WO2013154983A1

WO2013154983A1 - Molecular markers of endothelial dysfunction in gestational diabetes

Info

Publication number: WO2013154983A1
Application number: PCT/US2013/035597
Authority: WO
Inventors: Kathleen E. Rodgers; Gere S. Dizerega; Stan LOUIE; Nick MORDWINKIN
Original assignee: Rodgers Kathleen E; Dizerega Gere S; Louie Stan; Mordwinkin Nick
Priority date: 2012-04-09
Filing date: 2013-04-08
Publication date: 2013-10-17

Abstract

The present invention provides methods for identifying subjects at risk of or having gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D), or identifying and/or treating subjects having GDM or pre-diabetes.

Description

Molecular markers of endothelial dysfunction in gestational diabetes

Cross-Reference

This application claims priority to U.S. Provisional Patent Application Serial No. 61/621,762 filed April 9, 2012, incorporated by reference herein in its entirety.

Statement of U.S. Government Interest

This work was funded by grant number 5RO1HL082722-02 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention. Introduction

Gestational diabetes (GDM) occurs in up to 14% of pregnant women. Uncontrolled GDM can cause a complicated pregnancy and can predispose both the mother and baby to increase risk of type 2 diabetes. Treatment or prevention of GDM or early diagnosis could reduce the consequences of this diabetic disorder.

Summary of the invention

In a first aspect, the present invention provides methods for identifying subjects at risk of gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D), or identifying subjects having GDM or pre-diabetes, comprising determining, compared to control, one or more of the following in samples obtained from a pregnant female or a person suspected of having pre-diabetes:

(a) increase in circulating svCAM-1 (soluble vascular cell adhesion molecule 1) and/or siCAM-1 (soluble intercellular adhesion molecule 1);

(b) decrease in circulating EPC (endothelial progenitor cells) counts in maternal blood;

(c) decrease in SOD2 and/or SOD3 (superoxide dismutase) isoform mRNA expression in maternal blood and/or umbilical cord blood; and

(d) increase in eNOS (endothelial nitric oxide synthase) mRNA expression in maternal blood and/or umbilical cord blood; wherein one or more of the recited increases or decreases indicates a risk of GDM, CVD, and/or T2D in the pregnant female, or the presence of gestational diabetes in the pregnant female, or the presence of pre-diabetes in the subject.

In one embodiment, the subject is a pregnant female. In another embodiment, the one or more recited increases or decreases indicates a risk of GDM in the pregnant female.

In another aspect, the present invention provides methods for monitoring efficacy of treatment for gestational diabetes, comprising determining, compared to control, one or more of the following in samples obtained from a pregnant female with gestational diabetes:

(d) increase in eNOS (endothelial nitric oxide synthase) mRNA expression in maternal blood and/or umbilical cord blood;

wherein one or more of the recited increases or decreases indicates the efficacy of treatment for gestational diabetes in the pregnant female.

In a third aspect, the present invention provides methods for treating GDM, comprising administering to a subject with GDM an amount effective to treat the GDM of a polypeptide comprising or consisting of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof, All AT₂ type 2 receptor agonists, or an agonist (polypeptide or otherwise) of the MAS receptor, or pharmaceutically acceptable salts thereof.

Description of the Figures

Figure 1. Maternal and cord blood EPC% in non-diabetic and GDM patients. A significant reduction in the percentage of circulating EPC was seen in GDM vs. non-diabetic patients. Figures 2. Graphs of maternal and cord blood nitrite levels (A) and eNOS mRNA expression (B) in non-diabetic and GDM patients. Mean nitrite levels from maternal plasma were significantly lower in women diagnosed with GDM compared to non-diabetic women. In addition, maternal and cord blood eNOS mRNA expression were significantly higher in women diagnosed with GDM compared to non-diabetic women. Figures 3. Graphs of maternal and cord blood SOD (A-B) and p22-phox mRNA expression (B) in non-diabetic and GDM patients. Maternal blood SOD2 and SOD3 expression were significantly decreased in women with GDM compared to non-diabetic women. Maternal blood p22-phox expression was increased in women with GDM, however this difference was not statistically significant. Surprisingly, p22-phox mRNA expression in cord blood from women with GDM was significantly decreased compared to non-diabetic women.

Figures 4. Graphs of maternal and cord plasma sVCAM- 1 (A) and sICAM- 1 (B) in non- diabetic and GDM patients. In maternal plasma, both sVCAM-1 and sICAM-1 levels were significantly higher in GDM patients as compared to non-diabetic women. While there was a significant increase in sVCAM-1 levels in cord blood from women with GDM, there was no significant difference in sICAM-1 levels in cord blood.

Figures 5. Maternal blood circulating EPC counts correlated negatively with HbAi_c (A) and maternal plasma sVCAM-1 (B) and sICAM-1 levels (not shown) correlated positively with maternal HbAi_c. maternal blood eNOS mRNA expression also correlated positively with maternal HbAi_c (C) and cord blood SOD3 mRNA expression demonstrated an inverse correlation with maternal HbAi_c (D).

Figure 6. Conceptual model hypothesizing the potential mechanisms by which both maternal and fetal endothelial dysfunction may occur.

Figure 7. (A) Graph of % EPC in maternal and cord blood in normal, GDM, and pre-diabetic patients; (B) Graph of uM nitric oxide in maternal and cord plasma in normal, GDM, and pre- diabetic patients; (C) Graph of p22 phox mRNA expression as a % of control in maternal and cord blood in normal, GDM, and pre-diabetic patients; (E) Graph of eNOS mRNA expression as a % of control in maternal and cord blood in normal, GDM, and pre-diabetic patients

Detailed Description of the Invention

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al, 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2" Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise.

All embodiments disclosed herein can be combined unless the context clearly dictates otherwise.

(c) decrease in SOD2 and or SOD3 (superoxide dismutase) isoform mRNA expression in maternal blood and/or umbilical cord blood; and

wherein one or more of the recited increases or decreases indicates a risk of GDM, CVD, and/or T2D in the pregnant female, or the presence of gestational diabetes in the pregnant female, or the presence of pre-diabetes in the subject.

As demonstrated in the examples that follow, the recited methods can be used for early detection of gestational diabetes, and thus for early treatment to reduce the

complications caused by maternal hyperglycemia, including but not limited to fetal hyperinsulinemia, adverse long-term maternal outcomes (e.g. development of type 2 DM and atherosclerotic CVD), increased perinatal morbidity (e.g. macrosomia, birth trauma, preeclampsia), and long-term sequelae in offspring (e.g. childhood overweight, and metabolic factors that may increase risk of CVD). The methods may also be used for early detection of pre-diabetes, particularly in pregnant females. Currently, the severity of diabetes is measured by blood glucose levels, which is not accurate in pregnant women. Thus, the methods of the present invention provide a significant improvement over prior art methods.

