VDAC1在K5诱导血管内皮细胞凋亡中的关键作用及机制研究
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摘要
研究背景
血管新生（Angiogenesis）的概念，最早被JudahFolkman于1971年提出并被沿用至今。认为血管新生是从已存在的毛细血管网上大量生成新生血管的过程。血管新生在创伤和外周循环闭塞情况下具有恢复血供和促进伤口愈合的积极作用，但在更多的情况下血管新生则是一持续、无控性的过程，已成为肿瘤和血管增生性眼病等众多疾病的主要病理特征之一。内源性血管新生抑制因子和刺激因子之间的平衡失调是血管新生的基本分子机制。应用血管抑制因子和阻断血管新生刺激因子来阻断新生血管形成为治疗恶性肿瘤和其他血管增生性疾病提供了新的视野。
在已发现的众多血管抑制因子中，人纤溶酶原K5是目前发现的分子量小、性质稳定、活性较强的血管增生抑制剂之一。人纤溶酶原（plasminogen）含有5个环状结构域（Kringles），每个环由80个氨基酸残基组成，含3个二硫键，形成双环状构象。水解后可产生一组具有抑制血管增生作用的小分子片段：Angiostatin（Kringles1-4），Kringles1-5， Kringles1-3和Kringle5（K5）。血管抑素（Angiostatin）用于肿瘤的治疗已进入I期临床试验阶段。较之血管抑素（分子量约45kD），K5具有分子量小（16kD）、性质稳定且活性更强的优点。因为它的高效率、细胞选择性,和短的氨基酸序列,K5可作为治疗血管新生性疾病的候选药物。
K5抑制血管新生主要通过诱导血管内皮细胞凋亡实现，但其诱导内皮细胞凋亡的机制和信号通路尚需深入研究。重组K5蛋白被证明能够诱导增殖的内皮细胞凋亡；我们课题组之前的研究显示, K5在氧诱导的大鼠视网膜病变模型中，下调视网膜VEGF表达、上调PEDF表达，纠正VEGF/PEDF比率，恢复血管因子之间的平衡从而抑制视网膜血管新生并减少血管渗漏；在小鼠肝癌移植瘤模型上，K5可显著地增加切割的caspase3的含量诱导内皮凋亡，通过抑制血管新生来抑制肿瘤生长。
参与K5诱导凋亡的一些分子被陆续报道。先前的研究已经表明葡萄糖调节蛋白78（GRP78）和电压依赖性阴离子通道1(VDAC1)参与K5诱导肿瘤细胞和内皮细胞凋亡并可能起K5受体的作用,但尚存在争议。电压依赖性电离子通道（VDAC1）是多功能通道蛋白，主要位于线粒体外膜，调控一系列的生理和病理活动，如细胞Ca2+的平衡，能量代谢和细胞凋亡；VDAC1也可定位于细胞膜，作为膜受体介导信号转导。VDAC1在K5诱导内皮细胞凋亡中的作用尚缺乏系统研究。
研究目标
阐明K5诱导血管内皮细胞凋亡的信号通路；明确VDAC1在K5诱导血管内皮细胞凋亡中的关键作用和机制。为K5防治血管增生性疾病提供理论依据，同时关键分子的发现也为提供新的干预靶点提供线索。
研究内容与结果
第一章K5诱导内皮细胞凋亡的信号通路
1、高纯度K5重组蛋白的获取
诱导表达的含氨苄青霉素抗性的工程菌，按照提取可溶性K5重组蛋白的方法，采用Ni2+-His Bind Resin进行亲和层析得到与Ni柱结合的含有6个组氨酸的K5重组蛋白。采用SDS-PAGE电泳，将凝胶进行考马斯亮蓝染色显示获得高纯度可溶性蛋白。用凝胶成像系统对凝胶进行灰度扫描，结果显示纯化后的K5重组蛋白纯度为90%以上。
2、 K5抑制激活的HUVEC增殖和凋亡诱导作用
K5对激活的内皮细胞有明显的抑制增殖的作用，且存在浓度-效应关系；Hoechst33258染色初步显示随着K5浓度的增加，凋亡的内皮细胞逐渐增多；Annexin V/PI staining检测细胞凋亡率，与对照组相比，浓度变化在160~1280nmol/L各K5处理组细胞凋亡率分别为25.55±0.83%、33.4±3.11%、40.11±1.41%、45.04±0.99%，与阴性对照组和阳性对照组相比，均表现出能明显诱导血管内皮细胞凋亡的作用。
3、 K5通过线粒体凋亡通路诱导内皮细胞凋亡
3.1K5激活caspase7,8,9，降低线粒体膜电位，促进细胞色素C（Cyt c）释放
经640nmol/L K5处理72h后，Western blotting分析结果显示引起了明显的caspase7, caspase8和caspase9的切割；此外采用流式细胞仪检测K5处理后的线粒体膜电位的变化，线粒体膜电位降低，示内皮细胞线粒体膜被破坏；提取线粒体和胞浆蛋白，K5能明显地促进Cyt c从线粒体释放至胞浆，提示线粒体凋亡通路的激活。
3.2K5主要通过线粒体凋亡通路诱导内皮细胞凋亡
总caspase抑制剂（Z-VAD-FMK）, caspase8抑制剂（Z-IETD-FMK）或者caspase9抑制剂（Z-LEHD-FMK）预处理1h后，然后加入K5检测内皮细胞凋亡率，与caspase8抑制剂相比，caspase9抑制剂能更加有效地抑制K5诱导的内皮细胞凋亡，证实K5主要通过线粒体凋亡通路诱导内皮细胞凋亡。
3.3死亡受体凋亡通路对线粒体凋亡通路无交叉调控作用
此外，caspase8的抑制剂对K5引起的Bak分布的调控无影响，推论K5引起的Bcl-2家族蛋白细胞分布变化不受死亡受体凋亡通路的调控。
4、K5通过对Bcl-2家族蛋白的调控来引起Cyt c的释放，诱导凋亡
K5处理血管内皮细胞24h后，检测Bcl-2家族蛋白Bcl-2, Bcl-xL, Bax and Bak的分布，K5均不影响这四种蛋白的总量；然而K5明显地增加线粒体上Bak、减少Bcl-xL的量，与之对应的减少胞浆中Bak、增加Bcl-xL的量；推测K5通过调控Bak/Bcl-xL的比值引起Cyt c的释放来诱导凋亡。
第二章VDAC1在K5诱导内皮细胞凋亡中的作用
1、VDAC1在K5激活线粒体凋亡通路中的关键作用
1.1K5促进VDAC1与Bak结合增多
免疫共沉淀（IP）实验显示，K5上调磷酸化的VDAC1（p-VDAC1）,增加Bak与VDAC1的结合，降低Bcl-xL与VDAC1的结合。反向IP实验也证明，K5仍可增加VDAC1与Bak的结合。
1.2VDAC1与Bak结合促进线粒体通透性转换孔道（mPTP）的开放
应用MitoProbe TransitionPore试剂盒流式分析仪进行mPTP开放的检测，线粒体内钙黄绿素荧光的降低，表明膜通道孔的开放增加。经640nmol/LK5处理24h后，K5组线粒体内钙黄绿素荧光平均强度减低为对照组细胞的55.53±2.87%，差异有统计学意义。证明K5处理后线粒体膜被破坏、mPTP开放。
1.3VDAC1与Bak结合上调Cyt c的表达，激活caspase3
用160-1280nM浓度的K5处理HUVECs，Western blotting结果显示总蛋白中Cyt c出现剂量依赖性的上调；K5处理72h后，出现明显的caspase3的激活。
2、VDAC1作为细胞膜受体介导K5诱导的HUVECs凋亡
2.1抗体阻断实验对K5诱导内皮细胞凋亡的调控的影响
GRP78N端抗体和VDAC1mouse来源的抗体孵育HUVECs30min后，640nmol/L的K5作用72h后，流式检测凋亡率；WB检测caspase3的切割变化。结果显示使用GRP78的抗体后，K5仍能诱导HUVECs凋亡；使用VDAC1的抗体后，K5诱导HUVECs凋亡的作用被阻断。提示VDAC1抗体与K5竞争结合K5的受体----VDAC1。
2.2小分子RNA干扰实验对K5诱导内皮细胞凋亡的调控的影响
此外，RNAi实验用来证实VDAC1的关键作用。VDAC1和GRP78的siRNA，用HiPerFect转染HUVECs12h后，640nmol/L的K5作用72h，WB检测caspase3的切割变化；Promega公司的Caspase-Glo3/7Assay用于检测caspase3/7的活性。结果显示siVDAC1后，K5无激活caspase3/7的作用；然而si GRP78后，K5仍有激活caspase3/7的作用。提示VDAC1而非GRP78在K5诱导的凋亡中其关键作用。
2.3双分子荧光互补实验（BiFC）显示K5与VDAC1在细胞内存在直接结合
此外，构建双分子荧光互补N端和C端的两个质粒，pBiFC-K5VN155和pBIFC-VDAC1VC155。这两种质粒共转染293A细胞20h，全自动激光共聚焦显微镜显微镜拍照。BiFC实验阳性转染组细胞核内出现黄色荧光，共转染上述两质粒组出现黄色荧光，且荧光分别在线粒体和细胞膜，证明K5和VDAC1之间存在相互作用。已知VDAC1大部分定位线粒体，少部分定位细胞膜等，推论K5与VDAC1存在细胞膜和线粒体的共定位。
