WO2001001146A1 - Method of predicting an ability to develop new blood vessels in response to hypoxia or ischemia - Google Patents

Abstract

A method for determining a potential of a patient to form new blood vessels in response to hypoxia or ischemia, the method comprising the step of monitoring a vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue derived from the patient in response to oxygen deprivation.

Description

I

METHOD OF PREDICTING AN ABILITY TO DEVELOP NEW BLOOD VESSELS IN RESPONSE TO HYPOXIA OR ISCHEMIA

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to the use of VEGF response to hypoxia in cells or tissue derived from patients as a means of identifying patients likely to require intervention before and/or after occluded coronary arteries, retinopathy or cancer development.

Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.

The coronary artery collateral circulation is an anastomotic network of blood vessels that interconnects the three major epicardial coronary arteries. Coronary artery disease is frequently associated with a marked increase in the collateral circulation as demonstrated by angiography and autopsy studies. The collateral circulation is beneficial in limiting myocardial ischemia/infarction in the face of coronary artery stenosis and acute coronary occlusion [1-3].

Coronary collateral develop by the process of neo-angiogenesis or new blood vessel formation [4]. The stimulation of angiogenesis is the physiological response of a tissue to hypoxia or ischemia, which results in an increase in blood supply to the tissue [5]. It was first demonstrated in tumors that there are specific growth factors which can stimulate the process of angiogenesis [6]. The first such growth factor demonstrated to be hypoxia-inducible was vascular endothelial growth factor (VEGF) [7, 8]. VEGF was subsequently demonstrated to be induced by ischemia and hypoxia in non-malignant cells such as the cardiac myocyte and monocytes that are present in the ischemic myocardium [9-13]. Moreover, the monocyte has recently been demonstrated to play a critical role in the angiogenic response seen in a model of chronic vascular insufficiency [14]. A key problem in clinical cardiology is the inter-individual differences in the degree of collateral blood vessel formation among patients, with only approximately 50 % of patients with coronary artery stenosis developing collaterals [3]. Absence of collateral vessels in some patients might be explained by the occurrence of acute vascular syndromes resulting from rupture of a previously hemodynamically insignificant atherosclerotic plaque. However, this certainly cannot explain the marked heterogeneity in compensatory angiogenesis observed in many subjects with chronic stable coronary artery and peripheral vascular disease, and in hibernating myocardium.

While conceiving the present invention it was hypothesized that failure to generate new blood vessels such as collateral vessels with chronic vascular insufficiency could be associated with a failure to appropriately increase VEGF production with hypoxia or ischemia. Should this be the case, the correlation between VEGF response to hypoxia or ischemia can be used for identification of patients likely to require intervention as atherosclerosis develops in advance of any clinical symptoms and to alleviate myocardial ischemia. It will be appreciated in this respect that current clinical practice relies on catheterization of coronary arteries to provide data on collateral circulation. The catheterization procedure, although widely accepted, carries with it inherent risk.

There is thus a widely recognized need for, and it would be highly advantageous to have, a method for identifying in advance those patients at high risk to suffer from clinical cardiology problems in the future with the inherent risk involved in the catheterization procedure removed.

The natural history of non-proliferative and proliferative diabetic retinopathy (DR) has been well documented in several multicenter clinical trials [35-41]. Specific stages of DR, normally progressing in an orderly fashion, are characterized by defined clinical parameters on ophthalmological examination [35-41]. The prevalence and severity of non- proliferative and proliferative DR has been shown to be related to the age of diabetes onset [42-44], the duration of diabetes [41, 45, 46], the type of diabetes [47] and the level of glycemic control [40, 41, 48-54] in large population studies. However, it is well recognized that considerable variability exists between individuals in the development and progression of DR [36, 37, 55-57]. In type 1 diabetes mellitus (DM), non-proliferative DR peaks in incidence after 12-18 years of DM with approximately 98% of all patients demonstrating evidence of any DR after 20 years of exposure to DM [42, 58, 59]. In type II DM there is a peak in the incidence of non- proliferative DR 5-12 years after DM is diagnosed [45, 60]. The apparent shorter duration until onset of non-proliferative DR in type II DM patients may be attributed at least in part to the well-documented 5-7 year mean lag in the recognition and diagnosis of type II DM [60]. Over the past 5-6 years a mounting body of evidence has been generated that supports a major role for vascular endothelial growth factor (VEGF) in the pathogenesis of proliferative DR [61-68]. The stimulus for increased VEGF in the retina and ocular fluid of patients with proliferative retinopathy is due in large part to the production of retinal ischemia and hypoxia. VEGF has also been implicated in the development of early phases of non-proliferative diabetic retinopathy. Elevated levels of VEGF, directly resulting in an increase in vascular permeability, are increased in diabetic subjects before the onset of retinopathy [65, 69]. Injection of VEGF into normal non-human primate eyes induces many of the hallmark changes of non-proliferative retinopathy [70]. Retinal blood flow has been shown to be decreased significantly in the diabetic patient prior to the onset of any diabetic retinopathy [71] which may result in ischemia/hypoxia in the retina and in turn stimulate VEGF production. However, hypoxia may not be the most important stimulus for increased VEGF production in the retina of patients before the onset of non-proliferative DR [69]. A wide variety of cytokines and metabolites (glucose [72-74], advanced glycation end products [75, 76], IGF-I [77-79], insulin [80], angiotensin II [81-83]) are elevated in the diabetic patient and have been suggested to promote DR via stimulation of VEGF. Furthermore, many of these cytokines and metabolites induce VEGF through an similar if not identical signal transduction pathway as hypoxia [76, 80].

There is a widely recognized need for, and it would be highly advantageous to have, a method for identifying in advance those patients at high risk to suffer from retinal angiogenesis problems in the future. While conceiving the present invention is was hypothetized that diabetic patients with DR produce more VEGF in response to hypoxia than those diabetics who have not developed any evidence of DR.

