Studies On The Apoptosis Signal Pathway Mediated By Phosphatidylcholine-Specific Phospholipase C And Integrin β₄ In Vascular Endothelial Cell

BACKGROUND AND OBJECTIVE:Vascular endothelial cells play a critical role in angiogenesis and blood vessel degeneration [Pan X, et al, 2004]. Angiogenesis is necessary for normal physiological processes such as embryonic development and wound repair [Romagnani P, et al, 2004], but angiogenesis also represents an important step in tumor development and various inflammatory disorders [Griffioen AW, et al, 2004]. Apoptosis of vascular endothelial cells have important roles in anticancer therapeutics and in regulation of angiogenesis. Therefore, the importance of inhibiting and inducing apoptosis of vascular endothelial cells (VEC) in wound repair and cancer therapy is commonly accepted [Greenberger S, et al,.2004], and it is desirable to look for new drugs that induce or inhibit apoptosis of VEC.Even though studies on apoptosis of various cells have been extensive, the mechanism by which it involves in endothelial cells remains controversial, because these cells are resistant to apoptotic inducers such as Fas ligand [Wei P, et al, 2005], and it has also been difficult to induce apoptosis under normal condition without depletion of survival factors. It has been shown that rattlesnake venom can specifically induce apoptosis of vascular endothelial cells [Araki et al., 1993]. However, the molecular mechanism of apoptosis induced by rattlesnake venom remained undefined.The survival and proliferation of human vascular endothelial cells depend on adhesion to extracellular matrix. Integrins are transmembrane receptors implicated in mediating cell-substrate attachment or cell-cell adhesion, and in transducing signals that regulate the processes of cell proliferation, differentiation, apoptosis, etc. Integrin p4 is a key membrane protein in cellular signal transduction [Tang K et al., 1999; Sakakura C et al., 2002; Weaver V M et al., 2002]. When VECs are deprived of FGF, the expression of integrin p4 mRNA increases [Stepp, N. T. et al., 1994], and at the same time apoptosis occurs in these cells [Araki, S. et al., 1990]. hi previous studies, we found that the mAb against integrin p\t inhibited apoptosis induced by deprivation of serum and FGF. This result suggested that the integrin was involved in VEC apoptosis signaling [Miao J Y et al., 1997]. However, whether rattlesnake venom triggers apoptosis via integrin P4 in VEC is not clear.Phospholipase Cs are very important phospholipases in the intracellular signaling network, phosphatidylcholine-specific phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (PI-LC) are two kinds of isoforms of this enzyme. It has been well known that PC-PLC involved in Fas/APO-1-induced apoptotic signal pathway in lymophocytes [Cifone et al., 1995], but in vascular endothelial cells, Fas/APO-1 failed to induce apoptosis, Recently, it was shown that PC-PLC is implicated in several models of programmed cell death [Bjorkoy, et al. 1997, Machleidt, et al. 1996, Li, et al. 1998, Halstead, et al. 1995, Schutze, et al. 1992, Muller-Decker, 1989]. In our previous studies, we found that rattlesnake venom could induce apoptosis by stimulating PC-PLC in VEC [Miao et al., 1997b]. But the mechanisms of PC-PLC mediating apoptosis in VEC all remain unknown.These primary researches tell us that integrin P4 and PC-PLC may play important roles in apoptosis induced by rattlesnake venom in VEC. However, the mechanisms by which integrin p4 and PC-PLC regulate the apoptosis of VEC remain unclear. In this paper, in order to understand the signal transudation system that regulates apoptosis of human umbilical vein endothelial cells (HUVCE), we investigated the roles ofintegrin 04 and PC-PLC in apoptosis induced by rattlesnake venom and by deprivation of FGF and serum in this cell.It is well known that Akt/PKB is a well-established anti-apoptotic protein, this kinase plays a key role in PI3K/Akt signal pathway, which functions primarily in the normal response to a variety of survival signals, as well as in the aberrant survival of many types of cancer cells [Shaw L.M., et al, 1997]. In this study, we investigated the role of Akt/PKB in the apoptotic signal pathway mediated by PC-PLC in VEC treated with rattlesnake venom.Normal human cells could not divide forever in culture. After a defined number of passages, every culture enters a viable non-dividing state termed senescence. Three features distinguish senescent from presenescent cells, an irreversible block to cell proliferation, increased resistance to apoptotic death, and changes in differentiated functions [Faragher RG, 2000]. In previous studies, we found that suppressing PC-PLC with D609 could accelerate apoptosis in 36* passage VEC, which was contrary to the result in 16th passage VEC. Our study first indicated that apoptosis mediated by phosphatidylcholine-specific phospholipase C is associated with the aging of cell in vascular endothelial cells. In this thesis, we studied the relationship between apoptosis mediated by phosphatidylcholine-specific phospholipase C and the aging of