RNAi targeting embryonic myosin heavy chain isoform inhibited bound thrombin-induced migration of vascular smooth muscle cells.
To investigate the effect of bound thrombin, a complex of alpha-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell proliferation was measured by WST-1 reagent and migration was evaluated by counting migrated cells through pores of cell culture insert (8 mum size) after 48-hour treatment with bound thrombin (10 U/ml). To examine the role of an embryonic myosin heavy chain isoform (SMemb) in these effects by bound thrombin, the cells were subsequently treated for 48 h with an siRNA expression vector (ORF-2/pSilencer) directed against the open reading frame of SMemb mRNA. SMemb and plasminogen activator inhibitor-1 mRNA expressions were measured by Northern blot analysis. Bound thrombin significantly increased SMemb mRNA expression by 1.4 +/- 0.01-fold and significantly increased plasminogen activator inhibitor-1 mRNA expression by 2.65 +/- 0.69-fold (p < 0.01 vs. PBS treatment for each), which were abolished by treatment with ORF-2/pSilencer. Although bound thrombin had no effect on cell proliferation, bound thrombin significantly increased migration by 1.93 +/- 0.20-fold (p < 0.05). ORF-2/pSilencer treatment significantly reduced the bound thrombin-stimulated migration activity by 1.28 +/- 0.15-fold (p < 0.05). Thus, SMemb plays an important role in bound thrombin-induced cell migration activity of cultured vascular smooth muscle cells.
Sunagawa
Masanori
M
1st Department of Physiology, Unit of Physiological Science, School of Medicine, University of the Ryukyus, Nishihara, Japan. sunagam@med.u-ryukyu.ac.jp
Shimada
Seiji
S
Nakamura
Mariko
M
Kosugi
Tadayoshi
T
D000818:Animals; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D034622:RNA Interference; D011817:Rabbits; D013917:Thrombin / Q000494:pharmacology*
D020033:Protein Isoforms; D013917:Thrombin; D018995:Myosin Heavy Chains