第三章K5对VDAC1含量及线粒体和细胞膜转位的调节
1、 K5上调细胞内VDAC1的总量及其机制
1.1K5上调HUVECs内VDAC1的含量
160nmol/L~1280nmol/L K5处理HUVECs24h后，提取细胞总蛋白，Westernblotting分析结果显示，K5呈剂量依赖性增加总蛋白中的VDAC1含量。
1.2K5抑制VDAC1的泛素化降解
Q-PCR结果显示，K5不调控VDAC1的mRNA水平。推测K5通过抑制VDAC1的降解来上调VDAC1。
640nmol/LK5处理HUVECs4h后，1uM的MG132继续作用20h。细胞采用温和的裂解条件做IP，加入VDAC1的抗体结合细胞内的全部VDAC1。WB后，用Ub抗体检测出的蛋白即为泛素化的VDAC1，结果显示K5处理和K5+MG132处理后，泛素化的VDAC1的含量均降低。结果提示K5通过抑制VDAC1的泛素化降解来上调VDAC1。
1.3K5通过AKT-GSK3β通路促进VDAC1的磷酸化
Western blotting结果显示，经640nmol/L的K5处理HUVECs5h后，与对照组相比，K5明显抑制AKT Ser473位的磷酸化，不影响总的AKT的表达水平；K5明显抑制GSK3β Ser9位的磷酸化，上调总的GSK3β的表达。GSK3β Ser9位磷酸化代表GSK3β的活性抑制。上述结果显示K5抑制AKT的活性，激活GSK3β活性。
将终浓度为1uM/L的AKT抑制剂和GSK3β抑制剂预处理HUVEC30min后，K5继续作用24h。细胞采用温和的裂解条件做IP，加入VDAC1的抗体结合细胞内的全部VDAC1。WB后，检测总的和磷酸化的VDAC1。结果显示，K5上调总的和磷酸化的VDAC1，使用AKT抑制剂后，总的和磷酸化的VDAC1上调。与单独AKT抑制剂组相比，AKT抑制剂+K5组不影响总的VDAC1的表达，上调磷酸化的VDAC1的表达。使用GSK3β抑制剂后，总的和磷酸化的VDAC1下调。与单独GSK3β抑制剂组相比，GSK3β抑制剂+K5组不影响总的和磷酸化的VDAC1的表达。GSK3β干扰组出现与GSK3β抑制剂组相似的结果。
AKT的激活剂insulin预处理HUVEC30min后，K5继续作用24h。与单独insulin组相比，insulin+K5组不影响总的和磷酸化的VDAC1的表达。
以上结果显示，K5通过影响AKT-GSK3β通路来促进VDAC1的磷酸化，并进而影响其降解。
1.4VDAC1介导K5对AKT-GSK3β通路的调控
HK I为细胞内VDAC1天然高效的拮抗剂，可与VDAC1结合抑制其功能。HK I预处理HUVECs，K5继续作用5h。WB结果显示与单独的HK I处理组相比，HK I+K5组无抑制AKT活性，激活GSK3β的作用。干扰VDAC112h后，K5继续作用5h。WB结果出现与使用HK I抑制剂同样的结果。提示K5对细胞内信号通路的调控需要膜受体VDAC1的参与。
1.5K5通过促进VDAC1磷酸化抑制泛素化降解
构建VDAC1的四个磷酸化突变位点，将13位，52位，104位和107位磷酸化位点突变为丙氨酸。在转染相同量的质粒的情况下，无论在HUVEC还是293A细胞上，与单纯转染VDAC1过表达质粒组相比，S13A，S104A和T107A质粒转染组均降低外源性导入的VDAC1的含量。提示K5通过调控13位，104位和107位磷酸化来抑制其泛素化降解来上调VDAC1的表达。
2、K5促进VDAC1线粒体和细胞膜转位及机制
2.1K5促进VDAC1线粒体和细胞膜转位
K5处理HUVECs24h后，提取线粒体和胞浆蛋白。结果显示K5明显增加线粒体中的VDAC1，胞浆中未检测到VDAC1的表达。
采用文献报道的改良的IP方法来研究细胞膜上的VDAC1表达。K5处理24h后，细胞膜上的VDAC1明显增加。
2.2VDAC1总量的增多导致线粒体和细胞膜转位的增多
在HEK293A细胞过表达VDAC1，与转染阴性对照质粒组相比，出现和K5类似的上调VDAC1总量，及线粒体和细胞膜分布增加。提示VDAC1总量的增多导致了线粒体和细胞膜VDAC1分布的增多，即产生了线粒体和细胞膜的特定亚细胞转位。
2.3HK I拮抗K5对VDAC1细胞分布的调控
HK I预处理5min后，K5继续作用24h。提取总蛋白，线粒体和细胞膜蛋白，结果显示使用HK I后，K5对总蛋白，线粒体和细胞膜蛋白的VDAC1均无调控作用。免疫细胞化学的结果与此类似。提示K5对细胞内VDAC1量和分布的调控需要作为K5膜受体VDAC1本身的存在和参与。
结论及研究意义
1、K5通过线粒体凋亡通路诱导内皮细胞凋亡
首次阐明K5主要调节Bcl-2家族蛋白促凋亡分子Bak和抗凋亡分子Bcl-xL在HUVECs细胞组分中的分布，上调线粒体上Bak/Bcl-xL的比值来启动线粒体凋亡通路。随后降低线粒体膜电位，促进Cyt c的释放至胞浆，启动caspase9，导致内皮细胞凋亡。
2、VDAC1是介导K5诱导内皮细胞凋亡的关键调控分子
阐明VDAC1同时作为受体和线粒体膜通道蛋白在K5激活线粒体通路的关键作用，且此受体不受分子伴侣GRP78的调控，提示VDAC1为调控内皮细胞凋亡的有效干预靶点。
3、K5促进VDAC1磷酸化抑制其泛素化降解从而上调VDAC1
K5主要通过抑制AKT的活性，进而激活GSK3β来调控磷酸化的VDAC1，其作用的磷酸化位点有13位丝氨酸，104位丝氨酸和107位苏氨酸。本研究首次发现VDAC1的107位苏氨酸位点磷酸化可以抑制VDAC1的降解来上调VDAC1的含量。
4、K5通过上调VDAC1促进其线粒体和细胞膜转位
过表达VDAC1可促进VDAC1线粒体和细胞膜的转位，提示VDAC1总量的增多导致了细胞膜和线粒体VDAC1分布的增多，即K5通过上调VDAC1总量促进了细胞膜和线粒体的特定亚细胞转位。
5、K5通过正反馈调节环路放大对细胞凋亡的诱导作用
本研究的结果表明在配体K5和受体VDAC1之间存在正反馈调节环路。VDAC1作为受体介导K5对VDAC1自身含量的上调，而上调的VDAC1又进一步增加其在细胞膜的分布，放大了K5的作用。因此，这种正反馈调节环路发挥了对细胞凋亡的诱导放大功能。
BACKGROUND
Angiogenesis was first proposed by Judah Folkman in1971. Angiogenesis is theformation of new capillaries from preexisting vessels. In adults, neovasularizationfavours the blood flood and the wound healing during peripheral circulation andtrauma. However, angiogeneis is more often to be a persistent and uncontrolledprocess, which has been established as one of main pathological characteristics oftumor and other angiogenesis-related diseases. Angiogenesis is regulated by thebalance between endogenous angiogenic stimulators and inhibitors. In pathologicconditions, the balance is disrupted, and many diseases are driven. Recently, thestrategy of applying endogenous angiogenenic inhibitors and blocking endogenousangiogenic stimulators has been used to treat tumor and other angiogenesis diseases inclinical researches.
