpoint samples shown in Figure 6A). VEGF concentrations throughout pregnancy were very low and similar in controls and in cases before pre-eclampsia, with the exception of a significant reduction in case patients at 37-41 weeks. The mean concentrations of VEGF at 23-32 weeks in the cases excluding the samples obtained 5 weeks before pre-eclampsia did not differ significantly from the controls (11.6 vs 12.8 pg / ml), while the concentrations in the cases included the samples <; 5 weeks before delivery if they did (5.1 vs. 12.8 pg / ml, p <0.01). At 33-41 weeks the VEGF concentrations of cases > 5 o < 5 weeks before pre-eclampsia were higher and lower than the controls respectively (11.2 pg / ml and 8.3 versus 9.7 pg / ml), although these differences were not significant. Figure 7 represents sFlt-1 and PIGF at 23-32 weeks (Figure 7A) and 33-41 weeks (Figure 7B) by state of pre-eclampsia and severity. The graphs show that increases in sFlt-1 and reductions in PIGF before the onset of pre-eclampsia were associated with the severity of the disease, the time of onset and the presence of an SGA child. At 23-32 weeks, the levels of sFlt-1 and PIGF in the patients of cases with an SGA child before the onset of pre-eclampsia were significantly higher or lower, respectively, than the corresponding concentrations in the control patients with a SGA child. In addition, compared to control patients with preterm birth, patients in cases with preterm delivery had a higher sFlt-1 level and a significantly lower PIGF level. It was then determined whether circulating concentrations of PIGF and / or sFlt-1 could be used during the first trimester to identify women at risk of developing pre-eclampsia. At 8-20 weeks, after adjusting for gestational age, body mass index and sFlt-1, patients in cases with PIGF in the lowest quartile of distribution of control values had an almost 12-fold increase in eclampsia a < 34 weeks (odds ratio [OR] 11.7, p <0.05) compared to cases with PlGF in the three upper quartiles (Table 3). The risk of pre-eclampsia to < 34 weeks in the lower quartile, compared with the upper quartile increased almost 16 times (OR 15.8, p <0.01).

TABLE 3: Reasons for Inequality (OR) for Pre-eclampsia of Home Precocious by Control PIGF Distribution Quartiles at 8-20 Weeks Start of PE < 34 weeks Start of PE < 37 weeks PIGF Cases Controls OR Adj. * Cases Controls OR Adj * (pg / ml) (N) (N) (95% Cl) (N) (N) (95% Cl) Q4 > 267.5 2 30 1.0 Referent 4 30 1.0 Referent Q3 > 128 6-1 30 0.7 (0.1-8 .9) 4 30 1.3 (0.3-267.5-8) • Q2 > 70 .1- 30 2.3 (0.3-6 30 2.6 (0.6- 128. 6 19.3) 12.1) Ql 70 .1 30 15.8 (1.5- 17 30 22.3 (3.7 172.8) ** 135.6) *** * Inequality ratios adjusted for gestational age, body mass index, log sFlt-1 ** p < 0.01 *** p < 0.001 95% CI = 95% confidence limits These results demonstrate that sFlt-1 levels begin to rise dramatically approximately 5 weeks before the onset of pre-eclampsia symptoms. Parallel to the increase in sFlt-1, free PIGF levels and free VEGF levels are reduced, suggesting that the reduction in PIGF and VEGF may be due at least partially to antagonism by sFlt-1 and not due to a reduction in the placental production of PIGF and VEGF. Three subgroups of pre-eclampsia - severe pre-eclampsia, early onset of the disease and SGA children - had higher concentrations of sFlt-1 and lower concentrations of PIGF at 23-32 weeks and at 33-41 weeks than controls or women with mild pre-eclampsia. A small but significant reduction in the level of free PlGF that started early in the second trimester among women destined to develop pre-eclampsia has also been demonstrated. These results demonstrate that a reduction in PIGF levels can be a useful agent for predicting early onset pre-eclampsia. In the present invention, the gestational model of sFlt-1 in normal pregnancy is described for the first time, observing relatively stable levels throughout gestation followed by a constant increase starting at 33-36 weeks. This elevation corresponds to the last gestational reduction in PIGF observed in normal pregnancy by other authors (Torry et al., "Soc Gynecol Invest. 10: 178-188, 1998, Taylor et al., Am. J." Obset. Gynecol 188: 177-182, 2003) and in the results described in this document. The temporal association, together with the knowledge that sFlt-1 interferes with the ELISA measurement of PlGF (Maynard et al., Supra) suggests that the reduction of free PIGF levels during the latter part of gestation may be due to the elevation of the levels of sFlt-1. During the first and second trimesters, when placental growth is needed to respond to increasing fetal demands, PIGF concentrations are elevated and sFlt-1 concentrations are low, creating a relatively pro-angiogenic state. Later in pregnancy, when it may be necessary to tune and stop the placental vascular growth, an elevation occurs in the anti-angiogenic sFlt-1 and a resulting reduction in PIGF. In women with pre-eclampsia, the elevation of sFlt-1 begins earlier in pregnancy, approximately 5 weeks before the onset of symptoms, to approximately 29-32 weeks of gestation on average. Thus, in the case of pre-eclampsia, anti-angiogenic "brakes" can be applied too early and too forcefully, resulting in an exaggeration of a normal physiological process that stops placental growth. It seems clear that the pathological placental changes that characterize pre-eclampsia occur early in pregnancy (10-14 weeks), before the dramatic elevation in sFlt-1. The resulting placental ischemia itself may potentiate the production of sFlt-1, ultimately triggering a burst in sFlt-1. In addition to the large differences observed in the 5 weeks prior to the development of clinical symptoms, women destined to develop pre-eclampsia had small but statistically significant reductions in free PIGF levels already at 13-16 weeks of gestation. This reduction in PIGF was generally not accompanied by a reciprocal increase in sFlt-1 levels. Nevertheless, there was a trend towards slightly higher sFlt-1 levels during the first trimester although it was not statistically significant (for example, in the window 17-20 weeks, the mean levels of sFlt-1 in the cases were 865.77 pg / ml versus to 795.25 in the controls). This reduction in PIGF levels early in pregnancy may reflect reduced placental PIGF production in pregnancies compromised by conditions such as pre-eclampsia or SGA. Importantly, in patients with pre-eclampsia complicated by SGA, a statistically significant increase was found both in the elevation of sFlt-1 and in the reduction of PIGF before the presentation of the disease. It is also possible that there is no change in the placental production of PIGF in pre-eclamptic patients and that the elevation of local sFlt-1 levels in the placenta may contribute to the reduction of circulating free PIGF levels. This is confirmed by the finding that the placental PIGF, measured by immunohistochemistry, is not altered in pre-eclampsia (Zhou et al, Am J. Pathol, 160: 1405-1423, 2002). In summary, we have shown that sFlt-1 begins elevation in pre-eclampsia at least 5 weeks before the onset of clinical disease that is accompanied by levels of free PlGF and free circulating VEGF. The reduction of PIGF during the first trimester can serve to predict pre-eclampsia and the elevation of sFlt-1 can serve to predict the proximity of the clinical disease. These data together with the work in animals described above demonstrating that sFlt-1 alone induces symptoms similar to those of pre-eclampsia in rodents suggest a probable etiologic role of sFlt-1 in the pathogenesis of pre-eclampsia. Our limited data on SGA children and preterm birth in controls, compared to patients in the case group, suggests that the greatest alterations in protein levels observed in pre-ecplastic pregnancies with a SGA child are more substantial than the difference due only to the restriction of uterine growth or premature delivery in the absence of pre-eclampsia. Diagnosis The present invention characterizes diagnostic tests for the detection of pre-eclampsia, eclampsia or the propensity to develop such conditions. The levels of VEGF, PIGF or sFlt-1, free or total, are measured in a sample of a subject and used as an indicator of pre-eclampsia, eclampsia or the propensity to develop such conditions. In one embodiment, a measurement system is used to determine if a relationship between the levels of at least two of the proteins is indicative of pre-eclampsia or eclampsia. Conventional methods can be used to measure levels of VEGF, PIGF or sFlt-1 polypeptide in any body fluid, including but not limited to urine, serum, plasma, saliva, amniotic fluid or cerebrospinal fluid. Such methods include immunoassay, ELISA, Western blot using antibodies directed to VEGF, PIGF or sFlt-1, and quantitative enzymatic immunoassay techniques such as those described in Ong et al. (Obstet, Gynecol. 