While not being bound by any mechanism of action, the inventors believe that alterations in endothelial function are already present (prior to onset of hyperglycemia) in both mothers and their fetuses in GDM pregnancies. If these measures remain elevated, it is possible that these alterations can confer an increased risk for the development of CVD and type 2 DM in both mother and offspring.

As used herein, "gestational diabetes" (GDM) is a condition in which women without previously diagnosed diabetes exhibit hyperglycemia during pregnancy. GDM affects 3-8% of pregnancies in the United States and up to 12-14% in some high-risk populations.

In one embodiment, the subject at risk is any pregnant female. In another

embodiment, the subject at risk is a pregnant female who has one or more risk factors selected from the group consisting of obesity (i.e.: body mass index over 30), had GDM in a previous pregnancy, sugar in the urine, family history of diabetes, previously given birth to a baby weighing 4000 grams or more, had an unexplained stillbirth, had a baby with a birth defect, high blood pressure, over 35 years of age, or a member of a racial group with a high prevalence of diabetes (Hispanic, African, Native American, South or East Asian, Pacific Islander, and indigenous Australian ancestry).

As used herein, "pre-diabetes" is indicated by one or both of (a) a fasting blood glucose level of 100 to 125 mg/dl (5.6 mM to 6.9 mM); and (b) glycated hemoglobin of between 5.7 and 6.4%..

Subjects at risk of pre-diabetes may have one or more signs or symptoms including, but not limited to, constant hunger, unexplained weight loss or weight gain, flu-like symptoms, blurred vision, slow healing of cuts or bruises, tingling or loss of feeling in hands or feet, recurring gum or skin infections, and recurring vaginal or bladder infections.

In one embodiment, a marker level in each of groups (a)-(d) is measured (i.e.: (a) at least one of circulating svCAM-1 and/or siCAM; (b) circulating EPC; (c) SOD2 and/or SOD3 mRNA expression (preferably SOD3) if only one is measured) in at least one of maternal blood or umbilical cord blood; and (d) eNOS mRNA expression in at least one of maternal blood or umbilical cord blood. In another embodiment, all of the recited marker levels are measured.

In various embodiments, the presence of 2, 3, 4, 5, 6, 7, 8, or all 9 of the recited increases or decreases indicates a risk of GDM or the presence of GDM in the pregnant female. In a further embodiment, the presence of decreased maternal circulating EPC counts compared to control, increased soluble adhesion molecules in maternal blood compared to control, decreased SOD2 and/or SOD3 expression in both maternal and cord blood compared to control, and/or increased eNOS expression in both maternal and cord blood compared to control indicates a risk of GDM or the presence of GDM in the pregnant female.

In all embodiments of the methods disclosed herein, any suitable control can be used for comparison of marker levels, such as previously identified normal measurements of the various markers in normal subjects (i.e.: that do not have diabetes or GDM), or concurrent measurements from a normal control or non-pregnant individuals diagnosed with diabetes.

In a further embodiment of all of these methods, the methods may further comprise treating the subject based on the determined marker levels. Any suitable treatment may be administered as determined by an attending physician, including but not limited to insulin or an angiotensin analog, as described below. In a second aspect, the present invention provides methods for monitoring efficacy of treatment for gestational diabetes, comprising determining, compared to control, one or more of the following in samples obtained from a pregnant female with gestational diabetes:

As detailed in the examples that follow, the methods of the invention can also be used to monitor the effectiveness of treatment strategies on women being treated for gestational diabetes, including but not limited to dietary changes, exercise regimens, and

pharmacological management.

In one embodiment, a marker level in each of groups (a)-(d) is measured (i.e.: (a) at least one of circulating svCAM-1 and/or siCAM; (b) circulating EPC; (c) SOD2 and/or SOD3 mRNA expression in at least one of maternal blood or umbilical cord blood; and (d) eNOS mRNA expression in at least one of maternal blood or umbilical cord blood. In another embodiment, all of the recited marker levels are measured.

In various embodiments, the presence of 2, 3, 4, 5, 6, 7, 8, or all 9 of the recited increases or decreases indicates an efficacy of the therapeutic regimen.

In these embodiments, levels of the one or more markers may be obtained at any desirable interval to monitor treatment efficacy. In one non-limiting embodiment, a base line marker level is obtained prior to initiation of therapy, and the marker level(s) is(are) subsequently obtained at one or more time points after initiation of therapy. In one embodiment, the presence of decreased maternal circulating EPC counts compared to baseline control, increased soluble adhesion molecules in maternal blood compared to baseline control, decreased SOD2 and/or SOD3 expression in both maternal and cord blood compared to baseline control, and/or increased eNOS expression in both maternal and cord blood compared to baseline control indicates poor efficacy of the therapeutic regiment. In such a case, and attending physician might, for example, order a further determination of marker level(s) at a later time to provide more time for therapeutic efficacy to manifest itself, or might modify the therapeutic regiment based on the marker level(s) and other information relevant for determining an appropriate course of treatment for a given patient. In another embodiment, the presence of increased maternal circulating EPC counts compared to baseline control, decreased soluble adhesion molecules in maternal blood compared to baseline control, increased SOD2 and/or SOD3 expression in both maternal and cord blood compared to baseline control, and/or decreased eNOS expression in both maternal and cord blood compared to baseline control indicates positive efficacy of the therapeutic regiment. In such a case, and attending physician might, for example, order a further determination of marker level(s) at a later time to verify therapeutic efficacy over time, or might modify the therapeutic regiment based on the marker level(s) and other information relevant for determining an appropriate course of treatment for a given patient.

Detection of circulating svCAM-1 and/or siCAM-1 can be accomplished using standard techniques, such as use of commercial kits and antibodies against the relevant protein that are available for such purposes, as noted in the examples that follow. The amino acid sequences of sICAM- 1 and sVCAM- 1 are provided in SEQ ID NO: 15 and SEQ ID NO: 51, respectively.

