Statins, nitric oxide and neovascularization

Several landmark clinical trials suggest that 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) have additional cardiovascular protective activity that may function independently of their ability to lower serum cholesterol. The cardiovascular protective effects of statins are partly caused by the activation of postnatal neovascularization. At therapeutic doses, statins promote proliferation, migration and survival of endothelial cells, induce mobilization and differentiation of bone marrow-derived endothelial progenitor cells by stimulating the serine/threonine protein kinase Akt (also known as protein kinase B) and nitric oxide (NO) signal pathway. However, at excessive doses, statins may decrease protein isoprenylation as well as inhibit endothelial cell growth and migration. NO is an important signaling molecule that regulates a wide range of physiological and pathological processes in different tissues. There is substantial evidence that effective neovascularization requires endothelium-derived NO. Statins have pleiotropic effects on the expression and activity of endothelial nitric oxide synthase (eNOS) and lead to improved NO bioavailability. NO plays an important role in the effects of statins on neovascularization. In this review, we focus on the effects of statins on neovascularization and highlight specific novel targets, such as endothelial progenitor cells and NO.     

PGC-1α ameliorates AngiotensinII-induced eNOS dysfunction in human aortic endothelial cells

Increasing evidences support that PGC-1α participates in regulating endothelial homeostasis, in part by mediating endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the molecular mechanisms by which PGC-1α regulates eNOS activity are not completely understood. In the present study, we investigated the effects of PGC-1α on eNOS dysfunction and further explore the underlying mechanisms. The results showed that PGC-1α expression was downregulated after AngiotensinII (AngII) treatment and paralleled with the decreased NO generation in human aortic endothelial cells. Overexpression of PGC-1α with adenovirus or pharmacological agonist ameliorated AngII-induced the decrease of NO generation, evidenced by the restoration of cGMP and nitrite concentration. Rather than affecting eNOS expression and uncoupling, PGC-1α inhibited AngII-induced decrease of eNOS serine 1177 phosphorylation through activation of PI3K/Akt signaling. In addition, PGC-1α overexpression suppressed AngII-induced the increase of PP2A-A/eNOS interaction and PP2A phosphatase activity, with a concomitant decrease in PP2A phosphorylation, leading to eNOS serine 1177 phosphorylation. However, pharmacological inhibition of PI3K/Akt signaling blunted the observed effect of PGC-1α on PP2A activity. Taken together, our findings suggest that PGC-1α overexpression improves AngII-induced eNOS dysfunction and that improved eNOS dysfunction is associated with activated PI3K/Akt pathway, impaired PP2A activity and reduced PP2A-A/eNOS association. These date indicate that forced PGC-1α expression may be a novel therapeutic approach for endothelial dysfunction.     

Influence of statin use on endothelial function: from bench to clinics

Endothelial dysfunction has been shown to be a prognostic factor for cardiovascular disease and improvement of endothelial dysfunction prevents cardiovascular event presentation. Endothelial dysfunction is associated to a reduced nitric oxide (NO) bioactivity, as a result of the impairment of NO synthesis/release by the endothelial NO synthase (eNOS) or by inactivation of NO. Endothelial dysfunction measurements are valuable surrogate markers to assess the effectiveness of interventions addressed to prevent or treat coronary heart disease (CHD). Dyslipemia and other cardiovascular risk factors promote endothelial dysfunction and life style changes and pharmacological treatment, particularly HMG-CoA reductase inhibitors (statins), have shown early improve of endothelial-dependent vasomotion. Statins efficiently reduce plasma LDL cholesterol, an effect that may account for their beneficial effect on endothelial function, but they also reduce cellular levels of isoprenoid compounds relevant for the bioavailability of NO. Statins restore NO production by several mechanisms, including up-regulation of eNOS mRNA and protein levels and preservation of NO inactivation by reactive oxygen species (ROS). These effects are mediated, at least in a part, through mechanisms independent of their lipid lowering effect (pleiotropic effects). In this article we discuss the relevance of endothelium-dependent effects on the early and delayed clinical benefit of statins, as well as the multiple ways by which statins may restore endothelial function acting not only on the endothelium but also on endothelial progenitor cells (EPC), which likely could contribute to both ischemia-induced neovascularization and endothelial regeneration after injury.     

