Nitric Oxide In Lung Development And CLD
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5U01HL063399-08 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Nitric oxide (NO) is critically involved in numerous functions in the developing lung. NO is produced by neuronal NO synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have shown that all three isoforms are upregulated in the third trimester in fetal baboon lung. Chronic Lung Disease (CLD) is a condition which disrupts preterm human lung development, resulting in airway, parenchymal and pulmonary vascular dysfunction. We have shown in the baboon model of CLD that lung nNOS and eNOS are markedly decreased, that lung NO production remains low during the early course, and that inhaled NO gas (iNO) improves pulmonary hemodynamics, compliance and resistance. iNO also partially normalizes surfactant function, it stimulates lung growth, and it prevents certain structural features of CLD. Using physiologic, biochemical and histologic/morphologic readouts, the overall objective of this proposal is to determine if interventions which upregulate endogenous NO production, or which replace NO in a manner favoring physiologic NO metabolism, result in greater prevention of CLD. Studies will be done in preterm baboons born at 125d gestation (term = 185d) and ventilated for 14d. Aim 1 is to determine the effects of postnatal estradiol (E2) treatment on lung NO production, and on the development of CLD. E2 upregulates nNOS and eNOS expression and activity in some paradigms, and with preterm birth there is withdrawal from placentally-derived E2. Aim 2 is to determine if there is greater impact on the genesis of CLD with iNO plus glutathione (GSH), or with O-nitrosoethanol (ENO), versus iNO alone. Whereas endogenous NO forms biologically active S-nitrosothiols (SNO), iNO gas yields toxic higher oxides of nitrogen (NOX) and peroxynitrite. GSH, which is deficient in prematurity, is the primary substrate for lung SNO. In contrast to iNO, ENO preferably forms SNO and generates less NOX and peroxynitrite in mature models tested thus far. Aim 3 is to delineate the changes in pulmonary and systemic NO metabolism in CLD and in the intervention groups. In initial work, blood NOX rose while SNO fell postnatally in CLD, and iNO raised NOX further with scant impact on SNO. It is hypothesized that E2, iNO + GSH, or ENO will increase SNO and cause little change in NOX and peroxynitrite. These studies will increase our knowledge of the role of NO in CLD and also test three novel, mechanistically-based therapies.
Keywords: bronchopulmonary dysplasia, enzyme induction /repression, hormone therapy, isozyme, lung development, nitric oxide synthase, nitrogen metabolism, nonhuman therapy evaluation, cell type, cooperative study, disease /disorder model, enzyme activity, estradiol, glutathione, hemodynamics, messenger RNA, nitric oxide, nitroso compound, pathologic process, respiratory airway pressure, respiratory circulation, respiratory epithelium, baboon, echocardiography, tissue /cell culture
Project start date: 1999-09-01
Project end date: 2008-08-31
5U01HL063399-08 (2006): $540011
Sponsored Links Excellgen http://Excellgen.com
NITRIC OXIDE SYNTHASES IN LUNG DEVELOPMENT AND BRONCHOPU
Philip W Shaul, Professor
Pediatricsuniversity Of Texas Sw Med Ctr/dallas
dallas, Tx 753909105
Grant 5U01HL063399-03 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: The signaling molecule nitric oxide (NO) is critically involved in airway and vascular function in the developing lung. NO is produced by three isoforms of NO synthase (NOS), neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our preliminary work in normal baboon fetuses indicates that all three isoforms are expressed in airway epithelium, and that nNOS and eNOS are upregulated during late gestation to optimize NO production in the perinatal period. Bronchopulmonary dysplasia (BPD) is an inflammatory condition which disrupts the development of the preterm human lung, and it is characterized by airway and pulmonary vascular dysfunction. Our initial studies in the baboon BPD model indicate that lung NO production and lung nNOS and eNOS expression are markedly attenuated during the genesis of BPD, and that NO replacement by inhalation (iNO) results in a sustained improvement in oxygenation index. The overall objective of this proposal is to investigate the role of alterations in NOS expression in the pathophysiology of BPD in the baboon model. The primary hypothesis is that pulmonary nNOS and eNOS expression are downregulated during the development of BPD, leading to diminished NO production and abnormal airway and vascular structure and function. The secondary hypothesis is that iNO reverses these abnormalities. Aim 1 is to define the normal ontogeny and changes in NOS protein and mRNA expression in early BPD, using approaches including laser capture microdissection to evaluate NOS mRNA levels in specific cell types harvested from frozen sections. Aim 2 is to determine the role of each NOS isoform in airway and vascular function in studies of NOS antagonism in intact animals. Aim 3 is to reveal the changes in exhaled NO levels with fetal development and BPD, as well as the contribution of each NOS isoform to exhaled NO. Aim 4 is to determine the effects of iNO on airway and vascular function, on the pulmonary course of early BPD, and on lung histology. Aim 5 is to determine the mechanisms underlying constitutive nNOS and eNOS gene expression in cultured baboon airway epithelium, and the role of cytokines in their downregulation. The results obtained will increase our knowledge of the role of NO in normally successful postnatal pulmonary adaptation and in the pathophysiology of BPD, thereby possibly leading to novel therapies for this devastating disorder
Keywords: bronchopulmonary dysplasia, enzyme induction /repression, isozyme, lung, nitric oxide synthase cell type, cooperative study, disease /disorder model, enzyme activity, histogenesis, messenger RNA, nitric oxide, pathologic process, respiratory airway pressure, respiratory circulation, respiratory epithelium baboon, echocardiography, embryo /fetus cell /tissue, tissue /cell culture
Project start date: 1999-09-01
Project end date: 2003-08-31
5U01HL063399-03 (2001): $283214
5U01HL063399-02 (2000): $259551
Nitric Oxide In Lung Development And CLD
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5U01HL063399-07 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Nitric oxide (NO) is critically involved in numerous functions in the developing lung. NO is produced by neuronal NO synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have shown that all three isoforms are upregulated in the third trimester in fetal baboon lung. Chronic Lung Disease (CLD) is a condition which disrupts preterm human lung development, resulting in airway, parenchymal and pulmonary vascular dysfunction. We have shown in the baboon model of CLD that lung nNOS and eNOS are markedly decreased, that lung NO production remains low during the early course, and that inhaled NO gas (iNO) improves pulmonary hemodynamics, compliance and resistance. iNO also partially normalizes surfactant function, it stimulates lung growth, and it prevents certain structural features of CLD. Using physiologic, biochemical and histologic/morphologic readouts, the overall objective of this proposal is to determine if interventions which upregulate endogenous NO production, or which replace NO in a manner favoring physiologic NO metabolism, result in greater prevention of CLD. Studies will be done in preterm baboons born at 125d gestation (term = 185d) and ventilated for 14d. Aim 1 is to determine the effects of postnatal estradiol (E2) treatment on lung NO production, and on the development of CLD. E2 upregulates nNOS and eNOS expression and activity in some paradigms, and with preterm birth there is withdrawal from placentally-derived E2. Aim 2 is to determine if there is greater impact on the genesis of CLD with iNO plus glutathione (GSH), or with O-nitrosoethanol (ENO), versus iNO alone. Whereas endogenous NO forms biologically active S-nitrosothiols (SNO), iNO gas yields toxic higher oxides of nitrogen (NOX) and peroxynitrite. GSH, which is deficient in prematurity, is the primary substrate for lung SNO. In contrast to iNO, ENO preferably forms SNO and generates less NOX and peroxynitrite in mature models tested thus far. Aim 3 is to delineate the changes in pulmonary and systemic NO metabolism in CLD and in the intervention groups. In initial work, blood NOX rose while SNO fell postnatally in CLD, and iNO raised NOX further with scant impact on SNO. It is hypothesized that E2, iNO + GSH, or ENO will increase SNO and cause little change in NOX and peroxynitrite. These studies will increase our knowledge of the role of NO in CLD and also test three novel, mechanistically-based therapies.
