江西省啮齿动物携带立克次体分子流行病学研究

目的 调查江西省部分县区啮齿动物携带的立克次体。方法 在江西省南丰县、铜鼓县、上犹县、浮梁县、上高县、上饶市广信区捕获啮齿动物,运用形态学结合分子生物学方法鉴定其种类。采用巢式PCR方法检测捕获的啮齿动物的脾脏组织样本中的柯克斯体属、斑点热群、东方体属和无形体属立克次体。结果 捕获啮齿动物6种共393只,包括黑线姬鼠、褐家鼠、黄毛鼠、黄胸鼠、社鼠和臭鼩鼱,黑线姬鼠为优势鼠种(311/393)。从19只黑线姬鼠检出柯克斯体(6.11%),从2只黑线姬鼠检出东方体(0.64%),从1只黑线姬鼠检出斑点热群立克次体(0.32%)。对扩增阳性基因片段测序和对获得基因序列做同源性比对,结果浮梁县黑线姬鼠检出新塔拉萨维奇立克次体、恙虫病东方体、贝氏柯克斯体;铜鼓县和上饶市广信区黑线姬鼠检出贝氏柯克斯体;南丰县黑线姬鼠检出恙虫病东方体。除黑线姬鼠外,其它5种啮齿动物均未检出立克次体。结论 江西省部分地区的黑线姬鼠携带贝氏柯克斯体、新塔拉萨维奇立克次体、恙虫病东方体等立克次体病原体,人群存在相关立克次体感染的风险,疾控和医疗机构应加强立克次体病的监测。     

云南省泸西县啮齿动物携带立克次体调查

目的 调查云南省泸西县啮齿动物携带恙虫病东方体、无形体和埃立克体的状况,了解该类病原体在当地自然界中的保存状况和基因特征。方法 用鼠笼和鼠夹在云南省泸西县捕鼠,将捕获的动物种类鉴定后解剖取脾脏,活鼠取血。采用巢式PCR扩增脾脏的恙虫病东方体groEL基因,无形体和埃立克体的16S rRNA基因特异片段;测定PCR扩增阳性产物的DNA序列,对获得序列进行序列比对和系统进化分析。IFA法检测鼠血清中恙虫病东方体IgG抗体。结果 在泸西县共捕获啮齿动物10种225只。其中黄胸鼠36.89%(83/225)、大绒鼠35.11%(79/225)和中华姬鼠13.78%(31/225)为优势鼠种。获得鼠血清85份。鼠脾脏中检测到5株东方体groEL基因阳性标本,带毒鼠种为黄胸鼠2.41%(2/83)和大绒鼠3.80%(3/79)。同源性比较显示,这5株东方体的相似性在99.02%~100%之间,他们分别与GenBank中已知立克次体序列的相似性在98.75%~100%。系统发生树显示,5株OT与来自日本、泰国和中国安徽的菌株位于同一分支。3份16S rRNA阳性标本,其中1份埃立克体阳性,来源于大绒鼠;1份沃尔巴克氏体和1份巴尔通体阳性均来源于黄胸鼠。无形体均为阴性。埃立克体株序列比对显示与来自美国、中国和巴西的埃立克体基因同源性为98.0%~100%,并与分离自美国野外工作者皮肤的伊文氏埃立克体在同一分支。鼠血清恙虫病IgG抗体阳性7份,阳性率8.24%(7/85)。结论 该地区存在以黄胸鼠和大绒鼠为主要宿主的恙虫病自然疫源地。埃立克体、巴尔通体和沃尔巴克氏体在啮齿动物中也存在感染,需注意防控。     