Similarly, there is a widely recognized need for, and it would be highly advantageous to have, a method for identifying in advance those patients at high risk to suffer from tumor angiogenesis.

U.S. Pat. No. 5,332,671, teaches production of recombinant VEGF in recombinant protein expression systems via the use of VEGF DNA sequences. It further teaches anti-VEGF antibodies. Therefore, the possibility of delivering VEGF to a patient as a means of inducing collateral circulation or the possibility of delivering anti-VEGF antibodies to a patient as a means of reducing retinal or tumor angiogenesis are practical. U.S. Pat. No. 5,332,671 is incorporated herein by reference for all purposes as if fully set forth herein.

Thus, should failure to generate new blood vessels such as collateral coronary vessels with chronic vascular insufficiency be associated with a failure to appropriately increase VEGF production with hypoxia or ischemia, VEGF could be employed as a novel means of therapy for increasing coronary artery collateral blood flow by augmenting the ability of a patient to form coronary collateral blood vessels. SUMMARY OF THE INVENTION

Data submitted in support of the present invention demonstrates that failure to generate collateral vessels in patients with chronic vascular insufficiency is associated with a failure to appropriately increase VEGF production with hypoxia or ischemia. The VEGF response to hypoxia in monocytes harvested from patients with coronary artery stenosis correlates with the presence of coronary artery collaterals in the same patients. Patients with increased hypoxic induction of VEGF exhibit more collateral circulation than those patients with lower VEGF induction in response to hypoxia.

The VEGF response to hypoxia in monocytes harvested from patients with coronary artery stenosis correlates with the presence of coronary artery collaterals in the same patients. Patients with increased hypoxic induction of VEGF exhibit more collateral circulation than those patients with lower VEGF induction in response to hypoxia.

Thus, according to the present invention there is provided a method for determining a potential of a patient to form new blood vessels in response to hypoxia or ischemia, the method comprising the step of monitoring a vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue derived from the patient in response to oxygen deprivation.

According to further features in preferred embodiments of the invention described below, the vascular endothelial growth factor expressing cells or tissue are selected from the group consisting of monocytes, fibroblasts, lymphocytes and tissue biopsy specimens from heart, muscle, tumor or retina.

According to still further features in the described preferred embodiments monitoring the vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue is effected by monitoring expression of vascular endothelial growth factor messenger RNA.

According to still further features in the described preferred embodiments monitoring the expression of vascular endothelial growth factor messenger RNA is effected by an assay selected from the group consisting of quantitative reverse-transcriptase polymerase chain reaction and RNase protection.

According to still further features in the described preferred embodiments monitoring the vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue is effected by monitoring expression of vascular endothelial growth factor protein.

According to still further features in the described preferred embodiments monitoring the expression of vascular endothelial growth factor protein is effected by an assay selected from the group consisting an antibody based assay and an activity assay.

According to still further features in the described preferred embodiments the new blood vessels are coronary collateral arteries.

According to still further features in the described preferred embodiments the method further comprising the step of grading the potential of the patient to form the coronary collateral arteries in response to hypoxia or ischemia by comparing the vascular endothelial growth factor response in the vascular endothelial growth factor expressing cells or tissue to a grade reference.

According to still further features in the described preferred embodiments the new blood vessels are retinal blood vessels.

According to still further features in the described preferred embodiments the new blood vessels are blood vessels feeding a tumor.

According to still further features in the described preferred embodiments the method further comprising the step of grading the potential of the patient to form the blood vessels in response to hypoxia or ischemia by comparing the vascular endothelial growth factor response in the vascular endothelial growth factor expressing cells or tissue to a grade reference.

The present invention successfully addresses the shortcomings of the presently known configurations by providing an identification of high risk patients based on VEGF response to hypoxia or ischemia. For example, current clinical practice relies on catheterization of coronary arteries to provide data on collateral circulation. The catheterization procedure, although widely accepted, carries with it more inherent risk than harvest of a blood sample. In addition to medical risk, catheterization is costly relative to drawing blood. Furthermore, patients are likely to be more agreeable to the idea of periodic blood draws than to catheterizations conducted on a similar schedule. In addition, in cases of diabetic retinopathy, should proliferation of blood vessels in the retina be due to too much VEGF being produced in response to retinal ischemia as identified by the method of the present invention, then the patient may be treated in such a way so as to minimize the amount of VEGF that his or her retina will produce. This might involve tight glucose control, photocoagulation therapy and potentially anti-VEGF antibodies to block retinal angiogenesis which leads to blindness.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein: FIGs. la-b show representative frames from a patient with 2+ collaterals visualized by angiography. Figure la demonstrates total occlusion (arrow) of the mid-right coronary artery upon selective injection of the right coronary artery with contrast media. Figure 2b demonstrates complete filling by coronary artery collaterals (arrows) of the distal right coronary artery (right posterolateral and posterior descending arteries) after injection of the left coronary system with contrast media.

