Hong-hai Zhang

Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

Caveolin-1 Orchestrates Fibroblast Growth Factor 2 Signaling Control of Angiogenesis in Placental Artery Endothelial Cell Caveolae

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration‐ and time‐ dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2‐stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2‐stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co‐fractionated with caveolin‐1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl‐β‐cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin‐1. FGF2 stimulated dissociation of FGFR1 from caveolin‐1. Downregulation of caveolin‐1 significantly attenuated the FGF2‐induced activation of AKT1 and ERK1/2 and inhibited FGF2‐induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin‐1 scaffolding domain peptide to mimic caveolin‐1 overexpression also inhibited these FGF2‐induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2‐induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin‐1 interacts with FGFR1 and paradoxically regulates FGF2‐induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis. J. Cell. Physiol. 227: 2480–2491, 2012.

Estradiol-17β Stimulates Specific Receptor and Endogenous Nitric Oxide-Dependent Dynamic Endothelial Protein S-Nitrosylation: Analysis of Endothelial Nitrosyl-Proteome

Covalent adduction of a nitrosyl group to cysteines [S-nitrosylation (S-NO)] is emerging as a key route for nitric oxide (NO) to directly modulate protein functions. Here, we studied the effects of estrogens on endothelial protein S-NO and analyzed the nitrosyl-proteomes by biotin/CyDye switch technique combined with two-dimensional fluorescence difference gel electrophoresis and identified nitrosoproteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Estradiol-17beta (E2) rapidly stimulated protein S-NO in human umbilical vein endothelial cells, maximizing within 10- to 30-min post-E2 (10 nm) exposure. E2-BSA also rapidly stimulated protein S-NO. Both E2 and E2-BSA-induced protein S-NO was blocked by ICI 182,780 and N-nitro-l-arginine-methylester. Human umbilical vein endothelial cells expressed estrogen receptor (ER)alpha and ERbeta; both seemed to be required for E2 stimulation of protein S-NO because: 1) neither ERalpha or ERbeta agonist alone, but their combination, stimulated protein S-NO; and 2) either ERalpha or ERbeta antagonist blocked E2-induced protein S-NO. Numerous nitrosoproteins (spots) were observed on two-dimensional fluorescence difference gel. One hundred spots of interest were picked up; 58 were identified and, of which 15 were novel nitrosoproteins, 28 were up-regulated, 11 were decreased, and the rest were unchanged by E2. Pathway analysis suggested that nitrosoproteins are involved in regulating various endothelial functions, including apoptosis, cell structure and metabolism, redox homeostasis, etc. Thus, estrogens stimulate dynamic endothelial protein S-NO via mechanisms linked to specific ERs possibly on the plasma membrane and endogenous NO. These findings signify a critical next step for the understanding of the biological targets of enhanced NO production by estrogens.

Analysis of Nitroso-Proteomes in Normotensive and Severe Preeclamptic Human Placentas

Nitric oxide (NO) plays a key role in placental biology, and placental dysfunction is the main pathogenesis pathway for preeclampsia, yet the direct placental targets of NO actions have not been determined. Covalent adduction of an NO moiety to cysteines, termed S-nitrosylation (SNO), is emerging as a key route by which NO can directly modulate protein functions. This study was conducted to analyze global S-nitroso (SNO)-proteins in human placentas and to determine if their levels differ in normotensive versus severe preeclamptic placentas. Although total nitrite/nitrate increased, total levels of SNO-proteins and nitrosylated forms of endothelial NO synthase and heat shock protein 90 were decreased by preeclampsia. We further compared normotensive and preeclamptic placental nitroso-proteomes (total SNO-protein profiles) by using a biotin and CyDye switch test combined with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and identified SNO-proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Numerous SNO-proteins were displayed as spots on 2D-DIGE gels. One hundred spots of interest were excised; 46 spots were identified, of which 8 spots were novel SNO-proteins; levels of 15 spots were increased, and 6 spots were decreased, and the rest were unchanged by preeclampsia. Pathway analysis suggested that placental SNO-proteins are involved in regulating various cellular functions including protein synthesis, cell movement and metabolism, cell signaling, and other functions. These data therefore show for the first time that SNO is a crucial mechanism by which NO directly regulates placental proteins linked to various biological pathways. The significantly altered placental nitroso-proteome in preeclampsia suggests that SNO plays a role in the placental pathophysiology in preeclampsia.

Estrogen-responsive nitroso-proteome in uterine artery endothelial cells: role of endothelial nitric oxide synthase and estrogen receptor-β.

Covalent adduction of a NO moiety to cysteines (S‐nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol‐17β (E2) rapidly stimulated protein SNO that maximized within 10–30 min post‐E2 exposure. E2‐bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2‐induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2‐induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2‐stimulated SNO. E2‐induced SNO was inhibited by N‐nitro‐L‐arginine‐methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen‐induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso‐proteomes by CyDye switch technique combined with two‐dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry. Among the 54 identified, nine were novel SNO‐proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen‐responsive nitroso‐proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation. J. Cell. Physiol. 227: 146–159, 2012.