实时荧光定量PCR检测恙虫病东方体

目的建立检测恙虫病东方体的实时荧光定量PCR(quantitative real-ti me PCR)方法。方法根据恙虫病东方体56kD外膜蛋白基因序列设计引物和探针,以克隆的56kD基因片段作DNA模板,建立实时荧光定量PCR检测方法。结果建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.999)。荧光定量PCR检测恙虫病东方体的灵敏度约为套式PCR的100倍,并且具有良好的重复性。用该定量PCR检测其它相关立克次体和病原菌DNA样本,检出结果均为0。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肺脏、肝脏标本,结果脾脏中东方体出现最早和检出量最多,肝脏和肺脏次之。血中的恙虫病东方体量较低。结论本研究建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于东方体感染早期血样本的快速检测作恙虫病感染早期诊断,并且可以定量分析评价恙虫病东方体感染的程度。     

云南省永善县恙虫病流行病学调查

目的 对云南省永善县恙虫病开展流行病学调查。方法 应用胶体金免疫试验对发热患者血清进行恙虫病东方体(Orientia tsutsugamsushi)IgG和IgM抗体检测,采用夜夹法诱捕调查地区鼠类,用巢氏PCR方法对鼠脾脏做groEL基因片段扩增和基因序列分析。结果 2015年5-10月在永善县发现恙虫病患者34例,其中实验室确诊病例21例,临床诊断病例13例;主要发病于8月,占总病例数的32.35%;40—49岁年龄组发病最多,占总病例数的32.35%;农民发病数最多,占总病例数的79.41%;有焦痂或溃疡的病例占总病例数的94.12%,其中位于腹股沟最多(占有焦痂或溃疡患者的31.25%);有皮疹的病例占总病例数的50.00%。在捕获的39份黄胸鼠(Rattus flavipectus)脾组织样品中,恙虫病东方体groEL基因片段扩增阳性9份(阳性率23.08%)。groEL基因片段序列进化树分析显示本次检测到的YSP30株与的HSB1、FAR1、UAP4等Saitama型菌株的遗传进化关系最密切, 检测到的其余8株与UT213、UT221、SH205等Karp型菌株的遗传进化关系最密切。结论 血清学和分子生物学方法证实了永善县存在恙虫病疫源地,当地存在Karp和Saitama相关的2种基因型恙虫病东方体。     

厦门市恙虫病流行病学特征与防控策略

目的 总结和分析厦门市恙虫病流行特征和基因型,探讨防控策略。方法 收集厦门市1954-2016年人间恙虫病疫情资料,对2006-2016年人间恙虫病疫情进行流行病学调查,收集患者和宿主动物开展恙虫病基因型检测。结果 厦门市人间恙虫病疫情报告始于1954年,1954-2016年累计报告病例950例,其中1954-1988年发病率波动在0~6.75/10万,1989年后不再列为法定报告传染病,近10年发病呈上升趋势,2006-2016年累计报告690例。病例以思明区和湖里区为主,时间主要集中在夏季和秋季,男女性别比1.02∶1;年龄以20~65岁为主;职业以农民最多。宿主动物以褐家鼠为主,恙螨以地里纤恙螨为优势螨种,基因型以Karp为主。结论 厦门市存在恙虫病疫源地,应开展以鼠螨控制为重点的综合防控。     

银杏叶提取物对内皮细胞的保护作用

目的 探讨银杏叶提取物(GBE)对血管紧张素Ⅱ(Ang Ⅱ)致体外培养人脐静脉内皮细胞(HUVEC)损伤的保护作用.方法 将处于对数生长期的HUVEC分为正常对照组、AngⅡ组及高、中、低3个GBE处理组,其中正常对照组用含0.5%小牛血清的DMEM培养基培养,Ang Ⅱ组在正常对照组培养基基础上含Ang Ⅱ 10-8mol/L,GBE处理组在AngⅡ组基础上分别加GBE 50、100、200 mg/L做为GBE低、中、高组,采用MTT法测定培养细胞增殖能力.并应用Western印迹法检测各组细胞中磷酸化Akt表达.结果 MTT法检测结果表明,Ang Ⅱ组细胞增殖能力明显高于正常对照组(P＜0.05),除GBE低剂量组外,GBE高、中剂量组A值均明显高于Ang Ⅱ组(P＜0.05);Western 印迹法检测各组培养细胞磷酸化Akt表达结果显示,与正常对照组比较,Ang Ⅱ组磷酸化Akt蛋白表达量明显降低(P＜0.05),GBE高剂量组磷酸化Akt表达明显高于Ang Ⅱ组(P＜0.05),且与正常对照组比较,差异无统计学意义,GBE中、低剂量组磷酸化Akt蛋白表达量明显降低(P＜0.05),但仍显著高于Ang Ⅱ组(P＜0.05).结论 Ang Ⅱ导致血管内皮细胞增殖能力下降可能与其抑制磷酸化Akt表达量有关,GBE可通过上调Akt表达抵抗Ang Ⅱ引起的细胞损伤.     

