Gautam Chaudhuri - US grants

We propose to test the following hypothesis. (1) local release of PGI2 NO contributes to the relaxation of umbilical and fetoplacental vascular smooth muscle. (2) A dimunition in the formation of these endothelial derived vasodilators under increase 02 tension promotes contraction of umbilical and fetoplacental vascular smooth muscle. (3) Reduced formation of endothelial derived vasodilators modulates the sensitivity of fetoplacental vessels to endogenous vasoconstrictor substances and may contribute to the increase in placental resistance which is thought to occur in some diseased states. Three specific aims are proposed to achieve our objective and each aim corresponds to a part of the hypothesis. (1) To study the release and actions of PGI2 and NO on human umbilical and fetoplacental vessels obtained from normal pregnancies. (2) To study the interrelationship of PGI2 and NO release and action on human umbilical and fetoplacental vessels under different 01 tensions. (3) To study the release and action of PGI2 and NO on umbilical and fetoplacental vessels obtained from some abnormal pregnancies and evaluate whether these substances modulate the actions of angiotension II and serotonin. Long term objectives. The generation and actions of EDRF and PGI2 and their interrelationship may have important basic and clinical implications. The results of these studies may indicate whether diminished formation or actin of these substances may contribute to the closure of the umbilical artery immediately after birth and form the basis for future studies. The results would also lay the basis for future studies to assess the role of PGI2 and EDRF in the pathophysiology of certain abnormal pregnancies where the fetoplacental circulation is adversely affected.

We propose to test the following hypothesis: 1) The pregnancy related increase in uterine blood flow mediated by release of nitric oxide (NO) is flow dependent; 2) The mechanism by which this flow-dependent alteration of the vascular tone occurs is partly mediated by release of bradykinin (Bk) by endothelial cells, which in turn releases NO; 3) The mechanism of the flow dependent alteration of the vascular tone during pregnancy is also partly dependent on the release of NO by activation of the potassium channel; 4) The increase in serum concentrations of estradiol and progesterone during pregnancy is also responsible for the flow related release of NO by the uterine vascular bed. Five specific aims are proposed to achieve our objective. Specific Aim 1 We will assess whether the decrease in tone of th uterine vascular bed during pregnancy is mediated by flow-dependent release of NO. This specific aim will test hypothesis 1. Specific Aim 2 We will assess the modulating role of endothelium derived bradykinin in the flow dependent release of NO from the perfused uterine vascular bed in vitro. This specific aim will test hypothesis 2. Specific Aim 3 We will assess the role of K+channel activation on the flow related release of NO from the perfused uterine vascular bed. This specific aim will test hypothesis 3. Specific Aim 4 We will assess the role of estradiol and progesterone in modulating tone in the uterine microvasculature and also the cellular and molecular mechanisms involved. This specific aim will test hypothesis 4. Specific Aim 5 We will assess the mechanism of increase in uterine blood flow during pregnancy utilizing an in vivo model. This specific aim will test some key parts of hypothesis 1-4 in vivo. An understanding of the basic mechanism involved in modulating uterine blood flow (UBF) during pregnancy will have important basic and clinical implications. The results of these studies will indicate the local regulatory mechanism by which No is released and how sex steroids can modify this effect. The results of these studies would lay the foundation for our understanding of how these mechanisms may be altered in certain disease states associated with pregnancy such as hypertension, diabetes, and intrauterine growth retardation conditions where the fetus may be adversely affected.