Keywords: bronchopulmonary dysplasia, enzyme induction /repression, hormone therapy, isozyme, lung development, nitric oxide synthase, nitrogen metabolism, nonhuman therapy evaluation, cell type, cooperative study, disease /disorder model, enzyme activity, estradiol, glutathione, hemodynamics, messenger RNA, nitric oxide, nitroso compound, pathologic process, respiratory airway pressure, respiratory circulation, respiratory epithelium, baboon, echocardiography, tissue /cell culture
Project start date: 1999-09-01
Project end date: 2007-08-31
5U01HL063399-07 (2005): $542620
5U01HL063399-06 (2004): $532540
Grants awarded to Philip W Shaul
NITRIC OXIDE SYNTHASES IN LUNG DEVELOPMENT AND BRONCHOPU
Philip W Shaul, Professor
Pediatricsuniversity Of Texas Sw Med Ctr/dallas
dallas, Tx 753909105
Grant 1U01HL063399-01 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: The signaling molecule nitric oxide (NO) is critically involved in airway and vascular function in the developing lung. NO is produced by three isoforms of NO synthase (NOS), neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our preliminary work in normal baboon fetuses indicates that all three isoforms are expressed in airway epithelium, and that nNOS and eNOS are upregulated during late gestation to optimize NO production in the perinatal period. Bronchopulmonary dysplasia (BPD) is an inflammatory condition which disrupts the development of the preterm human lung, and it is characterized by airway and pulmonary vascular dysfunction. Our initial studies in the baboon BPD model indicate that lung NO production and lung nNOS and eNOS expression are markedly attenuated during the genesis of BPD, and that NO replacement by inhalation (iNO) results in a sustained improvement in oxygenation index. The overall objective of this proposal is to investigate the role of alterations in NOS expression in the pathophysiology of BPD in the baboon model. The primary hypothesis is that pulmonary nNOS and eNOS expression are downregulated during the development of BPD, leading to diminished NO production and abnormal airway and vascular structure and function. The secondary hypothesis is that iNO reverses these abnormalities. Aim 1 is to define the normal ontogeny and changes in NOS protein and mRNA expression in early BPD, using approaches including laser capture microdissection to evaluate NOS mRNA levels in specific cell types harvested from frozen sections. Aim 2 is to determine the role of each NOS isoform in airway and vascular function in studies of NOS antagonism in intact animals. Aim 3 is to reveal the changes in exhaled NO levels with fetal development and BPD, as well as the contribution of each NOS isoform to exhaled NO. Aim 4 is to determine the effects of iNO on airway and vascular function, on the pulmonary course of early BPD, and on lung histology. Aim 5 is to determine the mechanisms underlying constitutive nNOS and eNOS gene expression in cultured baboon airway epithelium, and the role of cytokines in their downregulation. The results obtained will increase our knowledge of the role of NO in normally successful postnatal pulmonary adaptation and in the pathophysiology of BPD, thereby possibly leading to novel therapies for this devastating disorder
Keywords: bronchopulmonary dysplasia, enzyme induction /repression, isozyme, lung, nitric oxide synthase cell type, cooperative study, disease model, enzyme activity, histogenesis, messenger RNA, nitric oxide, pathologic process, respiratory airway pressure, respiratory circulation, respiratory epithelium baboon, echocardiography, embryo /fetus cell /tissue, tissue /cell culture
Project start date: 1999-09-01
Project end date: 2003-08-31
1U01HL063399-01 (1999): $312424
Cholesterol And Endothelial NO Synthase
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5R01HL058888-09 from National Heart, Lung, And Blood Institute IRG: PTHA
Abstract: The mechanisms by which high density lipoprotein (HDL) cholesterol is atheroprotective are not well understood. We have shown that HDL is a potent agonist for endothelial NO synthase (eNOS) via binding to scavenger receptor-BI (SR-BI) in both cultured and intact endothelium. Further recent work indicates that HDL also stimulates endothelial cell migration, and that common upstream signaling events may be involved. The OBJECTIVE of this proposal is to determine the molecular mechanisms by which HDL and SR-BI mediate endothelial cell signaling and migration, and the role of these processes in reendothelialization in vivo. Four Aims will be addressed in cultured cell models and mice. We have preliminary evidence that in contrast to SR-BI, the splice variant SR-BII and CD36 are incapable of stimulating eNOS. Aim 1 is to determine the domains of SR-BI required for signaling by HDL by testing mutant or chimeric receptors cotransfected with eNOS in COS-M6 cells. Based on preliminary findings, including evidence of photocholesterol binding to the SR-BI C-terminal transmembrane (TM) domain, we will test the hypothesis that the C-terminal TM domain and cytoplasmic tail are critical to signaling by HDL. We also have initial evidence that signaling to eNOS involves the PDZ domain-containing protein PDZK1, which is known to interact with the SR-BI C-terminus. We have recently found that PDZK1 is expressed in endothelium. Aim 2 is to determine the role of PDZK1 in HDL-SR-BI signaling in gain-of-function and loss-of-function studies in cultured cells, and in studies of endothelium-dependent relaxation with HDL in arteries from PDZK1 /- mice. Aim 3 is to determine the basis for HDL activation of endothelial migration in a cultured cell wound model. Initial studies indicate mediation by PI3 kinase, which is also required for signaling to eNOS, but the migration is NO- independent. We will test the hypothesis that HDL activation of migration is through SRBI coupling to downstream kinases and small GTPases. Aim 4 is to determine the role of HDL in reendothelialization in vivo, and this will be done in studies of the mouse carotid artery following perivascular electric Injury. Reendothelializatlon will be compared in wild-type versus apoA-/- , SR-BI-/- and PDZK1 -/- mice receiving liver-directed gene transfer of apoA-I or control virus. The hypotheses raised are that HDL enhances reendothelialization, and that this process is mediated by SR-BI and PDZK1. These studies will provide valuable new information about novel direct actions of HDL and SR-BI in endothelium, thereby expanding our knowledge of the role of HDL in vascular health and disease.
Keywords: biological signal transduction, cell migration, cholesterol, high density lipoprotein, nitric oxide synthase, scavenger receptor, vascular endothelium, atherosclerosis, guanosinetriphosphatase, intracellular transport, membrane protein, mitogen activated protein kinase, CHO cell, laboratory mouse, tissue /cell culture
Project start date: 1997-07-15
Project end date: 2009-01-31
5R01HL058888-09 (2007): $332812
5R01HL058888-08 (2006): $342752
5R01HL058888-07 (2005): $351000
2R01HL058888-06A2 (2004): $351000
CRP, ENOS AND ENDOTHELIAL DYSFUNCTION
Philip W Shaul
University Of Texas Sw Med Ctr/dallas, Dallas, Tx 75390-9105
Grant 5R01HL075473-06 from National Heart, Lung, And Blood Institute
Abstract: The circulating level of C-reactive protein (CRP) is a strong predictor of endothelial dysfunction and hypertension in humans. We have demonstrated in transgenic CRP mice (TG-CRP) that CRP causes endothelial NO synthase (eNOS) downregulation, endothelial dysfunction and hypertension. Cell culture studies indicate that CRP also impairs insulin signaling to eNOS, and in preliminary studies we have discovered that TG-CRP mice are insulin resistant. Further work in double transgenic mice indicates that CRP-induced hypertension is mediated by the inhibitory IgG Fc? receptor (Fc?R), Fc?RIIB, and we now have direct evidence of endothelial cell expression of Fc?RIIB protein. Considering the important link between endothelial dysfunction and insulin resistance in numerous inflammatory conditions, the overall hypothesis to be tested in the proposed research is that CRP actions via endothelial Fc?RIIB cause both hypertension and insulin resistance. Aim 1 is to determine the role of endothelial Fc?RIIB in CRP-induced hypertension. The receptor will be deleted in endothelium by crossing floxed Fc?RIIB mice (Fc?RIIBflox/flox) with mice expressing tamoxifen-inducible Cre-recombinase under the regulation of the vascular endothelial cadherin promoter (VECad-Cre-ERT2). Fc?RIIBflox/floxVE-Cad-Cre-ERT2 will be crossed with TG-CRP mice, and BP will be measured by radiotelemetry. Related changes in vascular gene expression will also be assessed, and the novel mechanisms by which Fc?RIIB potentially modulates endothelial gene expression will be investigated. Aim 2 is to determine the basis for CRP-induced insulin resistance. We will perform glucose- and