Plasminogen kringle5(K5), a proteolytic fragment of plasminogen with smallmolecular and stable property, displays potent angiogenesis inhibitory activity asother endogenous angiogenic inhibitors. Plasminogen contains five kringles.Eachkringle consists of80amino acids and3disulfide bonds forming double loop structuredomain. Series of small molecular pieces emerge after hydrolyzation of plasminogen:Angiostati（nKringles1-4），Kringles1-5，Kringles1-3和Kringle5(K5). Angiostatinwas applied into phase I clinical trials to cure tumor. Contrast to angiostatin (45kD), K5possessed smaller molecular weight, higher activity in inhibiting angiogenesis. SoK5could be a candidate drug to treat angiogenesis-related diseases.
K5inhibited angiogenesis mainly through inducing endothelial cells apoptosis,but the mechanism and signal pathway involved in endothelial cells apoptosis neededto be thoroughly discussed. Recombinant K5has also been shown to induce apoptosisin proliferating endothelial cells. Our previous study has shown that K5inhibitsretinal neovascularization by decreasing the expression of VEGF, increasing theprotein level of PEDF and correcting the ratio of VEGF/PEDF to restore theangiogenesis balance and reduces vascular leakage in oxygen-induced rat retinopathymodel. Meanwhile, our study demonstrated that K5induced endothelial cellsapoptosis by increasing the cleavage of caspase3, and suppressed hepatocarcinomagrowth by anti-angiogenesis in HepA-grafted and Bel7402-xenograftedhepatocarcinoma mouse models.
Several molecules were identifed to be involved in K5-induced apoptosis.Previous studies have suggested that K5-induced apoptosis of tumor cells andendothelial cells was related to glucose-regulated protein78and Voltage dependentanion channel (VDAC1). However, there existed controversy. VDACs, also known asmulti-functional mitochondrial porins, are abundant proteins found in the outermitochondrial membrane (OMM). VDAC1regulate series of physiology andpathology events, such as the balance of celluar Ca2+, energy metabolism and cellapoptosis; VDAC1locates to plasma membrane too, as the receptor for K5to deliversignal. There still lacked systematicly research about the function of VDAC1involedin K5-induced endothelial cells apoptosis.
OBJECTIVES
To elucidate the signaling pathways involved in K5-induced endothelial cellsapoptosis, and further confirm the key role and the mechanism of VDAC1in thisprocess. On the basis of that, it provided a theoretical reason for K5in the treatmentof angiogenesis-related diseases; and the discovery of the key molecular would offerclue for the novel intervention targets.
EXPERIMENTAL RESULTS
Chapter1Research on the signaling pathways by which K5-inducedendothelial cells apoptosis
1, the acquisition of high purity K5recombinant protein
Expression was induced by the addition of Isopropyl-β-D-thiogalactopyranoside(IPTG) form the bacterias with ampicillin resistance construction plasmid. Solubleproteins were extracted under native conditions, and the recombinant peptide waspurified by passing through the His-Bind column. The purity of K5processed bythin-layer protein scanning of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) gel with Genegenius gel image system (Gene Co.England) were all over90%.
2, K5inhibits proliferation and induces apoptosis of activatedHUVECs
K5exhibited selective inhibitory effect of K5on activated HUVECs withsignificant dose-effect correlation. Hoechst33258assay preliminarily demonstratedan enhanced effect on inducing cell apoptosis with the increased concentration of K5.In comparison with PBS-treated group, Annexin V/PI analysis showed that apoptoticratio of160,320,640and1280nmol/l. K5-treated groups were25.55±0.83,33.4±3.11,40.11±1.41, and45.04±0.99%. Compared to negative and positive controls,K5induced the apoptosis of HUVECs in a dose-dependent manner.
3, K5induces endothelial cell apoptosis via mitochondrial pathway
3.1K5activated caspase7,8,9, decreased the mitochondrial me mbranepotential and promoted the release of cytochrome c
Western blotting analysis showed signifcant increase in the amounts ofcleavedcaspase7, cleavedcaspase8and cleavedcaspase9by K5treatment. To furtherinvestigate whether the mitochondria pathway is involved in the K5-induced apoptotic process, the mitochondrial membrane potential (MMP) was analyzed by fowcytometry. K5reduced the MMP of HUVECs. Futher more, western blotting analysisshowed that K5stimulated the release of cytochrome c from mitochondrial intocytosol. Mitochondria apoptosis pathway was involved in K5-induced apoptosis.
3.2K5induces endothelial cells apoptosis mainly through mitochondrialpathway
Apoptotic rate of HUVECs induced by K5in the presence of Z-VAD-FMK(Pan-caspaseinhibitor), Z-IETD-FMK (caspase8inhibitor) or Z-LEHD-FMK(caspase9inhibitor) was assessed, respectively. The results showed Z-VAD-FMK,Z-IETD-FMK or Z-LEHD-FMK could attenuate the K5-induced apoptosis. Itdemonstrated that K5induces endothelial cell apoptosis mainly through mitochondrialpathway.
3.3Death receptor apoptosis pathway had no cross regulation withmitochondrial apoptosis pathway
Further, Z-IETD-FMK, inhibitor of caspase8, had no effect on the K5-inducedtranslocation of Bak in HUVECs. We deduced that death receptor apoptosis pathwayhad no cross regulation with mitochondrial apoptosis pathway.
4, Effect of Bcl-2family proteins in K5-induced cytochrome c releaseand apoptosis
We analyzed the distribution of Bcl-2family proteins (Bcl-2, Bcl-xL, Bax andBak) in HUVECs after treatment with K5for24h by Western blotting analysis. Theseresults showed that K5had no effects on all the protein levels in total cell lysates.However, K5signifcantly increased the level of Bak and reduced the amount ofBcl-xL in mitochondria portion. These results inferred that the increased ratio of Bakto Bcl-xL on mitochondria was responsible for mitochondrial depolarization,cytochrome c release and consequently for the activation of caspase9.
Chapter2The implication of VDAC1in the apoptosis of endothelialcells induced by human plasminogen Kringle5
1, The important role of VDAC1involved in the K5-activatedmitochondrial apoptosis pathway
1.1K5increased the interaction between VDAC1and Bak
Co-immunoprecipitation (IP) assay performed that K5added the phosphorylationof VDAC1, increased the interaction between VDAC1and Bak and decreased theinteraction of VDAC1and Bcl-xL. Reverse IP assay still exerted that K5stillup-regulated the interaction of VDAC1and Bak.
1.2The inte raction within VDAC1and Bak promoted the opening ofmitochondrial permeability transition pore (mPTP)
The opening of mPTP was detected by MitoProbe Transition Pore kit andFCM. Decreased fluorescent intensity of Calcein AM in the mitochondria exerted theincrease opening of mPTP. In compared to control, the fluorescent intensity ofCalcein AM was reduced to55.53±2.87%and there had statistical significance.Results indicated that the mitochondrial membrane was disrupted and the mPTP wasopened in addition to K5treatment.
1.3The inte raction within VDAC1and Bak increased the Cyt c and activatedcaspase3
As shown in chapter one, K5stimulated the release of cytochrome c frommitochondrial into cytosol. Interestingly, K5could added the total amounts ofVDAC1in a dose-dependently manner. Moreover, K5obviously activated caspase3.
2, VDAC1was as the plasma membrane receptor in K5-inducedHUVECs apoptosis
2.1Antibody blockade assay affected K5-induced HUVECs apoptosis
GRP78N-terminal antibody and VDAC1antibody incubated HUVECs for30min, then cells were treated with K5for another72h, then we used Annexin V/PIassay to detect the apoptotic rate and the change of caspase3by western blottingassay. All assays showed that K5still induced HUVECs apoptosis in addition to GRP78N-terminal antibody, but VDAC1antibody blocked the K5-induced HUVECsapoptosis. It indicated that VDAC1antibody and K5had competitive binding with thereceptor of K5: VDAC1.
2.2Small RNA interference assay affected K5-induced HUVECs apoptosis
RNA interference assay was exploited to demonstrate the pivotal role of VDAC1.We transfected VDAC1si RNA or GRP78si RNA with HiPerFect reagent intoHUVECs for12h,640nM K5was utilized for another72hours. Then the change ofcaspase3was detected by western blotting and the activity of caspase3/7wasdetected by Caspase-Glo3/7Assay in Promega company. Results displayed that K5did not have activating function to caspase3/7after siVDAC1, K5remainedactivating caspase3/7with GRP78knockdown. The research pointed out thatVDAC1more than GRP78played an important role in K5-induced apoptosis.
