Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N(6)-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) > or = WT with l-NIL or iNOS(-/-) > eNOS(-/-) > or = eNOS(-/-) with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS(-/-) mice but not in eNOS(-/-) mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.     

Thromboxane A2 receptor signaling inhibits vascular endothelial growth factor-induced endothelial cell differentiation and migration

Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TPbeta, but not TPalpha, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TPbeta costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the alpha(v)beta3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.     

Hepatocyte growth factor-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase expression

OBJECTIVES: Hepatocyte growth factor (HGF) is an angiogenic mitogen which stimulates migration in various cell types and has been shown to induce the production of nitric oxide (NO) in epithelial cells. Conflicting data exist on the effect of NO on endothelial cell migration. The aim of this study was to investigate a possible role for NO in HGF-stimulated endothelial cell motility. METHODS: The study was performed primarily using an endothelial cell line derived from adult human saphenous vein. Transient transfection experiments were additionally performed using an adult human coronary artery endothelial cell line. Nitric oxide synthase expression was examined by western blot analysis. Time-lapse digital image microscopy was used to measure cell motility. A DNA construct was used in transient transfections to over-express inducible nitric oxide synthase (iNOS) as an N-terminal fusion to enhanced green fluorescent protein (EGFP). RESULTS: HGF upregulated the expression of iNOS but not constitutive endothelial nitric oxide synthase (eNOS). Treatment of cells with the specific iNOS inhibitor 1400 W revealed that functional iNOS was required for HGF-stimulated endothelial cell motility. HGF-induced iNOS expression was partially abrogated in the presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but not the Src kinase inhibitor, PP1. Endothelial cell motility increased significantly (P<0.0001) in the presence of the exogenous NO donor spermine-NO and cells expressing the iNOS-EGFP fusion protein exhibited significantly greater (P=0.0038) motility than those expressing EGFP alone. CONCLUSIONS: These combined data show that elevated NO production is sufficient to stimulate endothelial cell motility and link HGF and NO, both previously implicated in modulating motility, in a common signalling pathway.     

Hepatocyte growth factor stimulates nitric oxide production through endothelial nitric oxide synthase activation by the phosphoinositide 3-kinase/Akt pathway and possibly by mitogen-activated protein kinase kinase in vascular endothelial cells.

Hepatocyte growth factor (HGF) has recently been the focus of attention due to its angiogenic effects, which are similar to those of vascular endothelial growth factor (VEGF); because of these effects, HGF is considered to be a novel therapeutic agent against vascular disorders, including atherosclerotic angiopathies. Although nitric oxide (NO), which is derived from vascular endothelial cells (ECs), is also involved in angiogenesis, little is known regarding the interactions between HGF and NO. We therefore examined the effects of HGF on NO production as well as endothelial NO synthase (eNOS) phosphorylation, and investigated their mechanisms. In bovine aortic ECs, HGF induced a rapid (5 min) increase of NO production measured by diaminofluorescein-2 diacetate. Moreover, HGF rapidly (2.5 min) stimulated eNOS phosphorylation (Ser-1179) as determined by Western immunoblot analyses. Both of these effects were almost completely suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and were partially suppressed by the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126. HGF also stimulated Akt phosphorylation (Ser-473), which was completely suppressed by LY294002 and was partially suppressed by U0126. Moreover, HGF stimulated extracellular signal-regulated kinase 1/2 phosphorylation (Thr-202/Tyr-204), which was completely suppressed by U0126 and was partially suppressed by LY294002. Taken together, these results indicate that HGF not only phosphorylates eNOS through the PI3K/Akt pathway, but also partially through the MAPK pathway, and that these two pathways may interact. Compared with VEGF, HGF was more potent in both NO production and eNOS phosphorylation. Our study thus demonstrates a novel activity of HGF-the stimulation of NO production-which occurs via eNOS phosphorylation that may in turn be mediated by cross-talk between the PI3K/Akt and MAPK pathways.     