FIG. 2 shows representative ribonuclease protection assays demonstrating inter-individual differences in the hypoxic induction of VEGF mRNA in monocytes from different patients. The ribonuclease assay for the quantitative determination of VEGF mRNA levels in the monocytes was performed as described in the Examples section below. Treatment indicates whether the monocytes were cultured under hypoxic conditions (H) or normoxic conditions (N). The upper band for VEGF represents the protected fragment specific for the 165 amino acid isoform of VEGF. The second lower VEGF band represents the protected fragment specific for the 189 amino acid isoform of VEGF. A simultaneous ribonuclease protection assay using 18S rRNA was performed to allow for sample normalization. Quantitation of the VEGF mRNA 165 amino acid isoform and 18S band intensity was performed using a phosphorimager. The fold induction of VEGF by hypoxia was determined by dividing the VEGF/18S ratio under hypoxic conditions by that obtained under normoxic conditions. For the representative patients 1-4 shown the hypoxic induction of VEGF mRNA was 3.3, 2.9, 1.7, and 2.4 fold respectively. FIG. 3 shows a scattergram of the fold induction of VEGF by hypoxia in all of the patients included in the study described in the Examples section separated by collateral score without adjustment for any of the covariates described. For the group with 0+ collaterals the mean fold hypoxic induction of VEGF mRNA was 1.9 ± 0.2, for 1+ collaterals it was 2.8 ± 0.4 and for 2+ collaterals it was 3.4 ± 0.3. There was a statistically significant difference in the fold induction of VEGF mRNA between the group with 0+ collaterals and 2+ collaterals (p<0.0001) and between the group with 0+ and 1+ collaterals (p<0.04). DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a method for determining a potential of a patient to form new blood vessels, such as coronary collateral arteries, retinal blood vessels or tumor feeding blood vessels, in response to hypoxia or ischemia which can be used to grade patients into risk groups. Specifically, the present invention can be used to non-invasively grade the potential of an individual suffering from obstructive coronary artery disease to form an adequate collateral circulation. Still specifically, the present invention can be used to non-invasively grade the infiltration of blood vessels into tumor masses, or to grade asymptomatic individuals into risk groups of such infiltration. In addition, the present invention can be used to non-invasively grade the infiltration of blood vessels into the retina in cases of diabetic retinopathy, or to grade asymptomatic individuals into risk groups of such infiltration. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. While reducing the present invention to practice, as further detailed in the Examples section that follows, it was found that the ability to respond to progressive coronary artery stenosis by growing coronary artery collaterals is correlated to, and mediated by, the ability to induce VEGF expression in response to hypoxia. The presence of collaterals is well correlated to induction of VEGF mRNA in monocytes. This response is graded such that the mean fold induction of VEGF is greater in patients with 2+ as compared to 1+ collaterals. When patients were divided into only two: no collaterals (0+ collaterals) vs. some collaterals (patients with

1+ or 2+ collaterals), the level of statistical significance increased. This is important because while a collateral score of 1+ or 2+ might be subjective, the presence of none versus some collaterals is objective.

The significance of the difference in the induction of VEGF between patients with varying levels of collateral circulation was maintained even after adjustment of the data for a number of variables (age, sex, prior revascularization, hypertension, hypercholesterolemia, cigarette smoking and diabetes) that may have an affect on collateral density [17-22]. None of these variables have previously been shown to have an effect on the hypoxic regulation of VEGF in monocytes.

Although the patients differ markedly in the hypoxic induction of VEGF mRNA, normoxic levels of VEGF are not significantly different between patients with different degrees of collateral vessel formation. This is significant since regulation at the steady state mRNA level is known to be of central importance in determining the VEGF response to hypoxia [8-9]. The increase in VEGF mRNA in response to hypoxia is due to both an increase in the transcription of the VEGF gene and to an increase in the stability of VEGF mRNA [23-24]. Specific trans-acting nucleic acid binding proteins HIF-1 [25] and HuR [26] mediate this regulation by hypoxia [23-27]. HIF-1 has recently been shown to be sensitive to post- translational modifications that inhibit its ability to transactivate target genes [28]. These or other modifications may explain the striking inter- individual differences in the hypoxic induction of VEGF.

These data have several conceptual and clinical implications. First, they provide a potential explanation for the variability in collateral formation in patients with coronary artery disease. Patients identified as low VEGF responders may benefit more from treatment with parenteral recombinant VEGF to enhance collateral growth [30-32]. Second, a simple in vitro assay to identify low and high responders without catheterization is provided. Third, low responders may be amenable to pharmacological intervention tailored to augment VEGF production or to introduction of recombinant VEGF. Finally, the inter-individual variability in the hypoxic induction of VEGF has implications beyond the cardiovascular system because VEGF is a key mediator of the pathological angiogenesis seen in tumors [33] and in diabetic retinopathy [34]. In both instances, hypoxia in the tumor or retina has been shown to play an important role in this response.

Further while reducing the present invention to practice, the relationship of the VEGF response to hypoxia in monocytes harvested from diabetic patients with the presence or absence of DR was assessed. The results revealed a highly significant correlation, with increased hypoxic induction of VEGF in those patients with DR in comparison to those patients who do not develop DR even after many years of DM.

It is shown and exemplified herein that patients with evidence of any DR have a significantly higher hypoxic induction of VEGF in their monocytes compared to patients with no evidence of any DR and a long duration of DM. Patients with no evidence of any DR and a relatively short duration of DM were not found to have a significantly different VEGF hypoxic induction compared to patients with DR. One reason for this may be that the vast majority of these patients without evidence of DR will eventually develop DR as shown by numerous epidemiological studies [42, 45, 58-60].

Inter-individual differences in the regulation of VEGF might be expected to influence the natural history of many disease processes involving VEGF such as ischemic vascular disease [93], tumor angiogenesis [94-96], rheumatoid arthritis [97], ovarian hyperstimulation syndrome 98], inflammatory bowel disease [99] and wound healing [84]. It is shown herein that individuals who are low inducers of VEGF with hypoxia are less likely to develop any DR in the setting of DM.

It remains to be determined why individuals differ in their hypoxic induction of VEGF. Many of the non-hypoxic modulators of VEGF expression, such as insulin and IGF-1 [79], as well as hyperglycemia and AGE products [41], appear to be synergistic with hypoxia. Thus inter- individual differences in the plasma levels of these modulators may account for a portion of the observed inter-individual heterogeneity in the hypoxic induction of VEGF in monocytes cultured in vitro in autologous plasma. Thus, according to the present invention there is provided a method for determining a potential of a patient to form new blood vessels, such as, but not limited to, coronary collateral arteries, retinal blood vessels or blood vessels feeding a tumor, in response to hypoxia or ischemia, the method comprising the step of monitoring a vascular endothelial growth factor (VEGF) expression in vascular endothelial growth factor expressing cells or tissue derived from the patient in response to oxygen deprivation.