2.3Bimolecular fluorescence complementation (BiFC) assay showed the directinteraction of K5and VDAC1
We constructed the N–terminal and C-terminal plasmids of BiFC assay,pBiFC-K5VN155and pBIFC-VDAC1VC155. Plasmids for BiFC assay weretransfected into HEK293A cells for20h, we observed and photographed with fullautomatic laser confocal microscopy. There had yellow fluorescence in the nuclear inthe positive BiFC control, and transfected the above two plasmids group showedyellow fluorescence too in both mitochondria and plasma membrane. Resultsperformed that there had direct interaction within K5and VDAC1. It is known thatmost of VDAC1located in mitochondria, only small number of VDAC1located inplasma membrane and so on, we inferred that K5and VDAC1had co-localization inboth mitochondria and plasma membrane.
Chapter3K5regulated the amount and transposition of VDAC1inmitochondria and plasma membrane
1, The mechanism involved in K5-aroused increase of VDAC1.
1.1K5increased the total amount of VDAC1
HUVECs were treated with160nM~1280nM K5for24h, total protein were extracted and the Western blotting assay showed that K5dose-dependently increasedthe total expression level of VDAC1.
1.2K5restrained ubiquitin degradation of VDAC1
Further, Q-PCR assay showed that K5had no influence on the mRNA expressionof VDAC1. So we deduced that K5suppressed the degradation to up-regulateVDAC1protein level.
First640nM K5treated HUVECs for4hours,1uM MG132was added to theculture medium for another20hours. Then cells were lysed with RIPA buffer for IPassay and the VDAC1antibody was used to pull all protein of VDAC1. IP assayshowed that the ubiquitin protein represents ubiquitin VDAC1. Our results showedthe level of ubiquitin VDAC1was decreased in addition to K5treatment orK5+MG132treatment. It indicated that K5suppressed the ubiquitin chemicaldegradation to increase the total protein of VDAC1.
1.3K5regulated the expression of VDAC1via AKT-GSK3β pathway
HUVECs were managed with640nM K5for5h, contrast to the control groups,K5obviously restrained the activity of AKT in the ser473phosphorylation site, butthe total expression of AKT was not influenced by K5; K5clearly stopped the activityof GSK3β in the ser9phosphorylation site, and evidently raised the expression oftotal GSK3β. When phosphorylated on Ser9, GSK3β is inactivated. Results showedthat K5suppressed the activity of AKT and activated GSK3β.
HUVECs were pre-treated with either1umol/L Akt inhibitor (Akt inhibitor IV)or1umol/L GSK3β inhibitor and then with K5for another24h. VDAC1wasimmunoprecipitated from HUVECs lysates and its phosphorylation is determined byphosphoserine/threonine/tyrosine antibody. As shown in chapter one, K5increased thetotal protein and phosphorylation of VDAC1. AKT inhibitor could lead to the sameresults like K5. Compared to alone AKT inhibitor group, AKT inhibitor along with K5group up-regulated the phosphorylation of VDAC1, but did not affect the totalamount of VDAC1. The total protein and phosphorylation of VDAC1were decreasedby GSK3β inhibitor and siGSK3β. Compared to alone GSK3β inhibitor and siGSK3βgroup, GSK3β inhibitor and siGSK3β along with K5group did not affect the total amount and phosphorylation of VDAC1.
HUVECs were pre-treated with either AKT activator insulin and then with K5for another24h. Compared with alone insulin group, insulin along with K5group didnot affect the total amount and phosphorylation of VDAC1.
Our results displayed that K5promoted the phosphorylation of VDAC1viaAKT-GSK3β pathway to restrain ubiquitindegradationof VDAC1.
1.4VDAC1was mediated in the AKT-GSK3β pathway
HK I was the high effect agonist of VDAC1in kinds of cells, and HK I inhibitedVDAC1by the interaction. HUVECs were pre-treated with HK I and then with K5foranother5h. Compared to alone HK I group, HK I along with K5group did notrestrain the AKT activity and activate the GSK3β activity. VDAC1was interferedwith HiPerFect reagent in HUVECs for12h, K5treated HUVECs for another5h.Results were the same as HK I blockade assay. It hinted that VDAC1as the receptorfor K5was mediated in the AKT-GSK3β pathway.
1.5K5promoted the phosphorylation of VDAC1to inhibit its ubiquitindegradation
To further investigate the phosphorylated sites of VDAC1, we constructed aseries of VDAC1mutants, which disturbed the potential serine and tyrosinephosphorylation on VDAC1. These mutants, including S13A, T52A, S103A, andS107A, were transfected into HUVECs and HEK293A cells, and their protein levelswere detected by Western blotting assay. Three VDAC1mutants, S12A, S103A andS107A, could effectively reduce the protein level of VDAC1. These results indicatethat dephosphorylation of VDAC1may impair its protein level elevation.
2, The mechanism involved in K5-caused transposition of VDAC1inmitochondria and plasma membrane
2.1K5facilitated the translocation of VDAC1in mitochondria and plasmame mbrane
HUVECs were treated with K5for24h and then the mitochondrial and cytosolfraction were extracted. Results displayed that K5significantly increased the mitochondrial protein level of VDAC1, but there had no VDAC1protein in thecytosol.
We exploited the modified IP assay to detect the plasma membrane of VDAC1according to previously description. Our data showed that the plasma membraneprotein level of VDAC1was obviously added in addition to K5treatment for24h.
2.2The augment amount of VDAC1increased the transposition in mitochondriaand plasma me mbrane
VDAC1was transfected into HEK293A cells for24h. Compared to pcDNA3.1plasmid transfection group, VDAC1overexpression group added the amount andpromoter transposition of VDAC1in mitochondria and plasma membrane. Our datainferred that the augment amount of VDAC1increased the transposition inmitochondria and plasma membrane, as to say, resulted in the mitochondria andplasma membrane special subcellular translocation of VDAC1.
2.3HK I blocked the regulation of VDAC1by K5
HUVECs were pre-treated with HK I for5min and then with K5for another24h. We extracted the total protein, mitochondrial and cytosol fraction. Our data showedthat K5had no regulation to the total protein, mitochondrial and cytosol fraction ofVDAC1. It performed that the regulation of VDAC1by K5needed the receptorVDAC1.CONCLUSIONS
1, K5induces endothelial cells apoptosis via mitochondrial apoptosis pathway
Our results for the first time demonstrated that K5regulated the distribution ofBcl-2family members Bak and Bcl-xL in HUVECs, increased the ratio of Bak/Bcl-xLin the mitochondria to initiate mitochondrial apoptosis pathway. Subsequently, K5decreased the MMP, promoted the release of Cyt c to cytosol and activated caspase9to induce HUVECs apoptosis.
2, The important role of VDAC1involved in the K5-triggered HUVECsapoptosis
Our data elucidated the key role of VDAC1both as the receptor and mitochondrial membrane channel protein in activating the mitochondrial apoptosispathway, and the function of VDAC1did not affected by the molecular chaperoneprotein GRP78. It indicated that VDAC1was an effective intervention target toregulate endothelial cells apoptosis.
3, K5increased the VDAC1by promoting the phosphorylation of VDAC1toinhibit its ubiquitin degradation
K5suppressed the activity of AKT and activated GSK3β. Our results displayedthat K5promoted the phosphorylation of VDAC1via AKT-GSK3β pathway torestrain ubiquitin degradation of VDAC1. Dephosphorylation of VDAC1involved inS13, T52, S103, and S107site may impair its protein level elevation. For the first time,we discovered that the phosphorylation of VDAC1in its S107site suppressed thedegradation to increase the total amount of VDAC1.
4, K5facilitated the translocation of VDAC1in mitocho ndria and plasmame mbrane by up-regulating VDAC1
Our data inferred that the augment amount of VDAC1increased thetransposition in mitochondria and plasma membrane. So we deduced that K5promoted the mitochondria and plasma membrane special subcellular translocation ofVDAC1through up-regulating its total amount.
5, K5blowed up the apoptosis effect by positive feedback regulation of VDAC
Our results showed that there existed positive feedback loop between the ligandof K5and the receptor of VDAC1. As the receptor, VDAC1participated in itsregulation induced by K5, the increase of VDAC1further increased its plasmamembrane distribution to enlarge the function of K5. Since the positive feedback loopamplified the apoptosis influence.