98: 608-611, 2001) and Su et al. (Obset, Gynecol., 97: 898-904, 2001). ELISA assays are the preferred method for measuring levels of VEGF, PIGF or sFlt-1. Serum levels of sFlt-1 greater than 2 ng / ml are considered a positive indicator of pre-eclampsia. In addition, any detectable alteration in the levels of sFlt-1, VEGF or PlGF with respect to normal levels is indicative of eclampsia, pre-eclampsia or the propensity to develop such conditions. Preferably sFlt-1 is measured, more preferably the measurement of VEGF and PIGF are combined with this measurement and even more preferably the 3 proteins (or mRNA levels indicative of protein levels) are measured. In another embodiment, the PAAI (sFlt-l / VEGF + PIGF) is used as an anti-angiogenic index that is diagnostic of pre-eclampsia, eclampsia or the propensity to develop such conditions. If the PAAI is greater than 20 then it is considered that the subject has pre-eclampsia or has an imminent risk of developing it. The ratio of PAAI (sFlt-l / VEGF + PlGF) is simply an example of a useful measurement system that can be used as a diagnostic indicator. It does not intend to limit the invention. As a diagnostic indicator, practically any measurement system that detects an alteration in the anti-angiogenic index in the subject having eclampsia with respect to a normal control can be used. The expression levels of particular nucleic acids or polypeptides can be correlated with a particular disease state (e.g., pre-eclampsia or eclampsia) and thus are useful in diagnosis. As a probe, oligonucleotides or larger fragments from a nucleic acid sequence of sFlt-1, PlGF or VGF can be used as a probe not only to control expression, but also to identify subjects having a genetic variation, mutation, or polymorphism in a molecule of sFlt-1, PIGF or VEGF that are indicative of a predisposition to develop the conditions. Such polymorphisms are known to those skilled in the art and are described by Parry et al. (Eur. J Immunogenet, 26: 321-3, 1999). Such genetic alterations may be present in the promoter sequence, an open reading frame, intron sequence or 3 'untranslated region of a sFlt-1 gene. Information related to genetic alterations can be used to diagnose a subject having pre-eclampsia, eclampsia or a propensity to develop such conditions. As indicated throughout this document, specific alterations in the levels of biological activity of sFlt-1, VEGF and / or PIGF can be correlated with the predisposition or predisposition to pre-eclampsia or eclampsia. As a result, a person skilled in the art, having detected a given mutation, can assay one or more systems for measuring the biological activity of the protein to determine whether the mutation produces or increases the likelihood of pre-eclampsia or eclampsia. In one embodiment, a subject having pre-eclampsia, eclampsia or a propensity to develop such conditions will show an increase in the expression of a nucleic acid encoding sFlt-1 or an alteration of the PIGF or VEGF levels. Methods for detecting such alterations are conventional in the art and are described in Ausubel et al., Supra. In one example, Real-time Northern blot or PCR is used to detect sFlt-1, PIGF or VEGF mRNA levels. In another embodiment, hybridization can be used with PCR probes that are capable of detecting an sFlt-1 nucleic acid molecule, including genomic sequences, or closely related molecules, to hybridize with a nucleic acid sequence derived from a subject that have pre-eclampsia or eclampsia or at risk of developing such conditions. The specificity of the probe, whether it is made from a very specific region, for example, the 5 'regulatory region, or from a less specific region, for example as a conserved motif, and the stringency of hybridization or amplification (maximum, high, intermediate or low) determine whether the hybrid probe with a natural sequence, allelic variants or other related sequences. Hybridization techniques can be used to identify mutations indicative of a pre-eclampsia or eclampsia in an sFlt-1 nucleic acid molecule, or they can be used to control the expression levels of a gene encoding a sFlt-1 polypeptide (e.g. by Northern analysis, Ausubel et al., supra). In another embodiment, humans can be diagnosed with a propensity to develop pre-eclampsia or eclampsia by direct sequence analysis of a nucleic acid molecule of sFlt-1, VEGF or PIGF. A subject who has pre-eclampsia, eclampsia or a propensity to develop such conditions will show an increase in the expression of a sFlt-1 polypeptide. An anti-body that binds specifically to a sFlt-1 polypeptide can be used to diagnose pre-eclampsia or eclampsia or to identify a subject at risk of developing such conditions. Various protocols for measuring an alteration in the expression of such polypeptides are known, including immunological methods (such as ELISA and RIA) and provide a basis for diagnosing pre-eclampsia or eclampsia or a risk of developing such afflictions. Again, an increase in the level of the polypeptide is diagnostic of a subject having pre-eclampsia, eclampsia or a propensity to develop such conditions. In one embodiment, the level of polypeptide or nucleic acid of sFlt-1, VEGF or PIGF, or any combination thereof, is measured at at least two different times and an alteration in levels compared to the reference levels normal over time is used as an indicator of pre-eclampsia, eclampsia or the propensity to develop such conditions. The level of sFlt-1, VEGF or PIGF in the bodily fluids of a subject having pre-eclampsia, eclampsia or the propensity to develop such conditions can be altered in an amount as small as 10%, 20%, 30% 40% or in an amount as large as 50%, 60%, 70%, 80% or 90% with respect to the level of sFlt-1, VEGF or PIGF in a normal control. The level of sFlt-1 present in the bodily fluids of a subject having pre-eclampsia, eclampsia or the propensity to develop such conditions may increase 1.5 times, twice, 3 times, 4 times or even up to 10 times or more with respect to the levels of a normal control subject. In one embodiment, a sample of a body fluid from the subject (e.g., urine, plasma, serum or amniotic fluid) is collected in the first part of pregnancy prior to the onset of pre-eclampsia symptoms. In another example, the sample may be a tissue or cell collected in the first part of pregnancy before the onset of pre-eclampsia symptoms. Non-limiting examples include placental tissue, placental cells, endothelial cells and leukocytes such as monocytes. In humans, for example, samples of maternal blood serum are collected from the ante-ulnar vein of pregnant women during the first, second or third trimesters of pregnancy. Preferably, the test is carried out during the first trimester, for example, at 4, 6, 8, 10 or 12 weeks or during the second trimester, for example at 14, 16, 18, 20, 22 or 24 weeks. Such trials can also be done at the end of the second trimester or at the beginning of the third trimester (approximately at 28 weeks). It is preferable to measure sFlt-1, VEGF or PIGF levels twice during this time period. For the diagnosis of pre-eclampsia or postpartum eclampsia, trials for sFlt-1, VEGF or PIGF can be performed after delivery. In a particular example, blood samples may be collected in series during pregnancy and the levels of soluble sFlt-1 may be determined by ELISA. In a study using this technique, the alternately-linked mRNA encoding sFlt-1 has high expression by trophoblast cells and the protein was easily detectable in the plasma of pregnant women. It was observed that sFlt-1 levels increased approximately 3 times between 20 and 36 weeks of gestation. It was observed that the levels were significantly higher in high-risk women who subsequently developed pre-eclampsia (Charnock-Jones et al., J. Soc. Gynecol.Research 10 (2): 230, 2003). In veterinary practice, tests can be performed at any time during pregnancy, but preferably they can be performed in the early stages of pregnancy, before the onset of pre-eclampsia symptoms. Since the term of pregnancies varies widely between species, the time of the test will be determined by a veterinarian, but will generally correspond to the testing times during a human pregnancy. The diagnostic methods described herein may be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence, severity or estimated time of onset of pre-eclampsia or eclampsia. In addition, the diagnostic methods described in this document can be used in combination with any other diagnostic method that is determined to be useful for the acute diagnosis of the presence, severity or estimated time of onset of pre-eclampsia or eclampsia.

