The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.     

Induction of cytoprotective autophagy in PC-12 cells by cadmium

Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.     

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2^−/− MEFs but not JNK1^−/− MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.     

Release of nitric oxide and expression of constitutive nitric oxide synthase of human endothelial cells: Enhancement by a 14-membered ring macrolide

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca²⁺]_i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca²⁺]_i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.     

YM155 sensitizes non-small cell lung cancer cells to EGFR-tyrosine kinase inhibitors through the mechanism of autophagy induction

Resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib and gefitinib, is a major clinical problem in the treatment of patients with non-small cell lung cancer (NSCLC). YM155 is a survivin small molecule inhibitor and has been demonstrated to induce cancer cell apoptosis and autophagy. EGFR-TKIs have been known to induce cancer cell autophagy. In this study, we showed that YM155 markedly enhanced the sensitivity of erlotinib to EGFR-TKI resistant NSCLC cell lines H1650 (EGFR exon 19 deletion and PTEN loss) and A549 (EGFR wild type and KRAS mutation) through inducing autophagy-dependent apoptosis and autophagic cell death. The effects of YM155 combined with erlotinib on apoptosis and autophagy inductions were more obvious than those of YM155 in combination with survivin knockdown by siRNA transfection, suggesting that YM155 induced autophagy and apoptosis in the NSCLC cells partially depend on survivin downregulation. Meanwhile, we found that the AKT/mTOR pathway is involved in modulation of survivin downregulation and autophagy induction caused by YM155. In addition, YM155 can induce DNA damage in H1650 and A549 cell lines. Moreover, combining erlotinib further augmented DNA damage by YM155, which were retarded by autophagy inhibitor 3MA, or knockdown of autophagy-related protein Beclin 1, revealing that YM155 induced DNA damage is autophagy-dependent. Similar results were also observed in vivo xenograft experiments. Therefore, combination of YM155 and erlotinib offers a promising therapeutic strategy in NSCLC with EGFR-TKI resistant phenotype.     

Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid that exerts potent anti-cancer effects in vitro and in vivo. It was shown to induce apoptosis via a direct effect on mitochondria. This is largely independent of proapoptotic BAK and BAX, but can be inhibited by cyclosporin A (CsA), an inhibitor of the permeability transition (PT) pore. Here we show that blocking apoptosis with general caspase inhibitors did not prevent cell death, indicating that alternative, caspase-independent cell death pathways were activated. BetA did not induce necroptosis, but we observed a strong induction of autophagy in several cancer cell lines. Autophagy was functional as shown by enhanced flux and degradation of long-lived proteins. BetA-induced autophagy could be blocked, just like apoptosis, with CsA, suggesting that autophagy is activated as a response to the mitochondrial damage inflicted by BetA. As both a survival and cell death role have been attributed to autophagy, autophagy-deficient tumor cells and mouse embryo fibroblasts were analyzed to determine the role of autophagy in BetA-induced cell death. This clearly established BetA-induced autophagy as a survival mechanism and indicates that BetA utilizes an as yet-undefined mechanism to kill cancer cells.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.     

Extracellular histones disarrange vasoactive mediators release through a COX‐NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.     

Neuromedin B induces angiogenesis via activation of ERK and Akt in endothelial cells

Neuromedin B (NMB) is one of the bombesin-like peptides in mammals. Recently, bombesin-like peptides have been characterized as growth factors in highly vascularized tumors. In this study, we report that NMB potently stimulates in vivo neovascularization in a mouse Matrigel plug and the sprouting of endothelial cells ex vivo in rat aortic rings. In addition, NMB increases the migration and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with NMB activates the extracellular signal-regulated kinase 1/2 (ERK_1/2), Akt, and endothelial nitric oxide synthase (eNOS) and increases the level of NO production in a dose- and time-dependent manner. Furthermore, ERK activation and angiogenic sprouting in response to NMB are significantly blocked by the MEK inhibitor. Inhibition of phosphatidylinositol 3-kinase (PI3K) suppresses the NMB-stimulated tubular formation of HUVECs, along with reduction in the phosphorylation of Akt and eNOS. Taken together, these results indicate that NMB is a novel angiogenic peptide, and its angiogenic activity is mediated by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent pathways. This study suggests that NMB may play important roles in mediating a variety of pathophysiological angiogenesis.     

