Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.     

Long-term swimming exercise does not modulate the Akt-dependent endothelial nitric oxide synthase phosphorylation in healthy mice

Molecular mechanisms by which exercise exerts cardiovascular benefits are poorly understood. Exercise-induced increase of endothelial NO synthase (eNOS) phosphorylation through the protein kinase Akt has been shown to be a key mechanism underlying the beneficial effect of exercise in coronary artery disease patients. We examined whether this protective pathway might also be activated in long-term-exercised healthy mice. C57BL/6 wild-type mice swam for 24 weeks. A group of sedentary animals were used as controls. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at ser473 (p-Akt), total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), and PECAM-1 (platelet endothelial cell adhesion molecule-1) were assessed by Western blotting. Protein expressions of Akt, p-Akt, eNOS, p-eNOS, and PECAM-1 were not modulated by 24 weeks of exercise. The Akt-dependent eNOS phosphorylation did not seem to be a primary molecular adaptation in response to long-term exercise in healthy mice.     

3′-Oxo-tabernaelegantine A (OTNA) selectively relaxes pulmonary arteries by inhibiting AhR

BackgroundPulmonary arterial hypertension (PAH), characterized by pulmonary artery constriction and vascular remodeling, has a high mortality rate. New drugs for the treatment of PAH urgently need to be developed.PurposeThis study was designed to investigate the vasorelaxant activity of OTNA in isolated pulmonary arteries, and explore its molecular mechanism.MethodsPulmonary arteries and thoracic aortas were isolated from mice, and vascular tone was tested with a Wire Myograph System. Nitric oxide levels were determined with DAF-FM DA and DAX-J2™ Red. Cellular thermal shift assays, microscale thermophoresis, and molecular docking were used to identify the interaction between OTNA and aryl hydrocarbon receptor (AhR). The levels of PI3K, p-PI3K, Akt, p-Akt, eNOS, p-eNOS, and AhR were analyzed by Western blotting.ResultsOTNA selectively relaxed the isolated pulmonary artery rings in an endothelium-dependent manner. Mechanistic study showed that OTNA induced NO production through activation of the PI3K/Akt/eNOS pathway in endothelial cells. Furthermore, we also found that OTNA directly bound to AhR and activated the PI3K/Akt/eNOS pathway to dilate pulmonary arteries by inhibiting AhR.ConclusionsOTNA relaxes pulmonary arteries by antagonizing AhR. This study provides a new natural antagonist of AhR as a promising lead compound for PAH treatment.     

Acute Mechanical Stretch Promotes eNOS Activation in Venous Endothelial Cells Mainly via PKA and Akt Pathways

In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser¹¹⁷⁷, Ser⁶³³ and Ser⁶¹⁵ and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.     

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.     

Microvesicles-mediated communication between endothelial cells modulates,endothelial survival,and angiogenic function via transferring of miR-125a-5p

Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.     

