The Implication Of VDAC1in The Apoptosis Of Endothelial Cells Induced By Human Plasminogen Kringle5

BACKGROUNDAngiogenesis 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�'�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.OBJECTIVESTo 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 RESULTSChapter1Research on the signaling pathways by which K5-inducedendothelial cells apoptosis1, the acquisition of high purity K5recombinant proteinExpression 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 activatedHUVECsK5exhibited 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 pathway3.1K5activated caspase7,8,9, decreased the mitochondrial me mbranepotential and promoted the release of cytochrome cWestern 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 mitochondrialpathwayApoptotic 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 pathwayFurther, 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 apoptosisWe 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 Kringle51, The important role of VDAC1involved in the K5-activatedmitochondrial apoptosis pathway1.1K5increased the interaction between VDAC1and BakCo-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 activatedcaspase3As 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 apoptosis2.1Antibody blockade assay affected K5-induced HUVECs apoptosisGRP78N-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 apoptosisRNA 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 VDAC1We 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 membrane1, The mechanism involved in K5-aroused increase of VDAC1.1.1K5increased the total amount of VDAC1HUVECs 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 VDAC1Further, 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 pathwayHUVECs 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 pathwayHK 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 ubiquitindegradationTo 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 membrane2.1K5facilitated the translocation of VDAC1in mitochondria and plasmame mbraneHUVECs 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 mbraneVDAC1was 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 K5HUVECs 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.CONCLUSIONS1, K5induces endothelial cells apoptosis via mitochondrial apoptosis pathwayOur 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 HUVECsapoptosisOur 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 degradationK5suppressed 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 VDAC1Our 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 VDACOur 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.