Satohiko Araki

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation

Survival and proliferation of many types of vascular endothelial cells are influenced by fibroblast growth factor (FGF)1. Removal of FGF from the medium of human umbilical vein endothelial cells (HUVEC) in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide, an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in culture. The process of active death of vascular endothelial cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels.

Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells

Apoptosis in vascular endothelial cells is suppressed by fibroblast growth factor (FGF)1. In order to investigate the signal transduction system that regulates endothelial apoptosis, we studied the effects of several mitogenic factors. Apoptosis occurred in human vascular endothelial cells under serum-free conditions, and FGF inhibited apoptosis without a requirement of any cooperative factors, as distinct from the mitogenic response. Other mitogenic agents, such as epidermal growth factor, transferrin, transforming growth factor beta, and interleukin 1 etc., with the exception of dexamethasone, had no such inhibitory effects. The effect of FGF was mimicked by a phorbol ester and was prevented by an inhibitor of protein kinase C. The results suggest that the FGF and protein kinase C are important in endothelial apoptosis.

Crystal structures of catrocollastatin/VAP2B reveal a dynamic, modular architecture of ADAM/adamalysin/reprolysin family proteins.

Catrocollastatin/vascular apoptosis‐inducing protein (VAP)2B is a metalloproteinase from Crotalus atrox venom, possessing metalloproteinase/disintegrin/cysteine‐rich (MDC) domains that bear the typical domain architecture of a disintegrin and metalloproteinase (ADAM)/adamalysin/reprolysin family proteins. Here we describe crystal structures of catrocollastatin/VAP2B in three different crystal forms, representing the first reported crystal structures of a member of the monomeric class of this family of proteins. The overall structures show good agreement with both monomers of atypical homodimeric VAP1. Comparison of the six catrocollastatin/VAP2B monomer structures and the structures of VAP1 reveals a dynamic, modular architecture that may be important for the functions of ADAM/adamalysin/reprolysin family proteins.

Involvement of specific integrins in apoptosis induced by vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of vascular apoptosis-inducing protein 1 (VAP1) that specifically induces apoptosis in VEC. VAP1 belongs to the metalloprotease/disintegrin family. Yet the mechanism of inducing apoptosis by VAP1 is still not known. Since other various metalloproteases and disintegrins in snake venoms are known to influence extracellular matrix and cell adhesion, we investigated here the involvement of these adhesion molecules in VAP1-induced apoptosis. Consequently, VAP1 induced apoptosis without degrading extracellular matrix or inhibiting adhesion of VEC. However, VAP1-induced apoptosis was inhibited by antibodies for integrin alpha3, alpha6, beta1. Additionally, apoptosis was inhibited by antibody for CD9, an integrin associated protein. These results suggest that integrins are involved in VAP1-induced apoptosis by some specific role rather than that of adhesion to extracellular matrix.

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.

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.

Fragments of Amyloid β Induce Apoptosis in Vascular Endothelial Cells

Injury to cells that was triggered by fragments of amyloid β protein (Aβ) was studied in human vascular endothelial cells. A fragment of Aβ containing amino acids 25—35 promoted cell death, but fragments of Aβ containing amino acids 31—35 and 35—25 did not have such an effect. The fragment of Aβ containing amino acids 25—35 induced cell death that was characterized as programmed cell death (apoptosis) in view of the accompanying morphological changes, the fragmentation of DNA, and the requirement for protein synthesis.

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.