As used herein in the specification and in the claims section that follows, the term "hypoxia" refers to an abnormal condition of the body in which oxygen intake or use is inadequate. As used herein in the specification and in the claims section that follows, the term "ischemia" refers to local deficiency of blood supply produced by vasoconstriction or local obstacles to the arterial flow.

As used herein in the specification and in the claims section that follows, the term "to form" includes "to grow", "to develop" and the like. As used herein in the specification and in the claims section that follows, the phrase "vascular endothelial growth factor expressing cells or tissue" refers to cells or tissue that express VEGF. Such cells or tissue therefore transcribe VEGF mRNA and translate VEGF mRNA to VEGF protein. Such cells can also secrete the VEGF protein. The VEGF mRNA can be any alternatively spliced VEGF mRNA species or edited VEGF mRNA species. The VEGF protein can be any isoforms of VEGF, which may result from alternatively spliced or edited RNA, alternative initiation or termination of translation, and/or post translational modifications, such as signal peptide removal, glycosylation, acetylation, methylation, ubiqutination and the like. Examples of vascular endothelial growth factor expressing cells or tissue include monocytes fibroblasts (e.g., from a skin biopsy), lymphocytes (these may be from EBV transformed B cells derived from blood) and tissue biopsy specimens from heart, muscle, tumor or retina. As used herein in the specification and in the claims section that follows, the phrase "monitoring a vascular endothelial growth factor response" incudes direct or indirect detection of any one or more of the VEGF mRNA species and/or VEGF protein isoforms, their precursors and/or degradation products, within cells or tissue or in a conditioned medium following secretion from cells or tissue. Some examples for direct or indirect detection of VEGF mRNA species or VEGF protein isoforms are given hereinunder.

As used herein in the specification and in the claims section that follows, the phrase "oxygen deprivation" includes conditions in which oxygen is supplied in limited concentration as is compared to the atmosphere. Such conditions include the use of an artificial atmospheres including, for example, less than 15 % oxygen, preferably less than 10 % oxygen, more preferably less than 5 % oxygen, most preferably about 1 % or less than 1 % oxygen by volume. According to a preferred embodiment of the present invention the method further comprising the step of grading the potential of the patient to form new blood vessels, such as, but not limited to, coronary collateral arteries, retinal blood vessels and blood vessels feeding a tumor, in response to hypoxia or ischemia by comparing the vascular endothelial growth factor response in the vascular endothelial growth factor expressing cells or tissue to a grade reference.

It is shown in the Examples section that follows that the increase in the ratio of VEGF expression to expression of a house keeping molecule (rRNA in this case) in cells subjected to oxygen deprivation as compared to cells grown under oxygen non-limiting conditions correlates to a conventional collateral circulation grading (See Figure 3). This set of data can be used directly, or following expansion to additional subjects, to grade an examinee according the conventional grading system using the method of the present invention.

Thus, the present invention provides a non-invasive procedure for grading collateral circulation in cases of obstructive coronary diseases. It will be appreciated by one ordinarily skilled in the art that similar grading techniques can be employed in cases of diabetic retinopathy and cancer, in this case to predict the ability of developing new blood vessels which cause the disease or is related to poorer prognosis.

The method can also be used to grade individuals into risk groups prior to any apparent symptoms, to thereby alert individuals at risk of failing to adequately develop collateral circulation in response to hypoxia or ischemia associated with, for example, coronary artery sclerosis or any other obstructive coronary disease, to reduce exposure to known and controllable risk factors associated with such diseases, such as, but not limited to, hypertension, hypercholesterolemia, cigarette smoking and diabetes, and to undergo periodic testing. Similarly, the method can be used to grade individuals into risk groups prior to any apparent symptoms, to thereby alert individuals at risk of developing excessive retinal or tumor angiogenesis to undergo periodic testing and to take preventive measures. In cases of tumors, more severe and earlier applied treatment, such as chemotherapy can be used to improve prognosis. In cases of diabetic retinopathy, tight glucose control, earlier photocoagulation therapy and potentially anti-VEGF antibodies to block retinal angiogenesis which leads to blindness can be attempted.

Thus, the method according to the present invention would indicate in those patients with extensive coronary artery stenosis which patients would be able to grow there own coronary artery collaterals and which ones would not. Those patients who would not be able to grow new collaterals would be treated more aggressively with either conventional revascularization procedures such as CABG or PTCA or with newer technologies employing angiogenic factor application or angiogenic gene therapy.

Not only the present invention find uses in predicting risk factors and diagnosing patients suffering coronary obstructive diseases, it also provides means for predicting risk factors and diagnosing patients suffering from diabetic retinopathy or tumor angiogenesis. In the former case the present invention can be used to predict which diabetic patients would develop diabetic retinopathy and then blindness. This would cause the doctor and patient to become more aggressive in controlling blood glucose levels, to be more aggressive in monitoring disease progression and treating more aggressively with photocoagulation therapy. In the case of cancer this would predict which people with tumors would have tumors that could grow blood vessels more quickly and thus the tumor would grow more rapidly. This would then tell the physician to treat the patient more aggressively by taking a wider resection of the tumor and treating the patient with additional adjuvant chemotherapy for the cancer. According to one embodiment of the present invention monitoring the vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue is effected by monitoring expression of vascular endothelial growth factor messenger RNA (mRNA). Methods of isolating whole RNA or mRNA from cells are well known in the art. Such methods include, for example, differential extraction by organic solvents, and differential extraction by affinity methods (e.g., affinity column or magnetic beads).

Methods of quantifying a specific mRNA species are typically based upon hybridization thereof to a complementary sequence. Such methods include, but are not limited to, Northern blot, dot blot, RNase protection, and quantitative reverse-transcriptase polymerase chain reaction (RT-PCT), for each of which protocols have been developed over the years to enable not only detection of the presence of a specific RNA molecule but also to enable quantification or relative quantification thereof. Details describing the implementation of the above methods for quantification of RNA are provided in a variety of laboratory manuals, including, but not limited to, Sambrook et al., molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). The manual is hereinafter referred to as "Sambrook", and it is incorporated by reference as if fully set forth herein.

According to another embodiment of the present invention, monitoring the vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue is effected by monitoring expression of vascular endothelial growth factor protein. Such monitoring can be direct, e.g., by monitoring the protein itself or degradation products thereof in cells or tissue or in a medium containing such cells or tissue into which secretion has occurred, or indirect, e.g., via an activity assay, such as a receptor binding assay or a receptor response assay. Thus, according to a preferred embodiment of the present invention monitoring the expression of vascular endothelial growth factor protein is effected by an antibody based assay. Antibody based assays for quantification of specific proteins are well known in the art. U.S. Pat. No. 5,332,671, which is incorporated herein by reference, teaches anti-VEGF antibodies which can be used for VEGF quantification using an antibody- based assay.

A common antibody based assay for quantification of proteins in solution is the enzyme linked immunosorbent assay (ELISA). In principle, the substance to be measured is bound to a solid phase and then specifically detected by an enzyme- labeled antibody. The enzyme turns a colorless substrate into a product, the optical density (OD) of which is proportional to its concentration. Thus, with excess reagents the OD is proportional to the amount of substance bound to the solid phase. However, other methods, such as, but not limited to, denaturative and non-denaturative gel electrophoresis followed by, for example, Western detection, protein dot blots, affinity columns or beads and the like are also antibody based assays which are readily calibratable to provide protein quantification. These and other methods are further described in Sambrook. According to another embodiment of the present invention monitoring the expression of vascular endothelial growth factor protein is effected by an activity assay. Such an assay can be a binding assay such as a competitive binding assay in which the binding of labeled VEGF to its receptor is competed by non-labeled VEGF present in an analyzed sample, such as cell lysate or conditioned medium, which can be used to quantify the amount of VEGF in that sample. However, such an assay can also be a receptor response assay in which the response of cells to VEGF binding is monitored. Such an assay is described, for example, in U.S. Pat. No. 5,332,671 and include promoting growth of vascular endothelial cells in response to VEGF. Receptor binding and response assays are well known in the art and require no further description herein.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following Examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturers' specifications. These techniques and various other techniques are generally performed according to Sambrook et al., molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). The manual is hereinafter referred to as "Sambrook". Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Materials and Experimental Methods

Patient recruitment:

Patients giving informed consent were recruited consecutively from those undergoing diagnostic coronary artery catheterization at the Rambam hospital, Haifa, Israel over a three month period. The indications for catheterization in all patients were presence of stable or unstable angina pectoris or suspected significant myocardial ischemia. Only patients with at least one coronary stenosis of 70 % or greater by visual analysis of the angiogram were included in this study. Exclusion criteria were age less than 18 and the presence of anemia.

Over a three month period 81 consecutive patients were approached for inclusion in this study. Six patients declined to participate. Of the remaining patients 12 were found to have no coronary stenosis at angiography and hence were not included in the study. Twelve patients samples were discarded for technical reasons (bacterial contamination of culture in two patients and inadequate amount or poor quality of extracted RNA in 10 patients). The profile of the patients with 0+, 1+, or 2+ collaterals with regard to age, sex, hypertension, hypercholesterolemia, cigarette smoking, diabetes, family history, history of prior myocardial infarction, prior CABG, beta-blocker use and the number of diseased epicardial coronary vessels is given in Table 1. There was no statistically significant difference between the three groups in any of these variables. This meant that any observed difference in collateral circulation was attributable to some other factor.

TABLE 1

Collateral Score

0+ 1+ 2+ Chi

Covariate n 19 13 age 59±2 57±3 60+2 male sex 84% 77% 89% 0.9 0.63 prior MI 47% 31% 63% 3.27 0.19 prior CABG 5% 0% 21% 4.57 0.10 hypertension 68% 77% 63% 0.74 0.69 cigarette smoking 26% 31% 21% 0.39 0.82 diabetes 5% 31% 32% 4.76 0.09 family history 26% 46% 26% 1.77 0.41 beta-blockers 53% 62% 58% 0.26 0.87 hypercholesterolemia 58% 69% 63% 0.43 0.80

# diseased vessels 2.0 ± 0.2 2.2 ± 0.2 2.2 ± 0.2

Characteristics of patients in the 3 collateral groups. Statistical analysis was performed as described herein. For the covariates age and # of diseased vessels the 3 groups were not statistically different as assessed by one way analysis of variance (p>0.05). n = number of patients. Patient data collection:

Each patient's name, identification number, age, sex, previous revascularizations, history of hypertension, diabetes, cigarette smoking, family history of premature coronary artery disease or hypercholesterolemia were collected. Separate records of each patient's coronary anatomy (number of diseased vessels and collateral score (0, 1+, 2+)) was recorded by an experienced angiographer. The collateral scoring system used was modified from the TIMI system by grading from 0 to 2 rather than 1 to 3 but maintaining a 3 point scale [1]. The ranking from 0+ to 2+ was based on the presence of collateral vessels and opacification of the recipient vessel. A grade of 0+ for no visible collaterals; 1+, for visible collaterals but no filling of the recipient epicardial vessels; 2+, for filling (partial or complete) of the recipient epicardial vessel by collaterals. A representative frame from a patient with 2+ collaterals is shown in Figures la-b. Coronary anatomy and collaterals were reviewed again by a cardiologist blinded to the initial reading with a greater than 85 % concordance rate between the two reviewers in the collateral score. In instances of discrepancy between the two reviewers a third reviewer blinded to the readings of the first two reviewers was used and served as arbitrator. The coronary anatomy and collateral score was not revealed to those involved in the VEGF assay until after all patient samples had been analyzed for VEGF. This arrangement assured both objectivity in assigning collateral scores and independence of the assigned collateral scores from other collected data. Measurement of coronary artery collaterals: Coronary artery collaterals were initially scored by visual analysis by the physician performing the catheterization. All gradings were subsequently reviewed by a single cardiologist blinded to the first reading. There was a greater than 85 % concordance between the two reviewers. In those cases where there were disagreements between the two reviewers a third blinded reviewer was asked to review the film and served as arbitrator. Thirty seven percent of the patients had no collaterals, with 25 % of the patients having 1+ and 37 % having 2+ collaterals. This distribution of patients with and without collaterals is in agreement with previous studies in patients with obstructive coronary disease [16]. Concordance among reviewers and agreement with previous data show that scoring was accurate and objective.

Blood collection and monocyte culture:

Mononuclear cells were isolated from peripheral blood by a procedure initially described by Boyum using a mixture of polysaccharide and a radioopaque contrast medium [15]. Forty ml of blood was collected from the femoral venous catheter placed for the catheterization prior to commencing angiography. The blood was immediately placed in a 50 ml polypropylene heparinized tube (100 μl of 5000 u/ml heparin) and kept on ice before monocyte isolation. Blood was always used within 4 hours of collection. Twenty ml of heparinized blood was gently layered onto 10 ml of Histopaque-1077 (Sigma) in a fresh 50 ml polypropylene centrifuge tube. Tubes were centrifuged at 1800 rpm for 30 minutes at room temperature. Eight ml of plasma was removed from each tube and saved for later use. The middle phase (buffy coat) containing the monocytes was isolated and placed in a fresh 15 ml polypropylene centrifuge tube. The isolated mononuclear cells were washed twice with sterile phosphate buffered saline. The cell pellets were resuspended in Delbecco's Modified Eagle's Medium (Sigma) with 2 % fetal bovine serum (Sigma) and antibiotics. The cells were plated in two equal aliquots on two polystyrene 10 cm diameter tissue culture dishes (Corning) and incubated in a 95 % room air, 5 % C0₂ incubator (Forma) at 37 °C for one hour to allow for monocyte attachment. The medium from the two tissue culture dishes from a single patient was aspirated and replaced with 8 ml of autologous plasma on each dish. One of the tissue culture dishes was placed in a normoxic incubator 21 % 0₂, 5% C0₂ (Forma) and the other tissue culture dish from the same patient was placed in a hypoxia incubator 1 % 0₂, 5 % C0₂, 94 % N₂ (Triple Gas Incubator, Jouan). After 20 hours of exposure to either hypoxia or normoxia, RNA was extracted from the monocytes. This method allowed assay of VEGF induction by hypoxia. RNA isolation from monocytes:

Total RNA was isolated from the monocytes using the TRI Reagent (MRC, Inc.). Briefly, 1 ml of reagent was added to each dish with vigorous pipetting and transferred to a 1.5 ml eppendorf tube. Chloroform (200 μl) was added, and the tube vortexed and centrifuged at 14,000 rpm for 10 minutes. The RNA was precipitated with an equal volume of isopropranol and washed with 80 % ethanol. The RNA was air dried and resuspended in water treated with diethyl pyrocarbonate. The optical density of all of the samples was measured at 260 nm. On average 20 μg of RNA was obtained from both the normoxic and hypoxic monocytes. This amount was sufficxient for RNase protection assays.

Measurement of the fold induction of VEGF mRNA by RNase protection assay:

The quantity of VEGF mRNA was determined by RNase protection assay using a riboprobe to VEGF and to 18S rRNA to allow for sample normalization as previously described [9]. Quantitation of signal intensity was performed on a phosphorimager (Fujix). For each patient a VEGF/18S ratio was calculated for both the hypoxic and normoxic cells. The fold induction of VEGF with hypoxia was calculated by dividing the hypoxic by the normoxic value. Statistical Analysis:

Data are reported as the mean ± standard error. Analysis between groups for statistically significant differences in enumerative data such as gender, hypertension, hypercholesterolemia, diabetes, cigarette smoking, family history, beta-blockers, or prior CABG was performed using the chi- square test. Analysis between groups for continuous variable such as age and number of diseased vessels was performed using one-way analysis of variance. The fold induction of VEGF mRNA with hypoxia was compared between patients with collateral scores 0+, 1+ and 2+ by analysis of the covariance (ANCOVA) using age, number of diseased vessels, family history of heart disease, diabetes, smoking, hypertension, prior MI, and hypercholesterolemia as covariates. Bonferoni post-hoc comparisons were performed to compare the adjusted levels of VEGF between the 3 groups.

Experimental Results Measurement of VEGF mRNA induction with hypoxia:

A sensitive and quantitative RNase protection analysis was used to precisely quantitate the amount of VEGF mRNA in the samples. All values were normalized to 18S mRNA as previously described [9]. A representative RNase protection assay demonstrating a range of differences in the hypoxic induction of VEGF is shown in Figure 2. There was no significant difference in the mean basal (normoxic) level of VEGF mRNA between the three collateral groups. For patients with 0+ collaterals the mean normoxic VEGF/18S ratio was 0.019 ± 0.005, for patients with 1+ collaterals the mean normoxic VEGF/18S ratio was 0.023 ± 0.005 and for patients with 2+ collaterals the mean normoxic VEGF/18S ratio was 0.023 ± 0.006. A fold induction score was determined for each patient comparing the ratio of VEGF/18S under hypoxia and normoxia (Figure 3). The elevated fold induction score in patients with well developed collateral circulation indicates that VEGF plays a role in angiogenesis of collateral vessels in response to hypoxia.

Reproducibility of the VEGF mRNA induction with hypoxia in monocytes:

In order to use the fold induction of VEGF mRNA of a given patient as an indicator of a true phenotype for that particular patient the values obtained must be reproducible and consistent. We obtained blood samples on 2 or 3 different days from 5 normal volunteers. These samples were processed in an identical fashion to those from patients from the catheterization lab. The fold induction of VEGF mRNA with hypoxia from different blood samples drawn on different days from the 5 different normal volunteers were volunteer 1, 2.3, 3.1 and 4.4; volunteer 2, 1.6 and 1.7; volunteer 3, 1.0 and 1.5; volunteer 4, 1.6, 2.0 and 2.4; and volunteer 5, 5.5 and 8.1. These results indicate that the assay is reproducible and reliable.

Statistical analysis of data from patients with coronary artery disease: The unadjusted fold induction of VEGF mRNA for coronary artery disease patients with 0+, 1+ and 2+ collaterals were 1.9 ± 0.2, 2.8± 0.4 and 3.4 ± 0.3, respectively. The difference in the fold induction between patients with 0+ versus 2+ collaterals was highly statistically significant (pO.OOOl) as was the difference in the fold induction between patients with no collaterals (0+) and some collaterals (either 1+ or 2+) (1.9 ± 0.2 versus 3.2 ± 0.3; pO.OOOl). In addition there was a statistically significant difference between the fold induction of VEGF between the 0+ and 1+ collateral group (p<0.04). There was no statistical difference between the fold induction of VEGF between the 1+ and 2+ collateral group. Data from the three groups 0+, 1+ and 2+ collaterals were subjected to ANCOVA using the variables outlined above (i.e., age, sex, prior myocardial infarction, hypertension, family history, hypercholesterolemia, cigarette smoking, diabetes and number of diseased vessels) as covariates. This revealed an overall significant difference (F ratio 7.7, p<0.002). Bonferoni post-hoc comparison between the groups 0+ and 1+ and between the groups 0+ and 2+ collaterals revealed a statistically significant difference in VEGF induction with hypoxia (1.5 ± 0.4 vs. 2.6 ± 0.4, p<0.02 and 1.5 ± 0.4 vs. 3.2 ± 0.4, p< 0.0004, respectively). No significant difference was found between the groups 1+ and 2+ collaterals (2.6 ± 0.4 vs. 3.2 ± 0.4, p<0.3). Finally, combining groups +1 and +2 into a single group and repeating the ANCOVA for groups 0+ (no collaterals) and the combined group 1+ and 2+ (some collaterals) revealed a statistically significant difference between these two groups in the induction of VEGF with hypoxia (1.5 ± 0.4 vs. 2.9 ± 0.3, p<0.003).

EXAMPLE 2 Materials and Experimental Methods Patient recruitment: This study was approved by the Human Research Ethics Committee of the Rambam Medical Center. Informed consent was obtained from all patients. Patients were recruited consecutively from outpatients at the Rambam Hospital and its clinics in the Haifa area over a 8 month period. Patients with no evidence of retinopathy but with diabetes documented to be of less than 10 years duration were excluded from the study. Patient data collection:

For each patient, a data sheet was completed with the patient's code, age, sex, duration of diabetes, type of diabetes, medications and HbAjC.

All patients had a dilated fundal examination performed by a trained retinal surgeon and seven-field stereoscopic fundal photography 85-90] performed and read by an experienced reader using standardized criterion.

Determination of the presence or absence of DR was conducted in a masked fashion, without any knowledge of the VEGF response. Absence of DR was defined as complete absence of macular edema, hard exudates, blot hemorrhages, microaneurysms, venous beading, intraretinal microvascular abnormalities, cotton wool spots or neovascularization.

Blood collection:

Mononuclear cells were isolated from peripheral blood by a procedure initially described by Boyum [91] and recently modified [see Example 1]. Briefly, 40 ml of blood was collected by venipuncture and used within 4 hours. Twenty ml of blood was gently layered onto 10 ml of Histopaque-1077 in a fresh 50 ml polypropylene centrifuge tube. Tubes were centrifuged at 1800 rpm for 30 minutes at room temperature. Eight ml of plasma was removed from each tube and saved for later use. The middle phase (buffy-coat) containing the monocytes was isolated and placed in a fresh 15 ml polypropylene tube. The isolated mononuclear cells were washed twice with sterile phosphate buffered saline and plated in 2 equal aliquots on 2 polystyrene, 10 cm diameter tissue culture dishes and incubated in a 95% room air, 5% C0₂ incubator at 37 °C for 1 hour to allow for monocyte attachment. The medium from the 2 tissue culture dishes from a single patient was aspirated and replaced with 8 ml of autologous plasma on each dish. One of the dishes was placed in a normoxic incubator at 21% 0₂, 5% C0₂ and the other tissue culture dish was placed in a hypoxia incubator at 1% 0₂, 5% C0₂, 94% N₂. After 20 hours of exposure to either hypoxia or normoxia, total RNA was extracted from the cells.

RNA isolation from monocytes:

Total RNA from the monocytes was isolated from monocytes with the TRI reagent as described under Example 1. On average, 10-20 μg of RNA was obtained from both the normoxic and hypoxic monocytes. Measurement of VEGF mRNA by RNase protection assay:

The quantity of VEGF mRNA was determined by RNase protection assay using riboprobes to VEGF and 18S rRNA [92]. Quantification of signal intensity was performed on a phosphorimager (Fujix). For each patient, a VEGF/18S ratio was calculated for both the hypoxic and normoxic cells. The fold induction of VEGF with hypoxia was calculated by dividing the hypoxic VEGF/18S ratio by the normoxic VEGF/18S ratio.