The diagnostic methods described herein can also be used to control and treat pre-eclampsia or eclampsia in a subject. In one example, if a subject is determined to have a serum sFlt-1 protein level of 10 ng / ml and a serum free PIGF level of 10 pg / ml, then VEGF can be administered until the level of PIGF in serum is raised to approximately 400 pg / ml. In this embodiment, the levels of sFlt-1, PIGF and VEGF, or each and every one of these, are measured repeatedly as a method not only to diagnose the disease, but also to control the treatment of pre-eclampsia and eclampsia. Diagnostic Kit The invention also provides a diagnostic test kit. For example, the diagnostic assay kit may include anti-bodies against sFlt-1, VEGF or PIGF, and means to detect and more preferably evaluate the binding between the antibodies and the sFlt-1, VEGF or PIGF polypeptide. For detection, the anti-body or polypeptide sFlt-1, VEGF or PIGF is labeled, and the anti-body or polypeptide sFlt-1, VEGF or PlGF binds to the substrate, such that the sFlt polypeptide interaction -1, VEGF or PIGF-anti-body can be established by determining the amount of marker bound to the substrate after binding between the anti-body and the polypeptide sFlt-1, VEGF or PIGF. A conventional ELISA is a common method known in the art for detecting the anti-body-substrate interaction and can be provided with the kit of the invention. The sFlt-1, VEGF or PIGF polypeptides can be detected in virtually any body fluid including, but not limited to urine, serum, plasma, saliva, amniotic fluid or cerebrospinal fluid. A kit that determines an alteration in the level of polypeptide sFlt-1, VEGF or PIGF with respect to a reference, such as the level present in a normal control, is useful with diagnostic kit in the methods of the invention. Selection Tests As described above, the expression of a sFlt-1 nucleic acid or polypeptide is increased in a subject having pre-eclampsia, eclampsia or a propensity to develop such conditions. Based on these findings, the compositions of the invention are useful for the selection of low-throughput and high-throughput candidate compounds to identify those that modulate the expression of a sFlt-1, VEGF or PIGF nucleic acid or polypeptide molecule whose expression is altered in a subject who has pre-eclampsia or eclampsia. Several methods are available to perform screening assays to identify new candidate compounds that alter the expression of a nucleic acid molecule of sFlt-1, VEGF or PlGF. In a working example, candidate compounds at variable concentrations are added to the culture medium of cultured cells expressing a nucleic acid sequence of sFlt-1, VEGF or PlGF. The expression of the gene is then measured, for example, by micro-array analysis, Northern blot analysis (Ausubel et al., Supra), or RT-PCR using any appropriate fragment prepared from the nucleic acid molecule as a probe. hybridization. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate compound. A compound that promotes an alteration such as an increase in the expression of a VEGF or PIGF gene, a nucleic acid molecule or polypeptide, or a reduction in the expression of a sFlt-1 gene, nucleic acid molecule, polypeptide, or a functional equivalent thereof, is considered useful in the invention; such a molecule can be used, for example, as therapy to treat eclampsia or eclampsia in a subject. In another working example, the effect of candidate compounds can be measured at the level of polypeptide production using the same general strategy and conventional immunological techniques, such as Western blotting or immuno-precipitation with an anti-body specific for a sFlt-1 polypeptide., VEGF or PlGF. For example, immunoassays can be used to detect or control the expression of at least one of the polypeptides of the invention in an organism. Polyclonal or monoclonal antibodies (produced as described above) can be used, which are capable of binding to such a polypeptide in any conventional immunoassay format (e.g., ELISA, Western Blot, RIA assay) to measure the level of the polypeptide. In some embodiments, a compound that promotes an alteration such as an increase in the expression or biological activity of a VEGF or PIGF polypeptide or a reduction in the expression or biological activity of a sFlt-1 polypeptide is considered particularly useful. Again, such a molecule can be used, for example, as a therapy to delay, improve or treat pre-eclampsia or eclampsia, or the symptoms of pre-eclampsia or eclampsia in a subject. In another working example, candidate compounds can be selected to select those that specifically bind to a sFlt-1, VEGF or PIGF polypeptide. The efficacy of such a candidate compound depends on its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be easily assayed using any number of conventional binding techniques and functional assays (for example, those described in Ausubel et al., Supra). In one embodiment, a candidate compound can be tested in vi tro with respect to its ability to specifically bind to a polypeptide of the invention. In another embodiment, a candidate compound is tested for its ability to reduce the biological activity of a sFlt-1 polypeptide by reducing the binding of a sFlt-1 polypeptide and a growth factor, such as VEGF or PIGF. In another working example, a sFlt-1, VEGF or PIGF nucleic acid is expressed as a transcriptional or translational fusion with a detectable indicator, and is expressed in an isolated cell (e.g., mammalian cell or insect) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable indicator in that cell is compared to the expression of the detectable indicator in an untreated control cell. A candidate compound that reduces the expression of a detectable indicator of sFlt-1 or that increases the expression of a detectable VEGF or PIGF indicator is a compound that is useful for the treatment of pre-eclampsia or eclampsia. In preferred embodiments, the candidate compound alters the expression of a reporter gene fused to a nucleic acid or a nucleic acid. In a particular work example, a candidate compound that binds to a sFlt-1 polypeptide can be identified using a technique based on chromatography. For example, a recombinant polypeptide of the invention can be purified by conventional techniques from cells engineered to express the polypeptide (e.g., those described above) and can be immobilized on a column. A solution of candidate compounds is then passed through the column and a compound specific for the sFlt-1 polypeptide is identified based on its ability to bind to the polypeptide and is immobilized on the column. To isolate the compound, the column is washed to remove the bound molecules in a non-specific manner, and the compound of interest is released from the column and collected. Similar methods can be used to isolate a compound bound to a polypeptide microarray. If desired, the compounds isolated by this method (or any other suitable method) can be further purified (for example, by high performance liquid chromatography). In addition, these candidate compounds can be tested for their ability to reduce the activity of a sFlt-1 polypeptide or increase the activity of a VEGF signaling pathway (for example as described herein). Compounds isolated by this strategy can also be used, for example, as a therapy to treat pre-eclampsia or eclampsia in a human being. Particularly useful in the invention are compounds that are identified as compounds that bind to a polypeptide of the invention with an affinity constant less than or equal to 10 mM. Alternatively, any in vivo protein interaction detection system can be used, for example, any two-hybrid assay to identify compounds or proteins that bind to a polypeptide of the invention. Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids and anti-bodies that bind to a sFlt-1 nucleic acid sequence or a sFlt-1 polypeptide. DNA sequences of sFlt-1 can also be used in the discovery and development of a therapeutic compound for the treatment of pre-eclampsia and eclampsia. The encoded protein, after expression, can be used as a target for drug selection. In addition, DNA sequences encoding the amino-terminal region of the encoded protein or Shine-Dalgarno or other sequences that facilitate translation of the respective mRNA can be used to construct sequences that reduce the expression of a coding sequence of sFlt-1. Such sequences can be isolated by conventional techniques (Ausubel et al., Supra). Optionally, the compounds identified in any of the assays described above can be confirmed as compounds useful in an assay for compounds that reduce the biological activity of sFlt-1 or that increase the activity of a VEGF signaling pathway. The small molecules of the invention preferably have a molecular weight less than 2,000 Daltons, more preferably between 300 and 1,000 Daltons and even more preferably between 400 and 700 Daltons. It is preferred that these small molecules are organic molecules. Therapeutic Agents that Target the VEGF Signaling Route VEGF is a potent endothelial cell-specific mitogen that stimulates angiogenesis, vascular hyper-permeability and vasodilation. Three tyrosine kinase signaling receptors for VEGF have been identified. VEGF receptor binding induces a signaling cascade that results in tyrosine phosphorylation of phospholipase Cgl, producing increases in intracellular levels of inositol 1,4,5-triphosphate and increases in intracellular calcium that activate nitric oxide synthase to produce nitric oxide (NO). The formation of NO activates guanylate cyclase within vascular smooth muscle cells and endothelial cells, causing the production of cGMP. It is believed that this cascade of NO / cGMP mediates the vasoactive effects of VEGF. Another route that seems to be involved in mediating the vasoactive effects of VEGF is the route of prostacyclin release. VEGF induces the production of PGI2 through the activation of phospholipase A2 as a consequence of the initiation of the MAPK cascade. The highest levels of VEGF are useful for the treatment of pre-eclampsia and eclampsia. Therapeutic compounds that target the VEGF signaling pathways, or the components of a VEGF signaling pathway, and potentiate the activity of a VEGF signaling pathway are also useful for the treatment of pre-eclampsia and eclampsia. . Such compounds include sildenafil, prostacyclin analogues, such as Flolan, Remodulin and Tracleer. Test Compounds and Extracts In general, compounds capable of reducing the activity of a sFlt-1 polypeptide or increasing the activity of VEGF or PIGF are identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries or polypeptides or nucleic acids, according to methods known in the art. The specialists in the field of drug discovery and development will understand that the precise source of extracts or test compounds is not critical to the method of selection of the invention. The compounds used in the investigations may include known compounds (e.g., known therapeutic agents used for other diseases or disorders). Alternatively, any number of unknown chemical compounds or extracts can be investigated using the methods described herein. Examples of such extracts with compounds include, but are not limited to, plant, fungal, prokaryotic or animal extracts, fermentation broths and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available to generate random or targeted synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including but not limited to compounds based on saccharides, lipids, peptides and nucleic acids. Synthetic compound libraries are available on the market from Brandon Associates (Merrimack, New Hampshire, United States) and Aldrich Chemical (Milkwaukee, Wisconsin, United States). As an alternative, libraries of natural compounds in the form of bacteria, fungi, plants and animal extracts from various sources are available on the market, including Biotics (Sussex, United Kingdom).

United); Xenova (Slough, United Kingdom), Harbor Branch Oceangraphics Institute (Ft. Pierce, Florida, United States), and PharmaMar (Cambridge, Massachusetts, United States). In addition, natural and synthetic libraries are produced, if desired, according to methods known in the art, for example, by conventional extraction and fractionation methods. In addition, if desired, any library or compound is easily modified using conventional chemical, physical or biochemical methods. In addition, specialists in the field of drug discovery and development will readily understand that methods of de-replication (eg, taxonomic de-replication, biological de-replication and chemical de-replication, or any combination thereof) should be employed whenever possible. same) or the elimination of replicates or repetitions of materials already known for their activity of alteration of the molt. When it is discovered that a crude extract reduces the activity of a sFlt-1 polypeptide, or binds to a sFlt-1 polypeptide, additional fractionation of the positive extract is needed to isolate chemical constituents responsible for the observed effect. In this way, the objective of the extraction, fractionation and purification process is the characterization and careful identification of a chemical entity within the gross extract that reduces the activity of a sFlt-1 polypeptide. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds that have been shown to be useful as therapeutic agents for the treatment of pre-eclampsia or human eclampsia are chemically modified according to methods known in the art. Therapeutic Agents The present invention features methods for treating or preventing pre-eclampsia or eclampsia in a subject. Preferably, the therapeutic agent is administered during pregnancy for treatment or prevention of pre-eclampsia or eclampsia or after a pregnancy to treat pre-eclampsia or eclampsia after delivery. The techniques and dosages for administration vary depending on the type of compound (anti-body, anti-sense and nucleic acid vector, etc.) and are well known to those skilled in the art or are easily determined. The therapeutic compounds of the present invention may be administered with a pharmaceutically acceptable diluent, carrier or excipient in unit dosage form. Administration can be parenteral, intravenous, subcutaneous, oral or local by direct injection into the amniotic fluid. The preferred method for administering the therapeutic compounds of the present invention is intravenous administration by continuous infusion. The composition may be in the form of a pill, tablet, capsule, liquid or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration. Methods well known in the art for making formulations are found, for example in "Remington: The Science and Practice of Pharmacy" (20th edition, A. R. Gennaro, editor, 2000, Lippincott Williams &Wilkins, Philadelphia, Pennsylvania, United States). Formulations for parenteral administration may contain, for example, excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, vegetable oil or hydrogenated naphthalenes. Biocompatible and biodegradable polymers of lactide, lactide / glycolide copolymer, or polyoxyethylene polyoxypropylene copolymers can be used to control the release of the compounds. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion system and liposomes. The concentration of the compound in the formulation varies depending on several factors including the dosage of the drug to be administered and the route of administration. Optionally, the compound can be administered as pharmaceutically acceptable salts, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulphonic, toluenesulfonic or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethylcellulose or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid or the like. The metal complexes include zinc, iron and the like.