血管新生（Angiogenesis）的概念，最早被JudahFolkman于1971年提出并被沿用至今。认为血管新生是从已存在的毛细血管网上大量生成新生血管的过程。血管新生在创伤和外周循环闭塞情况下具有恢复血供和促进伤口愈合的积极作用，但在更多的情况下血管新生则是一持续、无控性的过程，已成为肿瘤和血管增生性眼病等众多疾病的主要病理特征之一。内源性血管新生抑制因子和刺激因子之间的平衡失调是血管新生的基本分子机制。应用血管抑制因子和阻断血管新生刺激因子来阻断新生血管形成为治疗恶性肿瘤和其他血管增生性疾病提供了新的视野。
在已发现的众多血管抑制因子中，人纤溶酶原K5是目前发现的分子量小、性质稳定、活性较强的血管增生抑制剂之一。人纤溶酶原（plasminogen）含有5个环状结构域（Kringles），每个环由80个氨基酸残基组成，含3个二硫键，形成双环状构象。水解后可产生一组具有抑制血管增生作用的小分子片段：Angiostatin（Kringles1-4），Kringles1-5， Kringles1-3和Kringle5（K5）。血管抑素（Angiostatin）用于肿瘤的治疗已进入I期临床试验阶段。较之血管抑素（分子量约45kD），K5具有分子量小（16kD）、性质稳定且活性更强的优点。因为它的高效率、细胞选择性,和短的氨基酸序列,K5可作为治疗血管新生性疾病的候选药物。
K5抑制血管新生主要通过诱导血管内皮细胞凋亡实现，但其诱导内皮细胞凋亡的机制和信号通路尚需深入研究。重组K5蛋白被证明能够诱导增殖的内皮细胞凋亡；我们课题组之前的研究显示, K5在氧诱导的大鼠视网膜病变模型中，下调视网膜VEGF表达、上调PEDF表达，纠正VEGF/PEDF比率，恢复血管因子之间的平衡从而抑制视网膜血管新生并减少血管渗漏；在小鼠肝癌移植瘤模型上，K5可显著地增加切割的caspase3的含量诱导内皮凋亡，通过抑制血管新生来抑制肿瘤生长。
参与K5诱导凋亡的一些分子被陆续报道。先前的研究已经表明葡萄糖调节蛋白78（GRP78）和电压依赖性阴离子通道1(VDAC1)参与K5诱导肿瘤细胞和内皮细胞凋亡并可能起K5受体的作用,但尚存在争议。电压依赖性电离子通道（VDAC1）是多功能通道蛋白，主要位于线粒体外膜，调控一系列的生理和病理活动，如细胞Ca2+的平衡，能量代谢和细胞凋亡；VDAC1也可定位于细胞膜，作为膜受体介导信号转导。VDAC1在K5诱导内皮细胞凋亡中的作用尚缺乏系统研究。
研究目标
阐明K5诱导血管内皮细胞凋亡的信号通路；明确VDAC1在K5诱导血管内皮细胞凋亡中的关键作用和机制。为K5防治血管增生性疾病提供理论依据，同时关键分子的发现也为提供新的干预靶点提供线索。
研究内容与结果
第一章K5诱导内皮细胞凋亡的信号通路
1、高纯度K5重组蛋白的获取
诱导表达的含氨苄青霉素抗性的工程菌，按照提取可溶性K5重组蛋白的方法，采用Ni2+-His Bind Resin进行亲和层析得到与Ni柱结合的含有6个组氨酸的K5重组蛋白。采用SDS-PAGE电泳，将凝胶进行考马斯亮蓝染色显示获得高纯度可溶性蛋白。用凝胶成像系统对凝胶进行灰度扫描，结果显示纯化后的K5重组蛋白纯度为90%以上。
2、 K5抑制激活的HUVEC增殖和凋亡诱导作用
K5对激活的内皮细胞有明显的抑制增殖的作用，且存在浓度-效应关系；Hoechst33258染色初步显示随着K5浓度的增加，凋亡的内皮细胞逐渐增多；Annexin V/PI staining检测细胞凋亡率，与对照组相比，浓度变化在160~1280nmol/L各K5处理组细胞凋亡率分别为25.55±0.83%、33.4±3.11%、40.11±1.41%、45.04±0.99%，与阴性对照组和阳性对照组相比，均表现出能明显诱导血管内皮细胞凋亡的作用。
3、 K5通过线粒体凋亡通路诱导内皮细胞凋亡
3.1K5激活caspase7,8,9，降低线粒体膜电位，促进细胞色素C（Cyt c）释放
经640nmol/L K5处理72h后，Western blotting分析结果显示引起了明显的caspase7, caspase8和caspase9的切割；此外采用流式细胞仪检测K5处理后的线粒体膜电位的变化，线粒体膜电位降低，示内皮细胞线粒体膜被破坏；提取线粒体和胞浆蛋白，K5能明显地促进Cyt c从线粒体释放至胞浆，提示线粒体凋亡通路的激活。
3.2K5主要通过线粒体凋亡通路诱导内皮细胞凋亡
总caspase抑制剂（Z-VAD-FMK）, caspase8抑制剂（Z-IETD-FMK）或者caspase9抑制剂（Z-LEHD-FMK）预处理1h后，然后加入K5检测内皮细胞凋亡率，与caspase8抑制剂相比，caspase9抑制剂能更加有效地抑制K5诱导的内皮细胞凋亡，证实K5主要通过线粒体凋亡通路诱导内皮细胞凋亡。
3.3死亡受体凋亡通路对线粒体凋亡通路无交叉调控作用
此外，caspase8的抑制剂对K5引起的Bak分布的调控无影响，推论K5引起的Bcl-2家族蛋白细胞分布变化不受死亡受体凋亡通路的调控。
4、K5通过对Bcl-2家族蛋白的调控来引起Cyt c的释放，诱导凋亡
K5处理血管内皮细胞24h后，检测Bcl-2家族蛋白Bcl-2, Bcl-xL, Bax and Bak的分布，K5均不影响这四种蛋白的总量；然而K5明显地增加线粒体上Bak、减少Bcl-xL的量，与之对应的减少胞浆中Bak、增加Bcl-xL的量；推测K5通过调控Bak/Bcl-xL的比值引起Cyt c的释放来诱导凋亡。
第二章VDAC1在K5诱导内皮细胞凋亡中的作用
1、VDAC1在K5激活线粒体凋亡通路中的关键作用
1.1K5促进VDAC1与Bak结合增多
免疫共沉淀（IP）实验显示，K5上调磷酸化的VDAC1（p-VDAC1）,增加Bak与VDAC1的结合，降低Bcl-xL与VDAC1的结合。反向IP实验也证明，K5仍可增加VDAC1与Bak的结合。
1.2VDAC1与Bak结合促进线粒体通透性转换孔道（mPTP）的开放
应用MitoProbe TransitionPore试剂盒流式分析仪进行mPTP开放的检测，线粒体内钙黄绿素荧光的降低，表明膜通道孔的开放增加。经640nmol/LK5处理24h后，K5组线粒体内钙黄绿素荧光平均强度减低为对照组细胞的55.53±2.87%，差异有统计学意义。证明K5处理后线粒体膜被破坏、mPTP开放。
1.3VDAC1与Bak结合上调Cyt c的表达，激活caspase3
用160-1280nM浓度的K5处理HUVECs，Western blotting结果显示总蛋白中Cyt c出现剂量依赖性的上调；K5处理72h后，出现明显的caspase3的激活。
2、VDAC1作为细胞膜受体介导K5诱导的HUVECs凋亡
2.1抗体阻断实验对K5诱导内皮细胞凋亡的调控的影响
GRP78N端抗体和VDAC1mouse来源的抗体孵育HUVECs30min后，640nmol/L的K5作用72h后，流式检测凋亡率；WB检测caspase3的切割变化。结果显示使用GRP78的抗体后，K5仍能诱导HUVECs凋亡；使用VDAC1的抗体后，K5诱导HUVECs凋亡的作用被阻断。提示VDAC1抗体与K5竞争结合K5的受体----VDAC1。
2.2小分子RNA干扰实验对K5诱导内皮细胞凋亡的调控的影响
此外，RNAi实验用来证实VDAC1的关键作用。VDAC1和GRP78的siRNA，用HiPerFect转染HUVECs12h后，640nmol/L的K5作用72h，WB检测caspase3的切割变化；Promega公司的Caspase-Glo3/7Assay用于检测caspase3/7的活性。结果显示siVDAC1后，K5无激活caspase3/7的作用；然而si GRP78后，K5仍有激活caspase3/7的作用。提示VDAC1而非GRP78在K5诱导的凋亡中其关键作用。
2.3双分子荧光互补实验（BiFC）显示K5与VDAC1在细胞内存在直接结合
此外，构建双分子荧光互补N端和C端的两个质粒，pBiFC-K5VN155和pBIFC-VDAC1VC155。这两种质粒共转染293A细胞20h，全自动激光共聚焦显微镜显微镜拍照。BiFC实验阳性转染组细胞核内出现黄色荧光，共转染上述两质粒组出现黄色荧光，且荧光分别在线粒体和细胞膜，证明K5和VDAC1之间存在相互作用。已知VDAC1大部分定位线粒体，少部分定位细胞膜等，推论K5与VDAC1存在细胞膜和线粒体的共定位。