insulin-tolerance tests, hyperinsulinemic-euglycemic clamps evaluating glucose infusion rate, glucose disposal rate and endogenous glucose output, and skeletal muscle glucose uptake studies in TG-CRP mice and in crosses of TG-CRP and Fc?RIIB-/- mice. Body weight and composition, food intake, and energy expenditure will also be evaluated. Aim 3 is to determine the mechanistic linkage between the endothelial actions of CRP and CRP-induced insulin resistance. Insulin-induced skeletal muscle bloodflow will be evaluated in TG-CRP mice, and euglycemic clamps will be done in the Fc?RIIBflox/floxVE-Cad-Cre-ERT2TG- CRP mice and controls from Aim 1. We will also determine if molsidomine or Sildenafil treatment to overcome diminished NO bioavailability in TG-CRP mice normalizes insulin sensitivity. By meeting these aims, we will increase our basic understanding of how CRP participates in the pathogenesis of hypertension and insulin resistance. The new knowledge gained about endothelial Fc?RIIB will also be valuable in understanding why immune complex-mediated diseases such as rheumatoid arthritis and lupus are complicated by both cardiovascular disease and insulin resistance. We anticipate that the proposed work will lead to new therapies capable of simultaneously optimizing endothelial cell function, blood pressure and glucose metabolism in a variety of clinical conditions. Elevations in the circulating level of C-reactive protein (CRP) are a strong predictor of the development of hypertension and insulin resistance. The proposed research program will determine how CRP and its cell surface receptors cause hypertension and insulin resistance
Keywords: (Z)-2-[4(1, 2-diphenyl-1-butenyl)-phenoxyl]-N, N-dimethylethanamine; 1-p-beta-dimethylamino-ethoxyphenyl-trans-1, 2-diphenylbut-1-ene; 7B4 Antigen; 7B4 protein; ANG-(1-8)Octapeptide; Address; AngII; Angiotensin II; Angiotensin Receptor; Angiotensin-(1-8) Octapeptide; Antigen-Antibody Complex; Atrophic Arthritis; Attenuated; Bioavailability; Biologic Availability; Biological; Biological Availability; Blood Pressure; Blood Pressure, High; Blood Vessels; Blood flow; Body Composition; Body Weight; C-reactive protein; CD144 Antigen; CDH5; Cardiovascular Diseases; Cell Culture Techniques; Cell Function; Cell Process; Cell Surface Receptors; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Chronic; Clamping, Glucose; Clinical; Conscious; Consciousness; D-Glucose; Development; Dextrose; Disease; Disorder; Down-Regulation; Down-Regulation (Physiology); Downregulation; Dysfunction; EC 1.14.13.39; EDRF Synthase; Eating; Endothelial Cells; Endothelium; Endothelium-Derived Growth Factor Synthase; Energy Expenditure; Energy Metabolism; Ethanamine, 2-(4-(1, 2-diphenyl-1-butenyl)phenoxy)-N, N-dimethyl-, (Z)-; Euglycaemic Clamp; Euglycemic Clamping; Fc Receptor; FcR; Food Intake; Functional disorder; Gamma Globulin, 7S; Gene Expression; Glucose; Glucose Clamp; Glucose Intolerance; Glucose tolerance test; Guanylyl Cyclase-Activating Factor Synthase; Health; Human; Human, General; Humulin R; Hypertension; INFLM; IPGTT; IVGTT; IgG; Immune Complex; Immunoglobulin G; Inflammation; Inflammatory; Inflammatory Arthritis; Infusion; Infusion procedures; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin Resistance; Insulin, Regular; Knockout Mice; Knowledge; L-Arginine, NADPH[{..}]oxygen oxidoreductase (nitric-oxide-forming); Lead; Link; Longitudinal Studies; Lupus; Mammals, Mice; Man (Taxonomy); Man, Modern; Measurement; Measures; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Metabolic; Metabolic Diseases; Metabolic Disorder; Methods and Techniques; Methods, Other; Mice; Mice, Knock-out; Mice, Knockout; Molecular; Molsidomine; Morsydomine; Murine; Mus; Muscle, Skeletal; Muscle, Voluntary; NADPH-Diaphorase; NO Synthase; Nitric Oxide Synthase; Nitric-Oxide Synthetase; Novolin R; Null Mouse; Pathogenesis; Pb element; Phenotype; Physiologic Availability; Physiopathology; Process; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein C; Proteins; Proteins, specific or class, C-reactive; Receptor Protein; Regulation; Research; Rheumatoid Arthritis; Risk Factors; Role; Skeletal Muscle Tissue; Skeletal muscle structure; Subcellular Process; Syd imine, N-(ethoxycarbonyl)-3-(4-morpholinyl)-; TAM; Tamoxifen; Techniques; Testing; Thesaurismosis; Transgenic Mice; Transgenic Organisms; VE-Cadherin; Vascular Endothelial Cadherin; Vascular Endothelial Cadherin 1; Vascular Hypertensive Disease; Vascular Hypertensive Disorder; Work; antibody receptor; base; bioavailability of drug; cadherin 5; cardiovascular disorder; disease/disorder; experiment; experimental research; experimental study; gene product; glucose disposal; glucose metabolism; glucose output; glucose uptake; heavy metal Pb; heavy metal lead; hyperpiesia; hyperpiesis; hypertensive disease; in vivo; insulin resistant; insulin sensitivity; insulin signaling; insulin stimulated glucose disposal; insulin tolerance; long-term study; meetings; metabolism disorder; novel; pathophysiology; programs; public health relevance; receptor; receptor expression; recombinase; research study; sildenafil; social role; transgenic; vascular
Relevance: Elevations in the circulating level of C-reactive protein (CRP) are a strong predictor of the development of hypertension and insulin resistance. The proposed research program will determine how CRP and its cell surface receptors cause hypertension and insulin resistance
Project start date: 2003-12-23
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
5R01HL075473-06 (2010): $543710
2R01HL075473-05A1 (2009): $543710
5R01HL075473-04 (2007): $332812
5R01HL075473-03 (2006): $342752
5R01HL075473-02 (2005): $351000
Sponsored Links Excellgen http://Excellgen.com
1R01HL075473-01 (2004): $375771
REGULATION OF NITRIC OXIDE SYNTHASE IN DEVELOPING LUNG
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5R01HD030276-13 from National Institute Of Child Health And Human Development IRG: ZRG1
Abstract: Scanned from s description) Nitric oxide (NO), produced by endothelial NO synthase (eNOS), plays a key role in pulmonary vasodilation at birth and in the genesis of persistent pulmonary hypertension of the newborn (PPHN). Studies in fetal sheep have shown that the hormone estrogen, which rises markedly in the fetal blood during parturition, causes pulmonary vasodilation due to nongenomic effects on NO production. Estrogen also reverses the vascular abnormalities in a lamb model of PPHN. We have shown in ovine fetal pulmonary artery endothelial cells (PAEC) that estradiol (E2) cause eNOS activation through novel nongenomic actions of estrogen receptor (ER) a and calcium-mediated signaling. The OBJECTIVE of this proposal is to determine the molecular mechanisms by which E2 causes nongenomic eNOS activation in fetal PAEC. Four Aims will be addressed in primary and immortalized PAEC, and transfected COS-7 cells. We have new evidence that a subset of ERa is localized to PAEC plasma membrane (PM) where they modulate eNOS activity. AIM 1 is to determine the mechanisms underlying ERa PM function, testing the hypotheses that E2 causes internalization of PM ERa, that processes needed for genomic ERa function are not required, and that specific ERa domains are involved in PM function. We also have preliminary evidence that fetal PAEC express ERb, and that eNOS stimulation by E2 Is greater after ERb overexpression. AIM 2 is to determine the role of ERb in eNOS activation, testing the hypotheses that a subset of PM ERb is capable of nongenomic function, and that neither ERa nor dimerization are needed for PM ERb action. In further initial studies, E2 activation of eNOS was blocked by pertussis toxin. AIM 3 is to determine the role of G proteins in eNOS activation by E2, testing the hypotheses that G proteins are critically involved, that downstream signaling is mediated by Gai, and that E2 causes ERa-Gai interaction on PM. Finally, in recent studies we have detected ERa protein in PAEC caveolae, which compartmentalize signaling molecules on the PM including eNOS, and have found that E2 causes potent, ER-dependent eNOS activation in isolated caveolae membranes. AIM 4 is to characterize an E2-eNOS signaling module in PAEC caveolae, testing the hypotheses that known signaling proteins are coupled in caveolae, and that a caveolae-associated calcium pool is released by E2 to activate eNOS. Unknown components will be identified by ERa immunoprecipitation and microsequencing and yeast two-hybrid screening. These studies will fill major gaps in our understanding of NO production in the developing lung as well as our knowledge of nongenomic E2 actions in the coronary and uterine circulation and in nonvascular cells. Ultimately, we may be able to take greater therapeutic advantage of the effects of E2 on the pulmonary and also coronary and uterine circulations, thereby optimizing the vascular health of both the fetus and the mother.