Formulations for oral use include tablets containing the active ingredient (s) in a mixture with non-toxic and pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (for example sucrose and sorbitol), lubricants, glidants and anti-adhesives (for example magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils or talc). . Formulations for oral use can also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent or as soft gelatin capsules where the active ingredient is mixed with water or an oily medium. The dosage and time of administration of the compound depend on several clinical factors that include the general health of the subject and the severity of the symptoms of pre-eclampsia. In general, once pre-eclampsia or a propensity to develop pre-eclampsia is detected, a continuous infusion of the purified protein is used to treat or prevent further progression of the condition. The treatment may be continued for a period of time ranging from 1 to 100 days, more preferably from 1 to 60 days and even more preferably from 1 to 20 days, or until the end of pregnancy. The dosages vary depending on each compound and the severity of the condition and are titrated to achieve a steady state blood serum concentration ranging from 1 to 500 ng / ml of VEGF or PlGF, or both, preferably from 1 to 100 ng / ml. ml, more preferably 5 to 50 ng / ml and even more preferably 5 to 10 ng / ml of VEGF or PIGF or both. Methods for Increasing the Expression of VEGF or PIGF Protein The present invention features methods for increasing the levels of VEGF and PIGF in a subject who has been diagnosed with pre-eclampsia or eclampsia. The highest levels of VEGF or PlGF can be achieved using several different methodologies that are described below, among others. Purified Proteins In a preferred embodiment of the present invention, purified forms of VEGF or PIGF or both are administered to the subject to treat or prevent pre-eclampsia or eclampsia. The purified VEGF or VEGF-like proteins include any protein with an amino acid sequence that is homologous, more desirably, substantially identical to the amino acid sequence of VEGF, or any member of the VEGF family, that can induce angiogenesis or that is capable of promoting the selective growth of endothelial, vascular or endothelial cells of the umbilical vein. An example of a purified VEGF compound is human recombinant VEGF from Genentech, Inc. (San Francisco, California, United States). Purified PIGF or PIGF-like proteins include any protein with an amino acid sequence that is homologous, more desirably substantially identical to the amino acid sequence of PlGF, or any member of the PIGF family, which can induce angiogenesis or which is capable of promote the selective growth of vascular endothelial cells or umbilical vein endothelial cells. An example of purified PIGF available in the market is human recombinant PIGF from R &D Systems (catalog number 264-PG, R &D Systems, Minneapolis, Minnesota, United States). TromboGenics Ltd is also developing a purified form of PIGF for the treatment of ischemic stroke; presumably this form of PIGF would be effective for the applications described in the present invention. Therapeutic Compounds That Increase the Activity of VEGF or PIGF The present invention provides the use of any compound that is known to stimulate or increase blood serum levels of VEGF or PIGF, or the biological activity of these polypeptides, for the treatment or prevention of pre-eclampsia in a subject. These compounds can be used alone or in combination with the purified proteins described above or any of the other methods used to increase the levels of VEGF or PlGF protein described herein. An example of a compound that has been shown to stimulate VEGF production is nicotine. Although the habit of smoking has many risks for the general health of a pregnant woman and her developing fetus, it is believed that nicotine itself is safer than cigarettes and can be used for short-term therapy in high-risk subjects. Examples include Nicorette (nicotine polacrilex), which is a product of nicotine chewing gum of advertising pharmaceutical specialties manufactured by SmithKline Beecham and NicoDerm CQ, which is a nicotine patch of advertising pharmaceutical specialties manufactured by Hoechst Marion Roussel Inc. (formerly Marion Merrell Dow). The nicotine released by tobacco is specifically excluded from the methods of the invention in which the patient has not been diagnosed using the methods of the invention. Nicotine is given after the diagnosis of pre-eclampsia or eclampsia using the patch or chewing gum. Dosages vary depending on the severity of the condition and the general health of the subject. In general, the manufacturer's instructions are followed to achieve a level of nicotine in serum ranging from 5 to 500 ng / ml, more preferably from 5 to 100 ng / ml, and even more preferably from 50 to 100 ng / ml.

Theophylline is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Theophylline is a broncho-dilator that is often used for the treatment of asthma and is available with many commercial names (eg, Aerolate Sr, Asmalix, El-xophyllin, etc.) as well as the generic one. The methods of administration and dosages will vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, daily doses range from 1 to 500 mg, more preferably from 100 to 400 mg and even more preferably from 250 to 350 mg administered twice a day to achieve a serum level of theophylline of 5 to 50 mg / ml. Adenosine is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Adenosine (Fujisawa Pharmaceutical Co.) is commonly used as an anti-hypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dose of 50 mg / kg administered twice a day is typical for adenosine. Nifedipine is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Nifedipine (Bayer Pharmaceuticals) is commonly used as an anti-hypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dosage of 1-2 mg / kg administered twice a day orally or subcutaneously is typical for nifedipine. Minoxidil is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Minoxidil (Pfizer, Inc.) is commonly used as an anti-hypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dosage of 0.25 to 1.0 mg / kg administered twice a day orally or subcutaneously is typical for minoxidil. Magnesium sulfate is another example of an additional compound that can be used to treat or prevent pre-eclampsia or eclampsia. Magnesium sulfate is a generic drug that is typically used as an anti-hypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dosage of 1-2 g administered intravenously every four hours is a typical dosage for magnesium sulfate.

In addition to the use of compounds that can increase serum levels of VEGF or PIGF, the invention provides for the use of any medication for chronic hypertension used in combination with any of the compounds directed to VEGF or PIGF. Medications used for the treatment of hypertension during pregnancy include methyldopa, hydralazine hydrochloride or labetalol. For each of these medications, modes of administration and dosages are determined by the physician and by the manufacturer's instructions. Acid Nucleic Therapeutics Recent work has shown that the provision of nucleic acid (DNA or RNA) capable of expressing a mitogen of endothelial cells such as VEGF at the site of a lesion of a blood vessel will induce the proliferation and re-endothelialization of the injured vessel . Although the present invention does not relate to blood vessel lesions, techniques for delivering nucleic acids encoding endothelial cell mitogens such as VEGF and PIGF used in these studies can also be employed in the present invention. These techniques are described in US Patents 5,830,879 and 6,258,787 and are incorporated herein by reference. In the present invention, the nucleic acid can be any nucleic acid (DNA or RNA) including genomic DNA, CDNA and mRNA, which encodes VEGF or PIGF or any member of the VEGF or PIGF family. The nucleic acid may also include any nucleic acid encoding a protein that has been shown to bind to the sFlt-1 receptor. The nucleic acids encoding the desired protein can be obtained using routine procedures in the art, for example recombinant DNA or PCR amplification. Acid Therapeutic Nucleic Acids that Inhibit sFlt-1 Expression The present invention relates to the use of anti-sense nucleobase oligomer to down-regulate the expression of sFlt-1 mRNA directly. By binding to the complementary nucleic acid sequence (the sense or coding strand), oligomers of anti-sense nucleobases can inhibit protein expression presumably by enzymatic cleavage of the RNA strand by RNase H Preferably, the anti-sense nucleobase oligomer can reduce the expression of the sFlt-1 protein in a cell that expresses excess levels of sFlt-1. Preferably, the reduction of the expression of the sFlt-1 protein is at least 10% with respect to the cells treated with a control oligonucleotide, more preferably 25% and even more preferably 50% or more. In the art, methods for selecting and preparing antisense nucleobase oligomers are well known. As an example of the use of antisense nucleobase oligomers to negatively regulate the expression of VEGF, see US Pat. No. 6,410,322, incorporated herein by reference. Methods for evaluating protein expression levels are also well known in the art and include Western blot, immuno-precipitation and ELISA. The present invention also relates to the use of RNA interference (RNAi) to inhibit the expression of sFlt-1. RNA interference (RNAi) is a recently discovered mechanism of post-transcriptional gene silencing (PTGS) in which a double-stranded RNA (dsRNA) corresponding to a gene or mRNA of interest is introduced into an organism resulting in degradation of the Corresponding mRNA. In the RNAi reaction, both the sense chain and the anti-sense strand of a dsRNA molecule are processed into small RNA fragments or segments that vary in length from 21 to 23 nucleotides (nt) and that have tails 3 'of two nucleotides. Alternatively, synthetic dsRNAs having a length of 21 to 23 nt and having 3 'tails of 2 nucleotides can be synthesized, purified and used in the reaction. These dsRNAs of 21 to 23 nt are known as "guide RNA" or "short interfering RNA" (siRNA). Then the siRNA duplexes bind to a nuclease complex composed of proteins that direct and destroy the endogenous mRNA that has siRNA homology within the complex. Although the identity of the proteins within the complex remains unclear, the function of the complex is to target the homologous mRNA molecule by means of base-pairing interactions between one of the siRNA strands and the endogenous mRNA. The mRNA is then cleaved at a distance of approximately 12 nt from the 3 'end of the siRNA and degraded. In this way, specific genes can be targeted and degraded, thereby achieving a loss of protein expression of the target gene. In the international publication WO 01/75164 (incorporated herein by reference) the specific requirements and modifications of dsRNA are described. Although dsRNA molecules can vary in length, it is most preferable to use siRNA molecules that are dsRNAs of 21 to 23 nucleotides with 3 'overhangs typically 2 to 3 nucleotides (21-deoxy) thymidine or uracil. The siRNAs typically comprise a 3 'hydroxyl group. Mono-catenary siRNAs as well as blunt-ended forms of dsDNA can also be used. In addition, to further improve the stability of RNA, the 3 'overhangs can be stabilized against degradation. In one of these embodiments, the RNA is stabilized including purine nucleotides, such as adenosine or guanosine. Alternatively, substitution of pyrimidine nucleotides by modified analogs is tolerated, for example, substitution of 2-nucleotide overhangs of uridine by (2'-deoxy) thymidine and does not affect the efficacy of RNAi. The absence of a 2 'hydroxyl group significantly improves nuclease resistance of the overhang in a tissue culture medium. Alternatively, siRNA can be prepared using any of the methods indicated in international publication WO 01/75164 (incorporated herein by reference) or by using conventional procedures for RNA in vitro transcription and dsRNA annealing procedures as