【作者】 徐晟杰；

【导师】 傅国胜；

【作者基本信息】 浙江大学 ， 内科学， 2012， 博士

【摘要】 越来越多的实验结果提示内皮祖细胞(EPCs)通过合成并分泌细胞因子来促进血管新生和内皮层的修复。其中血管内皮生长因子(VEGF)和白介素8(IL-8)在EPCs通过旁分泌因子促进血管新生和内皮修复的作用中扮演着主要的角色。血浆同型半胱氨酸(Hcy)水平升高作为缺血性心血管病的一个独立危险因素,参与缺血性心血管病发病的多个环节,其机制十分复杂。研究发现,Hcy损害外周血EPCs的数量和功能。相关文献报道Hcy减少EPCs数量并损害其增殖、迁移和黏附功能,且随着Hcy浓度增加及作用时间延长,EPCs的数量减少和功能损害明显加重。随后的在体研究显示高Hcy血症患者外周血EPCs数量减少、功能减退。进一步研究提示Hcy加速EPCs衰老,伴随EPCs增殖和集落形成能力的损害。Hcy加速EPCs衰老可能跟EPCs端粒酶活性下降以及Akt磷酸化水平的下降有关。尽管高Hcy引起EPC增殖、迁移等功能受损,但是,Hcy是否影响EPCs的旁分泌功能目前并不清楚。因此,我们拟进一步观察Hcy对EPCs旁分泌能力以及旁分泌细胞因子在体内皮修复能力的影响。另外值得关注的是,Hcy影响EPCs功能损害的确切机制目前仍有待明确。作为过氧化物酶体增殖物激活受体γ (PPARγ)激动剂的一种,匹格列酮(pioglitazone,PIO)已被证明能够调节细胞粘附,迁移以及新生血管能力。研究发现,PIO可以抑制EPCs的凋亡并增加小鼠体内的血管新生。PIO亦可以改善糖尿病和冠心病病人体内的EPCs的迁移和粘附能力。但是PIO对EPCs的治疗作用是否包括改善其旁分泌能力目前还有待进一步证实。因此,基于上述考虑,我们首先观察Hcy对于EPCs旁分泌细胞因子VEGF和IL-8的影响,EPCs旁分泌能力在体内对损伤内皮层的影响；PIO能否改善Hcy环境下EPCs的旁分泌,粘附和迁移能力及机制；PIO对NADPH酶亚基蛋白表达的影响。以下分三部分对本研究的方法、结果及结论作一简述。1同型半胱氨酸对内皮祖细胞旁分泌功能的影响目的：探讨Hcy体外干预EPCs对EPCs旁分泌功能的影响。方法：采用密度梯度离心法从健康人外周血获取单个核细胞,接种于人纤维连接蛋白包被的培养板,培养7d后,采用激光共聚焦显微镜检测FITC-UEA-I及DiI-acLDL双染色鉴定EPCs,采用流式细胞仪检测其表面标志(VEGFR-2,CD34, CD133),进一步鉴定EPCs。采用不含细胞因子的培养基EBM-2替代正常培养基培养EPCs24h,通过real-time PCR法检测EPCs细胞内细胞因子合成的变化,并采用ELISA法检测条件培养基中VEGF和IL-8水平。加入不同浓度的Hcy(10,50,100,200和500μmol/L)干预24h,观察量效关系。此外,采用200μmol/L的Hcy分别干预不同时间(3,6,12,24和36h),观察时效关系。采用球囊损伤法建立大鼠颈动脉损伤模型,雄性Sprague-Dawley大鼠在颈动脉损伤后分别按如下分组进行灌注：(1)正常对照组；(2)颈动脉损伤+单纯培养液；(3)颈动脉损伤+EPCs条件培养液；(4)颈动脉损伤+Hcy干预后EPCs条件培养液。一周后观察不同条件培养液对大鼠颈动脉内皮修复的影响。结果：与正常培养基相比,在不含细胞因子及血清的条件培养基培养环境中,EPCs大量合成VEGF和IL-8,而其他细胞因子如IGF, HGF, SDF-1,PDGF-BB和Tβ4的合成无明显变化。ELISA法检测EPCs条件培养基中VEGF, IL-8浓度分别为260.54±32.09pg/ml/1×106EPCs,15724.54±1817.24pg/ml/1×106EPCs。高浓度Hcy抑制EPCs分泌VEGF和IL-8。200μmol/L浓度Hcy干预EPCs时间依赖性抑制VEGF和IL-8的分泌。EPCs条件培养基显著促进大鼠颈动脉内皮层修复,体外受到Hcy干预的EPCs条件培养基的促内皮修复能力受到抑制。结论：EPCs合成分泌以VEGF和IL-8为主,通过旁分泌细胞因子可促进大鼠动脉内皮修复。Hcy显著减少EPCs旁分泌VEGF和IL-8,并损害EPCs通过旁分泌功能修复损伤动脉的作用。2匹格列酮通过抑制PKC的表达和NADPH酶的活性逆转同型半胱氨酸下调的内皮祖细胞旁分泌,粘附和迁移功能目的：探讨匹格列酮(PIO)对Hcy下调的EPCs旁分泌,粘附和迁移功能的作用以及相关机制。方法：从健康人外周血分离、培养循环EPCs7d。 EPCs条件培养液(EPCs-derived conditioned medium,EPC-CM)是体外培养循环EPCs用不含血清的EBM-2培养24h后获得。如前述,EPCs在Hcy环境下(200μmol/L)培养24h,并提前30min加入PIO(10μmol/L)共处理。采用ELISA和realtime-PCR分别检测VEGF和IL-8在条件培养液中的浓度和细胞内相应mRNA合成水平。Western blot检测Hcy对EPCs中的PKC磷酸化水平的影响,并加入PIO和PKC抑制剂预处理30min,观察PKC磷酸化的变化。细胞内活性氧(ROS)浓度通过在细胞内装载荧光探针H2DCF-DA后用流式细胞仪分析。NADPH酶活性采用以光泽精基础的化学发光检测法。观察预先加入PIO,NADPH抑制剂DPI和PKC抑制剂GF对Hcy诱导的EPCs内ROS和NADPH酶活性的影响。对于GF和DPI预处理的Hcy共培养的EPCs,通过ELISA和realtime-PCR观察抑制PKC和NAPDH酶后对EPCs旁分泌的影响。采用粘附能力测定实验和millicell小室分别观察PIO对Hcy诱导下的EPCs的粘附能力和迁移能力的影响。结果：PIO逆转Hcy下调的VEGF和IL-8的mRNA合成和蛋白分泌水平。Westernblot提示Hcy能时间依赖性激活P-PKC,以PKCβⅡ激活为主,而PIO能抑制PKC的磷酸化。化学发光检测法提示Hcy能时间依赖性激活NADPH酶,PIO, PKC抑制剂GF可以抑制NADPH酶的激活。通过荧光探针检测EPCs内ROS,PIO, NADPH酶和PKC抑制剂减少Hcy上调的活性氧生成。PKC抑制剂和NADPH酶抑制剂逆转Hcy下调的VEGF和IL-8水平。PIO可以改善Hcy诱导的EPCs的粘附迁移能力损伤。结论：PIO能显著改善Hcy对EPCs的旁分泌,粘附和迁移功能的损害。PIO通过抑制PKC的激活和NADPH酶的活性逆转Hcy所诱导的EPCs功能损伤。3匹格列酮对同型半胱氨酸干预下内皮祖细胞NADPH酶的影响目的：探讨PIO对Hcy干预下EPCs的NADPH酶亚单位的影响。方法：从健康人外周血分离、培养循环EPCS。培养第七天,采用不同浓度的Hcy(10,50,100和200μmol/L)干预EPCs24h,Western blot检测NADPH酶亚单位p67和Nox2。分别预先加入PIO (10μmol/L), NADPH酶抑制剂DPI和PKC抑制剂GF共培养,观察PIO对Hcy干预下NADPH酶亚单位表达的影响。采用p67-siRNA和Nox2-siRNA干扰NADPH酶相应亚基蛋白合成,观察亚基蛋白下调后对Hcy诱导的EPC旁分泌功能损伤的作用。