套式PCR应用于恙虫病鼠类标本的检测研究

目的　评价基因扩增法应用于检测鼠类标本调查恙虫病疫源地的作用。方法　用一定量恙虫病东方体人工感染昆明种小鼠 ,攻击后第 3、6、9天处死小鼠取血块及脾脏用特异性套式 PCR技术进行恙虫病东方体 DNA的检测 ,再用该技术测定现场采集的鼠类标本 ,以可从标本中扩增出一段长 88bp的 DNA片段作为实验或现场捕获鼠感染了恙虫病东方体的指标 ,继而估计该地区是否存在恙虫病疫源地。结果　实验感染恙虫病东方体 3天时在两类标本中均不能检出该病原 DNA,而第6天测定时均可发现含有恙虫病东方体 ;从福建闽西北地区采集的 111份鼠类脾脏中检出 1份阳性标本 ,另从江西送检的 2 9份野鼠血块中也检出 1份阳性标本 ,说明采集标本区域已成为恙虫病疫源地。结论　套式 PCR扩增技术具有很好的特异性和敏感性 ,可用于现场鼠类标本的恙虫病病原学的调查。     

高血压小鼠动脉血管平滑肌细胞PI3K/Akt/SREBP1信号活化及表型转化研究

目的 研究固醇调节元件结合蛋白1（SREBP1）在血管紧张素II（Ang II）诱导的血管平滑肌细胞（VSMC）表型转化中的作用。方法 将C57BL/6J小鼠分为对照组、处理组1（150 ng/kg/min Ang II）、处理组2（300 ng/kg/min Ang II）、处理组3（600 ng/kg/min Ang II）和缬沙坦组（600 ng/kg/min Ang II+40 mg/kg/d 缬沙坦）处理28 d。观测小鼠血压和动脉血管变化。实时荧光定量PCR和免疫印迹检测血管SREBP1的表达。将VSMC分为正常组、处理组1 （0.1×10-6 mol/L Ang II）、处理组2（0.5×10-6 mol/L Ang II）、处理组3（1×10-6 mol/L Ang II）、缬沙坦组（1×10-6 mol/L Ang II+1×10-6 mol/L 缬沙坦）、LY294002组（1×10-6 mol/L Ang II+10 ng/mL LY294002）、沉默对照组（1×10-6 mol/L Ang II+scramble siRNA）、沉默组（1×10-6 mol/L Ang II+SREBP1 siRNA）处理,免疫印迹检测细胞SREBP1、Akt、磷酸化Akt（p-Akt）、α平滑肌肌动蛋白（α-SMA）、骨桥蛋白（OPN）的表达。 结果 与对照组比较,Ang II处理组小鼠收缩压和舒张压明显升高,缬沙坦组与处理组比较血压降低。相比于对照组,处理组小鼠血管壁增厚、管腔增大,缬沙坦处理则抑制血管重塑。Ang II处理组小鼠主动脉血管SREBP1 mRNA和蛋白表达水平高于对照组和缬沙坦组。SREBP1、磷酸化Akt、OPN在处理组VSMC细胞中表达水平高于正常组,而在缬沙坦组、LY294002组和沉默组中低于处理组;α-SMA在处理组中表达降低,而在缬沙坦组、LY294002组和沉默组中升高。 结论 Ang II通过活化PI3K/Akt/SREBP1通路调控VSMC表型转化、诱导小鼠动脉血管重塑。     