Leishmaniasis, a human disease caused by the parasitic protozoa of the genus Leishmania, is a major scourge in the Tropical World. In contrast to its mammalian hosts, most of the mature mRNAs in Leishmania have a 39 nt signature sequence at their 5'-ends. Without this miniexon sequence, which is transspliced during the maturation of precursor RNAs, the transcripts are not translated into proteins. Phosphorothioate oligodeoxynucleotide (ODN) antisense to this signature sequence of the parasite cells has been shown to be lethal to the parasite proliferation in cultured macrophages, particularly when targeted to the parasitophorous vacuoles by receptor-mediated delivery. The specific aims of this project are to extend this study by (i) optimizing the chemistry of the ODN to effect entry through the parasite plasma membrane, to increase stability and to lessen toxicity to mammalian hosts; (ii) improving the modality of receptor-mediated delivery of this ODN by optimizing the method of conjugation of the ODN with maleylated serum albumin, an artificial but very effective ligand of macrophage scavenger receptors; (iii) evaluating the antileishmanial efficacy of this ODN with respect to its pharmacokinetics and potential toxicity in Leishmania infected mouse model; and (iv) testing the feasibility of the optimized antisense approach in inhibiting the function of specific genes in Leishmania. The long term goal of this study is to develop know-how for research and therapeutic applications of antisense oligonucleotides in the study of leishmanial parasites. Successful completion of this study will not only advance our knowledge towards developing an efficient and specific antileishmanial chemotherapeutic strategy but will also optimize an alternative technology for gene knockout experiments in Leishmania and related organisms.

DESCRIPTION (Adapted from the applicant's abstract): This proposal will examine the mechanism by which GI motility is modulated during pregnancy. Aim 1 will assess the amount of nitric oxide (NO) release following stimulation of the NANC nerves of the GI tract obtained from pregnant rats of different gestational stages using classical pharmacological techniques, and will quantitate the release of NO using a superfusion cascade bioassay. Aim 2 will assess whether the increased NO release from NANC nerves innervating the GI smooth muscles of the pregnant rat is mediated by estradiol, progesterone, or a combination of both. Aim 3 will assess whether the increased NO release following NANC nerve stimulation is due to an increase in the amount of neuronal NO synthase (nNOS), and what the cellular and molecular mechanisms involved in this phenomenon are. Aim 4 will assess whether the decrease in motility of the GI tract and delayed gastric emptying during pregnancy can be reversed by inhibition of NO synthesis.

NO is a free radical and is now thought to be an important intracellular signaling molecule. It in low concentrations, can act as a pro-oxidant presumably by forming peroxynitrite with superoxide, whereas at higher concentrations its antioxidant action predominates. There is convincing evidence that cellular pro-oxidant states-i.e., increased concentrations of active oxygen species and organic peroxides and radicals can promote initiated cells to neoplastic growth. On the other hand, pro- oxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense system and many antioxidants are anti-promoters and anti-carcinogens. It is, therefore, not surprising that NO has been shown to involve cellular transformation and stimulation of neoplastic growth, whereas other studies indicate that NO has a cytostatic and/or cytotoxic effect on tumor cells. The overall hypothesis to be tested is that "NO has a biphasic action on human breast cancer cell lines, i.e. at low steady state concentrations, it increases cell proliferation, whereas at high steady state concentrations, it decreases cell proliferation. This biphasic action is due to its actions on cyclin D1-cyclin dependent kinase (CDK)-retinoblastoma protein (pRG) cascade. NO also acts as an intermediary in some of the actions of estradiol leading to cell proliferation." This hypothesis will be tested under 4 specific aims. They are as follows: Specific Aim 1. We will determine the effects of different concentrations of NO on cell cycle and proliferation and DNA synthesis of human breast cancer (HBC) cell lines. Specific Aim 2. We will determine the effects of low steady state concentrations of NO to modulate phosphorylation of pRB in HBC cell lines, increase cyclin D1 expression, and elucidate the step(s) at which NO may be acting on the cyclin D1-CDK-pRB cascade. Specific Aim 3. We will determine the effects of high steady state concentrations of NO (determined under specific aim 1) to modulate the phosphorylation of pRB and elucidate the step(s) at which NO may be acting on the cyclin D1-CDK-pRB cascade in HBC cell lines. Specific Aim 4. We will determine the ability of inhibitors of NO synthesis to modulate the estradiol induced increase in breast cancer cell proliferation in vitro; shorten the G0/G1 phase of the cell cycle and the molecular mechanisms involved in this phenomenon.