Keywords: embryo /fetus, enzyme activity, estradiol, hormone regulation /control mechanism, lung, molecular dynamics, nitric oxide synthase, pulmonary hypertension, vascular endothelium, G protein, biological signal transduction, calcium, cell membrane, estrogen receptor, gene expression, growth /development, protein structure function, respiratory enzyme, immunoprecipitation, nucleic acid sequence, polymerase chain reaction, sheep, tissue /cell culture, yeast two hybrid system
Project start date: 1993-04-01
Project end date: 2006-03-31
5R01HD030276-13 (2005): $315900
5R01HD030276-12 (2004): $315900
5R01HD030276-11 (2003): $339611
5R01HD030276-10 (2002): $339890
2R01HD030276-09 (2001): $339206
OXYSTEROLS, ESTROGEN, RECEPTORS ANATGONISM AND VASCULAR DISEASE
Philip W Shaul, Professor Of Pediatrics
University Of Texas Sw Med Ctr/dallas, Dallas, Tx 75390-9105
Grant 5R01HL087564-04 from National Heart, Lung, And Blood Institute
Abstract: Recent randomized clinical trials suggest that estrogen therapy may not provide protection from cardiovascular disease in menopausal and postmenopausal women. There is strong evidence that treatment failure is more likely if it is initiated after atherosclerosis is present, suggesting that antagonistic mechanisms may be operative under those conditions. We have discovered that the oxysterol 27- hydroxycholesterol (27HC), which accumulates in atherosclerotic lesions, is a potent antagonist of estrogen receptor a (ERa) and ER¿ function, and that it attenuates estradiol (E2)-induced upregulation of endothelial NO synthase (eNOS). We have also found that female Cyp7b1-/- mice incapable of metabolizing 27HC have impaired E2-induced reendothelialization. The OBJECTIVE of the proposed research is to determine if 27HC is an endogenous antagonist of ER action in endothelium, thereby diminishing estrogen-related cardiovascular protection. Aim 1 is to determine if 27HC blunts E2-related protection from neointimal formation after vascular injury and from atherosclerosis. Studies of protection by endogenous E2 (manipulated by anastrazole treatment) and exogenous E2 will be done in female Cyp7b1+/+ versus Cyp7b1-/- mice undergoing a cuff model of vascular injury, and in females derived from crosses of Cyp7b1-/- and apoE-/- or LDL receptor-/- mice. Aim 2 is to determine if 27HC modifies membrane-associated ER function. The impact of 27HC on eNOS and upstream kinase activation by ER subtype selective agonists and a new estrogen dendrimer conjugate (EDC) directed at membrane ER will be determined in cultured endothelium. The impact of 27HC on endothelial cell phenotypes dictated by membrane ER, including the stimulation of migration and proliferation and the antagonism of monocyte adhesion, will also be evaluated. Aim 3 is to identify nuclear targets of ER function in endothelium which are modified by 27HC. Using RT-PCR and focused microarrays, changes in expression of known and unknown ER targets will be determined in endothelium sorted from Tie2-GFP;Cyp7b+/+ versus Tie2-GFP;Cyp7b-/- female mice after altering endogenous or exogenous E2 status. 27HC target genes downstream of membrane ER in endothelium will also be identified in vitro using EDC as agonist. The roles of 27HC target genes will be tested in gain- and loss-of-function studies of cultured endothelial cell phenotypes. Aim 4 is to elucidate the pharmacology of 27HC as a unique SERM. Whereas 27HC antagonizes ER function in vascular and MCF-7 cells, it enhances ER function in Hep G2 and Caco-2 cells. To determine the basis for cell specificity, peptide phage display will be employed to identify peptide binding to ERa or ER¿ which is uniquely modulated by 27HC versus E2 and known SERMs. The impact of identified peptides on inhibitory versus stimulatory effects of 27HC on ERE-luciferase, and also on 27HC actions via membrane ERa and ER¿ will be evaluated. By investigating a novel mechanism of endogenous ER antagonism, the proposed research will increase our fundamental understanding of the processes underlying estrogen treatment failure
Keywords: 2, 2`-(5-(1H-1, 2, 4-triazol-1-ylmethyl)-1, 3-phenylene)bis(2-methylpropionitrile); 2, 2`-[5-(1H-1, 2, 4-Triazol-1-ylmethyl)-1, 3-phenylene]di(2-methylpropionitrile); 27-hydroxycholesterol; 5-cholestene-3 beta, 27-diol; APOE [{C0003595}]; Adhesions; Affect; Agonist; Alpha, alpha, alpha`, alpha`-tetramethyl-5-(1H-1, 2, 4-triazol-1-ylmethyl)-1, 3-benzenediacetonitrile; Anastrozole (Arimidex); Androstenedione Aromatase Inhibitor; Aorta; Apo-E; ApoE; Apolipoprotein E; Aquadiol; Arimidex; Aromatase Inhibitors; Arterial Fatty Streak; Arteries; Astra brand of anastrozole; AstraZeneca brand of anastrozole; Atheroma; Atheromatous; Atheromatous degeneration; Atheromatous plaque; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Attenuated; Binding; Binding (Molecular Function); Blood Vessels; Blood monocyte; CNOS; Caco-2 Cells; Carcinoma of the Liver Cells; Cardiovascular; Cardiovascular Body System; Cardiovascular Diseases; Cardiovascular system; Cardiovascular system (all sites); Carotid Arteries; Cell Communication and Signaling; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Cell Signaling; Cell membrane; Cell model; Cells; Cellular model; Change of Life, Female; Conestron; Conjugated Estrogen - Premarin; Constitutive NOS; Corlutina; Corluvite; Cultured Cells; Cyclogest; Cytoplasmic Membrane; Dimenformon; Diogyn; Diogynets; EC 1.14.13.39; ECNOS; EDRF Synthase; ENOS; Electric Injuries; Endothelial Cells; Endothelial NOS; Endothelial Nitric Oxide Synthase; Endothelial Nitric Oxide Synthase 3; Endothelium; Endothelium-Derived Growth Factor Synthase; Estra-1, 3, 5(10)-triene-3, 17-diol (17beta)-; Estrace; Estradiol; Estradiol-17 beta; Estradiol-17beta; Estraldine; Estrogen Receptors; Estrogen Replacement Therapy; Estrogen Synthase Inhibitor; Estrogen Synthetase Inhibitor; Estrogen Therapy; Estrogenic Agents; Estrogenic Compounds; Estrogenic Hormones, Conjugated; Estrogenic Substances, Conjugated; Estrogens; Estrogens, Conjugated; Estrogens, Conjugated (USP); FLR; Failure (biologic function); Female; Femest; GFP; Gene Expression; Gene Targeting; Genetically Engineered Mouse; Gestagenic Agents; Gestagens; Gestiron; Gestone; Green Fluorescent Proteins; Guanylyl Cyclase-Activating Factor Synthase; HCC; Heart; Hepatocellular Carcinoma; Hepatocellular cancer; Hepatocyte Nitric Oxide Synthase; Hepatoma; Hormone Replacement Rx; Hormone replacement therapy; Hyperplasia; Hyperplastic; INOS; Immunologic, Luciferase; In Vitro; Incubated; Inducible Nitric Oxide Synthase; Injury; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Investigators; Knowledge; L-Arginine, NADPH[{..}]oxygen oxidoreductase (nitric-oxide-forming); Lead; Lipo-Lutin; Lipoprotein LDL Receptors; Low Density Lipoprotein Receptor; Luciferases; Luteohormone; Lutocyclin; Lutocylin M; Lutogyl; Lutromone; MCF-7; MCF-7 Cell; MCF7; MCF7 cell; Macrophage Nitric Oxide Synthase; Mammals, Mice; Marrow monocyte; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Membrane; Menopause; Mice; Mice, Transgenic; Modeling; Molecular Interaction; Murine; Mus; NADPH-Diaphorase; NO Synthase; NOS Type II; NOS Type III; NOS2; NOS2A; NOS2A protein, human; NOS3; NOS3 protein, human; NOSIII; Nitric Oxide Synthase; Nitric Oxide Synthase 2A; Nitric Oxide Synthase 3; Nitric-Oxide Synthetase; Nuclear; Nuclear Envelope; Nuclear Membrane; Nuclear Receptors; Organ System, Cardiovascular; Ovocyclin; Ovocylin; Pb element; Peptides; Phage Display; Pharmacology; Phenotype; Plasma Membrane; Post-Menopause; Post-menopausal Period; Postmenopausal Period; Postmenopause; Premarin; Prevention; Primary carcinoma of the liver cells; Process; Production; Progestagenic Agents; Progestasert; Progestational Agents; Progestational Compounds; Progestational Hormones; Progesterone Agents; Progestins; Progestogel; Progestogens; Progestol; Progeston; Programs (PT); Programs [Publication Type]; Progynon; Prolidon; Proluton; RT-PCR; RTPCR; Randomized Clinical Trials; Randomized Controlled Trials; Receptor Protein; Receptors, LDL; Research; Research Personnel; Researchers; Reverse Transcriptase Polymerase Chain Reaction; Role; SERMs; Selective Estrogen Receptor Modulators; Shock from electric current; Signal Transduction; Signal Transduction Systems; Signaling; Sodestrin; Sorting - Cell Movement; Specificity; Streaks, Arterial Fatty; Syngesterone; Targetings, Gene; Testing; Therapeutic Estradiol; Therapeutic Estrogen; Therapeutic Progestin; Time; Transgenic Mice; Treatment Failure; Trials, Randomized Clinical; Up-Regulation; Up-Regulation (Physiology); Upregulation; Utrogestan; Vascular Diseases; Vascular Disorder; Vascular remodeling; Vascular, Heart; Woman; Work; Zeneca brand of anastrozole; anastrazole; anastrozole; atheromatosis; atherosclerosis plaque; atherosclerotic lesions; atherosclerotic plaque; atherosclerotic vascular disease; attenuation; base; biological signal transduction; blood vessel disorder; cardiovascular disorder; cell sorting; cholest-5-ene-3 beta, 27-diol; circulatory system; colon cancer cell line; eNOS enzyme; electrical injury; endothelial constitutive nitric oxide synthase; failure; heavy metal Pb; heavy metal lead; human NOS2A protein; human NOS3 protein; iNOS enzyme; in vivo; interventional strategy; loss of function; membrane structure; menopausal; migration; monocyte; new therapeutics; next generation therapeutics; nitric oxide synthase, Type II; novel; novel therapeutics; plasmalemma; post-menopausal; postmenopausal; prevent; preventing; programs; randomized controlled study; receptor; response; reverse transcriptase PCR; social role; sorting; transcription factor; upstream kinase; vascular; vulnerable plaque