Effects of endomorphins on human umbilical vein endothelial cells under high glucose

The endomorphin-1 (EM1) and endomorphin-2 (EM2) are endogenous opioid peptides, which modulate extensive bioactivities such as pain, cardiovascular responses, immunological responses and so on. The present study was undertaken to investigate the effects of EM1/EM2 on the primary cultured human umbilical vein endothelial cells (HUVECs) damaged by high glucose. PI AnnexinV-FITC detection was performed to evaluate the apoptosis rate. Levels of nitric oxide (NO) and nitric oxide synthase (NOS) activity were measured by the Griess reaction and the conversion of 3H-arginine to 3H-citrulline, respectively. Endothelin-1 (ET-1) was evaluated by the enzyme-linked immunosorbent assay (ELISA). Cell proliferation was determined by the MTT viability assay. mRNA expression of endothelial nitric oxide synthase (eNOS) and ET-1 were measured by real-time PCR. Our data showed that EM1/EM2 inhibited cell apoptosis. The high glucose induced increase in expression of NO, NOS and ET-1 were significantly attenuated by pretreatment with EM1/EM2 in a dose dependent manner. In addition, EM1/EM2 suppressed the mRNA eNOS and mRNA ET-1 expression in HUVECs under high glucose conditions. Naloxone, the nonselective opioid receptor antagonist, did not influence the mRNA eNOS expression when it was administrated on its own; but it could significantly antagonize the effects induced by EM1/EM2. Furthermore, in all assay systems, EM1 was more potent than EM2. The results suggest that EM1/EM2 have a beneficial effect in protecting against the endothelial dysfunction by high glucose in vitro, and these effects were mediated by the opioid receptors in HUVECs.     

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.     

Adenosine diphosphate–sensitive P2Y11 receptor inhibits endothelial cell proliferation by induction of cell cycle arrest in the S phase and induces the expression of inflammatory mediators

Extracellular adenosine diphosphate (ADP) mediates a wide range of physiological effects as an extracellular signaling molecule, including platelet aggregation, vascular tone, cell proliferation, and apoptosis by interacting with plasma membrane P2 receptors. However, the effect of ADP on cell proliferation was contradictory. In this study, we found that ADP significantly inhibited cell proliferation of human umbilical vein endothelial cells at high concentrations (50 to 100 µM). Treatment with ADP did not induce cell apoptosis but instead induced cell cycle arrest in the S phase, which may be partly due to the downregulation of cyclin B1. The inhibition of cell proliferation was blocked by suramin, a nonspecific antagonist of the P2 receptors, and high concentrations of ADP significantly upregulated the messenger RNA (mRNA) and protein expression of P2Y11 in endothelial cells. Moreover, the downregulation of P2Y11 by RNA interference reversed the inhibition of cell proliferation. In addition, ADP (100 µM) can induce the formation of cytosolic autophagy in endothelial cells and a rapid phosphorylation of extracellular signal regulated kinase (ERK) 1/2, which is a canonical signal molecule downstream of P2Y receptors, accompanied by a mRNA expression of proinflammatory cytokines such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. Taken together, our study excludes a mechanism for extracellular ADP impairing endothelial cells proliferation via P2Y11 receptor by downregulating cyclin B1 and arresting cell cycle at the S phase, besides, ADP induces cell autophagy and mRNA expression of inflammatory cytokines, whether it is mediated by Erk signaling pathways needs further studies to confirm.     

Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan‐dependent mechanism

Accumulating evidences point to a significant role for the chromogranin A (CgA)‐derived peptide vasostatin 1 (VS‐1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS‐1 induces a PI3K‐dependent‐nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio‐suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high‐affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS‐1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K‐dependent, endocytosis‐coupled mechanism. In bovine aortic endothelial cells (BAE‐1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water‐soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS‐1‐dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS‐1 induces a marked increase in the caveolae‐dependent endocytosis, (115 ± 23% endocytotic spots/cell/field in VS‐1‐treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 ± 15% above control) and Wortmannin (Wm, 7 ± 22% above control). Heparinase, Wortmannin, and methyl‐β‐cyclodextrin (MβCD) abolish the VS‐1‐dependent eNOS phosphorylation (P^Ser1179eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K‐dependent eNOS phosphorylation. J. Cell. Biochem. 110: 70–79, 2010. © 2010 Wiley‐Liss, Inc.     

Genistein‐3′‐sodium sulphonate protects against lipopolysaccharide‐induced lung vascular endothelial cell apoptosis and acute lung injury via BCL‐2 signalling

Under septic conditions, Lipopolysaccharide (LPS)‐induced apoptosis of lung vascular endothelial cells (ECs) triggers and aggravates acute lung injury (ALI), which so far has no effective therapeutic options. Genistein‐3′‐sodium sulphonate (GSS) is a derivative of native soy isoflavone, which has neuro‐protective effects through its anti‐apoptotic property. However, whether GSS protects against sepsis‐induced lung vascular endothelial cell apoptosis and ALI has not been determined. In this study, we found that LPS‐induced Myd88/NF‐κB/BCL‐2 signalling pathway activation and subsequent EC apoptosis were effectively down‐regulated by GSS in vitro. Furthermore, GSS not only reversed the sepsis‐induced BCL‐2 changes in expression in mouse lungs but also blocked sepsis‐associated lung vascular barrier disruption and ALI in vivo. Taken together, our results demonstrated that GSS might be a promising candidate for sepsis‐induced ALI via its regulating effects on Myd88/NF‐κB/BCL‐2 signalling in lung ECs.     

Effect of water-soluble fraction of cigarette smoke on human aortic endothelial cells – a proteomic approach

Proteomic analysis is an important investigative tool used to systematically explore cellular proteins that are responsive to adverse environmental challenges. Tobacco smoking is the second major cause of death in the world. In this study, we utilized two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) technologies to explore protein changes in human aortic endothelial cells (HAECs) in response to cigarette smoke extracts (CSE). Among 389 individual proteins resolved using 2-DE, 43 had a 2- to 3-fold change in levels as measured by spot intensity and 32 had more than a 3-fold change. Sixteen of the 32 spots with sufficient amount of proteins were excised for identification by performing matrix-assisted laser desorption/ionization (MALDI)-MS analysis. Using a peptide mass fingerprinting (PMF) to search the nrNCBI database, we identified all these 16 proteins, which were either increased (n = 9) or decreased (n = 7) after CSE treatment. All these proteins have known functions, however, none have been reported to be altered after CSE treatment. The findings from our study suggest that utilizing a systemic investigative tool, such as the proteomic approach using 2-DE, may play an important role in discovering novel molecular mechanisms for cigarette smoking-induced pathological changes. Further investigation following the systemic discoveries must be further examined as they may potentially lead to new therapeutic approaches to smoking-induced diseases – a health issue affecting everyone in the world.     

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.     

胰岛素促进血管内皮细胞产生一氧化氮的实验研究

目的：探讨胰岛素对血管内皮细胞增殖、NO产生和NOS基因表达的影响。方法：培养牛主动脉内皮细胞,测定培养上清液中NO氧化产物NO2^－的水平并应用定量RT－PCR技术检测内皮细胞NOS mRNA的表达水平。结果：①胰岛素对大血管内皮细胞无细胞毒作用,也不影响细胞增殖;②在1－15μg/ml浓度范围内,胰岛素加强内皮细胞释放NO,且呈剂量依赖的方式,NOS特异性抑制剂L－NAME可阻抑之;③胰岛素轻度增加NOS mRNA表达水平,但无统计学意义。结论：胰岛素既不影响大血管内皮细胞增殖,也不影响内皮细胞NOS mRNA表达水平,但以剂量依赖的方式加强内皮细胞产生NO,推测其诱导NO产生的机制可能是通过酶活性的诱导,加速NO的合成。