Vanadate is a potent activator of endothelial nitric-oxide synthase: evidence for the role of the serine/threonine kinase Akt and the 90-kDa heat shock protein

We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure of bovine lung microvascular cells (BLMVEC) to vanadate increased the release of biologically active NO in endothelium/smooth muscle cocultures, as measured by the accumulation of its surrogate marker, cGMP. This release was sensitive to NO synthase (NOS) inhibition and was greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal and ionomycin-induced NO production, they failed to inhibit vanadate-induced NO release. Moreover, in the absence of calcium/calmodulin, cell lysates from vanadate-treated cells exhibited greater NOS activity compared with control cells. Vanadate activates the phosphoinositide3-kinase (PI3-K)/Akt pathway, which is known to increase endothelial NOS (eNOS) activity by direct phosphorylation of Ser-1179. Treatment of BLMVEC with vanadate resulted in phosphorylation of both Akt and endothelial NOS. In addition, wortmannin, a PI3-K inhibitor, blocked both the vanadate-induced phosphorylation of eNOS and the increase in cGMP accumulation. Similarly, adenovirus-mediated gene transfer of an activation deficient form of Akt (AA-Akt) blocked the release of NO brought about by vanadate. To further investigate the mechanism of action of vanadate, eNOS was immunoprecipitated and its association with proteins that alter eNOS activity was tested. Immunoblots demonstrated that the eNOS-caveolin interaction remained unaffected by vanadate, whereas vanadate promoted recruitment of the 90-kDa heat shock protein (hsp90) to eNOS. We conclude that vanadate causes NO release via a mechanism that involves Akt-induced eNOS phosphorylation and increased binding of the activator protein hsp90 to eNOS.     

Nitric oxide homeostasis as a target for drug additives to cardioplegia

The vascular endothelium of the coronary arteries has been identified as the important organ that locally regulates coronary perfusion and cardiac function by paracrine secretion of nitric oxide (NO) and vasoactive peptides. NO is constitutively produced in endothelial cells by endothelial nitric oxide synthase (eNOS). NO derived from this enzyme exerts important biological functions including vasodilatation, scavenging of superoxide and inhibition of platelet aggregation. Routine cardiac surgery or cardiologic interventions lead to a serious temporary or persistent disturbance in NO homeostasis. The clinical consequences are "endothelial dysfunction", leading to "myocardial dysfunction": no- or low-reflow phenomenon and temporary reduction of myocardial pump function. Uncoupling of eNOS (one electron transfer to molecular oxygen, the second substrate of eNOS) during ischemia-reperfusion due to diminished availability of L-arginine and/or tetrahydrobiopterin is even discussed as one major source of superoxide formation. Therefore maintenance of normal NO homeostasis seems to be an important factor protecting from ischemia/reperfusion (I/R) injury. Both, the clinical situations of cardioplegic arrest as well as hypothermic cardioplegic storage are followed by reperfusion. However, the presently used cardioplegic solutions to arrest and/or store the heart, thereby reducing myocardial oxygen consumption and metabolism, are designed to preserve myocytes mainly and not endothelial cells. This review will focus on possible drug additives to cardioplegia, which may help to maintain normal NO homeostasis after I/R.     

HMG-CoA reductase inhibitor protects against in vivo arterial thrombosis by augmenting platelet-derived nitric oxide release in rats

Acute coronary syndromes are caused by platelet-mediated thrombosis following rupture of a plaque. HMG-CoA reductase inhibitors (statins) reduce the incidence of events early after acute coronary syndromes, which are independent of its cholesterol-lowering effect. Accordingly, we investigated whether statins inhibit platelet-mediated arterial thrombus formation in vivo and, if so, the underlying mechanisms. Rats were divided into 4 groups. Group 1 was treated with the vehicle, whereas groups 2, 3, and 4 were treated with cerivastatin for 7 days (1, 2, and 5 mg/kg i.p., respectively). Cerivatatin did not change serum cholesterol levels. Carotid arterial thrombosis was created by perivascular FeCl3 delivery. Cerivastatin significantly prolonged the time to thrombotic occlusion of carotid artery. Cerivastatin significantly dose-dependently inhibited both ex vivo platelet P-selectin expression, a marker of platelet activation, and platelet aggregation. Cerivastatin significantly augmented platelet-derived nitric oxide (NO) release, and up-regulated platelet and endothelial nitric oxide synthase (NOS) mRNA expressions. N-nitro-L-arginine methylester abolished the effects of cerivastatin. This study demonstrates that in vivo administration of statin protects against platelet-mediated arterial thrombosis, possibly by augmenting platelet- and endothelium-derived NO releases via up-regulation of platelet and endothelial NOS.     

Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway

Salidroside (SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Given the importance of endothelial nitric oxide synthase (eNOS) in atherosclerosis, we sought to elucidate whether SAL could stimulate eNOS activation and also to explore its upstream signaling pathway. Six-week old apoE^−/− male mice were fed a high-fat diet for 8 weeks and then were administered with SAL for another 8 weeks. SAL significantly improved endothelial function associated with increasing eNOS activation, thus reduced the atherosclerotic lesion area. SAL increased eNOS-Ser1177 phosphorylation and decreased eNOS-Thr495 phosphorylation, indicative of eNOS activation in endothelium. The aortic sinus lesions in SAL treated mice displayed reduced inflammation. SAL significantly activated AMP-activated protein kinase (AMPK). Both AMPK inhibitor and AMPK small interfering RNA (siRNA) abolished SAL-induced Akt-Ser473 and eNOS-Ser1177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. High performance liquid chromatography (HPLC) analysis revealed that SAL decreased cellular ATP content and increased the cellular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease the mitochondrial membrane potential (ΔΨm), which is a likely consequence of reduced ATP production. The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide (NO) production, which was associated with mitochondrial depolarization and subsequent activation of the AMPK/PI3K/Akt/eNOS pathway. Taken together, our data described the effects of SAL on mitochondria, which played critical roles in improving endothelial function in atherosclerosis.     

Effects of endothelial nitric oxide synthase gene polymorphisms on platelet function,nitric oxide release,and interactions with estradiol

Impaired platelet-derived nitric oxide (NO) contributes to acute coronary syndromes by enhancing platelet recruitment and thrombus formation. Polymorphic variants of the endothelial NO synthase (eNOS) gene have been associated with cardiovascular diseases. To examine whether eNOS variants affect platelet-derived NO and platelet function, and to assess the effects of estradiol on platelet function, we studied platelets from 47 healthy caucasians who were genotyped for eNOS polymorphisms in the promoter region (T-786 C), in intron 4, and in exon 7 (Glu298Asp). Platelet aggregation, platelet-derived NO and superoxide production were measured in control samples and samples pretreated with 17-alpha-estradiol (10 nmol/l). The occurrence of variants in the promoter region (P = 0.002) or in exon 7 (P = 0.007), but not in intron 4 (P > 0.05), were associated with lower levels of platelet-derived NO. An increased (P = 0.047) release of superoxide was observed with platelets from subjects with the variant in the promoter region, but not with other eNOS genetic variants. The eNOS gene polymorphisms did not affect ADP-induced platelet aggregation (P > 0.05). However, estradiol significantly increased platelet aggregation (P = 0.004), and platelet-derived superoxide (P = 0.047) in individuals homozygous for the variant in exon 7, but not in subject with other genotypes. These data demonstrate that the eNOS variants in the promoter region and in exon 7 decrease platelet-derived NO and that estradiol significantly increases platelet aggregation in homozygous for the variant in exon 7 but not in subjects with other genotypes, suggesting that eNOS variants may influence the thrombotic response.     

Adverse balance of nitric oxide/peroxynitrite in the dysfunctional endothelium can be reversed by statins

Vascular endothelial dysfunction is a complex phenomenon that might be caused by a deficiency of nitric oxide (NO) and an overproduction of peroxynitrite (ONOO-). This study used a nanotechnological approach to monitor the in vitro effect of statins on the [NO]/[ONOO-] balance in normal and dysfunctional endothelial cells. NO and (ONOO-) were measured by electrochemical nanosensors in a single human umbilical vein endothelial cell (HUVEC) treated with atorvastatin or simvastatin for 24 hours in the presence or absence of 50 microg/mL oxidized-LDL. An imbalance between [NO]/[ONOO-] concentrations was used as an indicator of endothelial dysfunction and correlated with endothelial nitric oxide synthase (eNOS) expression. Ox-LDL induced dysfunction of the endothelium by uncoupling eNOS. NO concentration decreased from 300 +/- 12 to 146 +/- 8 nmol/L and (ONOO-) increased from 200 +/- 9 to 360 +/- 13 nmol/L. The [NO]/[ONOO-] balance decreased from 1.50 +/- 0.04 (control) to 0.40 +/- 0.03 for cells co-incubated with ox-LDL. Treatment with statins reversed eNOS uncoupling, induced by oxidized-LDL and significantly increased the [NO]/[ONOO-] balance to 1.2 +/- 0.1. These results demonstrate that statins can restore endothelial function by increasing eNOS expression, decreasing eNOS uncoupling, reducing the (ONOO-) level (nitroxidative stress), and shifting the [NO]/[ONOO-] balance towards NO.     