Adiponectin inhibits vascular endothelial growth factor-induced migration of human coronary artery endothelial cells

AIMS: Vascular endothelial growth factor (VEGF)-induced endothelial cell migration and angiogenesis are associated with the vascular complications of diabetes mellitus, and adiponectin is an abundant plasma adipokine that exhibits salutary effects on endothelial function. We investigated whether adiponectin suppresses VEGF-induced migration and related signal transduction responses in human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: Using a modified Boyden chamber technique and a monolayer 'wound-healing' assay, both the recombinant adiponectin globular domain and full-length adiponectin protein potently suppressed the migration of HCAEC induced by VEGF. Adiponectin did not increase endothelial cell apoptosis, as measured by terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labelling assay. Adiponectin also suppressed VEGF-induced reactive oxygen species generation, activation of Akt, the mitogen-activated protein kinase ERK and the RhoGTPase RhoA, and induction of the formation of actin stress fibres and focal cellular adhesions. VEGF-stimulated cell migration was inhibited by activation of adenylyl cyclase with forskolin, and adiponectin treatment increased cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) enzymatic activity. Pharmacological inhibition of either adenylyl cyclase or PKA significantly abrogated the effect of adiponectin globular domain to suppress VEGF-induced cell migration. CONCLUSION: Adiponectin suppresses VEGF-stimulated HCAEC migration via cAMP/PKA-dependent signalling, an important effect with implications for a regulatory role of adiponectin in vascular processes associated with diabetes and atherosclerosis.     

Role of endothelial nitric oxide synthase in endothelial cell migration.

Endothelium-derived nitric oxide (NO) and its precursor L-arginine have been implied to promote angiogenesis, but little is known about the precise mechanism. The inhibition of endogenous NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) but not its inactive enantiomer D-NAME (1 mmol/L) inhibited endothelial cell sprouting from the scratched edge of the cultured bovine aortic endothelial cell monolayer. Inhibition of endogenous NO release by L-NAME was confirmed by amperometric measurement using an NO-specific electrode. In the modified Boyden chamber, L-NAME (1 mmol/L) significantly inhibited endothelial cell migration, whereas L-NAME did not affect endothelial DNA synthesis as assessed by analysis of [3H]thymidine incorporation. We then examined alteration of endothelial cell adhesion molecule expression after the inhibition of NO by L-NAME in cultured human umbilical vein endothelial cells. In both normoxic and hypoxic conditions, L-NAME (1 mmol/L) inhibited surface expression of integrin alphavbeta3, which is an important integrin facilitating endothelial cell survival and angiogenesis. However, L-NAME did not affect the expression of platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, vascular endothelial adhesion molecule-1, gap junction protein connexin 43, and VE-cadherin, which have been reported to potentially affect angiogenesis. In summary, inhibition of endothelial NO synthase by L-NAME attenuated endothelial cell migration but not proliferation in vitro. Furthermore, endogenous endothelium-derived NO maintains the functional expression of integrin alphavbeta3, a mediator for endothelial migration, survival, and angiogenesis. Endothelium-derived NO, thus, may play an important role in mediating angiogenesis by supporting endothelial cell migration, at least partly, via an integrin-dependent mechanism.     

eNOS/NO 信号通路与心血管疾病关系的研究进展

一氧化氮（nitric oxide,NO）作为一种信号分子,在生理活动中起着重要作用,包括血压调节、血管张力维持、免疫系统调控等,尤其在心血管系统中发挥重要作用。NO产生异常是多种心血管疾病的诱因。内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）作为诱导合成NO的限速酶,主要在血管壁的调节中发挥重要作用,因此,其与心血管的正常生理活动密切相关。本文综述了eNOS的基本结构与功能、NO的理化性质,以及eNOS/NO信号通路与心血管系统疾病的关系。     

Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCalpha

OBJECTIVE: Oxidized low-density lipoprotein (ox-LDL) increases superoxide anion (O(2)(-)) production by the endothelial nitric oxide (NO) synthase (eNOS). We assessed whether the uncoupling of eNOS was associated with alterations in eNOS phosphorylation and/or the assembly of the eNOS signaling complex. METHODS AND RESULTS: In unstimulated human endothelial cells, eNOS Thr(495) was constitutively phosphorylated. ox-LDL, but not native LDL, enhanced the production of O(2)(-) by endothelial cells, an effect that was partially sensitive to NOS inhibition. ox-LDL, but not native LDL, induced a time- and concentration-dependent decrease in the phosphorylation of eNOS on Thr(495). Protein kinase C (PKC) has been reported to phosphorylate this residue, and the increase in the phosphorylation of Thr(495) induced by phorbol 12-myristate 13-acetate was attenuated in cells pretreated with ox-LDL. Moreover, the phosphorylation and activity of PKCalpha was attenuated by ox-LDL and paralleled the changes in eNOS phosphorylation. ox-LDL also induced the dissociation of eNOS from the plasma and Golgi membranes. In COS-7 cells, a T495A eNOS mutant generated significantly more O(2)(-) than a T495D mutant did, indicating that the dephosphorylation of Thr(495) alone can increase O(2)(-) production by eNOS. However, although the dephosphorylation of Thr(495) in histamine-stimulated endothelial cells enhanced the binding of calmodulin to eNOS, calmodulin no longer bound to eNOS from ox-LDL-treated endothelial cells. CONCLUSIONS: These results indicate that a decrease in the activity of PKCalpha in ox-LDL-treated endothelial cells is associated with the dephosphorylation of eNOS, dissociation of the eNOS signaling complex, and the enhanced production of eNOS-derived O(2)(-).     

血管内皮生长因子对培养内皮细胞合成一氧化氮的影响

目的 :探讨血管内皮生长因子 （VEGF） /血管渗透因子 （VPF）对内皮细胞 （EC）分泌一氧化氮 （NO）的影响。方法 :将培养的人脐静脉内皮细胞 （HUVEC）随机分为 6组 （n=6 /组 ） :1正常对照组 ;2 VEGF 1ng/ m l;3VEGF 10ng/ m l;4VEGF 10 0 ng/ m l;5低氧组 ;6低氧组 +VEGF 10 0 ng/ m l。采用硝酸还原酶法测定培养液中的 NO2 - ,NO3 -的含量以反映 NO水平。结果 :VEGF促进正常 EC分泌 NO,在一定浓度范围内呈剂量依赖性 ;低氧损伤 EC,使其分泌 NO减少 ;VEGF 10 0 ng/ ml预处理可保护 EC免受低氧损害 ,保持正常分泌 NO的功能。结论 :VEGF能够调节 EC的功能 ,为其促血管形成作用中 EC分裂、增殖、迁移奠定物质基础     

Fibroblast growth factor-2 stimulates endothelial nitric oxide synthase expression and inhibits apoptosis by a nitric oxide-dependent pathway in Nb2 lymphoma cells