Project start date: 2006-08-01
Project end date: 2010-05-31
Budget start date: 1-JUN-2009
Budget end date: 31-MAY-2010
5R01HL087564-04 (2009): $381118
5R01HL087564-02 (2007): $381118
1R01HL087564-01 (2006): $392500
REGULATION OF CYCLOOXGENASE GENES IN DEVELOPING LUNG
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5R01HL053546-09 from National Heart, Lung, And Blood Institute IRG: ZRG2
Abstract: Prostacyclin, or PGI/2, is a critical mediator of vasodilation in the developing lung produced in endothelium by cyclooxygenase (COX). We have shown that there is a 30-fold maturational rise in PGI/2 in ovine fetal and newborn intrapulmonary arteries due to increasing expression of the COX-1 isoform. The increase in COX-1 in the fetus may be induced by rising plasma estrogen (E/2) levels because we have shown that 48h exposure to E/2 up-regulates COX-1 in ovine fetal pulmonary artery endothelial cells (PAEC). Preliminary studies indicated that E/2 also causes rapid PGI/2 activation (15 min), and that an estrogen receptor (ER) subpopulation is associated with the plasma membrane. The OBJECTIVE of this proposal is to determine the molecular mechanisms by which prolonged E/2 exposure up- regulates COX-1 expression and by which E/2 also acutely activates PGI/2 in cultured ovine fetal PAEC. Aim 1 is to determine the mechanisms underlying E/2-induced COX-1 up-regulation in transient transfection studies focused on the roles of the ER subtypes, ERalpha and ERbeta, and tyrosine kinase (TK)/MAP kinase (MAPK) signaling. E/2-induced transcriptional activation will also be studied using COX-1 promoter- reporter gene constructs. Aim 2 is to determine the basis for acute PGI/2 activation by E/2, elucidating the step in the PGI/2 synthetic cascade hat is involved. The roles of ERalpha and ERbeta and TK/MAPK signalling in the acute response will also be discerned by pharmacologic approaches and cell transfection. Aim 3 is to identify the ER subtypes expressed in PAEC and to further delineate their localization in studies of plasma membrane caveolae and other cell fractions using immunodetection and radio-ligand binding. Results will be confirmed by immunofluorescence and immunoelectron microscopy in intact cells. Cell surface ER function will also be evaluated in studies of PGI/2 stimulation by immobilized E/2 ligand. Aim 4 is to determine the mechanisms regulating PAEC ER expression, following up on the observation that ERalpha abundance is potently up-regulated by E/2. The molecular basis for this process will be determined, including studies of transcriptional events using ERalpha promoter activity assays. The effects of E/2 on ERbeta expression will also be examined. These studies will provide fundamental new information about the regulation of pulmonary PGI/2 synthesis in the perinatal period, and about the roles of E/2 and ERs in vascular biology.
Keywords: developmental genetics, enzyme induction /repression, estradiol, estrogen receptor, lung, perinatal, prostacyclin, prostaglandin endoperoxide synthase, pulmonary artery, biological signal transduction, eicosanoid metabolism, genetic promoter element, genetic regulation, genetic transcription, hormone regulation /control mechanism, mitogen activated protein kinase, protein isoform, protein tyrosine kinase, receptor expression, vascular endothelium, vasodilation, embryo /fetus cell culture, immunofluorescence technique, polymerase chain reaction, sheep
Project start date: 1994-12-01
Project end date: 2004-11-30
5R01HL053546-09 (2003): $303701
Sponsored Links Excellgen http://Excellgen.com
5R01HL053546-08 (2002): $296877
5R01HL053546-07 (2001): $290257
5R01HL053546-06 (2000): $282816
2R01HL053546-05 (1999): $274577
REGULATION OF CYCLOOXYGENASE GENES IN DEVELOPING LUNG
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5R01HL053546-04 from National Heart, Lung, And Blood Institute IRG: HED
Abstract: Prostacyclin (PGI2) and prostaglandin E2 (PGE2) are critical mediators of vasomotor tone, airway reactivity, and surfactant synthesis in the developing lung. They are produced primarily in vascular cells by cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. We have demonstrated O2 modulation of PGI2 and PGE2 synthesis in ovine fetal and newborn intrapulmonary arteries (PA), with hypoxia causing decreased synthesis in fetal PA and increased synthesis in newborn PA. We have also demonstrated a 32-fold maturational rise in PGI2 synthesis. Both the O2- and age-associated alterations in synthesis are related to changes in COX protein expression. The OVERALL OBJECTIVE of this proposal is to determine the molecular mechanisms by which COX gene expression in pulmonary vascular cells is modified by changes in O2 and by maturation, focusing the latter studies on modulation by hormonal factors and shear stress. Aim No. 1 is to determine the effects of O2 on COX expression. Fetal and newborn ovine PA endothelial cells (PAEC) and vascular smooth muscle (VSM) cells will be subjected to varying O2 and changes in PGI2 and PGE2 synthesis, and COX-1 and COX-2 protein and mRNA levels will be assessed. COX protein synthesis and degradation, and COX gene transcription and mRNA stability will also be evaluated. We hypothesize that hypoxia downregulates COX expression in fetal cells and upregulates expression in newborn cells. Aim No. 2 is to determine the effects of estrogen and glucocorticoids on COX expression in fetal PAEC and VSM, using a similar approach. Estrogen enhances and glucocorticoids attenuate PGI2 synthesis in other cell types, and estrogen levels rise in the fetus and cortisol levels fall postnatally as PA COX protein increases. As such, we hypothesize that COX expression is upregulated by estrogen and downregulated by glucocorticoids. Aim No. 3 is to determine if COX expression is upregulated by prolonged shear stress, which increases PG2 synthesis in nonpulmonary endothelium. Fetal PAEC will be subjected to 24 h of shear, and changes in COX function, protein and mRNA expression will be assessed. Aim No. 4 is to test the hypothesis that there is cell- specific developmental regulation of COX expression. Immunohistochemical analyses and in situ hybridization for COX-1 and COX-2 will be performed in fetal, newborn and adult ovine lung. By revealing the molecular processes modulating PGI2 and PGE2 synthesis in the developing lung, these studies will increase our understanding of the mechanisms underlying successful pulmonary vascular, airway and alveolar function in the neonatal period.
Keywords: gene expression, growth /development, lung, oxygen tension, oxygenase, vascular endothelium, embryo /fetus cell /tissue, hormone regulation /control mechanism, messenger RNA, newborn animal, physiologic stressor, prostacyclin, prostaglandin E, protein biosynthesis, protein degradation, vascular smooth muscle, SDS polyacrylamide gel electrophoresis, immunocytochemistry, in situ hybridization, molecular cloning, northern blotting, polymerase chain reaction, radioimmunoassay, sheep, western blotting
Project start date: 1994-12-01
Project end date: 1998-11-30
5R01HL053546-04 (1998): $230838
5R01HL053546-03 (1997): $209031
Nitric Oxide In Lung Development And CLD
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 2U01HL063399-05 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Nitric oxide (NO) is critically involved in numerous functions in the developing lung. NO is produced by neuronal NO synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have shown that all three isoforms are upregulated in the third trimester in fetal baboon lung. Chronic Lung Disease (CLD) is a condition which disrupts preterm human lung development, resulting in airway, parenchymal and pulmonary vascular dysfunction. We have shown in the baboon model of CLD that lung nNOS and eNOS are markedly decreased, that lung NO production remains low during the early course, and that inhaled NO gas (iNO) improves pulmonary hemodynamics, compliance and resistance. iNO also partially normalizes surfactant function, it stimulates lung growth, and it prevents certain structural features of CLD. Using physiologic, biochemical and histologic/morphologic readouts, the overall objective of this proposal is to determine if interventions which upregulate endogenous NO production, or which replace NO in a manner favoring physiologic NO metabolism, result in greater prevention of CLD. Studies will be done in preterm baboons born at 125d gestation (term = 185d) and ventilated for 14d. Aim 1 is to determine the effects of postnatal estradiol (E2) treatment on lung NO production, and on the development of CLD. E2 upregulates nNOS and eNOS expression and activity in some paradigms, and with preterm birth there is withdrawal from placentally-derived E2. Aim 2 is to determine if there is greater impact on the genesis of CLD with iNO plus glutathione (GSH), or with O-nitrosoethanol (ENO), versus iNO alone. Whereas endogenous NO forms biologically active S-nitrosothiols (SNO), iNO gas yields toxic higher oxides of nitrogen (NOX) and peroxynitrite. GSH, which is deficient in prematurity, is the primary substrate for lung SNO. In contrast to iNO, ENO preferably forms SNO and generates less NOX and peroxynitrite in mature models tested thus far. Aim 3 is to delineate the changes in pulmonary and systemic NO metabolism in CLD and in the intervention groups. In initial work, blood NOX rose while SNO fell postnatally in CLD, and iNO raised NOX further with scant impact on SNO. It is hypothesized that E2, iNO + GSH, or ENO will increase SNO and cause little change in NOX and peroxynitrite. These studies will increase our knowledge of the role of NO in CLD and also test three novel, mechanistically-based therapies.