萝卜硫素通过Src/PI3K/Akt通路调控人脐静脉内皮细胞NO的生成机制

目的：研究萝卜硫素（SFN）促进人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）生成一氧化氮（NO）,参与内皮细胞修复的作用机制。方法：采用MTT法检测SFN对HUVEC细胞存活率的影响;检测SFN对HUVEC NO释放量的影响;采用Western blot法检测SFN对NO释放途径相关蛋白表达量的影响。结果：SFN浓度低于50 μmol·L^-1时,对HUVEC细胞无明显毒性（P<0.05）;SFN可显著增加NO释放量,呈剂量依赖性,eNOS特异性抑制剂L-NAME明显抑制NO生成（P<0.05）;SFN呈时间和剂量依赖性显著增加eNOS磷酸化（p-eNOS）水平,促使Akt磷酸化（p-Akt）及PI3K上游调节因子Src激酶的活化。结论：SFN通过Src/PI3K/Akt信号通路增强HUVEC细胞中eNOS的活性,促进NO生成参与内皮细胞修复,为SFN在预防内皮功能障碍、动脉粥样硬化和其他心血管疾病方面的应用提供实验依据。     

Diabetes mellitus and vascular endothelial dysfunction: current perspectives

Patients with diabetes mellitus (DM) have a high prevalence of coronary artery disease (CAD), as diabetes is implicated in the formation of atherosclerotic plaque. Endothelial dysfunction is one of the precursor key steps in the development of atherosclerosis in diabetic subjects. Decreased nitric oxide (NO) production, increased oxidative stress and impaired function of endothelial progenitor cells are the main mechanisms involved in the accelerated atherosclerotic process observed in type 2 DM patients. Therapeutic approaches including classic agents such as statins, angiotensinconverting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), antioxidants and novel agents such as tetrahydrobiopterin (BH4), asymmetric dimethylarginine (ADMA) and homocysteine (tHcy), have been implicated in order to ameliorate endothelial function of diabetic patients.     

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N^G-nitro-l-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease.     

Effects of resistin on insulin signaling in endothelial cells

The objective of this study was to investigate the effects of resistin on insulin signaling in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with recombinant human resistin (0–100 ng/mL) for 24 h. Akt and endothelial nitric oxide synthase (eNOS) phosphorylation levels of endothelial cells under basal or insulin stimulated conditions were measured by Western blot. Nitric oxide (NO) production of HUVECs was also detected. The results showed that resistin could significantly inhibit Akt and eNOS phosphorylation and NO production in endothelial cells under insulin stimulated conditions (P < 0.05 vs control). But under basal conditions, treatment with resistin could result in a decrease in eNOS phosphorylation (P < 0.05 vs control) but had no effect on NO production and Akt phosphorylation levels. These findings suggested that resistin exerted an inhibitory effect on NO production by inhibiting insulin signaling and eNOS phosphorylation in endothelial cells.     

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.     

Ginsenoside Rb1 prevents homocysteine-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition

Hyperhomocysteinemia (HHcy), a risk factor for cardiovascular disease, is associated with endothelial dysfunction. Ginsenoside Rb1, the major active constituent of ginseng, potently attenuates homocysteine (Hcy)-induced endothelial damage. However, the underlying mechanism remains unknown. In this study, we have investigated the effect of Ginsenoside Rb1 on Hcy-induced endothelial dysfunction and its underlying signal pathway in vivo and in vitro. Ginsenosides prevented Hcy-induced impairment of endothelium-dependent relaxation and Rb1 reversed Hcy-induced reduction of NO production in a dose-dependent manner as detected by nitrate reductase method. Rb1 activated serine-1177 phosphorylation of endothelial nitric oxide synthase (eNOS) and serine-473 phosphorylation of Akt, while inhibited threonine-495 phosphorylation of eNOS as detected by western blotting. Rb1-induced phosphorylation of serine-1177 was significantly inhibited by wortmannin, PI3K inhibitor or SH-5, an Akt inhibitor, and partially reversed by Phorbol 12-myristate 13-acetate (PMA), a PKC activator. PMA also stimulated phosphorylation of threonine-495 which was inhibited by Rb1. Here we show for the first time that Rb1 prevents Hcy-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.     

