Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O₂^•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N^G-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O₂^•− was measured using lucigenin chemiluminescence, and O₂^•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O₂^•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O₂^•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.     

Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 microM) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, a specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.     

Suppression of endothelial cell adhesion by XJP-1, a new phenolic compound derived from banana peel

The adhesion of monocytes to activated vascular endothelial cells is a critical event in the initiation of atherosclerosis. Adhesion is mediated by oxidized low-density lipoprotein (ox-LDL) which up-regulates inflammatory markers on endothelial cells. Here we report that (±) 7, 8-dihydroxy-3-methyl-isochromanone-4 (XJP-1), an inhibitor of ox-LDL-induced adhesion of monocytes to endothelial cells blocks cellular functions which are associated with adhesion. We show that XJP-1 down-regulates ox-LDL-induced over-expression of adhesion molecules (ICAM-1 and VCAM-1) in a dose-dependent manner in human umbilical vein endothelial cells (HUVECs), attenuates ox-LDL-induced up-regulation of low-density lipoprotein receptor (LOX)-1, decreases generation of reactive oxygen species (ROS), blocks translocation of nuclear factor-kappa B (NF-κB) activity, and prevents activation of c-Jun N-terminal kinase (JNK)/p38 pathways in endothelial cells. These findings suggest that XJP-1 may attenuate ox-LDL-induced endothelial adhesion of monocytes by blocking expression of adhesion molecules through suppressing ROS/NF-κB, JNK and p38 pathways.     

Protocatechuic Acid Induces Angiogenesis through PI3K‐Akt‐eNOS‐VEGF Signalling Pathway

In this study, we sought to elucidate whether protocatechuic acid contributes to induce angiogenesis as well as its mechanisms. To this end, we examined the role of protocatechuic acid on human brain microvascular endothelial cell line (HBMEC) proliferation, invasion and tube formation in in vitro. For the study of mechanisms involved, the phosphoinositide 3 kinase (PI3K)‐Akt inhibitor LY294002, the endothelial nitric oxide synthase (eNOS) inhibitor L ‐NAME, vascular endothelial growth factor (VEGF), antagonist sFlt‐1 and VEGF receptor blocker SU‐1498 were used. Proliferation of HBMEC was tested by MTT. Scratch adhesion test was used to assess the ability of invasion. A Matrigel tube formation assay was performed to test capillary tube formation ability. PI3K‐Akt‐eNOS‐VEGF pathway activation in HBMEC was tested by Western blot. Our data suggested that protocatechuic acid induces angiogenesis in vitro by increasing proliferation, invasion and tube formation. VEGF expression was increasing by protocatechuic acid and counteracted by VEGF antagonist sFlt‐1, LY294002 and L‐NAME in HBMEC. Tube formation was increased by protocatechuic acid and counteracted by VEGF receptor blocker‐SU1498, LY294002 and L‐NAME. These data suggest that protocatechuic acid may be a candidate therapy for stroke recovery by promoting angiogenesis via a programmed PI3K/Akt/eNOS/VEGF signalling axis.     

Ellagic acid protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized low-density lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. Previous studies have shown that the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase/nitric oxide (PI3K/Akt/eNOS/NO) pathway is involved in oxLDL-induced endothelial apoptosis. Ellagic acid, a natural polyphenol found in berries and nuts, has in recent years been the subject of intense research within the fields of cancer and inflammation. However, its protective effects against oxLDL-induced injury in vascular endothelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. Our results showed that pretreatment with ellagic acid (5-20 μM) significantly attenuated oxLDL-induced cytotoxicity, apoptotic features, and generation of reactive oxygen species (ROS). In addition, the anti-apoptotic effect of ellagic acid was partially inhibited by a PI3K inhibitor (wortmannin) and a specific eNOS inhibitor (cavtratin) but not by an ERK inhibitor (PD98059). In exploring the underlying mechanisms of ellagic acid action, we found that oxLDL decreased Akt and eNOS phosphorylation, which in turn activated NF-κB and downstream pro-apoptotic signaling events including calcium accumulation, destabilization of mitochondrial permeability, and disruption of the balance between pro- and anti-apoptotic Bcl-2 proteins. Those alterations induced by oxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of oxLDL-induced endothelial apoptosis by ellagic acid is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.     

dl-3n-Butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase signaling pathways

We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.     