第三章K5对VDAC1含量及线粒体和细胞膜转位的调节
1、 K5上调细胞内VDAC1的总量及其机制
1.1K5上调HUVECs内VDAC1的含量
160nmol/L~1280nmol/L K5处理HUVECs24h后，提取细胞总蛋白，Westernblotting分析结果显示，K5呈剂量依赖性增加总蛋白中的VDAC1含量。
1.2K5抑制VDAC1的泛素化降解
Q-PCR结果显示，K5不调控VDAC1的mRNA水平。推测K5通过抑制VDAC1的降解来上调VDAC1。
640nmol/LK5处理HUVECs4h后，1uM的MG132继续作用20h。细胞采用温和的裂解条件做IP，加入VDAC1的抗体结合细胞内的全部VDAC1。WB后，用Ub抗体检测出的蛋白即为泛素化的VDAC1，结果显示K5处理和K5+MG132处理后，泛素化的VDAC1的含量均降低。结果提示K5通过抑制VDAC1的泛素化降解来上调VDAC1。
1.3K5通过AKT-GSK3β通路促进VDAC1的磷酸化
Western blotting结果显示，经640nmol/L的K5处理HUVECs5h后，与对照组相比，K5明显抑制AKT Ser473位的磷酸化，不影响总的AKT的表达水平；K5明显抑制GSK3β Ser9位的磷酸化，上调总的GSK3β的表达。GSK3β Ser9位磷酸化代表GSK3β的活性抑制。上述结果显示K5抑制AKT的活性，激活GSK3β活性。
将终浓度为1uM/L的AKT抑制剂和GSK3β抑制剂预处理HUVEC30min后，K5继续作用24h。细胞采用温和的裂解条件做IP，加入VDAC1的抗体结合细胞内的全部VDAC1。WB后，检测总的和磷酸化的VDAC1。结果显示，K5上调总的和磷酸化的VDAC1，使用AKT抑制剂后，总的和磷酸化的VDAC1上调。与单独AKT抑制剂组相比，AKT抑制剂+K5组不影响总的VDAC1的表达，上调磷酸化的VDAC1的表达。使用GSK3β抑制剂后，总的和磷酸化的VDAC1下调。与单独GSK3β抑制剂组相比，GSK3β抑制剂+K5组不影响总的和磷酸化的VDAC1的表达。GSK3β干扰组出现与GSK3β抑制剂组相似的结果。
AKT的激活剂insulin预处理HUVEC30min后，K5继续作用24h。与单独insulin组相比，insulin+K5组不影响总的和磷酸化的VDAC1的表达。
以上结果显示，K5通过影响AKT-GSK3β通路来促进VDAC1的磷酸化，并进而影响其降解。
1.4VDAC1介导K5对AKT-GSK3β通路的调控
HK I为细胞内VDAC1天然高效的拮抗剂，可与VDAC1结合抑制其功能。HK I预处理HUVECs，K5继续作用5h。WB结果显示与单独的HK I处理组相比，HK I+K5组无抑制AKT活性，激活GSK3β的作用。干扰VDAC112h后，K5继续作用5h。WB结果出现与使用HK I抑制剂同样的结果。提示K5对细胞内信号通路的调控需要膜受体VDAC1的参与。
1.5K5通过促进VDAC1磷酸化抑制泛素化降解
构建VDAC1的四个磷酸化突变位点，将13位，52位，104位和107位磷酸化位点突变为丙氨酸。在转染相同量的质粒的情况下，无论在HUVEC还是293A细胞上，与单纯转染VDAC1过表达质粒组相比，S13A，S104A和T107A质粒转染组均降低外源性导入的VDAC1的含量。提示K5通过调控13位，104位和107位磷酸化来抑制其泛素化降解来上调VDAC1的表达。
2、K5促进VDAC1线粒体和细胞膜转位及机制
2.1K5促进VDAC1线粒体和细胞膜转位
K5处理HUVECs24h后，提取线粒体和胞浆蛋白。结果显示K5明显增加线粒体中的VDAC1，胞浆中未检测到VDAC1的表达。
采用文献报道的改良的IP方法来研究细胞膜上的VDAC1表达。K5处理24h后，细胞膜上的VDAC1明显增加。
2.2VDAC1总量的增多导致线粒体和细胞膜转位的增多
在HEK293A细胞过表达VDAC1，与转染阴性对照质粒组相比，出现和K5类似的上调VDAC1总量，及线粒体和细胞膜分布增加。提示VDAC1总量的增多导致了线粒体和细胞膜VDAC1分布的增多，即产生了线粒体和细胞膜的特定亚细胞转位。
2.3HK I拮抗K5对VDAC1细胞分布的调控
HK I预处理5min后，K5继续作用24h。提取总蛋白，线粒体和细胞膜蛋白，结果显示使用HK I后，K5对总蛋白，线粒体和细胞膜蛋白的VDAC1均无调控作用。免疫细胞化学的结果与此类似。提示K5对细胞内VDAC1量和分布的调控需要作为K5膜受体VDAC1本身的存在和参与。
结论及研究意义
1、K5通过线粒体凋亡通路诱导内皮细胞凋亡
首次阐明K5主要调节Bcl-2家族蛋白促凋亡分子Bak和抗凋亡分子Bcl-xL在HUVECs细胞组分中的分布，上调线粒体上Bak/Bcl-xL的比值来启动线粒体凋亡通路。随后降低线粒体膜电位，促进Cyt c的释放至胞浆，启动caspase9，导致内皮细胞凋亡。
2、VDAC1是介导K5诱导内皮细胞凋亡的关键调控分子
阐明VDAC1同时作为受体和线粒体膜通道蛋白在K5激活线粒体通路的关键作用，且此受体不受分子伴侣GRP78的调控，提示VDAC1为调控内皮细胞凋亡的有效干预靶点。
3、K5促进VDAC1磷酸化抑制其泛素化降解从而上调VDAC1
K5主要通过抑制AKT的活性，进而激活GSK3β来调控磷酸化的VDAC1，其作用的磷酸化位点有13位丝氨酸，104位丝氨酸和107位苏氨酸。本研究首次发现VDAC1的107位苏氨酸位点磷酸化可以抑制VDAC1的降解来上调VDAC1的含量。
4、K5通过上调VDAC1促进其线粒体和细胞膜转位
过表达VDAC1可促进VDAC1线粒体和细胞膜的转位，提示VDAC1总量的增多导致了细胞膜和线粒体VDAC1分布的增多，即K5通过上调VDAC1总量促进了细胞膜和线粒体的特定亚细胞转位。
5、K5通过正反馈调节环路放大对细胞凋亡的诱导作用
本研究的结果表明在配体K5和受体VDAC1之间存在正反馈调节环路。VDAC1作为受体介导K5对VDAC1自身含量的上调，而上调的VDAC1又进一步增加其在细胞膜的分布，放大了K5的作用。因此，这种正反馈调节环路发挥了对细胞凋亡的诱导放大功能。
BACKGROUND
Angiogenesis was first proposed by Judah Folkman in1971. Angiogenesis is theformation of new capillaries from preexisting vessels. In adults, neovasularizationfavours the blood flood and the wound healing during peripheral circulation andtrauma. However, angiogeneis is more often to be a persistent and uncontrolledprocess, which has been established as one of main pathological characteristics oftumor and other angiogenesis-related diseases. Angiogenesis is regulated by thebalance between endogenous angiogenic stimulators and inhibitors. In pathologicconditions, the balance is disrupted, and many diseases are driven. Recently, thestrategy of applying endogenous angiogenenic inhibitors and blocking endogenousangiogenic stimulators has been used to treat tumor and other angiogenesis diseases inclinical researches.