During aging in women, two important changes occur which affect the vascular system. The first is menopause which is associated with estrogen withdrawal and the other is endothelial dysfunction manifested by a decrease in endothelium dependent vasodilation, possibly a reflection of a decrease in NO production by endothelial cells. These two changes may be independent of each other or may be interlinked. We and others have demonstrated that estradiol (E2) increases NO production. Therefore, endothelial dysfunction seen during aging and menopause as manifested by a decrease in endothelium dependent vasodilation and decrease in NO production may be explained just on the basis of E2 withdrawal alone. Estrogen replacement to postmenopausal women has been utilized for potential benefits related to the cardiovascular system. However, the precise mechanism(s) by which estrogens exert a beneficial effect on the cardiovascular system is not known. On the other hand, a recent study indicates that administration of a combination of estrogen and a progestin to women with well-documented coronary artery disease was associated with an increase in the incidence of a second coronary event in the first year of the study compared to the placebo group. In this study, there was no group in which women received E2 alone. Therefore, the role of E2 in modulating atherogenesis, once the disease is established is not known. Similarly, post-menopausal women are occasionally administered testosterone (t) to increase their sexual function but the effect of T on atherogenesis is not known. The overall hypothesis to be tested is that "the mere withdrawal of E2 or T leads to a decrease in NO synthesis, and thereby, to a pro-inflammatory response of the vascular wall leading to changes seen early in atherogenesis. A corollary could be that the atherogenic process on the extreme might increase the production of OONO and under such conditions NO inhibition might actually be beneficial." We propose to mimic the postmenopausal state in experimental animals by ovariectomy (OVX). We propose to mimic endothelial dysfunction by pharmacological means by administering animals an inhibitor of NO synthesis. The hypothesis will be tested under three specific aims.

The overall theme of the Building Interdisciplinary Research Careers in Women's Health (BIRCWH) Center will be to train researchers, especially physicians/scientists to be independent investigators in research related to Women's Health. This program will be an institutional program crossing departmental barriers and will include the disciplines of developmental biology, molecular genetics, cell biology, behavioral sciences, cardiovascular sciences, women's cancer-related problems, clinical pharmacology, translational and clinical investigative research, as well as Health Services research. No core laboratory is being requested as these facilities will be made available for use to scholars in departments where the faculty mentors are situated or institutional core facilities. The training is divided into three phases to train physicians/investigators and institutional funds will be made available for the first two phases and BIRCWH Center grant will be utilized only for the third phase. The success of the program will be determined when the scholars obtain competitive independent peer reviewed funding like the ROI. The impact of the BIRCWH Center will extend beyond the New Program Development Awardees and will also be felt by the faculty members at UCLA. This will facilitate the recruitment of medical students, house officers and fellows into investigative basic, clinical and Health Services research related to Women's Health. In view of the multidisciplinary approach of the activities of the Center, we anticipate that faculty mentors from other departments who are a part of the mentor pool will better appreciate the problems related to Women's Health and later form a nucleus for the establishment of a Women's Health Research and Training Center at UCLA.