vascular endothelial cells.METHODS:Cell culture and apoptosis inducingFibroblast growth factor (FGF) was extracted from bovine brains by the method of Lobb and Fett (1984) in our laboratory. Human umbilical vascular endothelial cells (HUVECs) were isolated from sterile umbilical cord of healthy infant as described by Jaffe et al (1973). We induced cells apoptosis by deprivation of FGF and serum and by rattlesnake venoms.Apoptosis of cells was detected in those four ways:1. Apoptotic morphological changes in VEC and apoptotic bodies were observed under a phase-contrast microscope.2. Viability was determined by counting the cells that attached to dishes after treatments with a leukocytometer.3. Nuclear fragmentation was observed under laser scanning confocal microcopy (Zeiss, LSM510) after stained with acridinorange.4. DNA ladder analysisCells were treated with in these ways:Normal group, cells were cultured in MCDB131 medium contained FBS and FGF; Control group cells were cultured in basal MCDB131 medium; Apoptotic group, cells were incubated with apoptosis inducers, rattlesnake venom; D609 or P4 group, cells were incubated with rattlesnake venom and D609, or mAb of integrin p4.RESULTS:1. When the cells deprived of FGF and serum were incubated with rattlesnake venom, some cells gradually detached from the dish and apoptosis occurred. Specific morphological changes of apoptosis induced by the venom could be observed. Many apoptotic bodies were formed from these cells 6 h after the treatment. However, after exposure to mAb of integrin P4 or D609 for 6 h, the detachment and apoptosis in VEC were obviously inhibited, few apoptotic bodies were observed.2. The viability of cells treated with rattlesnake venom was 58.58 % (PO.05), while in the presence of mAb of integrin P4 and rattlesnake venom, the viability of cells was 93.87 % (PO.05). The viability of cells treated with both D609 and rattlesnake venom was 88.84 % (PO.05).3. After treatment, many nuclei became fragmentized and DNA fragmentation was observed in the cells of SV group, but in the presence of mAb of integrin P4 or D609 the nuclear fragmentation and DNA fragmentation were inhibited.4. In the cells of SV group, the levels of P53 and p4 expression were obvious higher than that in the cells of control groups, moreover, the changes in the subcelluar location of integrin P4 could be observed in the cells of this group, this integrin was translocated from cytoplasm to nuclear in these cells, but the phosphorylation of Akt/PKB was down-regulated markedly. The expression of P53 protein and integrin p\jwere suppressed evidently in the cells of D609 group, and the translocation of integrin p\? couldn't be observed in these cells; for the more, the activation of Akt/PKB kinase increased markedly, and this kinase centralized in the nuclear in these cells.5. There were two forms of PC-PLC in VEC, Ca2+-dependent PC-PLC and Ca2+-independent PC-PLC, moreover, the activity of Ca2+-independent PC-PLC was much higher than that of Ca2+-dependent PC-PLC. The two forms of PC-PLC distribute both at cell membrane system and in cytoplasm. The activity of PC-PLC decreased significantly during subculture in VEC. Before passages 14 in VEC, the activity of PC-PLC decreased dramatically, during passages 14 to 20, the changes in activity of this enzyme were very unobvious. The activity of PC-PLC in cells treated by rattlesnake venom increased observably, the activity of this enzyme have no obvious difference between the cells of D609 and control groups.6. The level of P53 protein expression increased significantly during subculture in VEC, and suppression PC-PLC could not inhibit this expression in 36th passage VEC.7. Both in the 16th passage and in the 36lh cultured VEC, rattlesnake venom could increase the levels of intracellular ROS evidently, on the contrary; D609 was able to suppress this change obviously.CONCLUSION:1. In the VEC treated with rattlesnake venom, PC-PLC mediated apoptosis signaling by regulating the expressions of integrin p4 and P53 protein and by translocating integrin pU from cell membrane and cytoplasm to nucleus. Integrin P4 wasinvolved14in this signal transduction by up-regulating the expression of P53. PC-PLC. integrin P4 and P53 protein were in the same apoptotic signal pathway.2. There was crosstalk between PI3K-Akt/PKB survival signal pathway and the apoptotic signal pathway mediated by PC-PLC in VEC induced by rattlesnake venom. PC-PLC could suppress the activation of Akt/PKB.3. The apoptotic signal pathway mediated by PC-PLC was related to the cell senescence in VEC treated with rattlesnake venom. PC-PLC may be an important enzyme in controlling apoptosis and cell senescence of VEC. It might regulate cell senescence and apoptosis through P53 protein inVEC.4. The effect of D609 and rattlesnake venom on the level of intracellular ROS attributes partly to the apoptosis of VEC, and this effect was not associated with cell senescence of VEC.5. There was at least one important component target to integrin P4 in crude rattlesnake (Crotalus atrox) venom, moreover, this component played its role in early phase of apoptosis.