Plasminogen kringle5(K5), a proteolytic fragment of plasminogen with smallmolecular and stable property, displays potent angiogenesis inhibitory activity asother endogenous angiogenic inhibitors. Plasminogen contains five kringles.Eachkringle consists of80amino acids and3disulfide bonds forming double loop structuredomain. Series of small molecular pieces emerge after hydrolyzation of plasminogen:Angiostati（nKringles1-4），Kringles1-5，Kringles1-3和Kringle5(K5). Angiostatinwas applied into phase I clinical trials to cure tumor. Contrast to angiostatin (45kD), K5possessed smaller molecular weight, higher activity in inhibiting angiogenesis. SoK5could be a candidate drug to treat angiogenesis-related diseases.
K5inhibited angiogenesis mainly through inducing endothelial cells apoptosis,but the mechanism and signal pathway involved in endothelial cells apoptosis neededto be thoroughly discussed. Recombinant K5has also been shown to induce apoptosisin proliferating endothelial cells. Our previous study has shown that K5inhibitsretinal neovascularization by decreasing the expression of VEGF, increasing theprotein level of PEDF and correcting the ratio of VEGF/PEDF to restore theangiogenesis balance and reduces vascular leakage in oxygen-induced rat retinopathymodel. Meanwhile, our study demonstrated that K5induced endothelial cellsapoptosis by increasing the cleavage of caspase3, and suppressed hepatocarcinomagrowth by anti-angiogenesis in HepA-grafted and Bel7402-xenograftedhepatocarcinoma mouse models.
Several molecules were identifed to be involved in K5-induced apoptosis.Previous studies have suggested that K5-induced apoptosis of tumor cells andendothelial cells was related to glucose-regulated protein78and Voltage dependentanion channel (VDAC1). However, there existed controversy. VDACs, also known asmulti-functional mitochondrial porins, are abundant proteins found in the outermitochondrial membrane (OMM). VDAC1regulate series of physiology andpathology events, such as the balance of celluar Ca2+, energy metabolism and cellapoptosis; VDAC1locates to plasma membrane too, as the receptor for K5to deliversignal. There still lacked systematicly research about the function of VDAC1involedin K5-induced endothelial cells apoptosis.
OBJECTIVES
To elucidate the signaling pathways involved in K5-induced endothelial cellsapoptosis, and further confirm the key role and the mechanism of VDAC1in thisprocess. On the basis of that, it provided a theoretical reason for K5in the treatmentof angiogenesis-related diseases; and the discovery of the key molecular would offerclue for the novel intervention targets.
EXPERIMENTAL RESULTS
Chapter1Research on the signaling pathways by which K5-inducedendothelial cells apoptosis
1, the acquisition of high purity K5recombinant protein
Expression was induced by the addition of Isopropyl-β-D-thiogalactopyranoside(IPTG) form the bacterias with ampicillin resistance construction plasmid. Solubleproteins were extracted under native conditions, and the recombinant peptide waspurified by passing through the His-Bind column. The purity of K5processed bythin-layer protein scanning of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) gel with Genegenius gel image system (Gene Co.England) were all over90%.
2, K5inhibits proliferation and induces apoptosis of activatedHUVECs
K5exhibited selective inhibitory effect of K5on activated HUVECs withsignificant dose-effect correlation. Hoechst33258assay preliminarily demonstratedan enhanced effect on inducing cell apoptosis with the increased concentration of K5.In comparison with PBS-treated group, Annexin V/PI analysis showed that apoptoticratio of160,320,640and1280nmol/l. K5-treated groups were25.55±0.83,33.4±3.11,40.11±1.41, and45.04±0.99%. Compared to negative and positive controls,K5induced the apoptosis of HUVECs in a dose-dependent manner.
3, K5induces endothelial cell apoptosis via mitochondrial pathway
3.1K5activated caspase7,8,9, decreased the mitochondrial me mbranepotential and promoted the release of cytochrome c
Western blotting analysis showed signifcant increase in the amounts ofcleavedcaspase7, cleavedcaspase8and cleavedcaspase9by K5treatment. To furtherinvestigate whether the mitochondria pathway is involved in the K5-induced apoptotic process, the mitochondrial membrane potential (MMP) was analyzed by fowcytometry. K5reduced the MMP of HUVECs. Futher more, western blotting analysisshowed that K5stimulated the release of cytochrome c from mitochondrial intocytosol. Mitochondria apoptosis pathway was involved in K5-induced apoptosis.
3.2K5induces endothelial cells apoptosis mainly through mitochondrialpathway
Apoptotic rate of HUVECs induced by K5in the presence of Z-VAD-FMK(Pan-caspaseinhibitor), Z-IETD-FMK (caspase8inhibitor) or Z-LEHD-FMK(caspase9inhibitor) was assessed, respectively. The results showed Z-VAD-FMK,Z-IETD-FMK or Z-LEHD-FMK could attenuate the K5-induced apoptosis. Itdemonstrated that K5induces endothelial cell apoptosis mainly through mitochondrialpathway.
3.3Death receptor apoptosis pathway had no cross regulation withmitochondrial apoptosis pathway
Further, Z-IETD-FMK, inhibitor of caspase8, had no effect on the K5-inducedtranslocation of Bak in HUVECs. We deduced that death receptor apoptosis pathwayhad no cross regulation with mitochondrial apoptosis pathway.
4, Effect of Bcl-2family proteins in K5-induced cytochrome c releaseand apoptosis
We analyzed the distribution of Bcl-2family proteins (Bcl-2, Bcl-xL, Bax andBak) in HUVECs after treatment with K5for24h by Western blotting analysis. Theseresults showed that K5had no effects on all the protein levels in total cell lysates.However, K5signifcantly increased the level of Bak and reduced the amount ofBcl-xL in mitochondria portion. These results inferred that the increased ratio of Bakto Bcl-xL on mitochondria was responsible for mitochondrial depolarization,cytochrome c release and consequently for the activation of caspase9.
Chapter2The implication of VDAC1in the apoptosis of endothelialcells induced by human plasminogen Kringle5
1, The important role of VDAC1involved in the K5-activatedmitochondrial apoptosis pathway
1.1K5increased the interaction between VDAC1and Bak
Co-immunoprecipitation (IP) assay performed that K5added the phosphorylationof VDAC1, increased the interaction between VDAC1and Bak and decreased theinteraction of VDAC1and Bcl-xL. Reverse IP assay still exerted that K5stillup-regulated the interaction of VDAC1and Bak.
1.2The inte raction within VDAC1and Bak promoted the opening ofmitochondrial permeability transition pore (mPTP)
The opening of mPTP was detected by MitoProbe Transition Pore kit andFCM. Decreased fluorescent intensity of Calcein AM in the mitochondria exerted theincrease opening of mPTP. In compared to control, the fluorescent intensity ofCalcein AM was reduced to55.53±2.87%and there had statistical significance.Results indicated that the mitochondrial membrane was disrupted and the mPTP wasopened in addition to K5treatment.
1.3The inte raction within VDAC1and Bak increased the Cyt c and activatedcaspase3
As shown in chapter one, K5stimulated the release of cytochrome c frommitochondrial into cytosol. Interestingly, K5could added the total amounts ofVDAC1in a dose-dependently manner. Moreover, K5obviously activated caspase3.
2, VDAC1was as the plasma membrane receptor in K5-inducedHUVECs apoptosis
2.1Antibody blockade assay affected K5-induced HUVECs apoptosis
GRP78N-terminal antibody and VDAC1antibody incubated HUVECs for30min, then cells were treated with K5for another72h, then we used Annexin V/PIassay to detect the apoptotic rate and the change of caspase3by western blottingassay. All assays showed that K5still induced HUVECs apoptosis in addition to GRP78N-terminal antibody, but VDAC1antibody blocked the K5-induced HUVECsapoptosis. It indicated that VDAC1antibody and K5had competitive binding with thereceptor of K5: VDAC1.
2.2Small RNA interference assay affected K5-induced HUVECs apoptosis
RNA interference assay was exploited to demonstrate the pivotal role of VDAC1.We transfected VDAC1si RNA or GRP78si RNA with HiPerFect reagent intoHUVECs for12h,640nM K5was utilized for another72hours. Then the change ofcaspase3was detected by western blotting and the activity of caspase3/7wasdetected by Caspase-Glo3/7Assay in Promega company. Results displayed that K5did not have activating function to caspase3/7after siVDAC1, K5remainedactivating caspase3/7with GRP78knockdown. The research pointed out thatVDAC1more than GRP78played an important role in K5-induced apoptosis.