[unreadable] DESCRIPTION (provided by applicant): The overall theme of the WRHR Center will be to emphasize a fundamental approach to the diseases of women that include the disciplines of developmental biology, molecular genetics and cell biology, behavioral science as well as translational and clinical investigative research. All of the proposed faculty mentors will be able to provide training to allow the WRHR scholars to establish their own independent, fundable research programs applicable to the clinical problems of women. No core laboratory in the WRHR Center is being requested as core laboratory facilities will be made available for use in departments where the faculty mentors are situated. The impact of the UCLA WRHR Center has extended beyond the New Program Development Awardees and has been felt by the faculty mentors in Obstetrics and Gynecology on each of the participating UCLA campuses. This has facilitated the recruitment of medical students, house officers and fellows into investigative basic and clinical research related to Women's Reproductive Health. In view of the multidisciplinary approach of the activities of the Center, we have observed that faculty mentors from other departments who are a part of the mentor pool better appreciate the problems related to Women's Reproductive Health and have been stimulated to work in this area of investigation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The overall theme of the BIRCWH Center has been to emphasize a fundamental approach to the diseases of women that include the disciplines of developmental biology, molecular genetics and cell biology, behavioral science, cardiovascular science, acquired immune deficiency disease, aging and its problems as it affects women, as well as translational and clinical investigative research. All of the proposed faculty mentors will be able to provide training to allow the BIRCWH Scholars to establish their own independent, fundable research programs applicable to the clinical problems of women. At present we have three Scholars; from Urology, Medicine (Rheumatology), and Obstetrics and Gynecology. All three are women, and one a Puerto Rican. All of them have been awarded peer-reviewed federal grants; one an R-01, one a VA Career Development Award, and one an R-21. All of this was possible because they were able to generate preliminary data while receiving BIRCWH grant support. One of our Scholars who graduated is an African-American who obtained an NIH K-01 award and is at present an Assistant Professor in the Department of Medicine (Cardiology). The impact of the BIRCWH Center has already extended beyond the New Program Development Awardees and has stimulated initiation and collaboration of research related to Women's Health between various faculty members transcending departmental boundaries and facilitated the recruitment of medical students, house officers, and fellows into investigative basic and clinical research related to Women's Health. In view of the multidisciplinary approach of the activities of the already established BIRCWH Center, and the fact that our institution is one of the national sites for the Women's Reproductive Health Research Career Development Center, as well as being awarded the National Center of Excellence Contract by the Office of Women's Health/Public Health Service, the foundations have been laid for the future creation of a "Women's Health Research Center" encompassing research, training, and teaching in matters related to Women's Health. The establishment of the BIRCWH and WRHR Centers has also helped our affiliated minority institution; the King/Drew Medical School. For example, the King/Drew Medical School and the David Geffen School of Medicine at UCLA applied for and received a grant entitled "Drew-UCLA Reproductive Sciences Research Center: Biologic Effects of Androgens in Men and Women". The BIRCWH grant will help train scientist from various disciplines to do research in Women's Health, which is an underserved area in public health.

[unreadable] DESCRIPTION (provided by applicant): A current hypothesis for the etiology of pre-eclampsia is that endothelial dysfunction is secondary to oxidative stress and some clinical studies have indicated that prophylactic administration of anti-oxidants may be useful in its prevention. A much larger trial utilizing the anti-oxidants vitamin C and vitamin E is, at present, being carried out by the Maternal-Fetal Network of the NICHD to assess their effectiveness in the prevention of pre-eclampsia. Endothelial cell activation is usually a constant accompaniment with this condition. Endothelial cell activation leads to endothelial cell dysfunction and is also accompanied by expression of adhesion molecules, i.e. vascular cell adhesion molecule-1 (VCAM-1), and other adhesion molecules. This leads to adhesion of monocytes to endothelial cells resembling early atherogenesis. There is evidence in the literature that tumor necrosis factor a (TNFa) may be