Detection of circulating EPC in maternal blood can be accomplished using standard techniques, such as flow cytometry as disclosed in the examples that follow Detection of SOD2 and/or SOD3 and eNOS mRNA expression in maternal blood and/or umbilical cord blood can be accomplished using standard techniques. In one none- limiting embodiment, total RNA may be extracted from whole blood and subjected to reverse transcription polymerase chain reaction (RT-PCR) using primers complementary to the relevant mRNA, such as real-time PCR, and resulting cDNA quantitated by standard means. The expression level of SOD and/or eNOS can be compared to any suitable control, including but not limited to SOD2 and/or SOD3 and/or eNOS expression in non-diabetic women. In other embodiments, hybridization assays can be carried out using hybridization probes complementary to the relevant mRNA sequence (SOD2: SEQ ID NO:53; SOD3 : SEQ ID NO: 54; eNOS: SEQ ID NO:52). Such hybridization probes and PCR primers can be from any portion of the relevant sequence and may be of any suitable length. In exemplary embodiments, RNA probes for hybridization are at least 15, 20, 25, 50, 100, 250, or more nucleotides with perfect complementarity to the relevant mRNA sequence

A pre-determined level or pre-determined range can be selected by calculating the value or range of values that achieves the greatest statistical significance for a given set of amounts or quantities for a particular biomarker. In some embodiments, the pre-determined level can be based on the variance of a sample of biomarker quantities from a population of control/normal subjects. For instance, the pre-determined level can be at least 2, 3, 4, or 5 standard deviations above the normal range for a particular biomarker. In one embodiment, the pre-determined level is at least 6 standard deviations above the normal range for the biomarker. In some embodiments, a pre-determined level or pre-determined range can be a ratio of levels of two different biomarkers measured from all subjects. A pre-determined level or pre-determined range can also be determined by calculating a level or range of biomarker quantities for which greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of patients having a quantity of biomarker within that level or range have gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D).

In one embodiment, the methods may result in a diagnosis of the subject as having gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D) based on the comparison. In another embodiment, the methods may result in providing the comparison to an entity for diagnosis of gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D). In these embodiments, the subject is at risk of gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D) but has not been definitively diagnosed with gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D). In various embodiments, the subject may present with one or more symptoms of gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D), as described above. "Diagnosing/diagnosis," as used herein, means identifying the presence or nature of gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D). Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

In another embodiment, the methods may result in determining a level of gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D) disease activity based on the comparison. In a further embodiment, the methods may result in providing the comparison to an entity for monitoring gestational diabetes (GDM), cardiovascular disease (CVD) and/or type 2 diabetes (T2D) disease activity.

Further details of the first and second aspects of the invention are detailed in the examples that follow.

In one embodiment, the polypeptides for use in the invention comprise or consist of a sequence of at least four contiguous amino acids of groups R^x-R⁸ in the sequence of general formula I

R^-R^-R^-R^R⁸ (SEQ ID NO: 1)

wherein R¹ is selected from the group consisting of H, Asp, Glu, Asn, Acpc (1- aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH₂) and Sue, or is absent, R² is selected from the group consisting of Arg, Lys, Ala, Cit, Orn, Ser(Ac), Sar, D- Arg and D-Lys,

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Lys, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(POs)2, Thr, Ser, homoSer, azaTyr, and Ala;

R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;

R⁶ is selected from the group consisting of His, Arg or 6-NH₂-Phe;

R⁷ is selected from the group consisting of Pro or Ala; and

R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group.

Exemplary AT2 agonists useful in the practice of the invention include the All analogues set forth above subject to the restriction that R⁶ is p-NEL-Phe.

In a further preferred embodiment of each of the above embodiments (SEQ ID NO:

16),

R¹ is selected from the group consisting of Asp and Glu, or is absent;

R² is selected from the group consisting of Arg, Lys, and Ala;

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Lys, and

Pro;

R⁴ is selected from the group consisting of Tyr and homoSer;

R⁶ is selected from the group consisting of His and Arg;

R⁷ is selected from the group consisting of Pro or Ala; and

R⁸ is selected from the group consisting of Phe, He, or is absent.

In alternate embodiments, the polypeptides comprise or consist of at least five, six, seven, or eight contiguous amino acids of groups R^x-R⁸ in the sequence of general formula I. In a further alternative, the polypeptides consist essentially of a sequence of at least four, five, six, seven, or eight contiguous amino acids of groups R^x-R⁸ in the sequence of general formula I.

Particularly preferred combinations for R¹ and R² are Asp-Arg, Asp-Lys, Glu- Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; A(l-7), Asp-Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(l-6), Asp-Arg-Val-Tyr-Ile- His [SEQ ID NO:8]; AII(l-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(l-4), Asp-Arg-Val- Tyr [SEQ ID NO: 10]; and AII(l-3), Asp-Arg-Val . Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 11] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 12]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 13].

In a most preferred embodiment, the polypeptides for use in the present invention comprise or consists of at least 5 contiguous amino acids of A(l-7) (SEQ ID NO:4), a peptide consisting of the amino acid sequence Asp-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO: 4). The A(l-7) may be linear or cyclized in any suitable manner, such as those described in

WO2008/018792, including but not limited to A(l-7) comprising a thioether bridge between positions 4 and 7, or other positions.

In various further embodiments, the peptide administered to the subject may be Asp- Arg-Val-Tyr-Ile (SEQ ID NO: 9), Asp-Arg-Val-Tyr-Ile-His (SEQ ID NO: 8), or most preferably Asp-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO: 4).

Other preferred embodiments comprise or consist of

Asp-Ar ξ-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 17

Asn-Ar ξ-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 18

Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe SEQ ID NO: 19

Glu-Arg ;-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 20

Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 21

Asp-Ar ξ-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO: 22

Asp-Ar ξ-Val-Thr-Ile-His-Pro-Phe SEQ ID NO: 23

Asp-Ar ξ-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO: 24

Asp-Ar ξ-Val-Tyr-Ile-Arg-Pro-Phe SEQ ID NO: 25

Asp-Ar ξ-Val-Tyr-Ile-His-Ala-Phe SEQ ID NO: 26

Asp-Ar ξ-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO: 27

Pro-Arg -Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 28

Asp-Ar ξ-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO: 13

Asp-Ar ;-Val-Tyr(P0₃)2-Ile-His-Pro-Phe SEQ ID NO: 29

Asp-Ar ξ-norLeu-Tyr-Ile-His -Pro-Phe SEQ ID NO: 30

Asp-Ar ξ-Val-Tyr-norLeu-His-Pro-Phe SEQ ID NO: 31 Asp-Arg-Val-homoSer-Tyr-Ile-His-Pro-Phe SEQ ID NO: 32

Asp-Arg-Nle-Tyr-Ile-His-Pro (SEQ ID NO: 33) Nle3 A( 1 - 7)

Another class of polypeptides of particular interest in accordance with the present invention are those of the general formula II:

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸ (SEQ ID NO: 14)

in which R² is selected from the group consisting of H, Arg, Lys, Ala, Orn, Citron, Ser(Ac), Sar, D-Arg and D-Lys;

are as defined above, and

excluding sequences including R⁴ as a terminal Tyr group..