Keywords: bronchopulmonary dysplasia, enzyme induction /repression, hormone therapy, isozyme, lung development, nitric oxide synthase, nitrogen metabolism, nonhuman therapy evaluation, cell type, cooperative study, disease /disorder model, enzyme activity, estradiol, glutathione, hemodynamics, messenger RNA, nitric oxide, nitroso compound, pathologic process, respiratory airway pressure, respiratory circulation, respiratory epithelium, baboon, echocardiography, tissue /cell culture
Project start date: 1999-09-01
Project end date: 2007-08-31
2U01HL063399-05 (2003): $550750
Estrogen Modulation Of Endothelial Cell Phenotype
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5R01HD030276-15 from National Institute Of Child Health And Human Development IRG: PN
Abstract: New blood vessel development in the ovary, uterus and placenta, which is critical to ultimate pregnancy outcome, is regulated by estradiol-17b (E2) acting through estrogen receptor a (ERa). The underlying processes include direct actions of E2 on endothelial cells to promote their growth and migration. These mechanisms also mediate cardiovascular protection by E2 remote from pregnancy. However, the basis for direct regulation of endothelial cell phenotype by E2 and ERa is poorly understood. We have shown that E2 stimulates endothelial NO synthase (eNOS) via the activation of plasma membrane-associated ERa, and that these receptors are G protein-coupled. In preliminary studies with a new estrogen-dendrimer conjugate (EDC), we have observed that G-protein-mediated membrane ERa activation of Src and eNOS underlies E2-induced endothelial cell growth and migration. The OBJECTIVE of the proposed research is to determine if estrogen regulates endothelial cell phenotype via direct coupling of membrane ERa to G proteins. In recent initial experiments we have identified dynamic protein-protein interactions between recombinant ERa and Gai and Gby. Aim 1 is to determine the molecular basis of ERa-G protein interaction and Src activation. The domains of ERa mediating G protein interactions will be determined, and their roles in signaling to Src will be tested. The ability of peptide sequences derived from the ERa interaction domains to inhibit signaling to Src will also be assessed. Aim 2 is to determine if membrane ERa coupling to G proteins regulates endothelial cell growth and migration in vitro. Using EDC as a membrane-directed ligand, the requirement for ERa will be determined by gain- and loss-of-function strategies, and additional interventions will test the requirements for Gai and Gby activation. Involvement of ERa-Gai and ERa-Gby interactions will be tested using interfering peptides identified in Aim 1. Aim 3 is to determine if membrane ERa coupling to G proteins regulates endothelial cell migration in vivo. In a nonpregnant female mouse model of carotid artery, reendothelialization, responses to E2 versus EDC will be evaluated in wild-type versus ERa mice. The requirement for Ga and Gby activation will be tested by local blockade of their functions in the carotid artery, and the requirement for ER-Gai and ER-Gby interactions will be assessed by endothelial delivery of interfering peptides. Aim 4 is to identify nuclear targets of membrane ERa-G protein activation in endothelium in vivo. Using focused microarrays and RT-PCR, changes in gene expression will be determined in endothelium sorted from Tie2-GFP;ERa+/+versus Tie2-GFP;Era mice treated with E2 versus EDC. Genes downstream of G protein activation will be identified by pertussis toxin treatment, and the requirement for ER-Gai and ER-Gby interactions will be tested by endothelial delivery of interfering peptides. The roles of identified target genes will be tested in gain- and loss-of-function studies of cultured endothelial cell phenotypes. By investigating the novel, most proximal processes by which E2 and ERa regulate endothelial cell phenotype, the proposed research will increase our fundamental understanding of how vascular actions of estrogen promote the reproductive and nonreproductive health of women.
Keywords: G protein, cell, estrogen, membrane, phenotype, adrenergic receptor, angiogenesis, aorta, artery, birth, blood vessel, carotid artery, caveola, cell migration, cell sorting, cell surface receptor, culture, estradiol, estrogen receptor, female, gas, gene, gene expression, gene targeting, genetically modified animal, green fluorescent protein, growth factor, health, heart, injury, laboratory mouse, lead, ligand, lipid raft, model, mutant, nitric oxide synthase, organ, ovary, peptide, pertussis, pertussis toxin, phosphorylation, placenta, plasma, posttranslational modification, pregnancy, protein protein interaction, protein sequence, receptor, recombinant protein, role, steroid hormone, tissue, tissue /cell culture, toxin, transfection, uterus
Project start date: 1993-04-01
Project end date: 2011-02-28
5R01HD030276-15 (2007): $245821
2R01HD030276-14 (2006): $253163
OXYSTEROLS, ESTROGEN RECEPTOR ANTAGONISM, AND VASCULAR DISEASE
Philip W Shaul, Professor Of Pediatrics
University Of Texas Sw Med Ctr/dallas, Dallas, Tx 75390-9105
Grant 2R01HL087564-05 from National Heart, Lung, And Blood Institute
Abstract: Estrogen has the potential to provide potent cardiovascular protection. However, there are mechanisms that modify the vascular actions of estrogen, particularly if disease is already present. In the current funding period we identified 27-hydroxycholesterol (27HC), which is elevated with hypercholesterolemia, as the first endogenous selective estrogen receptor modulator (SERM). We showed that 27HC antagonizes estrogen receptor (ER) function in endothelium and vascular smooth muscle (VSM), and that it causes impaired E2- induced reendothelialization in mice. Recent preliminary studies in mice null for Cyp7b1, which metabolizes 27HC, suggest that 27HC may also promote atherogenesis, attenuate E2-induced protection from neointima formation, and impair E2 regulation of body weight. The latter finding is also likely indicative of impaired glucose homeostasis. Our Overall Objective is to now determine how 27HC contributes directly and indirectly to vascular disease pathogenesis, and how 27HC levels and actions can be modified to lessen vascular disease. Aim 1 is to determine how administered 27HC impacts vascular disease pathogenesis. 27HC-induced changes in atherosclerotic lesion abundance and characteristics will be evaluated in apoE+/+ vs. apoE-/- male and ovariectomized placebo vs. E2-treated female mice. We will also determine how 27HC impacts E2-mediated protection from intimal hyperplasia, and effects of 27HC on E2 regulation of macrophage and VSM cell function will be investigated in culture. Aim 2 is to determine how 27HC impacts body weight regulation and glucose homeostasis, which secondarily influence cardiovascular health. Food intake, energy expenditure and adiposity will be quantified in control vs. 27HC-treated ovariectomized placebo vs. E2-treated female C57BL/6 mice placed on control vs. Western diet. Glucose tolerance and insulin sensitivity and the mechanisms that determine them will be evaluated. CNS vs. peripheral actions of 27HC on ER regulation of metabolism will be distinguished by intraventricular delivery of 27HC. Aim 3 is to determine how prevention of the elevation in 27HC that occurs with hypercholesterolemia impacts the protective actions of E2 on atherosclerosis and metabolism. Serum 27HC will be lowered by hepatic overexpression of Cyp7b1 by IV injection of adenovirus encoding the enzyme and also by creation of a liver-specific Cyp7b1 transgenic mouse. Aim 4 is to develop new strategies to attenuate serum 27HC levels and 27HC actions in target cells. Using a novel extranuclear SERM we developed for in vivo use in mice, the role of non-nuclear ER signaling in hepatic Cyp7b1 upregulation by E2 suggested in cell culture studies will be delineated in vivo. Using peptides identified by phage display to uniquely bind to 27HC-liganded ER, approaches for the selective blockade of 27HC actions mediated by nuclear as well as non-nuclear ER will be pursued in cultured cells. By accomplishing these aims, we will increase our basic understanding of the biology of the only known endogenous SERM in the context of vascular health and disease. 27-hydroxycholesterol is a metabolite of cholesterol that is elevated in the blood when circulating cholesterol levels are high, and it accumulates with cholesterol in diseased arteries. We have discovered that 27-hydroxycholesterol modifies the actions of estrogen in numerous cell types and organs. The proposed research program will determine how 27-hydroxycholesterol, through its impact on estrogen action, contributes to the development of blood vessel disease and to impaired body weight and glucose regulation