released by the placenta and activated leukocytes leading to activation of endothelial cells. The current proposal will mainly focus on mechanisms by which estrogens and anti-oxidants prevent TNFa-induced endothelial cell activation and apoptosis. We will, therefore, test the following four hypotheses. Hypothesis 1: Exposure of endothelial cells to TNFa leads, initially, to activation of the cells leading to VCAM-1 expression and increased synthesis of anti-apoptotic proteins, followed by stimulation of the survival pathway. Hypothesis 2: Estrogen and its metabolites prevent TNFa-induced activation, as well as apoptosis of HUVEC, by increasing the expression of eNOS and phosphorylation of Akt and ERK 1/2, in spite of the absence of migration of NFkB to the nucleus, thereby, directing the cells to the "survival pathway". Hypothesis 3: Anti-oxidants prevent endothelial cell activation and inhibit NFkB migration to the nucleus following exposure of endothelial cells to TNFa by an NO-independent Fak-mediated mechanism. Hypothesis 4: Anti-oxidants attenuate apoptosis in endothelial cells exposed to TNFa by upregulation of ERK 1/2 phosphorylation and downregulation of p38 phosphorylation. Results from these studies will also help explain the different mechanism(s) by which estrogens and anti-oxidants prevent activation of endothelial cells and apoptosis and, thereby, lay the scientific foundation by which anti-oxidants administered to women may prevent pre- eclampsia in women at risk of developing this condition. Pre-eclampsia is a condition that suddenly develops during pregnancy and is accompanied by high blood pressure, swelling of the body, and leakage of proteins. This condition can adversely affect both the mother and the fetus. Pre-eclampsia is more prevalent in Afro-Americans and is also seen in the lower socio-economic groups. Results from our studies will indicate to us which anti-oxidants are likely to be more effective than others and which can be used clinically to prevent this condition. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A current hypothesis for the etiology of pre-eclampsia is that endothelial dysfunction is secondary to oxidative stress and some clinical studies have indicated that prophylactic administration of anti-oxidants may be useful in its prevention. A much larger trial utilizing the anti-oxidants vitamin C and vitamin E is, at present, being carried out by the Maternal-Fetal Network of the NICHD to assess their effectiveness in the prevention of pre-eclampsia. Endothelial cell activation is usually a constant accompaniment with this condition. Endothelial cell activation leads to endothelial cell dysfunction and is also accompanied by expression of adhesion molecules, i.e. vascular cell adhesion molecule-1 (VCAM-1), and other adhesion molecules. This leads to adhesion of monocytes to endothelial cells resembling early atherogenesis. There is evidence in the literature that tumor necrosis factor a (TNFa) may be released by the placenta and activated leukocytes leading to activation of endothelial cells. The current proposal will mainly focus on mechanisms by which estrogens and anti-oxidants prevent TNFa-induced endothelial cell activation and apoptosis. We will, therefore, test the following four hypotheses. Hypothesis 1: Exposure of endothelial cells to TNFa leads, initially, to activation of the cells leading to VCAM-1 expression and increased synthesis of anti-apoptotic proteins, followed by stimulation of the survival pathway. Hypothesis 2: Estrogen and its metabolites prevent TNFa-induced activation, as well as apoptosis of HUVEC, by increasing the expression of eNOS and phosphorylation of Akt and ERK 1/2, in spite of the absence of migration of NFkB to the nucleus, thereby, directing the cells to the "survival pathway". Hypothesis 3: Anti-oxidants prevent endothelial cell activation and inhibit NFkB migration to the nucleus following exposure of endothelial cells to TNFa by an NO-independent Fak-mediated mechanism. Hypothesis 4: Anti-oxidants attenuate apoptosis in endothelial cells exposed to TNFa by upregulation of ERK 1/2 phosphorylation and downregulation of p38 phosphorylation. Results from these studies will also help explain the different mechanism(s) by which estrogens and anti-oxidants prevent activation of endothelial cells and apoptosis and, thereby, lay the scientific foundation by which anti-oxidants administered to women may prevent pre- eclampsia in women at risk of developing this condition. Pre-eclampsia is a condition that suddenly develops during pregnancy and is accompanied by high blood pressure, swelling of the body, and leakage of proteins. This condition can adversely affect both the mother and the fetus. Pre-eclampsia is more prevalent in Afro-Americans and is also seen in the lower socio-economic groups. Results from our studies will indicate to us which anti-oxidants are likely to be more effective than others and which can be used clinically to prevent this condition.