采用ELISA和real-time PCR检测VEGF和IL-8表达。采用粘附能力测定实验和millicell小室分别观察下调p67或Nox2亚单位对Hcy诱导下的EPCs的粘附能力和迁移能力的影响。预先加入PPARy受体特异性抑制剂GW9662以观察PIO是否通过该受体来拮抗Hcy的病理作用。结果：Hcy浓度依赖性增强NADPH亚单位p67和Nox2的表达。PIO抑制了Hcy上调的p67和Nox2蛋白的表达。NADPH酶抑制剂DPI和PKC抑制剂GF同样也抑制了NADPH酶亚单位蛋白的上调。p67-siRNA和Nox2-siRNA转染后显著抑制p67和Nox2蛋白表达。单独下调p67亚单位蛋白拮抗Hey诱导的EPCs旁分泌因子减少,单独下调Nox2亚单位蛋白统计学上无明显差异。下调p67或Nox2蛋白表达都可以逆转Hcy诱导的EPCs粘附能力和迁移能力损伤。我们使用了PPARγ抑制剂GW9662并发现其不能抑制匹格列酮带来的有利效应。结论：PIO能显著改善Hcy诱导下EPCs中NADPH酶亚单位蛋白的上调,这种作用通过抑制PKC的激活获得与NADPH酶抑制剂DPI相同的效果。转染p67-siRNA后逆转了Hcy诱导的EPCs旁分泌VEGF和IL-8的减少。单独下调p67或Nox2亚基都可以拮抗Hcy损伤的EPCs粘附和迁移能力。PIO改善EPCs旁分泌功能不依赖于PPARγ受体的激活。更多还原

【Abstract】 Increasing evidence suggests endothelial progenitor cells (EPCs) improve neovascularization and endothelium regeneration via the production of paracrine factors. Vascular endothelial growth factor (VEGF) and interleukin-8(IL-8) are major cytokines for EPC-based angiogenesis and re-endothelialization.Hyperhomocysteinemia has been recognized as a major risk factor of cardiovascular diseases. Although the mechanism by which Hey damages the vessels wall and induces atherosclerosis is complicated. Hey may injure EPC function and therefore inhibit the therapeutic effects of EPCs. Hey reduced EPC numbers and impairs their functional activity including proliferative, migratory, adhesive and in vitro vasculogenesis capacity. Hey accelerated the onset of EPC senescence, which involved inhibition of telomerase activity and Akt phosphorylation in EPCs. However, whether Hey cloud affect the paracrine function of EPCs needs further investigation. Therefore, we aimed to explore the effect of Hey on EPC paracrine function and the effect of paracrine factors from EPCs on in vivo post-injury reendothelialization in rats. Moreover, the mechanism of Hey involved in Hcy-induced EPC paracrine impairment is also under investigation. Pioglitazone (PIO), the peroxisome proliferator-activated receptors (PPARs) agonist, could improve the migratory response and the adhesive capacity of EPCs in patients with diabetes mellitus and coronary artery disease. PIO was proved to prevent apoptosis of EPCs and increase in vivo neoangiogenesis in mice. In addition, PIO ameliorated Ang Ⅱ-induced senescence of EPC. However, none of previous studies have investigated the effect of PIO on EPC cytokine production and related mechanisms.Based on these considerations, we aimed to investigate the expression and the secretion of cytokines in EPCs under high Hcy, and to study the effects of PIO on Hcy-injured EPCs and possible mechanism that involved in this progress. Finally, we observed the effect of knockdown of p67and Nox2on EPC function.Part1The effect of high Hcy on paracrine function of endothelial progenitor cellsObjective:To investigate the effect of high Hcy on paracrine function of endothelial progenitor cells.Methods:EPCs, isolated from peripheral blood of healthy volunteer, were cultured on fibronectin-coated dishes. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding under a laser scanning confocal microscope. They were further documented by demonstrating the expression of VE-cadherin, KDR, CD34and AC133by flow cytometry. EPCs-derived conditioned medium (EPC-CM) was obtained from culture EPCs subjected to trophic deprivation24h. The expression of cytokine mRNAs was measured by realtime PCR. VEGF and IL-8in cell culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). Cells were treated with Hcy (to make a series of final concentrations:10,50,100,200and500μmol/L) for24h. EPCs were also stimulated by200μmol/L Hey for respective time points (3,6,12,24and36h). After balloon injury, male Sprague-Dawley rats were divided into four groups to assay the re-endothelialization effect of EPC-CM:(1) normal rats as control;(2) balloon-injured vessels treated with EBM-2;(3) balloon-injured vessels treated with EPC-CM (4) balloon-injured vessels treated with EPC-CM collected from Hcy-stimulated EPCs. After treatment with EPC-CM for1week, the re-endothelialized area in balloon-injured arteries was observed.Result:compared to normal EGM-2, EBM-2, which is without cytokines or FBS (fetal bovine serum), stimulated EPCs to secrete a great quantity of VEGF and IL-8, but other cytokines such as IGF, HGF, SDF-1, PDGF-BB and Tβ4remained unchanged. Concentrations of VEGF and IL-8in EPC-CM were260.54±32.09pg/ml/1×106EPCs,15724.54±1817.24pg/ml/1×106EPCs, respectively. High Hey inhibited the secretion of VEGF and IL-8from EPCs.200μmol/L Hey inhibited VEGF and IL-8secretion in a time-dependent manner. The re-endothelialized area in balloon-injured arteries was increased significantly by EPC-CM. However, EPC-CM from Hcy-treated EPCs could not promote the reendothelialization process.Conclusion:VEGF and IL-8were the major cytokines secreted by EPCs. These paracrine cytokines could promote the reendothelialization of balloon-injured arteries. Hey induced a significant inhibition of VEGF and IL-8from EPCs, and inhibited the indirect therapy of EPCs on balloon-injured vessels. Part2Pioglitazone ameliorates Hcy-induced dysfunctions of EPCs via inhibiting the activation of PKC and NADPH oxidase Objective:To investigate the effect and mechanism of pioglitazone on Hcy-treated EPCsMethods:EPCs, isolated from peripheral blood of healthy volunteer, were cultured on fibronectin-coated dishes for7d. EPCs-derived conditioned medium(EPC-CM) was obtained from culture EPCs subjected to trophic deprivation24h. Cells were treated with Hcy (200μM) for24h. PIO (10μM) was added30min before Hcy treatment. The expression and secretion of VEGF and IL-8were measured by realtime-PCR and ELISA. The expression of PKC-α/βⅡ、p-PKC-α/βⅡ、PKC-α、p-PKC-α、PKC-βⅡ and p-PKC-βⅡ were detected by Western blot. EPCs were pre-incubated with PKC inhibitor GF (5μM) for30min before the addition of Hcy (200μM) for24h. Intracellular ROS levels were measured by flow cytometry with the fluorescent probe, H2DCF-DA. The lucigenin-derived enhanced chemiluminescence assay was used to determine NADPH oxidase activity. EPCs were pre-incubated with PIO (10μM), NADPH oxidase inhibitor DPI (5μM) and GF (5μM) for30min before the addition of Hcy (200μM) for24h. We observed the effect of PIO, DPI and GF on ROS and NADPH