Possible involvement of m-calpain in vascular smooth muscle cell proliferation.
Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.
Ariyoshi
H
H
Second Department of Surgery, Osaka University Medical School, Suita, Japan.
Okahara
K
K
Sakon
M
M
Kambayashi
J
J
Kawashima
S
S
Kawasaki
T
T
Monden
M
M
D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007536:Isomerism; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism
D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D004151:Dipeptides; D016376:Oligonucleotides, Antisense; D011480:Protease Inhibitors; C071482:calpeptin; C033668:calpastatin; D004247:DNA; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D002154:Calpain