Statistical analysis:

Data are reported as mean +/- SEM. Analysis between groups for statistically significant differences in the levels of VEGF (normoxic or fold induction) was performed using Student's unpaired t-tests. Experimental Results

Over an eight-month period, 95 consecutive patients were enrolled in the study. Of 50 patients with type I DM, 25 had evidence of any DR. Of 45 patients with type II DM, 38 had evidence of any DR. There were 25 type I DM and 7 type II DM patients with no DR and DM of at least 10 years duration. Five type I DM patients with no DR were identified with a duration of diabetes > 20 years. Of those type II patients without DR, 5 patients were identified with duration of diabetes > 15 years.

It is shown in Example 1 above that in normal volunteers the measurement of the fold hypoxic induction of VEGF is reproducible and consistent by assaying blood samples obtained on 2 or 3 separate occasions. The assay was further validated by repeating the VEGF mRNA measurement from blood drawn on two separate occasions in 3 of the patients included in this study with the replicates in fold induction of VEGF mRNA being 2.4, 2.3; 2.5, 2.1 ; and 2.5, 2.5.

In the 95 patients described here the range of the fold hypoxic induction of VEGF mRNA was 0.7-20.9. The median value for the hypoxic induction of VEGF was 2.4 and the mean was 4.1. There was no significant association between fold hypoxic VEGF induction and sex, age and glycemic control.

It was found that there was no significant difference between patients with and without DR in the mean basal (normoxic) level of VEGF, even when patients were grouped according to type and duration of DM. The mean normoxic VEGF mRNA level of type I patients with DR was 0.042+/- 0.015 and of type II patients with DR was 0.044+/-0.010. The mean normoxic VEGF mRNA level in type I patients without DR for more than 10 or 20 years was 0.028+/-0.004 (p<0.37), and 0.031+/-0.012 (p<0.58), respectively. The mean normoxic VEGF mRNA level in type II patients without DR for more than 10 or 15 years was 0.036+/-0.009 (p<0.56) and 0.032+/-0.010 (p<0.42), respectively. There was no significant difference in the mean normoxic level of all patients with DR compared to those patients without DR for long duration (> 20 years type I and > 15 years type II (0.043+/-0.008 versus 0.032+/-0.008, p<0.31). The mean hypoxic VEGF induction of type I patients with DR was

3.7+/-0.8 and of type II patients with DR was 4.8+/-0.8. Compared to type I patients with DR, there was no significant difference in the hypoxic induction of VEGF mRNA in type I patients without DR for more than 10 years (3.84+/-0.6, p<0.6) (Figure la). In patients with type I and no DR after > 20 years there was a decrease in the hypoxic induction of VEGF of borderline statistical significance (1.98+/-0.5, p<0.076). In patients with type II and no DR after more than 10 years of diabetes there was a decrease in the hypoxic induction of VEGF of borderline statistical significance (2.44+/-0.5, p<0.063). In patients with type II DM and no DR after > 15 years of diabetes there was a statistically significant decrease in the hypoxic induction of VEGF compared to type II patients with DR (1.76+/-0.2, p< 0.0004)(Figure lb). The difference in the mean hypoxic induction of VEGF in all patients with DR (n=63) versus all those patients without DR and DM of long duration (type I > 20 years and type 2 ≥ 15 years, n=10) was highly statistically significant (4.35+/-0.55 versus 1.87+/-0.3, pO.OOOl 2).

Patients with DR can be further subdivided into those with or without proliferative retinopathy. Of the patients with evidence of DR, 31% were found to have active proliferative retinopathy. Patients with proliferative retinopathy were found to have a significantly longer duration of DM compared to those patients with only background DR (23.4 vs. 18 years, pθ.04) but were not significantly different for HbAlC , age, sex, or type of DM. There was no significant difference in the mean normoxic level (0.053 vs. 0.041, pθ.53) or in the fold hypoxic induction of VEGF (3.0 vs. 4.5, pθ.12) in patients with or without proliferative retinopathy

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A method for determining a potential of a patient to form new blood vessels in response to hypoxia or ischemia, the method comprising the step of monitoring a vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue derived from the patient in response to oxygen deprivation.

2. The method of claim 1, wherein said vascular endothelial growth factor expressing cells or tissue are selected from the group consisting of monocytes, fibroblasts, lymphocytes and tissue biopsy specimens from heart, muscle, tumor or retina.

3. The method of claim 1, wherein monitoring said vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue is effected by monitoring expression of vascular endothelial growth factor messenger RNA.

4. The method of claim 1, wherein monitoring said expression of vascular endothelial growth factor messenger RNA is effected by an assay selected from the group consisting of quantitative reverse-transcriptase polymerase chain reaction and RNase protection.

5. The method of claim 1, wherein monitoring said vascular endothelial growth factor response of vascular endothelial growth factor expressing cells or tissue is effected by monitoring expression of vascular endothelial growth factor protein.

6. The method of claim 1, wherein monitoring said expression of vascular endothelial growth factor protein is effected by an assay selected from the group consisting an antibody based assay and an activity assay.

7. The method of claim 1, wherein said new blood vessels are coronary collateral arteries.

8. The method of claim 7, further comprising the step of grading the potential of the patient to form said coronary collateral arteries in response to hypoxia or ischemia by comparing said vascular endothelial growth factor response in said vascular endothelial growth factor expressing cells or tissue to a grade reference.

9. The method of claim 1, wherein said new blood vessels are retinal blood vessels.

10. The method of claim 9, further comprising the step of grading the potential of the patient to form said retinal blood vessels in response to hypoxia or ischemia by comparing said vascular endothelial growth factor response in said vascular endothelial growth factor expressing cells or tissue to a grade reference.

11. The method of claim 1, wherein said new blood vessels are blood vessels feeding a tumor.

12. The method of claim 11, further comprising the step of grading the potential of the patient to form said blood vessels feeding a tumor in response to hypoxia or ischemia by comparing said vascular endothelial growth factor response in said vascular endothelial growth factor expressing cells or tissue to a grade reference.