广州增江沿岸地区鼠形动物及携带病原体调查

目的 了解广州增江沿岸地区鼠形动物及携带伯氏疏螺旋体、钩端螺旋体、恙虫病东方体、埃立克体、新型布尼亚病毒和汉坦病毒6种病原体的情况。方法 使用笼夜法在广州市增城区捕获鼠形动物,利用荧光定量PCR方法检测鼠形动物脏器中的伯氏疏螺旋体、钩端螺旋体、恙虫病东方体、埃立克体、新型布尼亚病毒和汉坦病毒。采用χ²检验和Fisher确切概率法比较不同种属、雌雄和调查点鼠形动物病原体的感染情况。结果 捕获臭鼩鼱和褐家鼠共69只,鼠形动物密度为13.29%。褐家鼠中检出钩端螺旋体为57.89%、恙虫病东方体为31.58%、汉坦病毒为21.05%和伯氏疏螺旋体为5.26%;臭鼩鼱中仅检出伯氏疏螺旋体为2.00%。埃立克体、新型布尼亚病毒均未检出。此外,褐家鼠中存在4种病原体的双重感染,其双重感染率为36.84%。结论 广州增江沿岸地区鼠形动物以臭鼩鼱和褐家鼠为主且密度较高;褐家鼠中病原体感染率高于臭鼩鼱且存在多种病原体的复合感染。当地应加强对鼠形动物尤其是褐家鼠的监测和控制、降低鼠形动物密度。     

1952-1989年和2006-2017年中国大陆恙虫病流行及时空分布特征

目的 研究并阐明中国大陆恙虫病流行趋势和时空分布特征, 为恙虫病的预防和控制提供参考依据。方法 根据1952-1989年和2006-2017年中国大陆恙虫病疫情报告数据,采用描述性流行病学方法、空间自相关分析和ArcGIS 10.4软件的可视化技术等,全面系统研究中国大陆恙虫病流行及时空分布特征,并确定高风险地区。结果 在1952-1989年和2006-2017年期间,我国累计报道恙虫病病例156 234例,死亡180例。1952-1989年的年均发病率0.13/10万。2006年以后,年均发病率急剧上升,由2006年的0.09/10万上升到2017年的1.62/10万,增长了18倍,年平均增长率为33%。流行季节仍以夏季和秋冬为主,多数病例主要集中在10月,女性发病率高于男性(χ²=168.34, P<0.001)。云南、安徽、广东、福建、江苏、山东、广西和四川8个省(自治区)的病例数最多,占全国总病例数的91.31%。全局空间自相关分析结果表明,在全国范围内恙虫病整体上存在着空间正相关,具有空间聚集性(I=0.085, P<0.05)。局部空间自相关分析结果显示,广西、福建及其周边地区为“热点区域”,是恙虫病高发区。结论 1952-1989年和2006-2017年我国恙虫病发病率存在逐年升高趋势,病例以夏季型和秋冬型为主。空间自相关分析可以及时发现该病的聚集情况并确定高发区和危险区。     

Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-Kinase/Akt signal transduction pathway

Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase) and increased PI 3'-kinase activity in a dose-dependent manner. The PI 3'-kinase-specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3'-kinase-dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3'-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.     