described above. describes in Elbashir et al., (Genes &; .Dev. , 15: 188-200, 2001). RNAi is also obtained as described in Elbashir et al. By incubation of dsRNA which corresponds to a sequence of the target gene in a Drosophila lysate without cells derived from Drosophila embryos of blastoderm sincitial under conditions in which dsRNA is processed to generate siRNA from about 21 to about 23 nucleotides, which are then isolated using techniques known to those skilled in the art. For example, gel electrophoresis can be used to separate RNAs of 21-23 nt and RNAs can then be levigated from gel slices. In addition, chromatography (eg, size exclusion chromatography), glycerol gradient centrifugation and affinity purification with antibodies can be used to isolate RNAs from 21 to 23 nt. In the present invention, the dsRNA or siRNA is complementary to the sFlt-1 mRNA mRNA sequence and can reduce or inhibit the expression of sFlt-1. Preferably, the reduction of the expression of the sFlt-1 protein is at least 10% with respect to the cells treated with dsRNA or control siRNA, more preferably 25% and even more preferably at least 50%. Also known in the art are methods for evaluating protein expression levels and include Western blot, immuno-precipitation and ELISA. In the present invention, the nucleic acids used include any modification that improves the stability or function of the nucleic acid in any way. Examples include modifications to the phosphate backbone, the internucleotide linkage or the sugar moiety. To simplify the handling and handling of the nucleic acid encoding the sFlt-1 binding protein, the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter. The promoter must be capable of directing expression of the sFlt-1 binding protein in the desired target host cell. The selection of appropriate promoters can be easily performed. Preferably, a high expression promoter would be used. An example of a suitable promoter is the cytomegalovirus (CMV) promoter of 763 base pairs. The Rous Sarcoma Virus (RSV) promoter (Davis, et al., Hum, Gene Ther 4: 151-159, 1993) and the mouse mammary tumor virus (MMTV) promoter can also be used. Certain proteins can be expressed using their native promoter. Other elements that can increase expression (eg, enhancers or a system that results in high levels of expression such as a tat gene and a tar element) can also be included. The recombinant vector can be a plasmid vector such as pUClld, pBR322 or other known plasmid vectors including, for example, an E. coli origin of replication (see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press , 1989). The plasmid vector may also include a selective marker such as the β-lactamase gene for ampicillin resistance, provided that the polypeptide marker does not adversely affect the metabolism of the organism to be treated. The cassette can also be attached to a nucleic acid binding moiety in a synthetic delivery system, such as the system described in the international publication WO 95/22618. The nucleic acid can be introduced into the cells by any means appropriate to the vector employed. Many of these methods are well known in the art (Sambrook et al., Supra, and Watson et al., "Recombinant DNA", Chapter 12, 2nd edition, Scientific American Books, 1992). Recombinant vectors can be transferred by methods such as calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene gun, micro-injection, viral capsid mediated transfer, polybrene mediated transfer or protoblast fusion. As a review of the procedures for the preparation of liposomes, direction and supply of content, see Mannino and Gould-Fogerite, (Bio Techniques, 6: 682-690, 1988), Felgner and Holm (Bethesda Res. Lab. Focus, 11:21, 1989) and Maurer (Bethesda Res. Lab. Focus, 11:25, 1989). The transfer of the recombinant vector (the plasmid vector or viral vectors) can be carried out by means of direct injection into the amniotic fluid or intravenous supply. The supply of genes using adenoviral vectors or adeno-associated vectors (AAV) can also be used. Adenoviruses are present in a large number of animal species, are not very pathogenic and can replicate equally well in dividing and quiescent cells. As a general rule, adenoviruses used for gene delivery lack one or more genes required for viral replication. Recombinant adenoviral vectors with replication defects can be produced for the delivery of VEGF, PIGF or any sFlt-1 binding protein, according to methods known in the art (see Quantin et al., Proc. Nati. Acad. Sci. , 89: 2581-2584, 1992; Stratford-Perricadet et al., J Clin. Invest. , 90: 626-630, 1992; and Rosenfeld et al., Cell, 68: 143-155, 1992). As an example of the use of in utero gene therapy US Pat. No. 6,399,585. Several methods are available for transfection or introduction of dsRNA or oligonucleotides into mammalian cells. For example, there are several transfection reagents available in the market including, but not limited to: TransIT-TKO (Mirus, Cat. No. MIR 2150), Transmessenger (Qiagen, Cat. No. 301525), and Oligofectamine (Invitrogen, Cat No. MIR12252-011). Protocols are available for each transfection reagent at the manufacturer. Once transferred, the nucleic acid is expressed by the cells at the site of the lesion for a period of time sufficient to increase blood serum levels of VEGF, PIGF or any other sFlt-1 binding protein. As the vectors containing the nucleic acid are not normally incorporated into the genome of the cells, the expression of the protein of interest takes place only for a limited period of time. Typically, the protein is expressed at therapeutic levels for about two days to several weeks, preferably for about one to two weeks. A re-application of the DNA may be used to provide additional periods of expression of the therapeutic protein. Recent examples of gene therapy using VEGF for the treatment of vascular disease in mammals can be found in Deodato et al. (Gene Ther., 9: 777-785, 2002); Isner et al. (Human GeneTher., 12: 1593-1594, 2001); Lai et al. (Gene Ther., 9: 804-813, 2002); and reviewed in Freedman and Isner (Ann Intern. Med., 136: 54-71, 2002) and Isner JM (Nature, 415: 234-239, 2002). Assays for Gene and Protein Expression The following methods can be used to evaluate the expression of proteins or genes and determine the efficacy for any of the methods mentioned above for increasing the levels of VEGF, PIGF or any other sFlt-1 binding protein. , or to reduce the levels of the sFlt-1 protein. The levels of VEGF, PIGF or any protein ligand known to bind to sFlt-1 in the subject's blood serum are measured. The methods used to measure serum levels of proteins include ELISA, Western blot or immunoassays using specific anti-bodies. In addition, angiogenesis assays can be performed to determine if the subject's blood has been converted from an anti-angiogenic state to a pro-angiogenic state. Such assays are described above in Example 2. A positive result is considered an increase of at least 20%, preferably 30%, more preferably at least 50% and even more preferably at least 60% at serum levels of VEGF, PlGF or any protein ligand known to bind sFlt-1. A positive result can also be considered the conversion from an anti-angiogenic state to a pro-angiogenic state using the in vitro angiogenesis assay. There are several methods known in the art for evaluating gene expression. Some examples include the preparation of RNA from a subject's blood samples and the use of RNA for Northern blot, PCR-based amplification or RNase protection assays. Use of Anti-Bodies for Therapeutic Treatment The elevated levels of sFlt-1 found in serum samples taken from pregnant women suffering from pre-eclampsia suggests that sFlt-1 is acting as a "physiological fossa" to bind and reduce in the trophoblast cells and maternal endothelial cells functional VEGF and PIGF. The use of compounds, such as anti-bodies, to bind sFlt-1 and block the binding of VEGF or PIGF can help prevent or treat pre-eclampsia or eclampsia, producing an increase in VEGF or free PIGF levels. Such an increase would allow an increase in the proliferation of trophoblasts, migration and angiogenesis required for placental development and fetal nutrition, and for the health of systemic maternal endothelial cells. The present invention provides anti-bodies that specifically bind to the ligand binding domain of sFlt-1. The anti-bodies are used to inhibit sFlt-1 and it is believed that the most effective mechanism is by direct blocking of the binding sites for VEGF or PIGF, however, other mechanisms can not be ruled out. Methods for the preparation and use of anti-bodies for therapeutic purposes are described in several patents including patents US 6,054,297; 5,821,337; 6,365,157; and 6,165,464 and are incorporated herein by reference. The anti-bodies can be polyclonal or monoclonal; monoclonal anti-bodies are preferred. Monoclonal anti-bodies, particularly those derived from rodents including mice, have been used for the treatment of various diseases; however, there are limitations in its use including the induction of anti-mouse human immunoglobulin responses that result in rapid elimination and a reduction in treatment efficacy. For example, an important limitation in the clinical use of rodent monoclonal anti-bodies is an anti-globulin response during therapy (Miller et al., Blood, 62: 988-995, 1983; Schroff et al., Cancer Res., 45 : 879-885, 1985). The subject has tried to solve this problem by constructing "chimeric" anti-bodies in which a variable domain of binding to an animal antigen is coupled to a human constant domain (US Pat. No. 4,816,567; Morrison et al., Proc. Nati. Acad. Sci. USA, 81: 6851-6855, 1984; Boulianne et al., Nature 312: 643-646, 1984; Neuberger and collaborators, Nature, 314: 268-270, 1985). The production and use of such chimeric anti-bodies is described below. The competitive inhibition of the binding of a ligand to sFlt-1 is useful for the prevention or treatment of pre-eclampsia or eclampsia. The anti-bodies directed to sFlt-1 can block the binding of VEGF or PIGF to sFlt-1 resulting in higher levels of VEGF or PIGF. Such an increase can result in a rescue of endothelial dysfunction and a shift in the balance of pro-angiogenic / anti-angiogenic factors towards angiogenesis. A mixture of the monoclonal anti-bodies of the present invention can be used as an effective treatment for pre-eclampsia or eclampsia. The mixture may include only 2, 3 or 4 different anti-bodies or as many as 6, 8 or 10 different anti-bodies. In addition, the anti-bodies of the present invention can be combined with an anti-hypertensive drug (e.g., methyldopa, hydralazine hydrochloride or labetalol) or any other medication used to treat pre-eclampsia, eclampsia or the symptoms associated with pre-eclampsia. -eclampsia or eclampsia. Preparation of Anti-Bodies Monoclonal anti-bodies that bind specifically to the sFlt-1 receptor can be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein (Nature, 256: 495-497, 1975) and Campbell ("Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burton et al., Editors, Laboratory Techniques in Biochemistry and Molecular Biology, volume 13, Elsevier Science Publishers, Amsterdam, 1985), as well as by the recombinant DNA method described by (Science, 246, 1275-1281, 1989). Monoclonal anti bodies can be prepared from supernatants of poorly cultured hybrid cells or from ascites fluid induced by intra-peritoneal inoculation of hybrid cells in mice. The hybrid technique originally described by Kohler and Milstein (Eur. J. Immunol, 6, 511-519, 1976) has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal anti-bodies against many specific antigens. The route and immunization program of the host animal or of the cells producing cultured anti-bodies thereof, is generally in accordance with established and conventional techniques for the stimulation and production of anti-bodies. Typically, mice are used as the test model; however, any mammalian subject including human or anti-body-producing cells thereof, can be manipulated according to the processes of this invention to serve as the basis for the production of mammalian cell lines, including human.