S-Nitrosylation of Cofilin-1 Mediates Estradiol-17β-Stimulated Endothelial Cytoskeleton Remodeling

Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNO(Cys80) of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling.

S‐nitrosylation of Cofilin‐1 Serves as a Novel Pathway for VEGF‐Stimulated Endothelial Cell Migration

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) mediates vascular endothelial growth factor (VEGF)‐stimulated endothelial cytoskeleton remodeling and migration; however, the underlying mechanisms are elusive. Covalent adduction of a NO moiety (NO•) to cysteines called S‐nitrosylation (SNO) is a key NO signaling pathway. The small actin‐binding protein cofilin‐1 (CFL1) is essential for actin cytoskeleton remodeling. We investigated whether S‐nitrosylation regulates CFL1 function and endothelial cytoskeleton remodeling and migration upon VEGF stimulation. VEGF rapidly stimulated S‐nitrosylation of CFL1, which was blocked by NO Synthase inhibition and eNOS knockdown by specific eNOS‐siRNA. Cys80 and Cys139 were identified as the major SNO‐sites in CFL1 by LC‐MS/MS. The actin severing activity of recombinant SNO‐mimetic CFL1 (C80/139A DMA‐CFL1), but not SNO‐deficient CFL1 (C80/139S DMS‐CFL1), was significantly greater than that of wild‐type CFL1 (wt‐CFL1). When wt‐CFL1 and its mutants were overexpressed in endothelial cells, basal actin bound wt‐CFL1 was undetectable but significantly increased by VEGF; basal actin bound DMA‐CFL1 was readily high and basal actin bound DMS‐CFL1 was detectable but low, and both were unresponsive to VEGF. Treatment with VEGF significantly increased filamentous (F‐) actin and filopodium formation and cell migration in endothelial cells. Overexpression of wt‐CFL1 inhibited VEGF‐induced F‐actin formation. Overexpression of DMA but not DMS CFL1 decreased basal but not VEGF‐stimulated F‐actin formation. Overexpression of DMA but not DMS CFL1 suppressed VEGF‐stimulated filopodium formation and migration in endothelial cells. Thus, S‐nitrosylation of CFL1 provides a novel signaling pathway post‐NO biosynthesis via eNOS‐derived NO for endothelial cytoskeleton remodeling and migration upon VEGF stimulation. J. Cell. Physiol. 230: 406–417, 2015.

Compartmentalizing Proximal FGFR1 Signaling in Ovine Placental Artery Endothelial Cell Caveolae

ABSTRACT Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.

Augmented H2S production via cystathionine-beta-synthase upregulation plays a role in pregnancy-associated uterine vasodilation

Abstract Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-betasynthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy. Summary Sentence Augmented hydrogen sulfide biosynthesis via upregulating endothelium and smooth muscle cystathionine β-synthase expression plays a role in pregnancy-associated uterine vasodilation.

Endogenous NO Upon Estradiol-17β Stimulation and NO Donor Differentially Regulate Mitochondrial S-Nitrosylation in Endothelial Cells

Adduction of a nitric oxide (NO) moiety (NO(•)) to cysteines termed as S-nitrosylation (SNO) has emerged as a crucial mechanism for NO signaling crucial for mediating the vascular effects of estrogens. Mitochondrion is a known vascular risk factor; however, the effects of estrogens on mitochondrial SNO are incompletely understood. In this study we determined the effects of estradiol-17β (E2β) on mitochondrial protein SNO in primary human umbilical vein endothelial cells and compared the mitochondrial nitroso-proteomes in E2β- and a NO donor S-nitrosoglutathione (GSNO)-treated cells using a proteomics approach. Treatment with 10 nM E2β and 1 mM GSNO for 30 minutes significantly increased the levels of mitochondrial SNO-proteins. Subcellular localization of SNO-proteins showed mitochondria as the major cellular organelle for protein SNO in response to E2β and GSNO. E2β stimulated mitochondrial endothelial nitric oxide synthase (eNOS) phosphorylation and mitochondrial protein SNO that was enhanced by overexpression of mitochondrion or Golgi, but not membrane targeting eNOS constructs. We identified 11, 32, and 54 SNO-proteins in the mitochondria from the untreated, E2β-, and GSNO-treated human umbilical vein endothelial cells, respectively. Comparisons of the nitroso-proteomes revealed that common and different mitochondrial SNO-proteins were affected by endogenous NO on E2β stimulation and exogenous NO from donor. These SNO-proteins were associated with various mitochondrial functions, including energy and redox regulation, transport, iron homeostasis, translation, mitochondrial morphology, and apoptosis, etc. Collectively, we conclude that estrogens rapidly stimulate protein SNO in endothelial mitochondria via mitochondrial eNOS, providing a mechanism for mediating the vascular effects of estrogens.