Histone deacetylase 7 silencing alters endothelial cell migration, a key step in angiogenesis

Global inhibition of class I and II histone deacetylases (HDACs) impairs angiogenesis. Herein, we have undertaken the identification of the specific HDAC(s) with activity that is necessary for the development of blood vessels. Using small interfering RNAs, we observed that HDAC7 silencing in endothelial cells altered their morphology, their migration, and their capacity to form capillary tube-like structures in vitro but did not affect cell adhesion, proliferation, or apoptosis. Among several factors known to be involved in angiogenesis, platelet-derived growth factor-B (PDGF-B) and its receptor (PDGFR-beta) were the most upregulated genes following HDAC7 silencing. We demonstrated that their increased expression induced by HDAC7 silencing was partially responsible for the inhibition of endothelial cell migration. In addition, we have also shown that treatment of endothelial cells with phorbol 12-myristate 13-acetate resulted in the exportation of HDAC7 out of the nucleus through a protein kinase C/protein kinase D activation pathway and induced, similarly to HDAC7 silencing, an increase in PDGF-B expression, as well as a partial inhibition of endothelial cell migration. Collectively, these data identified HDAC7 as a key modulator of endothelial cell migration and hence angiogenesis, at least in part, by regulating PDGF-B/PDGFR-beta gene expression. Because angiogenesis is required for tumor progression, HDAC7 may represent a rational target for therapeutic intervention against cancer.     

Folic acid inhibits endothelial cell proliferation through activating the cSrc/ERK 2/NF-κB/p53 pathway mediated by folic acid receptor

Folate is important for normal cell division. Folate deficiency has been implicated in various diseases, including atherosclerosis, neural tube defects, and cancer. However, the effect of folate on angiogenesis was unclear. The aim of this study was to investigate the anti-angiogenic action of folic acid (FA). FA (0–10?μmol/L) concentration-dependently decreased DNA synthesis and proliferation in cultured human umbilical venous endothelial cells (HUVEC). Western blot analyses demonstrated that the levels of p21, p27 and p53 protein in HUVEC were increased by FA. The FA-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Knock-down of p53 prevented the FA-induced increases in p21 and p27 protein level. The levels of phosphorylated Src (p-Src) and p-Src-FA receptor (FR) complex in HUVEC were increased by FA. Knock-down of FR reduced the FA-induced increases of p-Src and p53. The FA-induced increases of p21, p27 and p53 protein levels were abolished when cSrc was knocked-down. FA also increased NF-κB nuclear translocation and binding onto the p53 promoter. The FA-induced up-regulation of the p53 promoter activity was prevented by knocked-down of ERK. Matrigel angiogenesis assay in mice demonstrate the anti-angiogenic effect of FA in vivo. In conclusion, our data indicate that FA bound to FR in HUVEC, subsequently activated the cSrc/ERK 2/NF-κB/p53 signaling pathway, which in turn up-regulated the expression of p21 and p27, and finally resulted in cell cycle arrest at the G0/G1 phase. In the present study, we uncover a completely novel role of FA for anti-angiogenesis.     

Hepatitis C virus-induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity

Chronic hepatitis C is characterized by iron accumulation in the liver, and excessive iron is hepatotoxic. However, the mechanism by which hepatitis C virus (HCV) regulates iron metabolism is poorly understood. Hepcidin plays a pivotal role as a negative regulator of iron absorption. The aim of the current study was to elucidate the mechanisms that govern hepcidin expression by HCV. Huh 7 cells, Huh7.5 cells, full-length HCV replicon cells established from Huh7.5 cells, and adenoviruses expressing HCV-core or HCV nonstructural proteins 3 through 5 (NS3-5) were used. Hepcidin expression was significantly lower in HCV replicon cells and in HCV core-expressing Huh7 cells. The expression was inversely correlated with the amount of reactive oxygen species (ROS) production. Anti-oxidants restored hepcidin expression in HCV replicon cells and Huh7 cells expressing HCV core. In HCV replicon cells, histone deacetylase (HDAC) activity was elevated at baseline and after exposure to hydrogen peroxide. Anti-oxidants reduced HDAC activity in a dose-dependent manner. HDAC inhibition increased hepcidin expression without affecting ROS production in HCV replicon cells. HCV-induced ROS stabilized the expression of two negative hepcidin regulators, HIF1alpha and HIF2alpha, and its expression was decreased by a HDAC inhibitor or an anti-oxidant. HCV-induced ROS also caused hypoacetylation of histones and inhibited binding of two positive regulators, C/EBPalpha and STAT3, to the hepcidin promoter, whereas anti-oxidant treatment of cells recovered C/EBPalpha and STAT3 binding to the hepcidin promoter. In addition, an HDAC inhibitor restored their binding to the hepcidin promoter via acetylation of histones. CONCLUSION: HCV-induced oxidative stress suppresses hepcidin expression through increased HDAC activity.     