oxidase. The effects of PKC inhibitor GF and NADPH oxidase inhibitor DPI on both protein and mRNA production of VEGF and IL-8by EPCs after co-cultured with Hcy were analyzed by ELISA and real-time RT-PCR. The effects of PIO, DPI and GF on EPC migration and adhesion were assayed in8.0-μm pore size transwells and fibronectin-coated culture dishes, respectively.Result:PIO normalized VEGF and IL-8secretion and expression which were decreased by Hcy. Hcy treatment increased phosphorylation of PKC in a time-dependent manner. We found Hcy stimulated PKCβⅡ phosphorylation but did not affect phosphorylation of PKCα. Moreover, PIO inhibited PKC activation induced by Hcy. PIO and GF pretreatment significantly reduced Hcy-induced ROS and NADPH oxidase activation, which was consistent with the results of DPI. Pretreatment with DPI and GF restored not only Hcy-mediated VEGF and IL-8reduction but also injured adhesion and migration of EPCs.Conclusion:Addition of PIO to Hcy treated EPCs attenuated dysfunctions. PIO attenuated EPC paracrine dysfunction induced by Hcy by inhibition of NADPH oxidase and PKC. Part3The effect of pioglitazone on NADPH oxidase of Hcy-treated EPCsObjective:To investigate the effect of pioglitazone on expression of NADPH oxidase subunits.Methods:EPCs were cultured on fibronectin-coated dishes for7d. Cells were treated with Hcy (10,50,100and200μM) for24h. EPCs were also pre-incubated with PIO (10μM), NADPH oxidase inhibitor DPI (5μM) and GF (5μM) for30min before the addition of Hcy (200μM) for24h. The expressions of p67and Nox2subunits were detected by Western blot. Transfection of EPCs with p67-siRNA and Nox2-siRNA resulted in abolishment of the expression of p67and Nox2. We observed the effect of down-regulation of NADPH subunits on EPC paracrine function. The productions of VEGF and IL-8by EPCs after transfection of siRNAs were analyzed by ELISA and real-time RT-PCR. The effects of down-regulation of p67and Nox2on EPC adhesion and migration were assayed in8.0-μm pore size transwells and fibronectin-coated culture dishes.Result:Western blotting data showed Hcy dose dependently increased expression of p67and Nox2. PIO may inhibit p67and Nox2by inhibition of PKC. Our data further demonstrated siRNAs against NADPH oxidase components, p67and Nox2, suppressed Hcy-mediated oxidative stress. P67-siRNA exerted a more significant effect than Nox2-siRNA on reversing paracrine dysfunction caused by Hcy. EPCs transfected with Nox2and p67siRNA showed apparently higher cell migration and adhesiveness than cells transfected with control siRNA under stimulation of Hey. Besides, PIO attenuated Hcy-induced EPC dysfunction, especially reduced expression and secretion of VEGF and IL-8by EPCs in a PPARy-independent manner.Conclusion:The mechanism of PIO-mediated down-regulation of p67and Nox2subunits involved inhibition of PKC. p67-siRNA reversed EPC paracrine dysfunction caused by Hey. EPCs transfected with Nox2or p67siRNA restored EPC migration and adhesiveness. Protective effect of PIO on cytokine secretion by EPCs could be unrelated to the activation of PPARy.更多还原