Keywords: 27-hydroxycholesterol; 5-cholestene-3 beta, 27-diol; APOE [{C0003595}]; Adenoviridae; Adenoviruses; Adipose tissue; Affect; Apo-E; ApoE; Apolipoprotein E; Aquadiol; Arterial Fatty Streak; Arteries; Atheroma; Atheromatous; Atheromatous degeneration; Atheromatous plaque; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Attenuated; Binding; Binding (Molecular Function); Biology; Blood; Blood Serum; Blood Vessels; Body Weight; C57BL/6 Mouse; Cardiovascular; Cardiovascular Body System; Cardiovascular Diseases; Cardiovascular system; Cardiovascular system (all sites); Cell Culture Techniques; Cell Function; Cell Process; Cell physiology; Cells; Cellular Expansion; Cellular Function; Cellular Growth; Cellular Physiology; Cellular Process; Characteristics; Cholest-5-en-3-ol (3beta)-; Cholesterol; Clamping, Glucose; Cultured Cells; D-Glucose; Development; Dextrose; Diet; Dimenformon; Diogyn; Diogynets; Disease; Disorder; Eating; Energy Expenditure; Energy Metabolism; Enzymes; Estra-1, 3, 5(10)-triene-3, 17-diol (17beta)-; Estrace; Estradiol; Estradiol-17 beta; Estradiol-17beta; Estraldine; Estrogen Nuclear Receptor; Estrogen Receptors; Estrogenic Agents; Estrogenic Compounds; Estrogens; Euglycaemic Clamp; Euglycemic Clamping; Fats; Fatty Tissue; Fatty acid glycerol esters; Female; Food Intake; Funding; Genetically Engineered Mouse; Glucose; Glucose Clamp; Health; Hepatic; Hypercholesteremia; Hyperplasia; Hyperplastic; Inflammatory Response; Infusion; Infusion procedures; Injection of therapeutic agent; Injections; Intermediary Metabolism; Intervention; Intervention Strategies; Intraventricular; Knockout Mice; Knowledge; Lead; Leiomyocyte; Ligands; Liver; METBL; Mammals, Mice; Measures; Mediating; Metabolic; Metabolic Processes; Metabolism; Mice; Mice, Knock-out; Mice, Knockout; Modeling; Molecular Interaction; Murine; Mus; Muscle, Skeletal; Muscle, Smooth, Vascular; Muscle, Voluntary; Myocytes, Smooth Muscle; Nuclear; Null Mouse; Obesity; Organ; Organ System, Cardiovascular; Ovocyclin; Ovocylin; PBO; Pathogenesis; Pb element; Peptide Signal Sequences; Peptides; Peripheral; Phage Display; Placebos; Prevention; Programs (PT); Programs [Publication Type]; Progynon; Receptor Signaling; Regulation; Research; Reticuloendothelial System, Blood; Role; SERMs; Selective Estrogen Receptor Modulators; Serum; Sham Treatment; Signal Peptide; Signal Sequences; Signal Sequences, Peptide; Skeletal Muscle Tissue; Skeletal muscle structure; Smooth Muscle Cells; Smooth Muscle Myocytes; Smooth Muscle Tissue Cell; Streaks, Arterial Fatty; Subcellular Process; Testing; Therapeutic Estradiol; Therapeutic Estrogen; Transgenic Mice; Up-Regulation; Up-Regulation (Physiology); Upregulation; Vascular Diseases; Vascular Disorder; Vascular Endothelium; Vascular remodeling; Vascular, Heart; Work; adipose; adiposity; atherogenesis; atheromatosis; atheroprotective; atherosclerosis plaque; atherosclerotic lesions; atherosclerotic plaque; atherosclerotic vascular disease; attenuation; base; blood glucose regulation; blood vessel disorder; body system, hepatic; cardiovascular disorder; cell growth; cell type; cholest-5-ene-3 beta, 27-diol; circulatory system; corpulence; corpulency; corpulentia; design and construction; disease/disorder; glucose control; glucose disposal; glucose homeostasis; glucose output; glucose regulation; glucose tolerance; glucose uptake; heavy metal Pb; heavy metal lead; hypercholesterolemia; in vivo; insulin sensitivity; insulin stimulated glucose disposal; insulin tolerance; interventional strategy; macrophage; male; migration; neointima formation; neointimal thickening; new approaches; new therapeutics; next generation therapeutics; non-genomic; nongenomic; novel; novel approaches; novel strategies; novel strategy; novel therapeutics; obese; obese people; obese person; obese population; organ system, hepatic; overexpression; prevent; preventing; programs; protein signal sequence; public health relevance; receptor function; sham therapy; social role; vascular; vulnerable plaque; white adipose tissue; yellow adipose tissue
Relevance: 27-hydroxycholesterol is a metabolite of cholesterol that is elevated in the blood when circulating cholesterol levels are high, and it accumulates with cholesterol in diseased arteries. We have discovered that 27-hydroxycholesterol modifies the actions of estrogen in numerous cell types and organs. The proposed research program will determine how 27-hydroxycholesterol, through its impact on estrogen action, contributes to the development of blood vessel disease and to impaired body weight and glucose regulation
Project start date: 2006-08-01
Project end date: 2015-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-070
2R01HL087564-05 (2010): $392500
Sponsored Links Excellgen http://Excellgen.com
REGULATION OF NITRIC OXIDE SYNTHASE IN DEVELOPING LUNG
Philip W Shaul, Professor
University Of Texas Sw Med Ctr/dallas Dallas, Tx 753909105
Grant 5R01HD030276-08 from National Institute Of Child Health And Human Development IRG: HED
Abstract: Endothelium-derived nitric oxide (NO) is a critical modulator of vasomotor tone in developing lung, having a key role in the normal transition to extrauterine life and in the pathophysiology of persistent pulmonary hypertension of the newborn (PPHN). The OVERALL OBJECTIVE of this proposal is to determine how pulmonary endothelial NO production is modified by changes in O2 and how it is regulated developmentally. Specific Aim No. 1 is to determine the direct effect of ACUTE alterations in O2 on NO production and the role of cytosolic free calcium (Cai 2+) homeostasis in this process. NO production in ovine fetal pulmonary endothelial cells will be assessed during acute changes in O2 by measuring cGMP- in detector smooth muscle cells. Studies with fura-2 will reveal O2-related alterations in endothelial Cai 2+, and pharmacologic methods will reveal the processes regulating Cai 2+ which mediate the changes in NO production. Aim No. 2 is to determine the effect of PROLONGED hypoxia on NO production and the role of changes in NO synthase activity and expression in this process. Chronic hypobaria in rats will be used to examine prolonged hypoxia in vivo, and ovine fetal pulmonary endothelial cells will be subjected to prolonged alterations in O2 in vitro. NO synthase activity will be determined by measuring 1-arginine conversion to 1-citrulline, the enzyme protein will be quantitated in immunoblots and by immunohistochemistry, and NO synthase mRNA expression will be assessed by ribonuclease protection assays and in situ hybridization. Aim No. 3 is to delineate the ontogeny of pulmonary endothelial NO production, correlating it with NO synthase activity, protein expression, and steady state MRNA levels. Whole lung, arterial segments, and endothelial cells from early and late third trimester fetal lambs and 1 week and 1 month old newborn lambs will be studied. Aim No. 4 is to determine the amount of constitutive versus inducible NO synthase in developing pulmonary endothelium and the putative factors regulating their expression. The dependence of NO synthase activity on calcium/calmodulin will be tested and inhibition profiles will be performed with 1-arginine analogues. The modulation of NO production by corticosteroids and cytokines will be examined and the basis of modulation will be determined in studies of NO synthase enzyme activity, protein quantification, and MRNA levels. By revealing for the first time the cellular and molecular processes regulating endothelial NO production in the developing pulmonary circulation, these studies will increase our fundamental knowledge of the mechanisms underlying successful cardiopulmonary transition and the pathophysiology of PPHN.
