C-reactive protein down-regulates endothelial nitric oxide synthase expression and promotes apoptosis in endothelial progenitor cells through receptor for advanced glycation end-products

Objective

C-reactive protein (CRP), the prototypic marker of inflammation, has been shown to be an independent predictor of atherosclerosis. CRP can regulate receptor for advanced glycation end-products (RAGE) expression in endothelial progenitor cells (EPCs). Endothelial nitric oxide synthase (eNOS) deficiency is a pivotal event in atherogenesis. It is believed that decreased eNOS bioactivity occurs early in atherogenesis. Therefore, we tested the hypothesis that CRP can alter eNOS expression and promote apoptosis in EPCs through RAGE.

Methods and results

EPCs, isolated from bone marrow, were cultured in the presence or absence of LPS-free CRP (5, 10, 15, 20, and50 μg/ml). RAGE protein expression and siRNA were measured by flow cytometric analysis. PCR was used to detect eNOS mRNA expression. eNOS protein expression was measured by Western blot analysis. A spectrophotometer was used to assess eNOS activity. A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure adhesiveness. A MTT assay was used to determine proliferation. Apoptosis was evaluated by annexin V immunostaining and TUNEL staining. Co-culturing with CRP caused a significant down-regulation of eNOS expression, inhibited the proliferation, migration, and adhesion of EPCs, and induced EPC apoptosis. In addition, these effects were attenuated during RAGE protein expression blockade by siRNA.

Conclusions

CRP, at concentrations known to predict cardiovascular event, directly quenches the expression of eNOS and diminishes NO production, and may serve to impair EPC function and promote EPC apoptosis through RAGE. These data further support a direct role of CRP in the development and/or progression of atherosclerosis and indicate a new pathophysiologic mechanism of disturbed vascular adaptation in atherosclerosis.     

Induction of endothelial nitric oxide synthase expression by IL-17 in human vascular endothelial cells: implications for vascular remodeling in transplant vasculopathy

IL-17 is a signature cytokine of Th17 cells, a recently described subset of effector CD4 T cells implicated in the development of several pathologies. We have examined the role of IL-17 in regulating endothelial NO synthase (eNOS) expression in human vascular endothelial cells (ECs) because of the key role of eNOS in determining the pathological outcome of immune-mediated vascular diseases. In cultured ECs, IL-17 increased expression of eNOS, eNOS phosphorylation at Ser(1177), and NO production. The induction of eNOS expression by IL-17 was prevented by the pharmacological inhibition of NF-κB, MEK, and JNK, as well as by small interfering RNA-mediated gene silencing of these signaling pathways. The expression of IL-17 was then examined by immunohistochemistry in human arteries affected by transplant vasculopathy (TV), a vascular condition that is a leading reflection of chronic heart transplant rejection. IL-17 was expressed by infiltrating leukocytes in the intima of arteries with TV, and the majority of IL-17-positive cells were T cells. The number of IL-17-positive cells was not correlated with the intima/media ratio, but was negatively correlated with the amount of luminal occlusion. There was also a significant positive correlation between the number of IL-17-positive cells and the density of eNOS-expressing luminal ECs in arteries with TV. Altogether, these findings show that IL-17 induces the expression of eNOS in human ECs and that this may facilitate outward expansion of arteries afflicted with TV.     

Busulphan-cyclophosphamide cause endothelial injury, remodeling of resistance arteries and enhanced expression of endothelial nitric oxide synthase

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system.     

Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection

The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.     

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO₂) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO₂ via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO₂-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO₂-FAs stimulated the phosphorylation of eNOS at Ser¹¹⁷⁹. These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO₂-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.     