霉酚酸酯及地塞米松对血管内皮细胞功能的影响

目的：研究霉酚酸酯,地寒米松对血管内皮细胞功能的影响,进一步探讨这两种免疫抑制剂抗炎作用的机制。方法：以脐静脉来源的的内皮细胞为研究对象,通过建立体外内皮细胞血管发生（ａｎｇｉｏｇｅｎｅｓｉｓ）模型,观察并比较了两种免疫抑制剂对内皮细胞血管发生,内皮细胞迁移能力（Ｓｃｒａｔｃｈ ｗｏｕｎｄ ｃｌｏｓｕｒｅ ａｓｓａｙ）和内皮细胞增生（＾３Ｈ－ＴｄＲ掺入）的影响。结果：霉酚酸酯对血管内皮细胞体外血管     

类表皮生长因子域7与动脉粥样硬化斑块内血管生成关系

目的 研究类表皮生长因子域7( Egfl7)在动脉粥样硬化斑块内的表达水平和小分子干扰RNA(siRNA)对内皮细胞株血管生长因子表达的影响. 方法 利用免疫组化和免疫荧光染色检测人髂动脉和小鼠无名动脉粥样硬化斑块内Egfl7的表达水平;并利用以Egfl7为靶标的siRNA,通过脂质体将siRNA转入人脐静脉内皮细胞株(HUVEC),以未转染siRNA细胞和转染无关siRNA细胞为对照,在干扰后0h,12 h,24 h,48 h,反转录聚合酶链反应(PT-PCR)检测Egfl7、血管内皮生长因子(VEGF)、血小板衍生生长因子A、B(PDGF-A、PDGF-B) mRNA水平的改变,免疫印迹法检测Egfl7、VEGF、PDGF、血管细胞黏附分子(VCAM)及细胞间黏附分子(ICAM)蛋白表达水平的变化.结果 人髂动脉和小鼠无名动脉粥样硬化斑块内Egfl7表达水平上调;HUVEC内Egfl7在siRNA干扰后0h、12 h、24 h、48 h时mRNA表达分别为(0.31±0.05)、(0.14±0.02)、(0.09±0.01)、(0.02±0.01),蛋白表达为(0.93±0.08)、(0.71±0.11)、(0.39±0.09)、(0.07±0.01).Egfl7、VEGF、PDGF-A、PDGF-B mRNA及其蛋白表达水平随干扰时间延长均下降或抑制,与siRNA干扰0h比较差异有统计学意义(均P＜0.05). 结论 动脉粥样硬化斑块内Egfl7表达水平提高;siRNA抑制HUVEC Egfl7表达水平的同时,其他血管生长因子和黏附分子表达水平亦下降.     

miR-320通过下调SRY相关高迁移率族蛋白4的表达抑制人脐静脉内皮细胞内皮间质转化

目的 探讨miR-320对人脐静脉内皮细胞(HUVEC)内皮间质转化(EndMT)的影响及其调控机制.方法 用miR-320 mimics或miR-320 inhibitor处理HUVEC.Western blot检测内皮细胞标记物血小板内皮细胞黏附分子1(CD31)、血管内皮钙黏蛋白(VE-Cadherin)及间质细...     

T-helper cell type 2 (Th2) memory T cell-potentiating cytokine IL-25 has the potential to promote angiogenesis in asthma

IL-25 (IL-17E) is a T-helper cell type 2 (Th2) cytokine best described as a potentiator of Th2 memory responses. Reports of expression of its receptor, IL-25R, on airways structural cells suggest a wider role for IL-25 in remodeling. We hypothesized that IL-25 stimulates local angiogenesis in the asthmatic bronchial mucosa. Immunoreactive IL-25(+), IL-25R(+), and CD31(+) (endothelial) cells in sections of bronchial biopsies from asthmatics and controls were detected by immunohistochemistry. The effect of IL-25 on angiogenesis was examined using an in vitro assay. Real-time PCR was used to detect expression of IL-25R and VEGF mRNA in cultured human vascular endothelial cells (HUVEC), and a cell proliferation kit (WST-8) was used to measure the effect of IL-25 on HUVEC proliferation. Immunostaining showed that IL-25(+), IL-25R(+), and CD31(+)/IL-25R(+) cells were significantly elevated in the bronchial mucosa of asthmatics compared with controls (P < 0.003). In asthmatics, the numbers of IL-25(+) cells correlated inversely with the forced expiratory volume in 1 s (r = -0.639; P = 0.01). In vitro, HUVEC constitutively expressed IL-25R, which was up-regulated further by TNF-α. IL-25 and TNF-α also increased expression of VEGF and VEGF receptors. IL-25 increased HUVEC proliferation and the number, length, and area of microvessel structures in a concentration-dependent manner in vitro. VEGF blockade, the PI3K-specific inhibitor LY294002, and the MAPK/ERK1/2 (MEK1/2)-specific inhibitor U0126 all markedly attenuated IL-25-induced angiogenesis, and the inhibitors also reduced IL-25-induced proliferation and VEGF expression. Our findings suggest that IL-25 is elevated in asthma and contributes to angiogenesis, at least partly by increasing endothelial cell VEGF/VEGF receptor expression through PI3K/Akt and Erk/MAPK pathways.     