Keywords: embryo /fetus, enzyme activity, gene expression, growth /development, lung, nitric oxide synthase, estrogen, pulmonary hypertension, respiratory enzyme, vascular endothelium, immunochemistry, northern blotting, nuclear runoff assay, sheep, statistics /biometry, tissue /cell culture, western blotting
Project start date: 1993-04-01
Project end date: 2001-03-31
5R01HD030276-08 (2000): $241557
2R01HD030276-05 (1997): $221059
5R01HD030276-03 (1995): $141636
5R01HD030276-02 (1994): $158331
1R01HD030276-01 (1993): $136862
5R01HD030276-07 (1999): $234521
5R01HD030276-06 (1998): $227690
CHOLESTEROL AND ENDOTHELIAL NO SYNTHASE TRAFFICKING
Philip W Shaul, Professor
Pediatricsuniversity Of Texas Sw Med Ctr/dallas
dallas, Tx 753909105
Grant 5R01HL058888-05 from National Heart, Lung, And Blood Institute IRG: PTHA
Abstract: Diminished production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a key role in the early pathogenesis of atherosclerosis. eNOS appears to be acylated and thereby targeted to cholesterol-enriched, plasmalemmal signal transduction domains called caveolae. Localization to caveolae is critical to eNOS function. The objectives of this proposal are to define the molecular mechanisms normally regulating eNOS trafficking to caveolae, and to determine if changes in cholesterol balance attenuate eNOS function by modifying eNOS trafficking to caveolae. Aim 1 will test the hypothesis that eNOS is first targeted to the Golgi apparatus by myristoylation, and then targeted to caveolae by palmitoylation. eNOS trafficking will be assessed in COS-7 cells transfected with wild-type or mutant eNOS cDNAs by immunoprecipitation of biosynthetically-labelled eNOS in subcellular fractions, and by immunofluorescence and, immunoelectron microscopy. Aim 2 will test the hypothesis that the integral membrane protein caveolin is necessary for eNOS trafficking to caveolae and for optimal eNOS function. Studies will be performed in permanently-transfected cell lines that are caveolin (+) or caveolin (-). Aim 3 will employ endothelial cells to test the hypothesis that components of the eNOS activation system are colocalized in caveolae. Receptor-mediated eNOS stimulation will be evaluated in isolated caveolae, and the existence of an eNOS activation complex will be assessed by coprecipitation. Aim 4 will test the hypothesis that eNOS trafficking to caveolae and eNOS function are attenuated when cholesterol content is abnormally low or high; experiments will be performed in transfected COS-7 cells, endothelial cells, and isolated plasma membranes. The studies planned will increase our basic knowledge of the regulation of endothelial NO production under normal conditions and during altered cholesterol balance, thereby revealing the pathogenetic link between hypercholesterolemia and endothelial dysfunction. In doing so, it is anticipated that the proposed work will lead to new preventative measures and treatment modalities for atherosclerosis that complement approaches aimed at cholesterol control
Keywords: atherosclerosis, cholesterol, intracellular transport, nitric oxide synthase, pathologic process, protein transport, vascular endothelium caveola, caveolin, membrane protein, myristate, palmitate immunoelectron microscopy, immunofluorescence technique, immunoprecipitation, tissue /cell culture
Project start date: 1997-07-15
Project end date: 2003-06-30
5R01HL058888-05 (2001): $278115
5R01HL058888-04 (2000): $272028
5R01HL058888-03 (1999): $264461
Sponsored Links Excellgen http://Excellgen.com
1R01HL058888-01 (1997): $249277
ESTROGEN MODULATION OF ENDOTHELIAL CELL PHENOTYPE
Philip W Shaul, Professor Of Pediatrics
University Of Texas Sw Med Ctr/dallas, Dallas, Tx 75390-9105
Grant 5R01HD030276-18 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development
Keywords: 17p; Adrenergic Receptor; Adrenoceptors; Agonist; Aorta; Aquadiol; Arteries; Biosynthetic Proteins; Blood Vessels; Body Tissues; COS-7 Cell; Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Carotid Arteries; Caveolae; Caveolas; Cell Communication and Signaling; Cell Isolation; Cell Locomotion; Cell Migration; Cell Movement; Cell Segregation; Cell Separation; Cell Separation Technology; Cell Signaling; Cell membrane; Cell model; Cellular Expansion; Cellular Growth; Cellular Migration; Cellular model; Chromosome 17 Proximal Arm; Chromosome 17 Short Arm; Chromosome 17p; Conestron; Conjugated Estrogen - Premarin; Coupling; Cultured Cells; Cytoplasmic Membrane; Development; Dimenformon; Diogyn; Diogynets; EC 1.14.13.39; EC 2.7; EDRF Synthase; Electric Injuries; Embryonic Tissue, Placenta; Endothelial Cells; Endothelium; Endothelium-Derived Growth Factor Synthase; Estra-1, 3, 5(10)-triene-3, 17-diol (17beta)-; Estrace; Estradiol; Estradiol-17 beta; Estradiol-17beta; Estraldine; Estrogen Receptors; Estrogenic Agents; Estrogenic Compounds; Estrogenic Hormones, Conjugated; Estrogenic Substances, Conjugated; Estrogens; Estrogens, Conjugated; Estrogens, Conjugated (USP); Female; Female Health; Femest; G Protein-Complex Receptor; G-Protein-Coupled Receptors; G-Proteins; GFAC; GFP; GTP-Binding Proteins; GTP-Regulatory Proteins; Gene Expression; Gene Targeting; Generalized Growth; Genes; Genetically Engineered Mouse; Genetics-Mutagenesis; Genital System, Female, Ovary; Genital System, Female, Uterus; Gestation; Green Fluorescent Proteins; Growth; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Guanylyl Cyclase-Activating Factor Synthase; Health; Heart; Histamine-Sensitizing Factor; IAP Pertussis Toxin; Immigrations; In Vitro; In-Migration; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Investigators; Islet-Activating Protein; Kinases; Knowledge; L-Arginine, NADPH[{..}]oxygen oxidoreductase (nitric-oxide-forming); Lead; Ligands; Lipid Rafts, Cell Membrane; Lymphocytosis-Promoting Factor; Mammals, Mice; Mediating; Membrane; Membrane Microdomains; Mice; Mice, Transgenic; Modeling; Molecular; Molecular Biology, Mutagenesis; Motility; Motility, Cellular; Murine; Mus; Mutagenesis; NADPH-Diaphorase; NO Synthase; Nitric Oxide Synthase; Nitric-Oxide Synthetase; Nuclear; Organ; Organ System, Cardiovascular; Ovary; Ovocyclin; Ovocylin; Pb element; Peptides; Pertussigen; Pertussis Toxin; Phenotype; Phosphorylation; Phosphotransferases; Placenta; Placenta-Tissue, Cells; Placentoma, Normal; Placentome; Plasma Membrane; Pregnancy; Pregnancy Outcome; Premarin; Process; Production; Programs (PT); Programs [Publication Type]; Progynon; Protein Phosphorylation; RT-PCR; RTPCR; Receptors, Epinephrine; Recombinant Proteins; Recombinants; Regulation; Research; Research Personnel; Researchers; Reverse Transcriptase Polymerase Chain Reaction; Role; Shock from electric current; Signal Transduction; Signal Transduction Systems; Signaling; Sodestrin; Sorting - Cell Movement; Sphingolipid Microdomains; Sphingolipid-Cholesterol Rafts; Targetings, Gene; Testing; Therapeutic Estradiol; Therapeutic Estrogen; Therapeutic Steroid Hormone; Time; Tissue Growth; Tissues; Transfection; Transgenic Mice; Transphosphorylases; Uterus; Vascular, Heart; Woman; Women`s Health; Work; adenoreceptor; aminoacid sequence of peptide; aminoacid sequence of protein; angiogenesis; base; biological signal transduction; cell determination; cell growth; cell motility; cell sorting; circulatory system; electrical injury; experiment; experimental research; experimental study; heavy metal Pb; heavy metal lead; in vivo; interventional strategy; lipid raft; loss of function; member; membrane structure; migration; mouse model; mutant; neovascularization; new therapeutics; next generation therapeutics; non-genomic; nongenomic; novel; novel therapeutics; ontogeny; peptide sequence; plasmalemma; programs; protein activation; protein aminoacid sequence; protein function; protein protein interaction; reproductive; research study; response; reverse transcriptase PCR; social role; sorting; steroid hormone; steroid hormone receptor; vascular; womb
Project start date: 1993-04-01
Project end date: 2011-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5R01HD030276-18 (2010): $238497
NITRIC OXIDE SYNTHASES IN LUNG DEVELOPMENT AND BRONCHOPULMONARY DYSPLASIA
Philip W Shaul, Professor
Southwest Foundation For Biomedical Res Box 760549 San Antonio, Tx 782450549
Grant 2P51RR013986-060162 from National Center For Research Resources IRG: ZRR1
Keywords: Primate, animal colony, bronchopulmonary dysplasia, lung development, nitric oxide synthase
Project start date: 2004-05-01
Project end date: 2005-04-30
REGULATION OF NITRIC OXIDE SYNTHASE IN DEVELOPING LUNG
Philip W Shaul, Professor
Pediatricsuniversity Of Texas Sw Med Ctr/dallas
dallas, Tx 753909105
Grant 5R01HD030276-04 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development IRG: HED
Project start date: 1993-04-01
Project end date: 1997-03-31
5R01HD030276-04 (1996): $147700
REGULATION OF CYCLOOXYGENASE GENES IN DEVELOPING LUNG
Philip W Shaul, Professor
Pediatricsuniversity Of Texas Sw Med Ctr/dallas
dallas, Tx 753909105
Grant 5R01HL053546-02 from National Heart, Lung, And Blood Institute IRG: HED
Project start date: 1994-12-01
Project end date: 1998-11-30
5R01HL053546-02 (1996): $210526