A particularly preferred subclass of the compounds of general formula II has the formula:

R²-R³-Tyr-R⁵-His-Pro-Phe [SEQ ID NO:34]

wherein R², R³ and R⁵ are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO:35] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:36].

In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. Other residues are abbreviated as follows:

TABLE 1

Abbreviation for Amino Acids

Sar N-methylglycyl (sarcosine)

Cit Citron

Orn Ornithine

NorLeu (Nle) NorLeucine

HomoSer HomoSerine (isotheronine)

Other angiotensin analogues that can be used in the methods of the present invention include the following:

TABLE 2

Angiotensin II Analog

Other embodiments of angiotensin analogues that can be used in the methods of the present invention include:

1GD Ala4-AII(l-7) DRVAIHP SEQ ID O:37

2GD Pro3-AII(l-7) DRPYIHP SEQ ID O:38

5GD Lys3-AII(l-7) DRKYIHP SEQ ID NO:39

9GD NorLeu-AII(l-7) DR(nor)YIHP SEQ ID NO:40

GSD 28 Ile⁸-AII DRVYIHPI SEQ ID NO: 41

Ala3aminoPhe6 All: RVAIHPF SEQ ID NO:42

Ala3-AIII RVAIHPF SEQ ID NO:43

GhZ-AII GRVYIHPF SEQ ID NO:44

NorLeu⁴-AIII — RVYnLHPF SEQ ID NO:45

Acpc³-AII DR(Acpc)YIHPF SEQ ID NO:46

GSD 37B Orn²-AII D(Orn)VYIHPF SEQ ID NO:47

GSD38B Citron²-AII D(Citron)VYIHPF SEQ ID NO:48

3GD Pro³Ala⁴-AII(l-7) DRPAIHP SEQ ID NO:49

8GD Hydroxy-Pro³-AII(l-7) DRP(OH)AIHP SEQ ID NO: 50

In another embodiment, the polypeptides may be any of those disclosed in

US20100055146, incorporated by reference herein in its entirety. In various embodiments, the polypeptide is:

a 4,7-cyclized analog of Angiotensin II (Ang(l-8), or any of its analogues disclosed herein;

a 4,7-cyclized analog of Angiotensin III (Ang(2-8)), or any of its analogues disclosed herein;

a 4,7-cyclized analog of Angiotensin IV (Ang(3-8)), or any of its analogues disclosed herein; or

a 4,7-cyclized analog of Ang(l-7), or any of its analogues disclosed herein.

In another embodiment, the methods comprise administering an agonist of the MAS receptor. Any suitable polypeptide or non-polypeptide agonist of the MAS receptor may be used, including but not limited to A(l-7) and analogues thereof, A779 (D-Ala A(l-7);

available from Sigma Chemical Co.) and AVE0991, (see, for example, Pinheiro et al, Hypertension. 2004 Oct;44(4):490-6. Epub 2004 Aug 23).

The polypeptides for use in the present invention may be linear or cyclized in any suitable manner, such as those described in WO2008/018792, including but not limited to polypeptides comprising a thioether bridge between positions 4 and 7, or other positions. While not being bound by any specific mechanism of action, the inventors believe that angiotensin peptides such as A(l-7) ameliorate oxidative stress and endothelial dysfunction in gestational diabetes.

The subject may be any suitable mammalian subject that can suffer from GDM, such as a human subject.

As used herein, "treat" or "treating" GDM means accomplishing one or more of the following: (a) limiting progression of GDM; (b) reversing effects of GDM; (c) limiting development of maternal CVD; (d) limiting development of maternal T2D; (e) limiting development of maternal and/or fetal hyperinsulinemia; (f) limiting perinatal morbidity, such as macrosomia, birth trauma, and pre-eclampsia); and (g) limiting long term risks in the offspring, including but not limited to excess weight/obesity, and metabolic factors that increase the risk of CVD.

In preferred embodiments, the peptide is administered in a dosage of 10 μg kg/day, 50 μg/day μg/kg/day, 100 μg/kg/day, 250 μg/kg/day, 500 μg kg/day, 1000 μg/kg/day or more. In various embodiments, the amount of peptide (such as A(l-7)) or pharmaceutical salt thereof is sufficient to provide a dosage to a patient of between 0.01 μg/kg and 10 mg/kg; 0.1 μg/kg and 5 mg/kg; 0.1 μg/kg and 1000 μg/kg; 0.1 μg/kg and 900 μg/kg; 0.1 μg/kg and 900 μg/kg; 0.1 μg/kg and 800 μg/kg; 0.1 μg/kg and 700 μg/kg; 0.1 μg/kg and 600 μg/kg; 0.1 μg/kg and 500 μg/kg; or 0.1 μg/kg and 400 μg/kg. Peptide can be administered as often as appropriate to achieve the desired result, including but not limited once per day, twice per day, every other day, three times per week, twice per week, or once per week.

In all aspects and embodiments of the invention, suitable acids which are capable of forming salts with peptide (such as A(l-7)) include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.

Suitable bases capable of forming salts with peptide (such as A(l-7)) include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like). Pharmaceutical compositions for use in the methods of the invention may be made up in a solid form (including granules, powders or suppositories), in aerosolized form, or in a liquid form (e.g., solutions, suspensions, or emulsions). The pharmaceutical compositions may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the peptide (such as A(l-7)), and are not harmful for the proposed application. In this regard, the compounds of the present invention are very stable but are hydro lyzed by strong acids and bases. The compounds of the present invention are soluble in organic solvents and in aqueous solutions at pH 5-8. The

pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants.

In other embodiments of all aspects of the invention, the pharmaceutical compositions of the present invention may further comprise one or more other therapeutics as needed by a given subject.

The peptide (such as A(l-7)) or salts thereof can further be derivatized to provide enhanced half-life, for example, by linking to polyethylene glycol or lipidized to increase oral bioavailability and/or prolong plasma half-life. The peptide (such as A(l-7)) or salts thereof may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various "designer" amino acids (e.g., β-methyl amino acids, Ca-methyl amino acids, and a-methyl amino acids, etc.) to convey special properties. In other embodiments, the N-terminus may be acetylated and/or the C-terminus may be amidated.

In addition, the peptide (such as A(l-7)) or salts thereof can have peptidomimetic bonds. For example, an A(l-7) peptide may be generated that incorporates a reduced peptide bond, i.e., Ri-CH₂-NH-R₂, where Ri and R₂ are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such polypeptides are resistant to protease activity, and possess an extended half-live in vivo.

