Junying Miao

Phosphatidylcholine-specific phospholipase C, p53 and ROS in the association of apoptosis and senescence in vascular endothelial cells

Previously, we found that phosphatidylcholine‐specific phospholipase C (PC‐PLC) participated in apoptosis signaling of vascular endothelial cells (VECs). Here, to explore whether PC‐PLC is involved in the association of apoptosis and senescence in VECs, we analyzed p53 expression and intracellular reactive oxygen species (ROS) levels in young and senescent VECs before and after inhibiting PC‐PLC activity. The results showed that suppressing PC‐PLC inhibited apoptosis and the elevation of p53 expression induced by apoptosis in young cells, but not in senescent cells, and that inhibiting PC‐PLC depressed intracellular ROS levels both in young and senescent cells. The data suggested that PC‐PLC was involved in the association of apoptosis and senescence. Its function might be closely related to the level of p53 in VECs.

Suppression of Apoptosis by Inhibition of Phosphatidylcholine-Specific Phospholipase C in Vascular Endothelial Cells

In order to clarify the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in the regulation of apoptosis in vascular endothelial cells (VEC), we investigated the effects of D609, a specific inhibitor of PC-PLC, on apoptosis that was induced by deprivation of fibroblast growth factor (FGF) and serum and also by rattlesnake venom. The early morphological changes (detachment of cells from dishes) and the fragmentation of DNA, which is a specific feature of apoptotic cell death, were clearly inhibited by D609 in these two apoptosis-inducing systems. Moreover, the production of diacylglycerol (DAG), which was stimulated in apoptotic VEC, was suppressed by D609. The effects of D609 on the activity of PC-PLC and on apoptosis of VEC were dose-dependent. Our results indicate that PC-PLC is involved in the apoptosis-inducing signal pathway in VEC and, that DAG, produced from phosphatidylcholine (PC), might be an important mediator in this signal-transduction pathway. Our results also suggest that rattlesnake venom, a strong promoter of apoptosis in VEC, might induce apoptosis by stimulating PC-PLC and, furthermore, that PC-PLC might play a significant role in anchorage-dependent signal transduction in VEC.

Apoptosis Mediated by Phosphatidylcholine-Specific Phospholipase C is Associated with cAMP, p53 Level, and Cell-Cycle Distribution in Vascular Endothelial Cells

In previous studies, the authors found that phosphatidylcholine-specific phospholipase C (PC-PLC) was implicated in apoptosis induced by deprivation of survival factors in vascular endothelial cells (VECs) (Miao et al. Endothelium, 5, 231-239, 1997). In order to understand which elements are involved in the apoptotic signal transduction mediated by PC-PLC, the authors examined cyclic adenosine monophosphate (cAMP) level, p53 expression, and the changes of cell cycle in VECs when PC-PLC activity was suppressed by D609 (tricyclodecan-9-yl-xanthogenate), a specific inhibitor of this enzyme. The results showed that cAMP level was reduced (p <.01), p53 expression was suppressed, and cell-cycle distribution was changed when apoptosis of VECs was inhibited by D609. The data indicate that cAMP and p53 are involved in this pathway, and that PC-PLC might regulate apoptosis by affecting the cell-cycle distribution of VECs.

Contrasting Effects of Phosphatidylinosital-and Phosphatidylcholine-Specific Phospholipase C on Apoptosis in Cultured Endothelial Cells

In the authors previous studies, they found that phosphatidylcholine-specific phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (PI-PLC) played contrary roles in the apoptosis of vascular endothelial cells (VECs), but the mechanism underlying the phenomenon remains unclear. To address this question, in this study, the authors investigated the changes of cell cycle distribution, the expression of P53, and the phosphorylation of Akt when PI-PLC was inhibited by its specific inhibitor compound 48/80, and they also examined the phosphorylation of Akt when VEC apoptosis was inhibited by D609, a specific inhibitor of PC-PLC. The results showed that suppression of PI-PLC promoted VEC apoptosis by inhibiting Akt phosphorylation, elevating P53 expression, and affecting the cell cycle distribution. Contrarily, suppression of PC-PLC promoted the phosphorylation of Akt. The data suggested that PI-PLC and PC-PLC might control the apoptosis by jointly regulating Akt phosphorylation, P53 expres...

cDNA Cloning and Some Additional Peptide Characterization of a Single-Chain Vascular Apoptosis-Inducing Protein, VAP2

Vascular apoptosis-inducing proteins (VAPs) from hemorrhagic snake venom are apoptosis-inducing toxins targeting vascular endothelial cells. Well-characterized VAPs consist of disulfide-bridged double chains (ddVAPs). The authors previously described a single-chain VAP (scVAP), VAP2 from Crotalus atrox, which also induces apoptosis in endothelial cells (Masuda et al., 1998, European Journal of Biochemistry, 253, 36-41). The authors report here the whole cDNA sequences and some additional peptide characteristics of VAP2. In addition to the apoptosis-inducing activity of VAP2, the toxin displays a cell-detaching activity after incubation in high-salt conditions. These observations indicate that the apoptosis and cell-detaching functions can be discriminated. Analysis of the cell-detaching activity also revealed that VAP2 consists of two similar peptides, VAP2A and VAP2B, which are members of the PIII-type snake venom metalloproteases (SVMPs). The VAP2A cDNA encodes a 609-amino acid protein. In contrast, the peptide sequences of VAP2B were identical to that of catrocollastatin, an inhibitor of platelet aggregation. VAP2A and VAP2B interact with each other to form a noncovalent dimer similar to the ddVAPs, which was detected by native polyacrylamide gel electrophoresis. These data show some new characteristics of VAPs, which are important to clarify the apoptotic pathways in vascular endothelial cells.

Relationships between phosphatidylcholine-specific phospholipase C and integrins in cell-substratum adhesion and apoptosis in vascular endothelial cells.

In order to understand the mechanism by which VEC control cell-substratum adhesion and apoptosis, we investigated relationships between PC-PLC and the integrins that are normally expressed in VEC. We found that promotion of cell-substratum adhesion by suppression of PC-PLC was almost completely blocked by a monoclonal antibody (mAb) against integrin beta1, and was partially blocked by a mAb against intergrin beta3. The production of diacylglycerol (DAG) which was inhibited by suppression of PC-PLC activity, was increased by mAbs against intergrin beta1 and beta3. When the mAb against integrin beta4 was added to the seeding medium, cell-substratum adhesion and spreading of cells were triggered, but the activity of PC-PLC was unaffected by this mAb. Furthermore, when both the mAb against integrin beta4 and a specific inhibitor (D609) of PC-PLC were present in the seeding medium, cell-substratum adhesion and spreading were promoted to a greater extent than when either of these agents was present alone. These data suggest that integrins beta1 and beta3 might regulate cell-substratum adhesion and apoptosis via a PC-PLC-dependent pathway, while integrin beta4 might regulate these phenomena via PC-PLC-independent pathway. These findings provide the first evidence of relationships between PC-PLC and integrins in cell-substratum adhesion and apoptosis.

Hemorrhagic activity of the vascular apoptosis-inducing proteins VAP1 and VAP2 from Crotalus atrox

Vascular apoptosis-inducing proteins (VAPs) from hemorrhagic snake venom are apoptosis-inducing toxins that target vascular endothelial cells. We now show that VAP1 and VAP2 from Crotalus atrox have hemorrhagic activity in mouse skin following intradermal injection. Following intravenous injection, VAP2 induced hemorrhage in the lung, intestine and kidney. Although the hemorrhagic activity was relatively weak, these apoptosis toxins may play a role in the complex mechanism of snake venom-induced hemorrhage.

Involvement of gene expressions in apoptosis of vascular endothelial cells induced by rattlesnake venom.

ABSTRACTFormation of apoptotic bodies is a typical character of apoptotic cell death, but how the processes are controlled is not known. In this study, we compared two apoptosis inducing systems in vascular endothelial cells (VEC). We found that the formation of apoptotic bodies during apoptosis induced by rattlesnake venom, which is an unique and specific apoptosis inducer to vascular endothelial cells, was much faster than that induced by deprivation of survival factors (aFGF and serum). When we blocked the synthesis of mRNAs in cells treated with rattlesnake venom by DRB (5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole), an inhibitor of transcription, the formation of apoptotic bodies was dramatically inhibited. We examined the expression of P53 gene and found that its expression was much higher in apoptosis induced by rattlesnake venom than that in apoptosis induced by deprivation of aFGF and serum. Our results suggest that gene expression is important and P53 gene may play a major role in inducing the formation of apoptotic bodies in VEC.