2.3Bimolecular fluorescence complementation (BiFC) assay showed the directinteraction of K5and VDAC1
We constructed the N–terminal and C-terminal plasmids of BiFC assay,pBiFC-K5VN155and pBIFC-VDAC1VC155. Plasmids for BiFC assay weretransfected into HEK293A cells for20h, we observed and photographed with fullautomatic laser confocal microscopy. There had yellow fluorescence in the nuclear inthe positive BiFC control, and transfected the above two plasmids group showedyellow fluorescence too in both mitochondria and plasma membrane. Resultsperformed that there had direct interaction within K5and VDAC1. It is known thatmost of VDAC1located in mitochondria, only small number of VDAC1located inplasma membrane and so on, we inferred that K5and VDAC1had co-localization inboth mitochondria and plasma membrane.
Chapter3K5regulated the amount and transposition of VDAC1inmitochondria and plasma membrane
1, The mechanism involved in K5-aroused increase of VDAC1.
1.1K5increased the total amount of VDAC1
HUVECs were treated with160nM~1280nM K5for24h, total protein were extracted and the Western blotting assay showed that K5dose-dependently increasedthe total expression level of VDAC1.
1.2K5restrained ubiquitin degradation of VDAC1
Further, Q-PCR assay showed that K5had no influence on the mRNA expressionof VDAC1. So we deduced that K5suppressed the degradation to up-regulateVDAC1protein level.
First640nM K5treated HUVECs for4hours,1uM MG132was added to theculture medium for another20hours. Then cells were lysed with RIPA buffer for IPassay and the VDAC1antibody was used to pull all protein of VDAC1. IP assayshowed that the ubiquitin protein represents ubiquitin VDAC1. Our results showedthe level of ubiquitin VDAC1was decreased in addition to K5treatment orK5+MG132treatment. It indicated that K5suppressed the ubiquitin chemicaldegradation to increase the total protein of VDAC1.
1.3K5regulated the expression of VDAC1via AKT-GSK3β pathway
HUVECs were managed with640nM K5for5h, contrast to the control groups,K5obviously restrained the activity of AKT in the ser473phosphorylation site, butthe total expression of AKT was not influenced by K5; K5clearly stopped the activityof GSK3β in the ser9phosphorylation site, and evidently raised the expression oftotal GSK3β. When phosphorylated on Ser9, GSK3β is inactivated. Results showedthat K5suppressed the activity of AKT and activated GSK3β.
HUVECs were pre-treated with either1umol/L Akt inhibitor (Akt inhibitor IV)or1umol/L GSK3β inhibitor and then with K5for another24h. VDAC1wasimmunoprecipitated from HUVECs lysates and its phosphorylation is determined byphosphoserine/threonine/tyrosine antibody. As shown in chapter one, K5increased thetotal protein and phosphorylation of VDAC1. AKT inhibitor could lead to the sameresults like K5. Compared to alone AKT inhibitor group, AKT inhibitor along with K5group up-regulated the phosphorylation of VDAC1, but did not affect the totalamount of VDAC1. The total protein and phosphorylation of VDAC1were decreasedby GSK3β inhibitor and siGSK3β. Compared to alone GSK3β inhibitor and siGSK3βgroup, GSK3β inhibitor and siGSK3β along with K5group did not affect the total amount and phosphorylation of VDAC1.
HUVECs were pre-treated with either AKT activator insulin and then with K5for another24h. Compared with alone insulin group, insulin along with K5group didnot affect the total amount and phosphorylation of VDAC1.
Our results displayed that K5promoted the phosphorylation of VDAC1viaAKT-GSK3β pathway to restrain ubiquitindegradationof VDAC1.
1.4VDAC1was mediated in the AKT-GSK3β pathway
HK I was the high effect agonist of VDAC1in kinds of cells, and HK I inhibitedVDAC1by the interaction. HUVECs were pre-treated with HK I and then with K5foranother5h. Compared to alone HK I group, HK I along with K5group did notrestrain the AKT activity and activate the GSK3β activity. VDAC1was interferedwith HiPerFect reagent in HUVECs for12h, K5treated HUVECs for another5h.Results were the same as HK I blockade assay. It hinted that VDAC1as the receptorfor K5was mediated in the AKT-GSK3β pathway.
1.5K5promoted the phosphorylation of VDAC1to inhibit its ubiquitindegradation
To further investigate the phosphorylated sites of VDAC1, we constructed aseries of VDAC1mutants, which disturbed the potential serine and tyrosinephosphorylation on VDAC1. These mutants, including S13A, T52A, S103A, andS107A, were transfected into HUVECs and HEK293A cells, and their protein levelswere detected by Western blotting assay. Three VDAC1mutants, S12A, S103A andS107A, could effectively reduce the protein level of VDAC1. These results indicatethat dephosphorylation of VDAC1may impair its protein level elevation.
2, The mechanism involved in K5-caused transposition of VDAC1inmitochondria and plasma membrane
2.1K5facilitated the translocation of VDAC1in mitochondria and plasmame mbrane
HUVECs were treated with K5for24h and then the mitochondrial and cytosolfraction were extracted. Results displayed that K5significantly increased the mitochondrial protein level of VDAC1, but there had no VDAC1protein in thecytosol.
We exploited the modified IP assay to detect the plasma membrane of VDAC1according to previously description. Our data showed that the plasma membraneprotein level of VDAC1was obviously added in addition to K5treatment for24h.
2.2The augment amount of VDAC1increased the transposition in mitochondriaand plasma me mbrane
VDAC1was transfected into HEK293A cells for24h. Compared to pcDNA3.1plasmid transfection group, VDAC1overexpression group added the amount andpromoter transposition of VDAC1in mitochondria and plasma membrane. Our datainferred that the augment amount of VDAC1increased the transposition inmitochondria and plasma membrane, as to say, resulted in the mitochondria andplasma membrane special subcellular translocation of VDAC1.
2.3HK I blocked the regulation of VDAC1by K5
HUVECs were pre-treated with HK I for5min and then with K5for another24h. We extracted the total protein, mitochondrial and cytosol fraction. Our data showedthat K5had no regulation to the total protein, mitochondrial and cytosol fraction ofVDAC1. It performed that the regulation of VDAC1by K5needed the receptorVDAC1.CONCLUSIONS
1, K5induces endothelial cells apoptosis via mitochondrial apoptosis pathway
Our results for the first time demonstrated that K5regulated the distribution ofBcl-2family members Bak and Bcl-xL in HUVECs, increased the ratio of Bak/Bcl-xLin the mitochondria to initiate mitochondrial apoptosis pathway. Subsequently, K5decreased the MMP, promoted the release of Cyt c to cytosol and activated caspase9to induce HUVECs apoptosis.
2, The important role of VDAC1involved in the K5-triggered HUVECsapoptosis
Our data elucidated the key role of VDAC1both as the receptor and mitochondrial membrane channel protein in activating the mitochondrial apoptosispathway, and the function of VDAC1did not affected by the molecular chaperoneprotein GRP78. It indicated that VDAC1was an effective intervention target toregulate endothelial cells apoptosis.
3, K5increased the VDAC1by promoting the phosphorylation of VDAC1toinhibit its ubiquitin degradation
K5suppressed the activity of AKT and activated GSK3β. Our results displayedthat K5promoted the phosphorylation of VDAC1via AKT-GSK3β pathway torestrain ubiquitin degradation of VDAC1. Dephosphorylation of VDAC1involved inS13, T52, S103, and S107site may impair its protein level elevation. For the first time,we discovered that the phosphorylation of VDAC1in its S107site suppressed thedegradation to increase the total amount of VDAC1.
4, K5facilitated the translocation of VDAC1in mitocho ndria and plasmame mbrane by up-regulating VDAC1
Our data inferred that the augment amount of VDAC1increased thetransposition in mitochondria and plasma membrane. So we deduced that K5promoted the mitochondria and plasma membrane special subcellular translocation ofVDAC1through up-regulating its total amount.
5, K5blowed up the apoptosis effect by positive feedback regulation of VDAC
Our results showed that there existed positive feedback loop between the ligandof K5and the receptor of VDAC1. As the receptor, VDAC1participated in itsregulation induced by K5, the increase of VDAC1further increased its plasmamembrane distribution to enlarge the function of K5. Since the positive feedback loopamplified the apoptosis influence.
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