The peptide (such as A(l-7)) or salts thereof may be chemically synthesized or recombinantly expressed or modified post expression, each of which can be accomplished using standard methods in the art.

The peptide (such as A(l-7)) or salts or ester analogs of the peptides thereof can be administered by any suitable route, including but not limited to dermal, subcutaneous, intradermal, transdermal (for example, by slow-release polymers), intramuscular, intraperitoneal, intravenous, oral, aural, epidural, anal or vaginal (for example, by suppositories), pulmonary route, intratracheal instillation, intranasal routes, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings

For administration, the pharmaceutical compositions are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.

Alternatively, the compositions of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, hydroxyethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art. Methods for the production of these formulations with the peptides or pharmaceutical compositions of the present invention are apparent to those of ordinary skill in the art.

Example 1. Alteration of endothelial function markers in women with gestational diabetes and their fetuses

ABSTRACT

We tested the hypothesis that women with GDM and their fetuses would demonstrate alterations in markers of eNOS uncoupling, oxidative stress, and endothelial dysfunction and that these changes would correlate with the levels of hyperglycemia. We conducted a pilot observational case-control study of women with GDM and their fetuses. We measured levels of soluble intercellular adhesion molecule- 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), C-reactive protein (CRP), nitric oxide (NO) levels, eNOS, p22- phox, and SOD gene expression, and endothelial progenitor cell (EPC) counts in both maternal and cord blood at the time of delivery in women with and without GDM. We demonstrated the presence of decreased maternal circulating EPC counts, increased soluble adhesion molecules in maternal blood, decreased SOD expression in both maternal and cord blood and increased eNOS expression in both maternal and cord blood in women with GDM. These data suggest that the molecular mechanisms behind oxidative stress in women with GDM and their fetuses appear similar to those hypothesized for non-pregnant adults with type 2 DM.

Introduction

Cardiovascular disease (CVD) is the main cause of death in the adult population in the

United States and many countries (1). CVD is responsible for 70% of the deaths in patients with diabetes mellitus (DM), whose risk is increased 2-4 times compared to non-diabetics. Endothelial dysfunction is now considered a key element in the development of CVD, and has also been strongly associated with obesity and insulin resistance, which are key features of type 2 DM (2). Impairment of endothelial function often occurs prior to diagnosis of DM (3), and endothelial dysfunction is often present years before any signs of microangiopathy are apparent (4). Such findings have generated the hypothesis that, in type 2 DM, endothelial dysfunction may precede the development of chronic hyperglycemia.

Once endothelial dysfunction has occurred, it may be too late to intervene to reverse existing damage, whereas early therapeutic interventions may prevent further damage to the endothelium. In this regard, it is critical to identify biomarkers that may appear in diabetes prior to permanent and irreversible damage to tissues.

The early mechanisms of endothelial dysfunction have not been thoroughly investigated in women with GDM. Such evidence would have implications, for example, in the diagnosis and treatment of affected mothers and their offspring. The purpose of this study was to test whether biomarkers of oxidative stress, eNOS uncoupling, and endothelial dysfunction may be altered in women with GDM and their fetuses.

Methods

Study setting, participants, and design

Patients were enrolled at two hospitals, both of which are affiliated with the Keck School of Medicine of the University of Southern California. We conducted a case-control observational study of women with GDM and their neonates using pregnant patients without a diagnosis of GDM as controls. Pregnant women with preterm and multiple gestations, maternal history of smoking, or chronic hypertension were excluded from the study. Case- patients were eligible for study enrollment if they had GDM as diagnosed by having two of the following: a fasting blood glucose >95 mg/dL, a 2-hour oral glucose tolerance test (OGTT) >180 mg/dL, or a 3-hour OGTT >140 mg/dL. Eligible case-patients were identified from outpatient clinics in their last month of pregnancy and enrolled upon hospital arrival prior to delivery. Eligible control-patients were identified upon hospital arrival for delivery. Maternal and venous cord blood was drawn to quantify markers of endothelial dysfunction which included sVCAM-1, sICAM-1, CRP, EPC counts, NO levels, and eNOS, p22-phox, and SOD mRNA expression. Maternal hemoglobin Ale (HbAlc) was tested to determine the level of glycemic control in both case and control patients. The study was approved by the University of Southern California Institutional Review Board, and complied with all patient protection criteria set forth therein.

Maternal venous blood was collected 3-12 hours before delivery. Venous cord blood was collected from the placental side of the cord after the delivery and cord clamping. All blood samples were centrifuged within 30 minutes after collection and processed

immediately, or stored at the appropriate temperature until analyzed.

Preparation of whole blood for flow cytometry

One hundred μΐ, of whole blood were incubated in test tubes for 30 minutes at room temperature in the dark with APC-, PE- and FITC-conjugated antibodies for mouse anti- human CD34, CD309 (KDR), and CD 133 added at the appropriate dilutions in staining buffer after mixing. Two mL of red blood cell lysis buffer (pre-warmed to room temperature) was added to each sample, mixed gently and incubated in the dark at room temperature for 10 minutes. Samples were centrifuged at 400 g at room temperature; after the supernatant was aspirated, it was washed one time with 2 mL of staining buffer. After an additional centrifugation, the stained cell pellets were re-suspended in 2% formaldehyde prior to analysis. Flow cytometric analysis was performed on a LSR II flow cytometer using

FACSDiva software (Becton Dickinson, Franklin Lakes, NJ) at the Flow Cytometry Core Facility at the University of Southern California School of Pharmacy.

Analysis of mRNA expression

Total RNA was extracted from whole blood using TRIzol (Invitrogen, Carlsbad, CA). For each sample, approximately 100 ng of RNA was reverse-transcribed using Maxima Reverse Transcriptase (Fermentas, Glen Burnie, MD). Real-time PCR was conducted to examine expression of eNOS, superoxide dismutase isoforms (SOD2 and SOD3), and p22- phox, a subunit of NADPH oxidase in maternal and cord blood. Quantitative amplification of the cDNA was performed using SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies, Carlsbad, CA) for 40 cycles consisting of heat denaturation, annealing and extension using an ABI 7300 (Applied Biosystems). Expression of eNOS, SOD2, SOD3, and p22-phox mRNA were normalized against 18S mRNA and reported as fold-change compared to non-diabetic women.

