title: Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells.
abstract: In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.
authors: Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka-City, Shizuoka, Japan. ohtat@u-shizuoka-ken.ac.jp
authors: Toshiro
authors: T
authors: Ohta
authors: Ryoji
authors: R
authors: Eguchi
authors: Akio
authors: A
authors: Suzuki
authors: Shinichi
authors: S
authors: Miyakaze
authors: Rie
authors: R
authors: Ayuzawa
authors: Kazuhiko
authors: K
authors: Kaji
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D002196:Capillaries / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism*; D061945:Caspase Inhibitors; D002467:Cell Nucleus / Q000473:pathology; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology*; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D017202:Myocardial Ischemia / Q000378:metabolism / Q000473:pathology; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014471:Umbilical Veins / Q000166:cytology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; C093642:SB 203580