After immunization, immune lymphoid cells are fused with myeloma cells to generate a hybrid cell line that can be grown and subcultured indefinitely, to produce large amounts of monoclonal anti-bodies. For the purposes of this invention, the immune lymphoid cells selected for fusion are lymphocytes and their normal differentiated progeny, extracted from lymph node tissue or from spleen tissue of immunized animals. The use of spleen cells is preferred, since they offer a more concentrated and convenient source of anti-body producing cells with respect to the mouse system. The myeloma cells provide the basis for a continuous propagation of the fused hybrid. Myeloma cells are tumor cells derived from plasma cells. Murine myeloma cell lines can be obtained, for example, from the American Type Culture Collection (ATCC, Manassas, Virginia, United States). Human myeloma and mouse-human heteromyeloma cell lines have also been described (Kozbort et al., J. Immunol., 133: 3001-3005, 1984; Brodeur et al; Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc. , New York, pp. 51-63, 1987). Hybrid cell lines can be maintained in vitro in cell culture medium. Once the hybridoma cell line has been established, it can be maintained in a variety of nutritionally suitable media such as the hypoxanthine-aminopterin-thymidine (HAT) medium. In addition, hybrid cell lines can be stored and stored in any number of conventional ways, including freezing and storage in liquid nitrogen. The frozen cell lines can be revived and cultured indefinitely with a resumed synthesis and secretion of monoclonal anti-body. The secreted anti-body is recovered from the tissue culture supernatant by conventional methods such as precipitation, ion exchange chromatography, affinity chromatography or the like. The anti-body can be prepared in any mammal, including mice, rats, rabbits, goats and humans. The anti-body can be a member of one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE and its sub-classes, preferably it is an anti-body IgG. Although the preferred animal for producing monoclonal anti-bodies is the mouse, the invention is not limited thereto; in fact, anti-human bodies can be used and it may be preferable. Such anti bodies can be obtained using human hybridomas (Colé et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss Inc., p 77-96, 1985). In the present invention, techniques developed for the production of chimeric anti-bodies can be used by splicing the genes of a mouse anti-body molecule of appropriate antigen specificity together with genes from an anti-human body molecule (Morrison and collaborators, Proc. Nati, Acad. Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454, 1985). Such anti-bodies are within the scope of this invention and are described below. As another alternative to the cell fusion technique, immortalized B cells of Epstein-Barr virus (EBV) are used to produce the monoclonal anti-bodies of the present invention (Crawford D. et al., J. of Gen. Virol. , 64: 697-700, 1983; Kozbor and Roder, J. Immunol., 4: 1275-1280, 1981; Kozbor et al., Methods in Enzymology, 121: 120-140, 1986). In general, the procedure consists of isolating the Epstein-Barr virus from a suitable source, usually a line of infected cells, and exposing the secretory cells of the anti-body supernatants containing the virus. The cells are washed and cultured in an appropriate cell culture medium. Subsequently, the cells transformed by the virus present in the cell culture can be identified by the presence of the Epstein-Barr virus nuclear antigen and the transformed anti-body secreting cells can be identified using conventional methods known in the art. Other methods for producing monoclonal anti-bodies such as recombinant DNA are also included within the scope of the invention. Preparation of Immunogens of sFlt-1 sFlt-1 can be used by itself as an immunogen, it can bind to a support protein or to other objects such as sepharose beads. sFlt-1 can be purified from cells known to express the endogenous protein such as human umbilical vein endothelial cells (HUVEC; Kendall et al., Biochem. Biophys. Res. Comm. , 226: 324-328, 1996). In addition, nucleic acid molecules encoding sFlt-1 or portions thereof can be inserted into vectors known for expression in host cells using standard recombinant DNA techniques. Host cells suitable for the expression of sFlt-1 include baculovirus cells (e.g. Sf9 cells), bacterial cells (e.g. E. coli) and mammalian cells (e.g., NIH3T3 cells). In addition, peptides can be synthesized and used as immunogens. Methods for making anti-peptide anti bodies are well known in the art and generally require coupling the peptide to a suitable support molecule, such as serum albumin. The peptides include any amino acid sequence that is substantially identical to any part of the amino acid sequence of sFlt-1 corresponding to the GenBanK accession number U01134.