Nitrite, HbAlc, sVCAM-1, sICAM-1, and CRP

Nitrite was determined using the Griess reaction, Griess Reagent System, (Promega,

Madison, WI). The remaining parameters were measured using commercial kits according to manufacturer's protocols (HbAlc: BioQuant, San Diego, CA; sVCAM-1, sICAM-1, and CRP: R&D Systems, Minneapolis, MN). Data collection and statistical analysis

Relevant patient information was retrieved from medical records using standardized tools. Data were analyzed using SAS statistical software (Cary, NC). Laboratory data were combined with the patient data for analysis. Univariate analyses were performed, and means are expressed ± the standard deviation (SD), with median and range. Analyses of continuous variables were performed with Kruskal-Wallis testing. For each marker, and for maternal and cord blood, data were examined comparing case- and control-patients. Correlations were determined using linear regression in GraphPad Prism version 5.0d for Mac OS X (GraphPad Software, San Diego, CA). Results

Sixteen patients were enrolled in this study, where nine patients met criteria classifying them as GDM, and seven were classified as non-GDM controls. Three control patients and one GDM patient were nulliparous. Only one GDM patient had a previous history of GDM, while no other patients had a previous history of any glucose abnormalities. Of the patients with GDM, six (67%) were classified as Al (fasting blood glucose >95 mg/dL), and three patients were classified as A2. In the GDM group, one had a cesarean in early labor, and one had an elective repeat cesarean (without labor); three of the non-GDM group had elective cesarean deliveries. All other patients delivered vaginally, with one control patient delivered by vacuum because of maternal exhaustion. There was no difference in BMI or birth weight (Table 1) between the two groups. A statistically significant difference in maternal age was detected between the two groups (p<0.05). When we analyzed the association between maternal age and HbAlc values and percent of circulating EPC, no significant correlation was detected (data not shown). Mothers diagnosed with GDM had significantly higher HbAlc as compared to the control subjects (Table 1 ; 7.6% versus 5.2%, respectively; p<0.01).

Table 1. Patient characteristics by GDM group. Results are expressed as mean + SD, median (range).

Maternal and cord blood EPC counts

EPC play an important role in the neovascularization process following injury, and have been shown to be significantly decreased in diabetes (10, 11). We therefore evaluated mean circulating EPC counts from maternal blood. A statistically significant reduction in the percentage of circulating EPC was seen in GDM versus non-diabetic patients (Figure 1 ; 0.26% versus 0.41%, respectively; p<0.05). Additionally, we measured EPC counts from cord blood of fetuses delivered. Umbilical cord EPC were higher from GDM patients than control patients, however this difference did not reach statistical significance (1.76% versus 1.46%, respectively).

Plasma nitrite levels and eNOS expression

In diabetes, decreased circulating NO levels are often accompanied by a paradoxical increase in eNOS expression, a molecular mechanism referred to as eNOS uncoupling. To determine circulating NO levels, the stable metabolite, nitrite, was measured using the Griess reaction. Mean nitrite levels from maternal plasma were significantly lower in women diagnosed with GDM compared to non-diabetic women (54 μΜ versus 28 μΜ, respectively; p<0.05; Figure 2A). Mean nitrite levels from umbilical cord plasma of fetuses delivered from women diagnosed with GDM were higher compared to those of non-diabetic women, however this difference did not reach statistical significance. In addition, maternal and cord blood eNOS mRNA expression were significantly higher in women diagnosed with GDM compared to non-diabetic women (Figure 2B; p<0.01). SOD and p22-phox mRNA expression

An imbalance in SOD isoform and NADPH oxidase expression is also observed in diabetes, which may explain increases in oxidative stress and concomitantly decreasing antioxidant mechanisms. SOD2 (mitochondrial SOD) and SOD3 (extracellular SOD) mRNA expression in the blood was measured using RT-PCR, where maternal SOD2 and SOD3 expression were significantly decreased in women with GDM compared to non-diabetic women (Figures 3A-B; p<0.01). In cord blood, SOD3 mRNA expression was significantly lower in women diagnosed with GDM compared to non-diabetic women, while there was no significant change in cord blood SOD2. Maternal blood p22-phox expression was increased in women with GDM, however this difference did not reach statistical significance (Figure 3C). Surprisingly, cord blood p22-phox mRNA expression was significantly decreased from women with GDM compared to non-diabetic women (p<0.05).

Plasma sVCAM-1, sICAM-1, and CRP levels

Levels of adhesion molecules such as sVCAM-1, sICAM-1, and CRP are associated with the inflammatory process, and can occur as a result of damage to tissue such as the endothelium. Increased levels of these adhesion molecules are observed in diabetes, and have been correlated with severity of disease. We evaluated maternal plasma for sVCAM-1 and sICAM-1, where both levels were significantly higher in GDM patients as compared to non- diabetic women (Figures 4A-B; p<0.05). There was a significant increase in cord blood sVCAM-1 levels from patients with GDM (p<0.01), however there was no significant difference in cord sICAM-1 levels. In addition, there were no significant differences between any groups in regards to plasma CRP levels, a marker of the overall inflammatory state (data not shown). Correlation with HbAlc

To determine whether a correlation exists between HbAlc as a measure of glycemic control and markers of oxidative stress or endothelial dysfunction exist, linear regression was performed on all parameters measured (Figures 5A-D). Maternal blood EPC correlated negatively with HbAlc, where an increase in HbAlc was associated with a decrease in circulating EPC numbers (R²=0.55; p<0.01). In addition, maternal plasma sVCAM-1 and sICAM-1 levels correlated positively with maternal HbAlc, where increased HbAlc was associated with increased sVCAM-1 and sICAM-1 levels (R²=0.79, p<0.01; R²=0.33, p<0.05 respectively). Maternal blood eNOS mRNA expression also correlated positively with maternal HbAlc, where increased HbAlc was associated with increased eNOS mRNA expression (R²=0.25; p<0.05). Lastly, cord blood SOD3 mRNA expression correlated inversely with maternal HbAlc (R²=0.60; p<0.01).

Discussion

GDM affects 3-8% of pregnancies in the United States and up to 12-14% in some high-risk populations, and is a form of hyperglycemia, occurring when the insulin supply cannot meet tissue demands for normal glucose regulation (13). Robust plasticity of β-cell function in the face of progressive insulin resistance is a principal feature of normal glucose regulation during pregnancy. The majority of women with GDM appear to have β-cell dysfunction that occurs on a background of chronic insulin resistance (14). Hyperglycemia, as determined by glucose tolerance testing at mid-pregnancy, has been the mainstay of GDM diagnosis for decades. Maternal hyperglycemia leads to fetal hyperinsulinemia and can lead to adverse long-term maternal outcomes (e.g. development of type 2 DM and atherosclerotic CVD), increased perinatal morbidity (e.g. macrosomia, birth trauma, pre-eclampsia), and long-term sequelae in offspring (e.g. childhood overweight, and metabolic factors that may increase risk of CVD).