Regulation of endothelial nitric oxide synthase by protein kinase C

Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M., Titani, K., and Taniguchi, H. (1996) Biochemistry 35, 14651-14658]. Here we report that bovine eNOS protein is phosphorylated at Thr497 in the calmodulin-binding domain by PKC both in vitro and in vivo, and that the phosphorylation negatively regulates eNOS activity. A specific antibody that recognizes only the phosphorylated form of the enzyme was raised against a synthetic phosphopeptide corresponding to the phosphorylated domain. The antibody recognized eNOS immunoprecipitated with anti-eNOS antibody from the soluble fraction of bovine aortic endothelial cells, and the immunoreactivity increased markedly when the cells were treated with phorbol 12-myristate 13-acetate. PKC phosphorylated eNOS specifically at Thr497 with a concomitant decrease in the NOS activity. Furthermore, the phosphorylated eNOS showed reduced affinity to calmodulin. Therefore, PKC regulates eNOS activity by changing the binding of calmodulin, an eNOS activator, to the enzyme.     

人eNOS复制缺陷型重组腺病毒的构建

目的:构建以巨型细胞病毒(CMV)为启动子的heNCS重组腺病毒转移载体.方法和结果:将heNOS cDNA全长亚克隆到穿梭质粒启动子CMV的下游,通过I-Ceu Ⅰ和PI-SceⅠ两个稀有酶切位点将目的基因heNOS与腺病毒质粒DNA(pAdeno-X)进行体外连接,获得重组腺病毒质粒DNA(pAdeno-heNOS),后者经限制性内切酶PacⅠ切割,利用脂质体转染法获得heNOS重组腺病毒.PCR双引物法鉴定是否成功构建heNCS重组腺病毒.结论:PCR双引物检测含有heNOS片段,表明成功构建了heNOS重组腺病毒转移载体AdhCMV-heNOS.     

Alternative polyadenylation sites of human endothelial nitric oxide synthase mRNA

The mRNA 3'-untranslated region (3'-UTR) has been shown to have important roles in the regulation of mRNA function. In this study, we investigated the human endothelial nitric oxide synthase (eNOS) 3'-UTR to evaluate its potential regulatory role. 3'-RACE analysis revealed that the human eNOS mRNA has multiple alternative polyadenylation sites. Apart from the proximal site (418bp downstream of the stop codon), we identified two additional distal sites approximately 770 and 1478bp downstream of the stop codon. In addition, Northern analysis showed that the usage of these sites differed among human tissues. Further, amounts of these eNOS mRNAs were changed during growth of cultured human aortic endothelial cells; mRNAs with long 3'-UTRs decreased more rapidly than total mRNA, as cells approached confluency. Thus, the 3'-UTRs of human eNOS results from alternative polyadenylation sites and differ across tissues and during cell growth.     

Protein kinase Cdelta regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.     

Hydrocortisone modulates the effect of estradiol on endothelial nitric oxide synthase expression in human endothelial cells.

The interaction between hydrocortisone and estradiol on the regulation of endothelial nitric oxide synthase (eNOS) expression was investigated in human umbilical vein endothelial cells (HUVECs). Following incubation in medium containing dextran-coated-charcoal-stripped serum (DCC-stripped medium) for 4 days, incubation of HUVECs with 0.1 nM estradiol for 24 hr in the absence of hydrocortisone increased levels of eNOS mRNA measured by ribonuclease protection assay above control (0 nM estradiol). 2 microM hydrocortisone applied for 24 hr preceding and during estradiol application inhibited the estradiol-elicited increase in eNOS mRNA levels, reducing mRNA levels from 134% +/- 14% of control to 85% +/- 5% of control. Significant (ANOVA, p<0.01) reductions of estradiol-mediated increases of mRNA levels occurred over a range of hydrocortisone concentrations (10 nM, p<0.05; 2 microM, p<0.05; n=3-12). In the presence of 2 microM hydrocortisone, 10 nM estradiol significantly reduced eNOS mRNA levels to 59% +/- 3% of control. The ability of hydrocortisone to block or reverse the estradiol-mediated increase in eNOS mRNA levels may provide a link between elevated hydrocortisone levels and decreased NO production, potentially contributing to the development of hypertension and cardiovascular disease in vivo and antagonizing cardioprotective effects of estrogens.     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis. (Mol Cell Biochem 264: 193–200, 2004)     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis.     

Vasomotor control in mice overexpressing human endothelial nitric oxide synthase

Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.