壮观霉素B1抑制过氧化物增殖激活受体共激活子类泛素修饰促高糖内皮细胞自愈

目的探讨过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)的小泛素相关修饰蛋白(SUMO)在糖尿病患者血管内皮修复能力下降过程中的作用,并寻找新的治疗方法。方法选取人脐静脉内皮细胞(HUVEC)第3~6代细胞随机分对照组、高糖组及壮观霉素B1治疗组(治疗组)3组(n=4),分别于1.0 g/L葡萄糖、4.5 g/L葡萄糖、4.5 g/L葡萄糖+20μmol/L壮观霉素B1培养液中作用48 h,用Western blot检测SUMO2/3、PGC-1α蛋白表达,RT-PCR方法检测核呼吸因子-2α(NRF-2α)和核受体雌激素受体相关受体α(ERRα)mRNA表达,细胞划痕实验检测HUVEC损伤修复功能,模拟血管形成实验检测HUVEC血管形成能力。结果高糖组和治疗组共价态SUMO2/3蛋白表达水平明显高于对照组,NRF-2α和ERRαmRNA表达水平明显低于对照组(P<0.05,P<0.01);治疗组共价态SUMO2/3蛋白表达水平明显低于高糖组,NRF-2α和ERRαmRNA表达水平明显高于高糖组(P<0.05)。高糖组游离态SUMO2/3水平低于对照组和治疗组(P<0.05)。3组PGC-1α蛋白表达水平比较,无统计学差异(P>0.05)。细胞划痕实验显示,高糖组和治疗组细胞迁移距离明显低于对照组[(19.47±4.04)μm vs(57.35±5.59)μm,P=0.017;(31.98±6.05)μm vs(57.35±5.59)μm,P=0.032],治疗组细胞迁移距离明显高于高糖组(P=0.037)。细胞模拟血管形成实验显示,治疗组能够改善网状交联情况,形成少量类血管结构。结论壮观霉素B1能够通过抑制PGC-1α蛋白的SUMO化修饰增加血管内皮细胞在高糖环境下的自愈能力。     

小泛素样修饰蛋白特异性蛋白酶1介导糖尿病性血管内皮细胞损伤修复的研究

目的利用小泛素相关修饰体(SUMO)特异性蛋白酶1(SENP1)解离过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)的小泛素样修饰蛋白1(SUMO1)修饰。方法人脐静脉内皮细胞培养传代后分对照组、高糖组及SENP1组;Western blot法检测SENP1、SUMO1、PGC-1α和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)水平;实时荧光定量PCR检测线粒体转录因子A(TFAM)、核呼吸因子2α(NRF-2α)和雌激素受体相关受体α(ERRα)的mRNA表达;ELISA测定乳酸脱氢酶(LDH);细胞划痕愈合方法、Transwell方法和体外血管模拟形成实验分别检测细胞自愈、迁移和形成血管拟态的能力。结果与对照组比较,高糖组共价SUMO1、PGC-1α和活化Caspase-3蛋白表达明显升高,SENP1蛋白表达明显降低,TFAM、NRF-2α和ERRα的mRNA表达明显降低,细胞划痕修复能力及模拟血管形成能力下降(P<0.05,P<0.01)。与高糖组比较,SENP1组SENP1蛋白表达明显升高,共价SUMO1、PGC-1α和活化Caspase-3蛋白表达明显降低,TFAM、NRF-2α和ERRα的mRNA表达明显升高,细胞划痕修复和迁移能力及模拟血管形成能力明显改善(P<0.05,P<0.01)。对照组、高糖组和SENP1组细胞上清液中LDH水平比较,差异有统计学意义[(24.66±6.39)ng/ml vs(302.45±30.54)ng/ml vs(174.08±21.03)ng/ml,P<0.01]。结论SENP1能够诱导PGC-1α发生去SUMO修饰,解除其对PGC-1α下游转录因子的抑制作用,改善线粒体功能,抑制高糖诱导的血管内皮细胞功能损伤作用。     

Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7

VEGF has been shown to regulate endothelial cell (EC) proliferation and migration. However, the nuclear mediators of the actions of VEGF in ECs have not been fully defined. We show that VEGF induces the phosphorylation of three conserved serine residues in histone deacetylase 7 (HDAC7) via protein kinase D, which promotes nuclear export of HDAC7 and activation of VEGF-responsive genes in ECs. Expression of a signal-resistant HDAC7 mutant protein in ECs inhibits proliferation and migration in response to VEGF. These results demonstrate that phosphorylation of HDAC7 serves as a molecular switch to mediate VEGF signaling and endothelial function.