In this case-control study of women diagnosed with GDM and their fetuses, we have demonstrated an increase in circulating adhesion molecules (sVCAM-1 and sICAM-1) in patients with GDM. Moreover, GDM subjects had significantly decreased circulating EPC counts in maternal blood, SOD isoform mRNA expression in both maternal and cord blood, and increased eNOS mRNA expression in both maternal and cord blood. These findings are consistent with the hypothesized mechanisms where hyperglycemia can lead to increased oxidative stress and endothelial dysfunction in diabetes. More importantly, our results suggest that alterations in endothelial function are already present in both mothers and their fetuses in GDM pregnancies. If these measures remain elevated, it is possible that these alterations can confer an increased risk for the development of CVD and type 2 DM.

Furthermore, the identification of disturbances at the molecular level (e.g. EPC counts, NO levels, SOD and NADPH oxidase activity, and other molecular markers for oxidative stress and NO production) enables the identification of potential new treatments, (e.g.

pharmaceuticals to prevent eNOS uncoupling and oxidative stress), but also should provide earlier targets for monitoring the effectiveness of current treatment strategies, including dietary changes, exercise regimens, and pharmacological management.

In spite of the historical use of hyperglycemia as the diagnostic criterion for GDM, it is now evident that hyperglycemia may be a late consequence of the disease process, becoming detectable only after the progressive loss of insulin secretion due to longstanding demands imposed by chronic insulin resistance (14). Even with strict glycemic control, women diagnosed with GDM are still at risk for adverse pregnancy outcomes. Therapeutic strategies that focus on tight glucose control often have limited success in avoiding accelerated fetal growth, particularly in pregnancies characterized by maternal obesity

(15, 16). Furthermore, adverse pregnancy outcomes have also been documented in women with mild glucose intolerance who do not meet current diagnostic criteria for GDM (17). In 2010, the International Association of Diabetes and Pregnancy Study Groups created a consensus panel to arrive at diagnostic criteria because there have been no obvious glucose thresholds at which risks of key outcomes are increased. Controversies regarding diagnostic criteria remain active, impeding the generation of new approaches to prevention and treatment.

We found a significant inverse correlation of HgAlC with EPC. Hyperglycemia promotes an environment prone to oxidative stress, which can result in tissue damage and also prevent the ability to repair existing damage. Interestingly, a correlation between HgAlC and sVCAM-1 as well as sICAM-1 has been demonstrated, suggesting that high glucose increases the adhesiveness of the circulating cells, which could explain the endothelial dysfunction often observed in diabetic patients.

For offspring, in utero exposure to a diabetic environment has been associated with obesity, type 2 DM, and dyslipidemia in the offspring, consistent with the concept of "fetal programming," wherein adult disease may find its origins in fetal development (21). Elevated levels of sICAM-1 and sVCAM-1 were found in obese, hypertensive, and diabetic children with a mean age of 15 years, suggesting that endothelial activation appears early in life, and that adhesion molecules are related to the earliest stages of atherosclerosis. Although the mechanisms by which exposure to a diabetic environment in utero causes metabolic abnormalities in the offspring are unknown, it is hypothesized that disturbed endothelial function may play a role (23). Figure 6 illustrates potential mechanisms by which both maternal and fetal endothelial dysfunction could occur.

In summary, our results have demonstrated the presence of maternal and fetal molecular markers of endothelial dysfunction and oxidative stress in women with GDM and their fetuses. We submit that continued advances in the understanding of the molecular mechanisms associated with endothelial dysfunction will have a substantial impact in the field of GDM, where the dynamics of the perinatal environment are remarkably complex. Given that current standards for strict glycemic control during pregnancy do not appear to eliminate the maternal and fetal repercussions of GDM, new treatment avenues are needed. The ability to identify and track early markers of vasculopathy in women with GDM, as we have demonstrated in this study, may provide a foundation for an improved understanding of the physiology of the disease process and its impact on both the mother and fetus at an early, and potentially reversible stage.

References

1. Lloyd- Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, Ferguson TB, Ford E, Furie K, Gillespie C, Go A, Greenlund K, Haase N, Halipern S, Ho PM, Howard V, Kissela B, Kittner S, Lackland D, Lisabeth L, Marelli A, McDermott MM, Meigs J, Mozaffarian D, Mussolino M, Nichol G, Roger VL, Rosamond W, Sacco R, Sorlie P, Stafford R, Thorn T, Wasserthiel-Smoller S, Wong ND, Wylie-Rosett J; American Heart Association Statistics Committee and Stroke Statistics Subcommittee (2010) Executive summary: heart disease and stroke statistics— 2010 update: a report from the American Heart Association. Circulation 121(7):948-954.

2. de Jagr J, Dekker JM, Kooy A, Kostense PJ, Nijpels G, Heine RJ, Bouter LM, Stehouwer CD (2006) Endothelial dysfunction and low-grade inflammation explain much of the excess cardiovascular mortality in individuals with type 2 diabetes: the Hoorn Study. Arterioscler Thromb Vase Biol 26(5): 1086-1093.

3. Calles-Escandon J and Cipolla M (2001) Diabetes and endothelial dysfunction: a clinical perspective. Endocr Rev 22(l):36-52.

4. Meigs JB, Hu FB, Rifai N, Manson JE (2004) Biomarkers of endothelial dysfunction and risk of type 2 diabetes mellitus. JAMA 291(16): 1978-1986.

5. Hod M and Yogev Y (2007) Goals of metabolic management of gestational diabetes: is it all about the sugar? Diabetes Care 30 Suppl 2:S180-187. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM (2002) Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation 105(14): 1656-1662.

Sheetz MJ and King GL (2002) Molecular understanding of hyperglycemia's adverse effects for diabetic complications. JAMA 288(20):2579-2588.

Bitar MS, Wahid S, Mustafa S, Al-Saleh E, Dhaunshi GS, Al-Mulla F (2005) Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes. Eur J Pharmacol 511(l):53-64.

Mordwinkin NM, Meeks CJ, Jadhav SS, Espinoza T, Roda N, diZerega GS, Louie SG, Rodgers KE (2012) Angiotensin^ 1-7) administration reduced oxidative stress in diabetic bone marrow. Endocrinology 153 (5):2189-2197.

Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275(5302):964-967.

Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC (2002) Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation 106(22):2781-2786.