木犀草素对子痫前期大鼠滋养层细胞凋亡的影响及其机制

目的探讨木犀草素（LUT）对子痫前期（PE）大鼠滋养层细胞凋亡的影响及其机制。 方法取妊娠10 d SD大鼠,按随机数字表法随机分为对照组、模型组、20、40、60 mmol/L LUT （LUT-L、LUT-M、LUT-H）组,每组各12只,模型组和给药组大鼠皮下注射100 mg/（kg·d）亚硝基左旋精氨酸甲酯建立PE大鼠模型,对照组大鼠皮下注射等量生理盐水,每天1次,注射6 d。妊娠16 d的大鼠分别予以20、40、60 mmol/L LUT腹腔注射,对照组、模型组大鼠腹腔注射等量生理盐水,每天1次,注射5 d。测量各组大鼠妊娠10、16、21 d尾动脉血压及24 h尿蛋白水平;妊娠21 d,原位末端标记法（TUNEL）检测滋养层组织细胞凋亡情况,Western blot法检测滋养层组织B淋巴细胞瘤-2 （Bcl-2）、Bcl-2相关X蛋（Bax）、磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K （p-PI3K）、蛋白激酶B （Akt）、磷酸化AKT （p-Akt）、内皮型一氧化氮合酶（eNOS）和磷酸化eNOS （p-eNOS）蛋白表达量。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。 结果妊娠10 d,各组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量差异无统计学意义;妊娠16 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量升高（P均< 0.05）;妊娠21 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组收缩压[（110.33±3.67）比（147.28±4.16）,（131.29±4.31）,（124.46±4.27）,（118.54±4.18）mmHg]、24 h蛋白尿、细胞凋亡率[（1.38±0.34）%,（43.45±3.72）%,（39.21±3.53）%,（27.86±3.41）%,（23.21±3.28）%]和Bax蛋白表达量均升高;Bcl-2、p-PI3K/PI3K （1.06±0.09比0.25±0.02,0.37±0.03,0.57±0.06,0.73±0.08）、p-Akt/Akt（0.87±0.08比0.11±0.01,0.23±0.03,0.56±0.07,0.78±0.06）和p-eNOS/eNOS蛋白表达水平（0.85±0.07比0.09±0.01,0.16±0.02,0.38±0.04,0.69±0.07）均降低（P均< 0.05）。与模型组比较,LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、滋养层组织细胞凋亡率和Bax蛋白表达量降低,Bcl-2、p-PI3K/PI3K、p-Akt/Akt和p-eNOS/eNOS蛋白表达量升高（P均< 0.05）。 结论LUT可抑制PE大鼠滋养层组织细胞凋亡,其机制可能与PI3K/Akt/eNOS信号通路激活,调控凋亡相关蛋白表达有关。     

黄芪甲苷抑制缺血再灌注诱导的大鼠心肌细胞凋亡

摘要 目的：本研究旨在探究黄芪甲苷（Astragaloside IV,Ast-IV）在缺血再灌注（ischemia-reperfusion,I/R）大鼠模型中的保护作用,并讨论黄芪甲苷在抑制I/R诱导心肌细胞凋亡过程中的作用。方法：通过左冠状动脉前降支结扎构建I/R大鼠模型;将40只SD大鼠分为4组：假手术组（Sham组）、模型组（I/R组）、黄芪甲苷干预组（Ast组）和黄芪甲苷+LY294002干预组（Ast+LY组）。使用试剂盒测定血清中心脏功能障碍标记物CPK、ALT、LDH和tropornin-T的表达水平;通过HE染色和TUNEL分别检测心肌组织病理学变化和心肌细胞凋亡情况;通过超氧化物荧光探针染色检测细胞内ROS水平;通过ELISA试剂盒测定心肌组织MDA、GSH和GSH-PX含量;免疫组织化学检测SOD2和HO-1蛋白表达水平,分析心肌氧化应激状态;通过Western blot检测PI3K、p-Akt、Akt、p-eNOS、eNOS、caspase-3、Bax和Bcl-2蛋白表达水平变化。结果：Ast组大鼠血浆CPK、ALT、LDH和tropornin-T酶活性均明显低于I/R组（P<0.05）。Ast组大鼠心肌纤维断裂,心肌细胞坏死和炎性细胞浸润等病变程度均低于I/R组。Ast组大鼠TUNEL阳性细胞数低于I/R组（P<0.05)。相较于I/R组,Ast组大鼠Caspase3和Bax表达水平均明显下调,Bcl-2和PI3K蛋白表达水平上调,p-Akt/Akt和p-eNOS/eNOS比值均显著上调（P<0.05）。Ast组大鼠DHE荧光强度显著低于Ast+LY组（P<0.05）。与I/R组相比,Ast组大鼠心肌组织中MDA含量降低,GSH、GSH-PX、SOD2和HO-1表达水平升高（P<0.05）;与Ast组相比,Ast+LY组大鼠心肌组织中MDA含量升高,GSH、GSH-PX、SOD2和HO-1表达水平降低（P<0.05）。结论：黄芪甲苷通过激活PI3K/Akt信号通路,抑制心肌细胞氧化应激反应,从而减少I/R诱导大鼠心肌细胞凋亡,缓解I/R后大鼠心肌损伤。     

Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.     