Angiopoietin/Tie2 pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension

RATIONALE: Angiopoietins are involved in blood vessel maturation and remodeling. OBJECTIVES: One consequence of endothelium-specific tyrosine kinase-2 (Tie2) receptor activation by angiopoietin-1 (Ang1) is the release of endothelium-derived growth factors that recruit vascular wall cells. We investigated this process in idiopathic pulmonary arterial hypertension (iPAH). METHODS: Ang1, Ang2, and total and phosphorylated Tie2 expression (mRNA and protein) was evaluated in human lung specimens and in cultured pulmonary artery smooth muscle cells (PA-SMCs) and pulmonary endothelial cells (P-ECs) isolated from patients with iPAH and control subjects. Media collected from Ang1-treated P-ECs were assessed for their PA-SMC growth-promoting effect. MEASUREMENTS AND MAIN RESULTS: Tie2 receptor was fourfold higher in lungs and P-ECs from patients with iPAH than in those from control subjects, with a parallel increase in phosphorylated lung Tie2 receptor. In contrast, Ang1 and Ang2 expression in lungs, P-ECs, and PA-SMCs did not differ. Incubation of PA-SMCs with medium collected from P-EC cultures induced marked proliferation, and this effect was stronger when using P-ECs from patients with iPAH than from control subjects. Ang1 pretreatment of P-ECs from either patients or control subjects induced a further increase in PA-SMC proliferation. Fluoxetine, an inhibitor of the mitogenic action of serotonin, reduced the growth-promoting effect of P-EC media. Ang1 added to P-ECs from patients with iPAH increased the production of endothelin-1 (ET-1) and serotonin, but not of platelet-derived growth factor-BB or epidermal growth factor, and increased the amount of mRNA encoding tryptophan hydroxylase-1 (the rate-limiting enzyme of serotonin synthesis), preproET-1, and ET-1-converting enzyme. CONCLUSIONS: The Ang1/Tie2 pathway is potentiated in iPAH, contributing to PA-SMC hyperplasia via increased stimulation of endothelium-derived growth factors synthesis by P-ECs.     

Hypoxia and vascular endothelial growth factor acutely up-regulate angiopoietin-1 and Tie2 mRNA in bovine retinal pericytes

The angiopoietin/Tie2 system is a predominant regulator of vascular development. This vascular development appears to be controlled and completed by the coordinated actions of two vascular cells, endothelial cells and their surrounding supporting cells, smooth muscle cells, or pericytes. The role of the angiopoietin/Tie2 system has been studied, but these studies are limited mostly to endothelial cells. In this study, using bovine retinal pericytes (BRP), we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF) treatment, on the regulation of the angiopoietin/Tie2 system. Hypoxia (2% O(2) concentration) was acquired by a hypoxia chamber. Both hypoxia and VEGF (10 ng/ml) treatment significantly increased angiopoietin-1 (Ang1) mRNA expression. This marked augmentation occurred acutely (maximal increase at 2 h) and subsequently decreased. In contrast, angiopoietin-2 (Ang2) mRNA expression was unaltered in BRP upon both treatment. Significant up-regulation of Tie2 mRNA expression was found and lasted up to 12 h. However, using bovine aortic endothelial cells (BAEC), we found that only Ang2 expression, but neither Ang1 nor Tie2, responded to these two angiogenic stimuli, which was consistent with many previous reports. In conclusion, our data suggest that both hypoxia and VEGF treatment differentially regulate the angiopoietin/Tie2 system in the two vascular cells and that, particularly in BRP, the regulation of Ang1, but not Ang2, and Tie2 expression may play an important role in vascular development.     

Roles of the receptor tyrosine kinases Tie1 and Tie2 in mediating the effects of angiopoietin-1 on endothelial permeability and apoptosis