Akt-dependent phosphorylation of serine 1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide

Hydrogen peroxide mediates vasodilation, but the mechanisms responsible for this process remain undefined. We examined the effect of H(2)O(2) on nitric oxide (NO*) production and the signaling events involved. NO* release from bovine aortic endothelial cells was detected with an NO*-specific microelectrode. The addition of H(2)O(2) caused a potent dose-dependent increase in NO* production. This was partially Ca(2+)-dependent because BAPTA/AM reduced NO* production at low (<50 microM) but not high (>100 microM) concentrations of H(2)O(2). Phosphatidylinositol (PI) 3-kinase inhibition [with wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], infection with a dominant-negative mutant of Akt, or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) inhibition (with PD98059 or U0126) partially attenuated, whereas inhibition of both PI 3-kinase and MEK1/2 abolished H(2)O(2)-dependent NO* production. ERK1/2 seemed necessary for NO* production early (<5 min) after H(2)O(2) addition, whereas PI 3-kinase/Akt was more important at later time points. Phosphorylation of endothelial nitric-oxide synthase (eNOS) at serine 1179 was observed >10 min after the addition of H(2)O(2), and this was prevented by wortmannin but not by PD98059. c-Src family tyrosine kinase(s) was found to be upstream of H(2)O(2)-dependent Akt and eNOS serine 1179 phosphorylation and subsequent NO* production. In summary, H(2)O(2) causes endothelial NO* release mediated by cooperative effects between PI 3-kinase/Akt-dependent eNOS serine 1179 phosphorylation and activation of MEK/ERK1/2. This may represent an acute cellular adaptation to an increase in oxidant stress.     

Poly(ADP‐ribose) polymerase 1 deficiency increases nitric oxide production and attenuates aortic atherogenesis through downregulation of arginase II

Poly (ADP ‐ribose) polymerase (PARP ) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular‐related diseases. However, whether PARP regulates nitric oxide (NO ), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP ‐1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E‐deficient (apoE^?/?) mice fed a high‐cholesterol diet for 12 weeks, PARP ‐1 inhibition via treatment with 3,4‐dihydro‐54‐(1‐piperindinyl) butoxy‐1(2H)‐isoquinoline (DPQ ) or PARP ‐1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II ) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAEC s), PARP ‐1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS ) was preserved in the aortas and MAEC s when PARP ‐1 was inhibited. Reduced NO production in vitro due to PARP ‐1 deficiency could be restored by treating the MAEC s with oxidized low‐density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP ‐1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.     

Benidipine, a dihydropyridine-calcium channel blocker, inhibits lysophosphatidylcholine-induced endothelial injury via stimulation of nitric oxide release.

Benidipine hydrochloride (benidipine), which is a long-lasting dihydropyridine calcium channel blocker, exerts antihypertensive action via inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels. In addition, benidipine is shown to restore endothelial function. However, the mechanisms whereby benidipine has protective effects on endothelium are poorly defined. Nitric oxide (NO), which is produced by endothelial NO synthase (eNOS), plays important roles in endothelial function. In this study, we examined effects of benidipine on NO production from human umbilical vein endothelial cells. Benidipine (0.3-10 microM) augmented eNOS expression and total eNOS enzymatic activities. Benidipine also promoted the production of NO and the accumulation of cGMP, a second messenger of NO. Lysophosphatidylcholine (lysoPC), a component of oxidized low-density lipoproteins, induced caspase-3 activation followed by apoptosis of endothelial cells. Benidipine (0.3-10 microM) prevented lysoPC-induced caspase-3 activation, which was canceled by Nomega-nitro-L-arginine-methyl ester (L-NAME) (250-2500 microM), an inhibitor of NOS. Moreover, diethylenetetraamine NONOate (30-100 microM), a NO donor, inhibited the caspase-3 activation. These results suggested that the increase in NO production by benidipine might be involved in the inhibition of caspase induction. The direct enhancement of endothelial NO release by benidipine may be in part responsible for amelioration of endothelial dysfunction.