Loomans CJ, de Koning EJ, Staal FJ, Rookmaaker MB, Verseyden C, de Boer HC, Verhaar MC, Braam B, Rabelink TJ, van Zonneveld AJ (2004) Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes 53(1): 195-199.

Jovanovic L and Pettit DJ (2001) Gestational diabetes mellitus. JAMA 286(20):2516- 2518.

Buchanan TA, Xiang AH, Peters RK, Kjos SL, Marroquin A, Goico J, Ochoa C, Tan S, Berkowitz K, Hodis HN, Azen SP (2002) Preservation of pancreatic beta-cell function and preservation of type 2 diabetes by pharmacological treatment of insulin resistance in high-risk Hispanic women. Diabetes 51(9):2796-2803.

Schaefer-Graf UM, Pawliczak J, Passow D, Hartmann R, Rossi, Buhrer C, Harder T, Plagemann A, Vetter K, Kordonouri O (2005) Birth weight and parental BMI predict overweight in children from mothers with gestational diabetes. Diabetes Care 28(7): 1745-1750.

Ouzounian JG, Hernandez GD, Korst LM, Montoro MM, Battista LR, Walden CL, Lee RH (201 1) Pre-pregnancy weight and excess weight gain are risk factors for macrosomia in women with gestational diabetes. J Perinatal 31(11):717-721.

17. Jensen DM, Damm P, Sorenson B, Molsted-Pedersen L, Westergaard JG, Klebe J, Beck-Nielsen H (2001) Clinical impact of mild carbohydrate intolerance in pregnancy: a study of 2904 nondiabetic Danish women with risk factors for gestational diabetes mellitus. Am J Obstet Gynecol 185(2):413-419.

18. Kautzky- Wilier A, Fasching P, Jilma B, Waldhausl W, Wagner OF (1997) Persistent elevation and metabolic dependence of circulating E-selectin after delivery in women with gestational diabetes mellitus. J Clin Endocrinol Metab 82(12):41 17-4121.

19. Krauss T, Emons G, Kuhn W, Augustin HG (2002) Predictive value of routine circulating soluble endothelial cell adhesion molecule measurements during pregnancy. Clin Chem 48(9): 1418-1425.

20. Bo S, Valpreda S, Menato G, Bardelli C, Botto C, Gambino R, Rabbia C, Durazzo M, Cassader M, Massobrio M, Pagano G (2007) Should we consider gestational diabetes a vascular risk factor? Atherosclerosis 195(2):72-79.

21. Hillier TA, Pedula KL, Schmidt MM, Mullen JA, Charles MA, Pettitt DJ (2007) Childhood obesity and metabolic imprinting: the ongoing effects of maternal hyperglycemia. Diabetes Care 30(9):2287-2292.

22. Glowinska B, Urban M, Peczynska J, Florys B (2005) Soluble adhesion molecules (sICAM-1, sVCAM-1) and selectins (sE selectin, sP selectin, sL selectin) levels in children and adolescents with obesity, hypertension, and diabetes. Metabolism 54(8): 1020-1026.

23. Sobngwi E, Boudou P, Mauvais-Jarvis F, Leblanc H, Velho G, Vexiau P, Porcher R, Hadjadj S, Pratley R, Tataranni PA, Calvo F, Gautier JF (2003) Effect of a diabetic environment in utero on predisposition to type 2 diabetes. Lancet 361(9372): 1861-

1865.

Example 2. Pre-diabetes

We analyzed the data obtained in Example 1 by demarcating the patients based on HbA ic levels (a measure of hyperglycemia over a prolonged period) into pre-diabetes vs diabetes based upon ADA guidelines. There was evidence of these markers being increased in pre-diabetes, as shown in Figures 7A-E.

Claims

We claim:

1. A method for identifying subjects at risk of gestational diabetes (GDM),

cardiovascular disease (CVD) and/or type 2 diabetes (T2D), or identifying subjects having GDM or pre-diabetes, comprising determining, compared to control, one or more of the following in samples obtained from a pregnant female or a person suspected of having prediabetes:

wherein one or more of the recited increases or decreases indicates a risk of GDM,

CVD, and/or T2D in the pregnant female, or the presence of gestational diabetes in the pregnant female, or the presence of pre-diabetes in the subject.

2. The method of claim 1, wherein the subject is a pregnant female.

3. The method of claim 1 or 2, wherein the one or more of the recited increases or decreases indicates a risk of GDM in the pregnant female.

4. The method of any one of claims 1-3, wherein the subject is a pregnant female who has one or more risk factors selected from the group consisting of obesity, had GDM in a previous pregnancy, sugar in the urine, family history of diabetes, previously given birth to a baby weighing 4000 grams or more, had an unexplained stillbirth, had a baby with a birth defect, high blood pressure, over 35 years of age, or a member of a racial group with a high prevalence of diabetes.

5. The method of any one of claims 1-4, wherein a marker level in each of groups (a)-(d) is measured.

6. The method of claim 5, wherein the level of each marker in groups (a)-(d) is measured.

7. The method of any one of claims 1 -6, wherein the presence of decreased maternal circulating EPC counts compared to control, increased soluble adhesion molecules in maternal blood compared to control, decreased SOD2 and/or SOD3 expression in both maternal and cord blood compared to control, and/or increased eNOS expression in both maternal and cord blood compared to control indicates a risk of GDM or the presence of GDM in the pregnant female.

8. The method of any one of claims 1-7, wherein the method further comprises treating the subject based on the determined marker levels.

9. A method for monitoring efficacy of treatment for gestational diabetes, comprising determining, compared to control, one or more of the following in samples obtained from a pregnant female with gestational diabetes:

10. The method of claim 9 wherein a marker level in each of groups (a)-(d) is measured.

1 1. The method of claim 10, wherein the level of each marker in groups (a)-(d) is measured.

12. The method of any one of claims 9-11, wherein the presence of decreased maternal circulating EPC counts compared to control, increased soluble adhesion molecules in maternal blood compared to control, decreased SOD2 and/or SOD3 expression in both maternal and cord blood compared to control, and/or increased eNOS expression in both maternal and cord blood compared to control indicates poor efficacy of treatment in the pregnant female.

13. A method for treating GDM, comprising administering to a subject with GDM an amount effective to treat the GDM of a polypeptide comprising or consisting of

angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof, All AT2 type 2 receptor agonists, or an agonist (polypeptide or otherwise) of the MAS receptor, or pharmaceutically acceptable salts thereof.

14. The method of claim 13, wherein the polypeptide comprises at least 5 contiguous amino acids of A(l-7) (SEQ ID NO:4). The method of claim 14, wherein the polypeptide comprises SEQ ID NO:4.