Angiopoietin-1 (Ang1) has key roles in development and maintenance of the vascular system. The ligand is a potent inhibitor of vascular leakage and suppresses endothelial apoptosis and vessel regression. Ang1 was originally identified as a ligand for the receptor tyrosine kinase Tie2. Recently however Ang1 has also been found to activate the related tyrosine kinase Tie1. The contribution of Tie1 to mediating the effects of Ang1 on endothelial function is not known. In this study we used an siRNA approach to investigate the relative importance of Tie1 and Tie2 in transducing the effects of Ang1 on monolayer permeability and induction of apoptosis in human endothelial cells. siRNA directed against either Tie1 or Tie2 suppressed expression of each respective receptor by more than 90%. Ang1 inhibited endothelial monolayer permeability and this effect was prevented by suppression of Tie2 expression. In contrast, Ang1 inhibition of permeability was not affected by suppression of Tie1 expression. The ability of Ang1 to inhibit induction of apoptosis in response to serum deprivation was completely blocked by suppression of Tie2 expression, but not diminished by suppression of Tie1 expression. Taken together these data demonstrate that Tie2 mediates the inhibitory effects of Ang1 on endothelial permeability and apoptosis. The data also demonstrates that Tie1 does not transduce anti-apoptotic or anti-permeability effects of Ang1 in endothelial cells.     

Hepatocyte growth factor mediates angiopoietin-induced smooth muscle cell recruitment

Communication between endothelial cells (ECs) and mural cells is critical in vascular maturation. Genetic studies suggest that angiopoietin/Tie2 signaling may play a role in the recruitment of pericytes or smooth muscle cells (SMCs) during vascular maturation. However, the molecular mechanism is unclear. We used microarray technology to analyze genes regulated by angiopoietin-1 (Ang1), an agonist ligand for Tie2, in endothelial cells (ECs). We observed that hepatocyte growth factor (HGF), a mediator of mural cell motility, was up-regulated by Ang1 stimulation. We confirmed this finding by Northern blot and Western blot analyses in cultured vascular endothelial cells. Furthermore, stimulation of ECs with Ang1 increased SMC migration toward endothelial cells in a coculture assay. Addition of a neutralizing anti-HGF antibody inhibited Ang1-induced SMC recruitment, indicating that the induction of SMC migration by Ang1 was caused by the increase of HGF. Interestingly, Ang2, an antagonist ligand of Tie2, inhibited Ang1-induced HGF production and Ang1-induced SMC migration. Finally, we showed that deletion of Tie2 in transgenic mouse reduced HGF production. Collectively, our data reveal a novel mechanism of Ang/Tie2 signaling in regulating vascular maturation and suggest that a delicate balance between Ang1 and Ang2 is critical in this process.     

The angiopoietin-tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis

OBJECTIVES: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery. METHODS: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. RESULTS: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. CONCLUSION: These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.     

Phosphatidylinositol 3-kinase/Akt regulates angiotensin II-induced inhibition of apoptosis in microvascular endothelial cells by governing survivin expression and suppression of caspase-3 activity

Angiotensin II (Ang II) plays essential roles in vascular homeostasis, neointimal formation, and postinfarct remodeling. Although Ang II has been shown to regulate apoptosis in cardiomyocytes and vascular smooth muscle cells, its role in vascular endothelial cells (ECs) remains elusive. To address this issue, we first performed TUNEL and caspase-3 activity assays with porcine microvascular ECs challenged by serum deprivation. Ang II significantly reduced the ratio of apoptotic cells and caspase-3 activity. The Ang II type 1 receptor (AT1) was responsible for these effects. Among the signaling molecules downstream of AT1, we revealed that PI3-kinase/Akt pathway plays a predominant role in the antiapoptotic effect of Ang II. Interestingly, the expression of survivin, a central molecule of cell survival, increased after Ang II stimulation. Overexpression of a dominant-negative form of Akt abolished both Ang II-induced antiapoptosis and survivin protein expression. In a murine model of hyperoxygen-induced retinal vascular regression, AT1a knockout mice showed a significant increase in retinal avascular areas. Our data indicate that Ang II plays a critical antiapoptotic role in vascular ECs by a mechanism involving PI3-kinase/Akt activation, subsequent upregulation of survivin, and suppression of caspase-3 activity.     

Interaction between Tie receptors modulates angiogenic activity of angiopoietin2 in endothelial progenitor cells

OBJECTIVE: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. METHODS AND RESULTS: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. CONCLUSIONS: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.     

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells

Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.