[unreadable] DESCRIPTION (provided by applicant): Leishmania is a group of parasitic protozoa that infect human macrophages and thrive inside the hostile environment of the phagolysosomes of these cells. The long-term objective of the proposed research is to identify the molecular events that must occur at the early stage of leishmanial interactions with the macrophages leading to the establishment of successful parasitism. Leishmania has long been known to 'renovate' the molecular environment of macrophages in order to establish infection inside their phagolysosomes. Leishmania specifically manipulates the expression of host macrophage genes in the early stages of their infection. Gene transcripts that are down regulated in macrophages upon exposure to Leishmania include 7SL RNA, the RNA component of the signal recognition particle (SRP). Since the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down regulation of 7SL RNA may be significant in the establishment of infection by Leishmania. More importantly, over expression of 7SL RNA in J774G8 or U937 cells confers resistance to Leishmania infection. These results demonstrate the biological significance of down-regulating 7SL RNA synthesis in the establishment of infection by Leishmania. Based on these findings, the hypothesis is that Leishmania-induced down regulation of the level of 7SL RNA in the macrophages favors in part the development of leishmaniasis in the mouse model. Exogenous compensation of 7SL RNA in their macrophages will thus make these mice resistant to Leishmania infection. Specific aims to test the hypothesis are the following: (1) Development of BALB/c mice derivatives with over expression of 7SL RNA in their macrophages by genetic manipulation of the bone marrow stem cells and transplantation. Over expression of 7SL RNA in the bone marrow derived macrophages will be done using already developed lentiviral constructs that will allow the expression to occur inside the cells of macrophage lineage. Genetically manipulated bone marrow stem cells will be transplanted into irradiated BALB/c mice, which are susceptible hosts for Leishmania. (2) Evaluation of the ability of Leishmania promastigotes to produce footpad lesions in mice over expressing 7SL RNA in their macrophages. (3) Evaluation of the effects of Leishmania exposure of macrophages isolated from the peritoneum of the transplanted mice on the levels of phagolysosomal proteins, surface membrane receptors and the levels of proteins secreted from these cells. We will measure the levels of cathepsins in lysosomes, and scavenger receptor, and CSF-1R on the cell surfaces of peritoneal macrophages isolated from transplanted mice after exposure of the macrophages for various time periods (0-12 h). These evaluations will be done by immunofluorescence microscopy and Western blotting analysis. Levels of cytokines and chemokines secreted from the macrophages with or without Leishmania exposure will be evaluated by cytokine antibody microarray analysis. PUBLIC HEALTH RELEVANCE: The proposed study will reveal a unique renovating mechanism employed by the parasitic protozoan Leishmania to establish infection in the macrophages. Development of the mouse model with over expression of 7SL RNA in its macrophages may be used to test whether macrophage vesicular protein transport is also critical for the parasitism of macrophages by other pathogens. Understanding host-parasite interaction mechanisms interplayed between macrophages and Leishmania in molecular details will help us in developing combat measures against this often deadly human pathogen. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Macrophages are a heterogeneous collection of terminally differentiated mononuclear phagocytes that are distributed all over the mammalian body to perform tissue clearing, tissue remodeling and other functions. These cells act in diverse capacities as the primary responders of our innate and adaptive immune systems. As a part of their tissue clearing roles, macrophages recruited at the site of breast tumor development ingest significant mass of cellular debris formed from the initially dying tumor cells. High levels of phagocytosis induce the production of reactive oxygen and nitrogen species which in turn elevates the expression of cytotoxic non- coding RNAs (ncRNAs) like Alu RNAs. If not interfered by the tumor cells, high levels of Alu RNA would produce severe inflammatory immune response through the activation of inflammasome and accelerated death of the overfed macrophages bringing collateral damage to the breast tumor cells. The preliminary data generated