The peptides may be of any length, preferably 10 amino acids or more, more preferably 25 amino acids or more, and even more preferably 40, 50, 60, 70, 80 or 100 amino acids or more. Preferably, the amino acid sequences have an identity of at least 60%, more preferably 85% and even more preferably 95% with the U01134 sequence. The peptides may be commercially available or manufactured using methods well known in the art, such as, for example, the Merrifield solid phase method (Science, 232: 341-347, 1985). The method can use commercially available synthesizers such as the Biosearth 9500 automatic peptide machine, cleavage of the blocked amino acids with hydrogen fluoride being achieved, and the peptides purified by preparative HPLC using a Waters Delta Prep 3000 instrument, in a column of 15 -20 μm Vydac C4 PrepPAK. Functional Equivalents of Anti-Bodies The invention also includes functional equivalents of the anti-bodies described in this specification. Functional equivalents include polypeptides with amino acid sequences substantially identical to the amino acid sequence of the variable or hyper-variable regions of the antibodies of the invention. The functional equivalents have binding characteristics comparable to those of the anti-bodies and include, for example, humanized and mono-catenary chimerized anti-bodies as well as their fragments. In the international publication WO 93/21319; patent application EP 0 239 400; international publication WO 89/09622; patent application EP 0 338 745; patent application EP 0 332 424; and US Patent 4,816,567; each of which is incorporated herein by reference, methods for producing such functional equivalents are described. The chimerized anti-bodies preferably have constant regions derived substantially or exclusively from constant regions of anti-human bodies and variable regions derived substantially or exclusively from the variable region sequence of a mammal other than a human. Such humanized anti-bodies are chimeric immunoglobulins, immunoglobulin chains or fragment thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antigen-binding sub-sequences of antibodies) containing the minimum derivative sequence of a non-human immunoglobulin. In the art, methods to humanize non-human bodies are well known (as reviewed see Vaswani and Hamilton, Ann Allergy Asthma Immunol., 81: 105-119, 1998 and Carter, Nature Reviews Cancer, 1: 118-129, 2001) . Generally, a humanized anti-body has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically taken from a variable import domain. Humanization can be carried out essentially following the methods known in the art (Jones et al., Nature, 321: 522-525, 1986; Riechmann et al., Nature, 332: 323-329, 1998 and Verhoeyen et al., Science, 239). : 1534-1536, 1988), substituting rodent CDRs or other CDR sequences for the corresponding sequences of a human anti-bodies. Accordingly, such humanized anti-bodies are chimeric anti-bodies in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species (see, for example, US Pat. No. 4,816,576). In practice, humanized antibodies are typically anti-human bodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites of rodent anti bodies (Presta, Curr. Op. Struct. Biol. 2: 593-596, 1992). Other methods for the preparation of humanized anti-bodies can be found in US Patents 5,821,337 as well as 6,054, 297, and Carter, (supra), which are incorporated herein by reference. The humanized anti-body is selected from any class of immunoglobulins, including IgM, IgG, IdD and IgE, and any isotope, including IgG1? IgG2, IgG3 and IgG4. When the cytotoxic activity is not needed, as in the present invention, the constant domain preferably is of the IgG2 class. The humanized anti-body may comprise sequences of more than one class or isotype, and it is within the usual experience in the art to select particular constant domains to optimize the desired effector functions. Anti-human bodies can also be produced using various methods known in the art, including phage display libraries (Marks et al., J. Mol. Biol., 222: 581-597, 1991 and Winter et al Annu, Rev. Immunol. , 12: 433-455, 1994). The techniques of Colé et al. And Boerner et al. Are also useful for the preparation of human monoclonal antibodies (Colé et al., Supra).; Boerner et al., J ". Immunol., 147: 86-95, 1991.) Suitable mammals other than humans include any mammal for which monoclonal antibodies can be manufactured.Examples of mammals other than humans include, for example, a council, rat, mouse, horse, goat or primate, a mouse is preferred The functional equivalents of anti-bodies also include fragments of mono-catenary anti-bodies, also known as mono-catenary anti-bodies (scFvs). fragments of mono-catenary anti-bodies are recombinant polypeptides that typically bind to antigens or receptors, these fragments contain at least one fragment of an anti-body variable heavy chain (VH) amino acid sequence chained to at least one fragment of a variable anti-body (VL) light chain sequence with or without one or more interconnect linkers-such a linker can be a short flexible peptide selected to ensure It should be noted that proper three-dimensional folding of the VL and VH domains occurs once they have been associated to maintain the binding specificity of the target molecule of the whole anti-body from which the mono-catenary anti-body fragment proceeds. Generally, the carboxyl terminus of the VL or VH sequence is covalently linked by such a peptide linker to the amino acid terminus of a complementary VL and VH sequence. Mono-catenary anti-body fragments can be generated by molecular cloning, anti-body phage display libraries or similar techniques. These proteins can be produced in eukaryotic cells or prokaryotic cells, including bacteria. The monocatenary anti-body fragments contain amino acid sequences having at least one of the variable or CDR regions of the whole anti-bodies described in this specification, but lacking some or all of the constant domains of those anti-bodies. bodies. These constant domains are not necessary for antigen binding, but they constitute a major portion of the entire antibody structure. Therefore, mono-catenary anti-body fragments can solve some of the problems associated with the use of anti-bodies that contain part or all of the constant domain. For example, monocatenary anti-body fragments tend to be free of unwanted interactions between biological molecules and the heavy chain constant region, or other unwanted biological activity. In addition, the fragments of mono-catenary anti-bodies are considerably smaller than the whole anti-bodies and, therefore, may have higher capillary permeability than the whole anti-bodies, allowing the anti-body mono-catenary fragments locate and bind to target antigen binding sites more efficiently. In addition, anti-body fragments can be produced on a relatively large scale in prokaryotic cells, thereby facilitating production. In addition, the relatively small size of the anti-body fragments. mono-catenaries makes them less likely to elicit an immune response in a recipient compared to whole antibodies. Functional equivalents also include fragments of anti-bodies that have the same binding characteristics or binding characteristics comparable to those of the whole anti-body. Such fragments may contain a Fab fragment or an F (ab ') 2 fragment or both fragments. Preferably, the anti-body fragments contain the six CDRs of the whole anti-body, although fragments containing less than all said regions such as three, four or five CDRs are also functional. In addition, the functional equivalents may be or may combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and their subclasses. Preparation of Anti-Body Functional Equivalents Anti-body equivalents are prepared by methods known in the art. For example, fragments of anti-bodies can be prepared enzymatically from whole anti-bodies. Preferably, antibody equivalents are prepared from DNA encoding such equivalents. The DNA encoding anti-body fragments can be prepared by removing all but the desired portion of the DNA encoding the full-length anti-body. The DNA encoding chimerized anti bodies can be prepared by recombinant DNA which encodes substantially or exclusively human constant regions and DNA encoding variable regions derived substantially or exclusively from the variable region sequence of a mammal other than a human. DNA encoding humanized anti-bodies can be prepared by recombination of DNA encoding constant regions and variable regions other than CDRs derived substantially or exclusively from the corresponding human anti-body regions and DNA encoding CDRs derived substantially or exclusively from a different mammal of a human being. Suitable sources of DNA molecules encoding anti-body fragments include cells, such as hybrid-mas, that express the full-length anti-body. The fragments can themselves be used as antibody equivalents, or can be recombined in equivalents as described above. The DNA deletions and recombinations described in this section can be performed by known methods, such as those described in the published patent applications indicated above. Research and Selection of Anti-Bodies Monoclonal anti-bodies are isolated and purified using conventional methods known in the art. For example, anti-bodies can be investigated using conventional methods known in the art such as ELISA against the sFlt-1 peptide antigen or Western blot analysis. Examples of such techniques are described in Examples II and III of US Patent 6,365,157, incorporated herein by reference. Anti-Body Therapeutic Uses When used in vivo for the treatment or prevention of pre-eclampsia or eclampsia, the anti-bodies of the present invention are administered to the subject in therapeutically effective amounts. Preferably, the anti-bodies are administered parenterally or intravenously by continuous infusion. The dose and the dosage regimen depend on the severity of the disease, and on the general state of health of the subject. The amount of antibody administered is typically in the range of about 0.01 to about 10 mg / kg of subject weight. For parenteral administration, the anti bodies are formulated in an injectable unit dosage form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are intrinsically non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can also be used as vehicles. The vehicle may contain minor amounts of additives such as substances that increase isotonicity and chemical stability, for example, buffers and preservatives. Anti-bodies are typically formulated in such vehicles at concentrations of about 1 mg / ml to 10 mg / ml. Therapeutic Compounds That Inhibit sFlt-1 Since levels of sFlt-1 are elevated in subjects who have pre-eclampsia, eclampsia or who are prone to develop such conditions, any agent that decreases the expression of a polypeptide or nucleic acid molecule of sFlt-1 is useful in the methods of the invention. Such agents include small molecules that can alter the binding of sFlt-1 to VEGF or PIGF, antisense nucleobase oligomers and dsRNA used to mediate RNA interference. Combination Therapies Optionally, a therapeutic agent for pre-eclampsia or eclampsia can be administered in combination with any other conventional therapy for pre-eclampsia or eclampsia; such methods are known to the person skilled in the art and are described in this document. A therapeutic agent for pre-eclampsia or eclampsia of the invention can be administered in combination with any compound that increases the activity of a VEGF pathway. Non-limiting examples of agents that also induce the production of endogenous VEGF include nicotine, minoxidil, nifedipine, adenosine, magnesium sulfate and theophylline. In one embodiment, the PIGF protein can be used in combination with any of the agents that induce the production of endogenous VEGF indicated above. Control of Subjects The disease or treatment status of a subject having pre-eclampsia, eclampsia or propensity to develop such a condition can be controlled using the methods and compositions of the invention. In one embodiment, the expression of a sFlt-1, VEGF or PIGF polypeptide present in a body fluid, such as urine, plasma, amniotic fluid or CFS, is controlled. Such control may be useful, for example, to evaluate the efficacy of a particular drug in a subject or to evaluate the progression of the disease. Particularly useful in the invention are therapeutic agents that reduce the expression of a sFlt-1 nucleic acid or polypeptide molecule that enhances the expression of a VEGF or PIGF polypeptide or nucleic acid molecule. Other Forms of Embodiment From the above description, it is evident that variations and modifications may be made to the invention described in this document to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims. All publications mentioned in this specification are incorporated herein by reference as if it were specifically and individually indicated that each publication or independent patent application is incorporated by reference. In addition, US 2004-0126828 and PCT publication WO 2004 / 008946A2 are hereby incorporated by reference in their entirety.

