RNAi targeting embryonic myosin heavy chain isoform inhibited bound thrombin-induced migration of vascular smooth...

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.