in the PI's laboratory revealed that during the breast tumor formation, inflammatory development of the recruited macrophages is prevented by the breast tumor cells through the reduction of the levels of Alu RNA in the tumor-associated macrophages converting them to accessory cells supporting tumor growth. We postulate that breast tumor cell-induced reduction in the level of cytotoxic Alu RNA in the tumor-associated macrophages creates a tissue microenvironment that fosters tumor progression. The long-term goal of the proposed research is to understand how breast tumor cells manipulate the macrophages to make them docile and supportive to the development of breast tumor. The central hypothesis is that as a part of the regulation of macrophage turnover, oxidative stress generated during intense phagocytosis by the macrophages at the site of tissue damage (e.g. breast tumor) induces the levels of cytotoxic Alu RNA in these cells. This response in the breast tumor-associated macrophages (TAMs) is suppressed by the tumor cells so that these longer-living immuno-suppressed macrophages in the tumor microenvironment can be utilized for further development and progression of the breast tumor. Specific Aim to test the hypothesis are: (a) Identification of the mediators secreted from the breast cancer cells that manipulate Alu RNA metabolism in the macrophages; and, (b) Evaluation of the mechanisms of manipulation of Alu RNA metabolism in the tumor-associated macrophages during breast tumor development. This research proposes an innovative basic mechanism to explain macrophage-assisted breast tumor development highlighting the critical importance of Alu RNAs in the maintenance and disposal of macrophages. The proposed research will make a significant contribution because it will not only highlight a new direction in the understanding of macrophage biology and the etiology of macrophage-associated breast tumor development but also will lead us towards the development of rational chemotherapy against such diseases of the breast.

Abstract Preeclampsia affects 3 to 6 % of pregnant women with devastating effects on the mother and the baby. However, there are no molecular targets that could drive drug discovery towards therapeutic success or early diagnostic markers that could predict the onset of this syndrome prior to development of symptoms. We have for the first time observed that gamma Aminobutyric acid (GABA) a central inhibitory neurotransmitter is synthesized and released by vascular endothelial cells into the systemic circulation and our findings therefore explain one important source of GABA in the systemic circulation. Furthermore, the level of GABA in the circulation is decreased in preeclampsia compared to non-pregnant women but the mechanism(s) involved is not known. The actions of GABA in the endothelial cells, and how a decrease in synthesis and release of GABA occurs in preeclampsia and its significance in the etiology of preeclampsia is not known. In preliminary experiments, we observed that human umbilical venous endothelial cells (HUVEC) obtained from pre- eclamptic pregnancies synthesize and release less GABA when compared with HUVEC obtained from normal pregnant women. We have also identified L-methionyl glutamate (LMG) in preeclamptic HUVEC which inhibits GABA synthesis by endothelial cells. Based on our novel observations, we HYPOTHESIZE that: ?a decrease in endothelial synthesis and release of GABA is responsible for endothelial dysfunction and contributes to the pathogenesis of pre- eclampsia? and our hypothesis will be tested by the following three specific aims. Specific Aim 1: We will assess whether HUVEC obtained from mothers with pre-eclampsia (PE-HUVEC) synthesize and release less GABA when compared to those obtained from mothers with normal pregnancy. Specific Aim 2: We will compare the role of GABA in maintaining the normal functions of HUVEC obtained from normal pregnancies with relation to free fatty acid (FFA) oxidation, pyruvate oxidation, ATP synthesis, regulation of adhesion molecules expression and reactive oxygen species (ROS) generation and how decreased synthesis of GABA in PE-HUVEC affects these processes to cause endothelial cell dysfunction. Specific Aim 3: (a) We will assess the effects of the endogenous GABA synthesis inhibitor LMG, which is high in PE-HUVEC, on some of the GABA mediated functions of NP-HUVEC and HAEC as assessed under specific aim 2 and (b) we will quantify the levels of GABA and LMG in the cord and maternal blood of normal pregnancy and correlate this with the cord and maternal levels in preeclamptic pregnancy.