Inhibitory effect of reinioside C on monocyte–endothelial cell adhesion induced by oxidized low-density lipoprotein via inhibiting NADPH oxidase/ROS/NF-κB pathway

Monocyte adhesion to activated vascular endothelial cells is the critical event in the initiation of atherosclerosis. Adhesion molecules are inflammatory markers, which are upregulated by oxidized low-density lipoprotein (ox-LDL) and play a pivotal role in atherogenesis. In present study, the effect of reinioside C, a major compound of Polygala fallax Hemsl., on adhesion of monocytes to endothelial cells induced by ox-LDL was investigated. The results showed that incubation of endothelial cells with ox-LDL (100?µg/mL) for 24 h markedly increased the expression of ICAM-1 and P-selectin and enhanced the adhesion of monocytes to endothelial cells. Pretreatment with reinioside C (1, 3, or 10?µM) dose-dependently decreased ox-LDL-induced upregulation of expression of ICAM-1 and P-selectin and the enhanced adhesion of monocytes to endothelial cells. To determine the role of NADPH oxidase/reactive oxygen species (ROS)/nuclear factor-κB (NF-κB) pathway, endothelial cells were treated with ox-LDL (100?µg/mL) for 2 h, and NADPH oxidase subunit (Nox 2 and p22^phox) mRNA expression, intracellular ROS level, and NF-κB activity were measured. The results showed that reinioside C attenuated ox-LDL-induced NADPH oxidase subunit (Nox 2 and p22^phox) mRNA expression, generation of ROS, and activation of NF-κB in endothelial cells in a dose-dependent manner; the two latter effects were inhibited by pyrollidine dithiocarbamate, the inhibitor of NF-κB. These findings suggest that reinioside C attenuates ox-LDL-induced expression of adhesion molecules (P-selectin and ICAM-1) and the adhesion of monocytes to endothelial cells by inhibiting NADPH oxidase/ROS/NF-κB pathway.     

Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation

BACKGROUND AND PURPOSE

Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS).

EXPERIMENTAL APPROACH

To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059).

KEY RESULTS

FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS.

CONCLUSIONS AND IMPLICATIONS

Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation.     

阿托伐他汀与氨氯地平对ox-LDL损伤的人脐静脉内皮细胞LOX-1和eNOS表达的影响

目的以人脐静脉内皮细胞(HUVECs)为载体,探讨阿托伐他汀与氨氯地平对氧化低密度脂蛋白(ox-LDL)损伤的HUVECs内血凝素样氧化型低密度脂蛋白受体(LOX-1)和内皮型一氧化氮合酶(eNOS)表达的影响。方法体外培养HUVECs,采用倒置显微镜观察内皮细胞形态变化,待细胞生长至融合状态时加入ox-LDL、阿托伐他汀、氨氯地平进行干预。随机分为:①对照组(培养基);②ox-LDL组(50mg/L);③阿托伐他汀组(10μmol/L);④氨氯地平组(10μmol/L);⑤阿托伐他汀+氨氯地平组。应用实时荧光定量聚合酶链反应(RT-PCR)检测各组LOX-1mRNA与eNOSmRNA表达的水平。结果与正常对照组比较,ox-LDL使LOX-1mRNA表达增加,eNOSmRNA表达减少;阿托伐他汀、氨氯地平均可使ox-LDL损伤的HUVECs内LOX-1mRNA表达下调,eNOSmRNA表达上调,且二者具有协同作用;混合刺激组与对照组比较,LOX-1mRNA、eNOSmRNA表达差异均无统计学意义(P>0.05)。结论阿托伐他汀和氨氯地平均可下调ox-LDL损伤的HUVECs内LOX-1mRNA的表达,上调eNOSmRNA的表达,且二者具有协同作用。     

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.     

Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: role of ETB receptor, NADPH oxidase and caveolin-1

1 Endothelin-1 (ET-1), an endothelium-derived vasoactive peptide, participates in the regulation of endothelial function through mechanisms that are not fully elucidated. This study examined the impact of ET-1 on oxidative stress, apoptosis and cell proliferation in human umbilical vein endothelial cells (HUVEC). HUVECs were challenged for 24 h with ET-1 (10 pM-10 nM) in the absence or presence of the ET(B) receptor antagonist BQ788 (1 microM) or the NADPH oxidase inhibitor apocynin (1 microM). Reactive oxygen species (ROS) were detected using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated with 4',6'-diamidino-2'-phenylindoladihydrochloride staining and by the caspase-3 assay. Cell proliferation was measured by a colorimetric assay. Expression of NADPH oxidase, Akt, pAkt, Bcl-2, Bax, IkappaB, caveolin-1 and eNOS was evaluated by Western blot analysis. 2 ET-1 significantly enhanced ROS generation and cell proliferation following 24-h incubation, both of which were prevented by BQ788 or apocynin, consistent with the ability of ET-1 to directly upregulate NADPH oxidase. ET-1 itself did not affect apoptosis but attenuated homocysteine-induced apoptosis through an ET(B) receptor-mediated mechanism. Western blot analysis indicated that ET-1 alleviated homocysteine (Hcy)-induced apoptosis, likely acting by antagonizing the Hcy-induced decreases in Akt, pAkt, pAkt-to-Akt, Bcl-2-to-Bax ratios and increases in Bax and caveolin-1 expression. Furthermore, ET-1 downregulated expression of caveolin-1 and eNOS, which was attenuated by BQ788 or apocynin. 3 In summary, our results suggest that ET-1 affects oxidative stress, proliferation and apoptosis possibly through ET(B), NADPH oxidase, Akt, Bax and caveolin-1-mediated mechanisms.     

Myricitrin attenuates endothelial cell apoptosis to prevent atherosclerosis: An insight into PI3K/Akt activation and STAT3 signaling pathways

Blood vessel endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) has been implicated in the pathogenesis of atherosclerosis and vasculopathy. The ox-LDL-elicited reactive oxygen species (ROS) release has been assumed to serve a critical function in endothelial damage. Myricitrin (from Myrica cerifera) is a natural antioxidant that has strong anti-oxidative, anti-inflammatory, and anti-nociceptive activities. However, the protective effect of myricitrin on ROS-induced endothelial cell injury and its related molecular mechanisms have never been investigated. This study demonstrates that myricitrin can inhibit ox-LDL-induced endothelial apoptosis and prevent plaque formation at an early stage in an atherosclerotic mouse model. The administration of myricitrin in vivo decreases the thickness of the vascular wall in the aortic arch of ApoE −/− mice. In vitro study shows that ox-LDL-induced human umbilical vein endothelial cell apoptosis can be reduced upon receiving myricitrin pre-treatment. Treatment with myricitrin significantly attenuated ox-LDL-induced endothelial cell apoptosis by inhibiting LOX-1 expression and by increasing the activation of the STAT3 and PI3K/Akt/eNOS signaling pathways. At the same time, our result demonstrates that myricitrin treatment optimizes the balance of pro/anti-apoptosis proteins, including Bax, Bad, XIAP, cIAP-2, and survivin. Our study suggests that myricitrin treatment can effectively protect cells from ox-LDL-induced endothelial cell apoptosis, which results in reduced atherosclerotic plaque formation. This result indicates that myricitrin can be used as a drug candidate for the treatment of cardiovascular diseases.     

Gomisin J from Schisandra chinensis induces vascular relaxation via activation of endothelial nitric oxide synthase