Retracted: Upregulated microRNA-132 rescues cardiac fibrosis and restores cardiocyte proliferation in dilated cardiomyopathy through the phosphatase and tensin homolog–mediated PI3K/Akt signal transduction pathway

Cardiac fibrosis is known to be present in dilated cardiomyopathy (DCM) and it predicts the occurrence of sudden death and congestive heart failure. The aim of our study is to investigate the expression of microRNA-132 (miR-132) and its effect on cardiocyte proliferation, apoptosis, and cardiac fibrosis by binding to phosphatase and tensin homolog (PTEN) through the phosphateidylinositol 3-kinase (PI3K)/protein kinase (Akt) signal transduction pathway in DCM rats. DCM rat models induced by doxorubicin were established and confirmed by an ultrasonic cardiogram. Epithelial cells were treated with inhibitors, activators, and small interfering RNAs to identify the mechanisms by which miR-132 controls cardiocyte activity and cardiac fibrosis. Angiotensin II (Ang II) and aldosterone (ALD) expressions were detected by an enzyme-linked immunosorbent assay. The relationship between PTEN and miR-132 was verified by a dual-luciferase reporter assay. Cell proliferation and apoptosis were tested by the MTT assay and flow cytometry. PTEN was determined to be the target gene of miR-132. Rat models of DCM exhibited a lower level of miR-132, PI3K, Akt, B-cell lymphoma 2, collagen I, and collagen III, but a higher level of PTEN, Bcl-2–associated X protein, and proliferating cell nuclear antigen as well as inflammatory response (Ang II and ALD), accompanied by declined cardiocyte proliferation and elevated apoptosis and cardiac fibrosis. Upregulated miR-132 or silenced PTEN activated the PI3K/Akt pathway, thus facilitating cardiocyte proliferation and repressing cardiocyte apoptosis and cardiac fibrosis, as well as inflammatory responses. Downregulated miR-132 reversed this tendency. These findings indicate that miR-132 activates the PI3K/Akt pathway by inhibiting PTEN expression, thus facilitating cardiocyte proliferation and inhibiting apoptosis and cardiac fibrosis in DCM rats.     

Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs

Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10~(-7)-10~(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P0.05, P0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.     

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.     

Exercise improves the dilatation function of mesenteric arteries in postmyocardial infarction rats via a PI3K/Akt/eNOS pathway-mediated mechanism

Myocardial infarction (MI) has been shown to induce endothelial dysfunction in peripheral resistance arteries and thus increase peripheral resistance. This study was designed to investigate the underlying mechanisms of post-MI-related dysfunctional dilatation of peripheral resistance arteries and, furthermore, to examine whether exercise may restore dysfunctional dilatation of peripheral resistance arteries. Adult male Sprague-Dawley rats were divided into three groups: sham-operated, MI, and MI + exercise. Ultrastructure and relaxation function of the mesenteric arteries, as well as phosphatidylinositol-3 kinase (PI3K), Akt kinases (Akt), endothelial nitric oxide synthase (eNOS) activity, and phosphorylation of PI3K, Akt, and eNOS by ACh were determined. Post-MI rats exhibited pronounced ultrastructural changes in mesenteric artery endothelial cells and endothelial dysfunction. In addition, the activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh were significantly attenuated in mesenteric arteries (P < 0.05-0.01). After 8 wk of exercise, not only did endothelial cells appeared more normal in structure, but also ameliorated post-MI-associated mesenteric arterial dysfunction, which were accompanied by elevated activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh (P < 0.05-0.01). Importantly, inhibition of either PI3K or eNOS attenuated exercise-induced restoration of the dilatation function and blocked PI3K, Akt, and eNOS phosphorylation by ACh in the mesenteric arteries. These data demonstrate that MI induces dysfunctional dilation of peripheral resistance arteries by degradation of endothelial structural integrity and attenuating PI3K-Akt-eNOS signaling. Exercise may restore dilatation function of peripheral resistance arteries by protecting endothelial structural integrity and increasing PI3K-Akt-eNOS signaling cascades.     

Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells

Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.     

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.     

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning

The opening of mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels has a significant role in delayed ischemic preconditioning, and nitric oxide (NO) is a well-known trigger for its activation. However, the source of NO remains unknown. Phosphorylation of endothelial NO synthase (eNOS) increases NO production and reduces apoptosis through the Akt signaling pathway. To elucidate the Akt signaling pathway involved in the opening and antiapoptotic effect of mitoKATP channel during delayed pharmacological preconditioning, the mitoKATP channel opener diazoxide (DE, 7 microg/kg i.p.) alone or DE plus Nomega-nitro-L-arginine methyl ester (L-NAME, 30 microg/kg i.v.), an inhibitor of NOS, or wortmannin (WTN, 15 microg/kg i.v.), an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), was administered to wild-type (WT) or eNOS(-/-) mice during DE treatment. Twenty-four hours later, hearts were isolated and subjected to 40 min ischemia and 30 min reperfusion (I/R). The effect of DE and other interventions on hemodynamic, terminal dUTP nick-end labeling staining and biochemical changes during I/R was assessed in mouse hearts. Treatment with DE resulted in a 2.2-fold increase in phosphorylation of Akt and a significant increase in eNOS and inducible NOS (iNOS) proteins. Akt is upstream of NOS and the mitoKATP channel as simultaneous pretreatment of WTN with DE abolished phosphorylation of Akt, which was not affected by L-NAME and 5-hydroxydecanoate. In hearts treated with DE, cardiac function was significantly improved after I/R, and apoptosis was also significantly decreased. WTN abolished the antiapoptotic effect of DE. Similarly, S-methylisothiourea, a specific iNOS inhibitor, when given to eNOS(-/-) mice that were pretreated with DE completely abolished the beneficial effects of DE on reduction of apoptotic death. DE was partially effective in eNOS(-/-) mice against the ischemic injury. It is concluded that DE activates Akt through the PI3 kinase signaling pathway and iNOS and eNOS is downstream of Akt.     

Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 μg/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 μg/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. l-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.     

Effects of astragalus polysaccharide on apoptosis of myocardial microvascular endothelial cells in rats undergoing hypoxia/reoxygenation by mediation of the PI3K/Akt/eNOS signaling pathway

The study explores the effect of astragalus polysaccharide (APS) mediating P13K/Akt/eNOS signaling pathway on apoptosis of myocardial microvascular endothelial cells (MMECs) in hypoxia/reoxygenation (H/R). MMECs were classified into blank, H/R, H/R + 25 mg/L APS, H/R + 50 mg/L APS, H/R + 100 mg/L APS, H/R + LY, and HR + 100 mg/L APS + LY groups. Cell viability was detected using MTT assay and apoptotic cell morphological changes by Hoechst staining. NO content, cell cycle and apoptosis, PI3K/Akt/eNOS signaling pathway proteins were detected using nitrate reductase assay, flow cytometry and Western blotting. An increased cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins, and a decreased apoptosis rate was observed in the H/R + 50 mg/L APS and H/R + 100 mg/L APS groups compared with the H/R and H/R + 25 mg/L APS groups. Compared with the H/R + 50 mg/L APS group, the apoptosis rate decreased, whereas the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins increased in the H/R + 100 mg/L APS group. The H/R + LY and HR + 100 mg/L APS + LY groups followed opposite trends. In comparison to the HR + 100 mg/L APS group, the apoptosis rate in the H/R + LY and HR + 100 mg/L APS + LY groups increased, and the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins decreased. Collectively, APS improves the damage caused by H/P by mediating PI3K/Akt/eNOS signaling pathway.