Claims (66)

1. CLAIMS 1. A method of monitoring the treatment of pre-eclampsia or eclampsia in a subject, comprising measuring the level of polypeptide SFlt-1, VEGF or PIGF in a sample of said subject. The method of claim 1, wherein said level measurement is performed on two or more occasions and a change in said levels between said measurements is indicative of pre-eclampsia or eclampsia. The method of claim 1, further comprising comparing said level with a pre-eclampsia reference, wherein a reduction in the sFlt-1 level relative to said pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia in said subject. The method of claim 1, further comprising comparing said level with the pre-eclampsia reference, wherein an increase in the level of VEGF or PIGF relative to said pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia in said subject. The method of claim 1, wherein said monitoring is used to determine the therapeutic dosage of a compound. 6. The method of claim 5, wherein said compound is administered in a dosage such that the level of sFlt-1 in said subject is less than 2 ng / ml. The method of claim 1, wherein said measurement is performed using an immunological assay. 8. The method of claim 1, wherein the level of sFlt-1 is the level of free, bound or total sFlt-1. The method of claim 1, wherein the level of sFlt-1 is the level of a polypeptide byproduct of a sFlt-1 polypeptide that has been enzymatically degraded or cut. 10. A method of monitoring the treatment of pre-eclampsia or eclampsia in a subject, comprising measuring the levels of at least two of the sFlt-1, VEGF and PIGF polypeptides in a sample of said subject and calculating the relationship between said levels using a metric The method of claim 10, wherein said metric is an anti-angiogenic index pre-eclampsia (P7AAI): [sFlt-1 / VEGF + PIGF]. 12. The method of claim 11, wherein a value PAAI less than 20 indicates an improvement in such pre-eclampsia or eclampsia. 13. The method of claim 12, wherein a PAAI value of less than 10 indicates an improvement in said pre-eclampsia or eclampsia. The method of claim 11, wherein said PAAI is used to determine the dosage of the therapeutic compound. 15. The method of claim 14, wherein the therapeutic compound is administered in a dose such that the PAAI is less than 20 after said therapeutic compound is administered. 16. The method of claim 15, wherein the therapeutic compound is administered in a dose such that the PAAI is less than 10 after said therapeutic compound is administered. 17. The method of claim 10, wherein said measurement is performed using an immunological assay. 18. The method of claim 10, wherein said level of sFlt-1 is the level of free, bound or total sFlt-1. The method of claim 10, wherein said level of sFlt-1 is the level of a polypeptide byproduct of a sFlt-1 polypeptide that has been enzymatically degraded or cut. The method of claim 10, wherein said level of VEGF or PIGF is the level of free VEGF or PIGF. 21. The method of claim 10, wherein a reduction in the level of the sFlt-1 polypeptide relative to a pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia. The method of claim 10, wherein an increase in the level of the free VEGF polypeptide relative to the pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia. 23. The method of claim 10, wherein an increase in the level of the free PIGF polypeptide relative to the pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia. The method of claim 10, wherein said level measurement is performed on two or more occasions and a change in said levels between measurements is a diagnostic indicator of pre-eclampsia or eclampsia. 25. A method of monitoring the treatment of pre-eclampsia or eclampsia in a subject, which comprises measuring the level of the nucleic acid molecule sFlt-1, VEGF or PIGF in a sample of said subject and comparing said level with a reference, where an alteration in said level in relation to said reference sample diagnoses pre-eclampsia or eclampsia in said subject. 26. The method of claim 25, wherein when the level of VEGF is measured, then the level of sFlt-1 or PIGF is also measured. The method of claim 25, wherein a reduction in the level of the sFlt-1 nucleic acid relative to a pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia. The method of claim 25, wherein an increase in the level of the VEGF nucleic acid relative to a pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia. 29. The method of claim 25, wherein an increase in the level of the PIGF nucleic acid relative to a pre-eclampsia reference indicates an improvement in said pre-eclampsia or eclampsia. 30. The method of claim 25, wherein said level measurement is performed on two or more occasions and a change in said levels between measurements is a diagnostic indicator of pre-eclampsia or eclampsia. 31. A method of diagnosing a subject as having, or having a propensity to develop, pre-eclampsia or eclampsia, said method comprising measuring the level of free PIGF in a urine sample of said subject. 32. The method of claim 31, wherein a level of free PIGF in said urine sample of less than 400 pg / ml measured during mid-gestation or late gestation is a diagnostic indicator of pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia. The method of claim 1, wherein a level of free PIGF in said urine sample of less than 200 pg / ml measured during mid-gestation or late gestation is a diagnostic indicator of pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia. -eclampsia or eclampsia. 34. The method of claim 31, further comprising said free PIGF level of said subject at the PIGF level of a reference sample. 35. The method of claim 34, wherein said reference sample is a previous sample taken from said subject. 36. The method of claim 34, wherein said reference sample is a sample taken from a subject who is pregnant but does not have pre-eclampsia or eclampsia, or a propensity to develop pre-eclampsia or eclampsia. 37. The method of claim 34, wherein a reduction in said free PIGF of said subject compared to said reference sample is a diagnostic indicator of pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia. 38. The method of claim 37, wherein said reduction is a reduction of at least 10% at said PIGF level of said subject sample as compared to said reference sample. 39. The method of claim 31, further comprising: (a) measuring the level of at least one polypeptide of Sflt-1, PIGF and VEGF in a sample of said subject, wherein said sample is a body fluid selected from the group consisting of in urine, blood, amniotic fluid, serum, plasma or cerebrospinal fluid; and (b) comparing said level of sFlt-1, PIGF or VEGF of said subject with the level of the polypeptide sFlt-1, PIGF or VEGF in a reference sample, where an increase in said level of sFlt-1 or a reduction in said level of the VEGF or PIGF polypeptide of said subject sample compared to said reference sample is a diagnostic indicator of pre-eclampsia or eclampsia, or a propensity to develop pre-eclampsia or eclampsia. 40. The method of claim 39, wherein the level of Sflt-1 of a serum sample of said subject is measured. 41. The method of claim 39, wherein the level of sFlt-1 and PIGF of a serum sample of said subject is measured. 42. The method of claim 39, further comprising calculating the ratio between the levels of at least one of sFlt-1, VEGF and PIGF from step (a) using a metric, where an alteration in the relationship between said levels in the sample of the subject in relation to a reference sample diagnoses pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia in said subject. 43. The method of claim 42, wherein said metric is an anti-angiogenic index of pre-eclampsia (PAAI): [sFlt-1 / VEGF + PIGF]. 44. The method of claim 43, wherein a PAAI value greater than 20 is a diagnostic indicator of pre-eclampsia or eclampsia. 45. The method of claim 31, wherein said measurement is performed using an immunological assay. 46. The method of claim 45, wherein said immunological assay is an ELISA assay. 47. A method of diagnosing a subject as having or having a propensity to develop pre-eclampsia or eclampsia, said method comprising (a) obtaining a urine sample from said subject; (b) contacting said sample with a solid support, wherein said solid support comprises a first immobilized PIGF binding agent, for a time sufficient to allow the ligature of said first binding agent of PIGF with free PIGF present in said sample; (c) contacting said solid support after step (b) with a preparation of a second marbling PIGF binding agent, for a time sufficient to allow the ligating of said second PIGF binding agent marinated to said bound free PIGF to said first immobilized PIGF ligation agent; (d) observing the binding of said second marginated PIGF binding agent to the immobilized PIGF binding agent linked to the free PIGF in the position where the PIGF binding agent is immobilized; and (e) comparing the ligature observed in step (d) with the ligature observed using a reference sample. 48. The method of claim 47, wherein said reference sample is recombinant PIGF at a concentration of 400 to 800 pg / ml and a reduction in the binding observed in step (d) compared to the binding observed using a sample of Reference in step (e) is a diagnostic indicator of pre-eclampsia or eclampsia or a propensity to develop pre-eclampsia or eclampsia. 49. The method of claim 47, wherein the tag is a colorimetric tag. 50. The method of claim 47, wherein said agent that binds to PIGF is an anti-body, or a purified fragment thereof, or a peptide. 51. The method of claim 50, wherein said anti-body or fragment thereof specifically binds to free PIGF. 52. The method of claim 47, wherein said agent which binds to a binding agent of PlGF is selected from the group consisting of: an anti-immunoglobulin anti-body, protein A, and protein G. 53. A method of diagnosing a subject as having or having a propensity to develop pre-eclampsia or eclampsia, said method comprising: (a) obtaining a urine sample from said subject; (b) contacting said sample with a solid support, wherein said solid support comprises an immobilized PIGF binding agent that is detectably marbined where said contact is for a sufficient time to allow the ligation of said PIGF binding agent. with free PIGF present in said sample; and (c) measuring said marketed PIGF binding agent linked to said free PIGF, wherein said measurement is capable of distinguishing between said bound and unbound PIGF binding agent. 54. The method of claim 53, wherein said tag is a fluorescent tag. 55. The method of claim 47 or 53, wherein said solid support is a membrane. 56. The method of claim 47 or 53, wherein said subject is a non-pregnant human being and said method diagnoses a propensity to develop pre-eclampsia or eclampsia. 57. The method of claim 47 or 53, wherein said subject is a pregnant human being. 58. The method of claim 47 or 53, further comprising measuring the level of nucleic acid or polypeptide sFlt-1, PIGF, or VEGF in a body fluid sample of said subject. 59. The method of claim 58, wherein said level of sFlt-1 polypeptide is measured in a serum sample of said subject. 60. The method of claim 47 or 53, wherein said level measurement is performed on two or more occasions and a change in said levels between measurements is a diagnostic indicator of pre-eclampsia or eclampsia. 61. A kit for diagnosing pre-eclampsia or eclampsia in a subject, which comprises a P1GF ligation agent for detecting the free PIGF polypeptide and instructions for its use for the diagnosis of pre-eclampsia or eclampsia or the propensity to develop pre-eclampsia or eclampsia in a subject. 62. The kit of claim 61, further comprising a component for an immunological assay, an enzymatic assay, a fluorescence polarization assay, or a colorimetric assay. 63. The kit of claim 61, wherein said PIGF binding agent is immobilized on a membrane. 64. The kit of claim 63, wherein said membrane is supported on a needle structure that is immersed and the sample is deposited on the membrane by placing the needle structure that is immersed in the sample. 65. The kit of claim 63, wherein said membrane is supported on a lateral flow cassette, and the sample is deposited on the membrane through an aperture in the cassette. 66. The kit of claim 61, wherein said kit is used to monitor said subject during therapy.