We recently reported that the rat Nb2 T lymphoma cells expressed messenger RNAs (mRNAs) encoding both fibroblast growth factor-2 (FGF-2) and the FGF receptor, suggesting possible paracrine and/or autocrine roles for FGF-2 in lymphoma cell function. We have also shown that the Nb2 cells expressed endothelial nitric oxide synthase (eNOS) and produced low levels of nitric oxide (NO) that inhibited apoptosis of PRL-deprived cells via a PRL-independent, bcl-2-mediated pathway. In this study the effects of PRL and FGF-2 on Nb2 cell survival and NO production were further investigated. The percentages of nonapoptotic cells in PRL-treated vs. PRL-deprived cultures after 6 days were 95% and 53%, respectively. Addition of FGF-2 to PRL-deprived Nb2 cells did not stimulate cell proliferation, but the onset of apoptosis was significantly inhibited, such that more than 85% of the cells remained nonapoptotic after 6 days. The steady state levels of bcl-2 and bag-1 mRNAs were low in PRL-deprived Nb2 cells, but were markedly increased by PRL or FGF-2. bcl-2 expression was induced within 1 h of PRL or FGF-2 addition and continued to increase to a level 20- to 25-fold above the control level within 24 h. bag-1 expression also increased within 1 h after the addition of PRL or FGF-2, was maximal within 8 h, and declined slowly thereafter. The levels of eNOS mRNAs were low but detectable in growth-arrested Nb2 cells, and PRL further down-regulated eNOS mRNA levels over the next 24 h. In contrast, FGF-2 significantly increased eNOS mRNA levels within 2 h to reach a peak 10-fold induction by 12 h. FGF-2 stimulation of eNOS mRNA was accompanied by a 2- to 3.5-fold increase in cellular levels of the eNOS protein and a 2.5-fold increase in serine-phosphorylated eNOS. However, the ratio of serine-phosphorylated eNOS vs. total cellular eNOS was unchanged, indicating that FGF-2 did not affect the serine phosphorylation status of eNOS. Nb2 cells produced low basal levels of NO, which increased with increasing L-arginine concentrations. PRL did not further increase NO release in the presence of L-arginine (0.1 or 1 mM), but FGF-2 significantly (P: 相似文献   

Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a key signaling molecule in vascular homeostasis. Loss of NO bioavailability due to reduced synthesis and increased scavenging by reactive oxygen species is a cardinal feature of endothelial dysfunction in vascular disease states. The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of eNOS activity: when BH4 availability is limiting, eNOS no longer produces NO but instead generates superoxide. In vascular disease states, there is oxidative degradation of BH4 by reactive oxygen species. However, augmentation of BH4 concentrations in vascular disease by pharmacological supplementation, by enhancement of its rate of de novo biosynthesis or by measures to reduce its oxidation, has been shown in experimental studies to enhance NO bioavailability. Thus, BH4 represents a potential therapeutic target in the regulation of eNOS function in vascular disease.     

雌激素对血管内皮细胞一氧化氮合酶活性调控的受体机制研究

目的　观察 17β 雌二醇对大鼠肺血管内皮细胞一氧化氮合酶 (NOS)活性的影响及雌激素受体在其中的作用。方法　用贴壁法和无酚红 16 40培养基培养大鼠肺血管内皮细胞 ,在不同浓度的 17β 雌二醇 (伴或不伴有雌激素受体拮抗剂代莫昔芬 )作用下 ,观察一定时间内内皮细胞的NOS活性及一氧化氮 (NO)的产量。放射配体结合分析技术检测内皮中的雌激素受体。结果　 (1) 1～ 10nmol的 17β 雌二醇作用 8～ 2 4h ,内皮细胞的NO产量显著增加 (vs对照 P<0 .0 5 ) ,10nmol的 17β 雌二醇作用 8～ 2 4hNOS活性显著增强 (与对照比 ,8h ,P<0 .0 5 ;16h ,2 4h ,P<0 .0 1)。 (2 )大鼠肺血管内皮细胞中存在雌激素受体。 (3)雌激素受体拮抗剂他莫昔芬能显著抑制雌激素的上述作用 (P <0 .0 1)。结论　 17β 雌二醇能增强大鼠血管内皮细胞的NOS活性和NO产量 ,该作用由雌激素受体介导 ,可能是雌激素降低血管阻力、抑制动脉粥样硬化作用的重要机理之一。     

SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase

Reduced caloric intake decreases arterial blood pressure in healthy individuals and improves endothelium-dependent vasodilation in obese and overweight individuals. The SIRT1 protein deacetylase mediates many of the effects of calorie restriction (CR) on organismal lifespan and metabolic pathways. However, the role of SIRT1 in regulating endothelium-dependent vasomotor tone is not known. Here we show that SIRT1 promotes endothelium-dependent vasodilation by targeting endothelial nitric oxide synthase (eNOS) for deacetylation. SIRT1 and eNOS colocalize and coprecipitate in endothelial cells, and SIRT1 deacetylates eNOS, stimulating eNOS activity and increasing endothelial nitric oxide (NO). SIRT1-induced increase in endothelial NO is mediated through lysines 496 and 506 in the calmodulin-binding domain of eNOS. Inhibition of SIRT1 in the endothelium of arteries inhibits endothelium-dependent vasodilation and decreases bioavailable NO. Finally, CR of mice leads to deacetylation of eNOS. Our results demonstrate that SIRT1 plays a fundamental role in regulating endothelial NO and endothelium-dependent vascular tone by deacetylating eNOS. Furthermore, our results provide a possible molecular mechanism connecting the effects of CR on the endothelium and vascular tone to SIRT1-mediated deacetylation of eNOS.     

Direct interaction of the cell division cycle 37 homolog inhibits endothelial nitric oxide synthase activity

Endothelial NO synthase (eNOS) via the production of NO in the endothelium plays a key role in cardiovascular biology and is tightly regulated by co- and posttranslational mechanisms, phosphorylation, and protein-protein interactions. The cell division cycle 37 homolog (Cdc37) is a key heat shock protein 90 (Hsp90) cochaperone for protein kinase clients, and Akt/Hsp90 interaction is dependent on Cdc37. Because both Hsp90 and Akt are key eNOS regulatory proteins, we hypothesized that Cdc37 interacts with eNOS as part of the regulatory complex. In the present study, we demonstrate by coimmunoprecipitation and affinity purification in bovine aortic endothelial cells (BAECs) that Cdc37 is complexed with eNOS, Hsp90, and Akt. In addition, cell fractionation data indicate that Cdc37 is found in caveolae with eNOS. Further analysis by in vitro binding assays reveals a direct interaction between purified Cdc37 and eNOS. Incubation of purified Cdc37 with purified wild-type eNOS decreases eNOS activity in vitro. Overexpression of wild-type Cdc37 in BAECs inhibits eNOS activity and NO release, whereas overexpression of S13A-Cdc37 mutant in BAECs increases eNOS activity and NO release. Taken together, these data suggest that Cdc37 has a direct regulatory interaction with eNOS and may play an important role in mediating the eNOS protein complex formation as well as subsequent eNOS phosphorylation and activation.     

Suppression of the nitric oxide pathway in metastatic renal cell carcinoma patients receiving vascular endothelial growth factor-signaling inhibitors

Therapies that target the vascular endothelial growth factor (VEGF) pathway cause hypertension, but the mechanism remains unknown. This cross-sectional study tested the hypothesis that VEGF inhibition causes hypertension by suppressing VEGF-mediated vasodilatory pathways. Urine was collected from 80 patients with metastatic renal cell carcinoma from 2002 to 2009, 40 at baseline and 40 while on VEGF inhibitors. Measured urinary biomarkers include albumin, metabolites of the nitric oxide (NO) pathway and its downstream effector cGMP, and prostaglandin pathway biomarkers prostaglandin E2, 6-keto prostaglandin F1α, and cAMP, all normalized to urinary creatinine. The mean age in both groups was 61.8 years, 76% were men, and urinary albumin was higher in patients receiving VEGF inhibitors (median: 18.4 versus 4.6 mg/g; P = 0.009). cGMP/creatinine was suppressed in patients on VEGF inhibitors (0.28 versus 0.39 pmol/μg; P = 0.01), with a trend toward suppression of nitrate/creatinine (0.46 versus 0.62 μmol/mg; P = 0.09). Both comparisons were strengthened when patients on bevacizumab were excluded, and only those receiving small molecule tyrosine kinase inhibitors were analyzed (cGMP/creatinine: P = 0.003; nitrate/creatinine: P = 0.01). Prostaglandin E2, 6-keto prostaglandin F1α, and cAMP did not differ between groups. These results suggest that hypertension induced by VEGF inhibitors is mediated by suppression of NO production. Prospective studies are needed to explore whether these biomarkers may be useful predictors of efficacy in patients receiving VEGF-targeted therapies.