Gomisin J (GJ) is a lignan contained in Schisandra chinensis (SC) which is a well-known medicinal herb for improvement of cardiovascular symptoms in Korean. Thus, the present study examined the vascular effects of GJ, and also determined the mechanisms involved. Exposure of rat thoracic aorta to GJ (1-30μg/ml) resulted in a concentration-dependent vasorelaxation, which was more prominent in the endothelium (ED)-intact aorta. ED-dependent relaxation induced by GJ was markedly attenuated by pretreatment with L-NAME, a nitric oxide synthase (NOS) inhibitor. In the intact endothelial cells of rat thoracic aorta, GJ also enhanced nitric oxide (NO) production. In studies using human coronary artery endothelial cells, GJ enhanced phosphorylation of endothelial NOS (eNOS) at Ser(1177) with increased cytosolic translocation of eNOS, and subsequently increased NO production. These effects of GJ were attenuated not only by calcium chelators including EGTA and BAPTA-AM, but also by LY294002, a PI3K/Akt inhibitor, indicating calcium- and PI3K/Akt-dependent activation of eNOS by GJ. Moreover, the levels of intracellular calcium were increased immediately after GJ administration, but Akt phosphorylation was started to increase at 20min of GJ treatment. Based on these results with the facts that ED-dependent relaxation occurred rapidly after GJ treatment, it was suggested that the ED-dependent vasorelaxant effects of GJ were mediated mainly by calcium-dependent activation of eNOS with subsequent production of endothelial NO.     

Raloxifene protects endothelial cell function against oxidative stress

Background and purpose:

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress.

Experimental approach:

Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis.

Key results:

In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Raloxifene also protected endothelial cell function against H₂O₂. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase.

Conclusion and implications:

Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.     

Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redox-sensitive activation of the PI3-kinase/Akt pathway

Red wine polyphenolic compounds (RWPCs) are potent inducers of endothelium-dependent relaxations of coronary arteries, which involve both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF). The EDHF-mediated relaxation to RWPCs is critically dependent on the formation of reactive oxygen species by a flavin-dependent enzyme. The aim of the present study was to determine the role of redox-sensitive protein kinases including p38 MAPK, ERK1/2 and PI3-kinase/Akt in RWPCs-induced EDHF-mediated relaxation. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension. Confluent cultures of porcine coronary artery endothelial cells were used to determine the phosphorylation level of p38 MAPK, ERK1/2 and Akt by Western blot analysis. All experiments were performed in the presence of indomethacin and Nomega-nitro-L-arginine. RWPCs caused pronounced endothelium-dependent relaxations, which were significantly reduced by wortmannin and LY294002, two inhibitors of PI3-kinase, and not affected by PD98059 (an inhibitor of ERK1/2 kinase kinase) and SB203580 (an inhibitor of p38 MAPK). In contrast, wortmannin did not affect relaxations to bradykinin or levcromakalim. RWPCs elicited within minutes a sustained and concentration-dependent phosphorylation of p38 MAPK, ERK1/2 and Akt in endothelial cells. The phosphorylation of Akt in response to RWPCs was abolished by wortmannin and LY294002, and by the membrane-permeant analogue of superoxide dismutase Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin. The present findings demonstrate that RWPCs cause EDHF-mediated relaxations of coronary arteries; these responses are critically dependent on the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells.     

Genistein ameliorated endothelial nitric oxidase synthase uncoupling by stimulating sirtuin-1 pathway in ox-LDL-injured HUVECs

Endothelial nitric oxidase synthase (eNOS) uncoupling plays a causal role in endothelial dysfunction in atherosclerosis. Genistein consumption has been associated with the prevention of atherosclerosis. However, the effect of genistein on eNOS uncoupling has not been reported. A model of oxidized low-density lipoprotein (ox-LDL)-induced injury on human umbilical vein endothelial cells (HUVECs) was established to evaluate the effect of genistein on eNOS uncoupling. We investigated the effect of genistein on NADPH oxidase-dependent superoxide production, NOX4 expression, BH4 synthesis and oxidation, the expression of GTP cyclohydrolase 1 (GCH1) and dihydrofolate reductase (DHFR). The results showed that genistein decreased superoxide production and NOX4 expression, enhanced the ratio of BH4/BH2, augmented the expressions of GCH1 and DHFR. Accompanied with genistein ameliorating eNOS uncoupling, genistein elevated the expression of sirtuin-1; furthermore, the effects of genistein on eNOS uncoupling were blunted with sirtuin-1 siRNA. The present study indicated that genistein ameliorated eNOS uncoupling was concerned with sirtuin-1 pathway in ox-LDL-injured HUVECs.     

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.