Mitochondrial reactive oxygen species and c-Src play a critical role in hypoxic response in vascular smooth muscle cells.
OBJECTIVE
Thickened atherosclerotic plaques are prone to be hypoxic because of poor perfusion. In this study, we tested (a) whether reactive oxygen species (ROS) and c-Src play roles in hypoxic induction of HIF-1alpha protein and PAI-1 gene expression in the rabbit aortic smooth muscle cell line C2/2 cells and primary cultures of rat aortic smooth muscle cells, and (b) how mitochondria act on the hypoxia-induced signaling mechanism.


RESULTS
Hypoxic exposure of C2/2 cells increased H2O2 generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. Catalase, a scavenger of H2O2, inhibited the hypoxia-induced ROS generation and PAI-1 gene expression. Src kinase inhibitors PP1 and PP2 inhibited hypoxia-induced HIF-1alpha protein and PAI-1 gene expression. Ablation of mitochondrial respiration by rotenone abolished hypoxia-induced ROS generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression.


CONCLUSIONS
Induction of HIF-1alpha protein and PAI-1 gene expression in response to hypoxia was regulated by ROS production and c-Src activation in vascular smooth muscle cells. Mitochondria linked the hypoxic signal to c-Src, which in turn led to HIF-1alpha protein and PAI-1 gene expression. These results provide evidence that hypoxia induces the ROS-mediated and c-Src-dependent signaling cascades which are closely associated with angiogenesis and thrombosis in atherosclerotic vasculature.
Sato
Hiroko
H
Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan.
Sato
Mahito
M
Kanai
Hiroyoshi
H
Uchiyama
Tsuyoshi
T
Iso
Tatsuya
T
Ohyama
Yoshio
Y
Sakamoto
Hironosuke
H
Tamura
Junichi
J
Nagai
Ryozo
R
Kurabayashi
Masahiko
M
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D002374:Catalase / Q000494:pharmacology; D015687:Cell Hypoxia; D002460:Cell Line; D005419:Flavonoids / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012402:Rotenone / Q000494:pharmacology; D000077191:Wortmannin
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; C412373:AG 1879; D000730:Androstadienes; D005419:Flavonoids; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D009281:Naphthalenes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D012333:RNA, Messenger; C061465:calphostin complex; D012402:Rotenone; D006861:Hydrogen Peroxide; D002374:Catalase; D016392:Proto-Oncogene Proteins pp60(c-src); C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin