SB-710411

Urotensin II (UII), a potent vasoconstrictive peptide, is able to stimulate phenotypic differentiation of adventitial fibroblasts. This study aimed to determine the effect of UII on monocyte chemoattractant protein-1 (MCP-1) expression in rat aortic adventitial fibroblasts, so as to explore possible mechanisms in the development of vascular inflammation.

Growth-arrested adventitial fibroblasts were incubated in serum-free medium with UII (10(-10)-10(-7) mol/L) and inhibitors of signal transduction pathways for 1 to 24 hours. MCP-1 mRNA and protein expression and secretion were determined by RT-PCR, Western blotting analysis and enzyme-linked immunosorbent assay (ELISA), respectively.

UII dose- and time-dependently promoted MCP-1 mRNA and protein expression and secretion in cells, with maximal effect at 10(-8) mol/L at 3 hours for mRNA expression, 24 hours for protein expression in the cells, and 12 hours for protein secretion from the cells. Furthermore, the UII effects were significantly inhibited by treatment with its receptor antagonist SB710411, Rho kinase inhibitor Y27632, protein kinase C (PKC) inhibitor H7, mitogen-activated protein kinase inhibitor PD98059, calcineurin inhibitor cyclosporine A, and the Ca(2+)channel blocker nicardipine.

UII may stimulate MCP-1 expression in rat aortic adventitial fibroblasts through its receptor and Rho kinase, PKC, mitogen-activated protein kinase, calcineurin and Ca(2+) channel signal transduction, thus contributing to adventitial inflammation.

Recent studies suggest that urotensin II (UII) and transforming growth factor-β1 (TGF-β1) both have critical roles in vascular remodeling. UII is a recently discovered vasoconstrictive peptide that is involved in the pathogenesis of atherosclerosis, restenosis and hypertension. TGF-β1 is an important factor that has a pivotal role in vascular fibrosis. This study aimed to explore whether TGF-β1 is involved in UII-induced collagen synthesis in rat aortic vascular smooth muscle cells (VSMCs) and examined the effects and mechanisms of UII on collagen synthesis and secretion in VSMCs.

VSMCs were prepared by the explant culture method. TGF-β1 and collagen I secretions from the cells were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of TGF-β1, collagen I, Smad2 and Smad3 were determined using Real-time RT-PCR and Western blotting.

UII dose-dependently promoted TGF-β1 protein expression and secretion from VSMCs, with maximal effect at 10(-8) mol/l at 24 h for protein expression and 10(-7) mol/l at 24 h for protein secretion (both P<0.01). Moreover, UII dose-dependently promoted Smad2 and Smad3 mRNA expression in VSMCs, with maximal effect at 10(-8) mol/l for 12 h (both P<0.01). The effects of UII were significantly inhibited by its receptor antagonists urantide (10(-6) mol/l) or SB-710411 (10(-6) mol/l), and by the mitogen-activated protein kinase (MAPK/ERK) inhibitor PD98059 (10(-6) mol/l). UII dose-dependently promoted collagen I mRNA expression and protein secretion in VSMCs, with maximal effect at 10(-8) mol/l at 12h for mRNA expression and 10(-6) mol/l at 24 h for protein secretion (both P<0.01). Collagen synthesis and secretion from VSMCs induced by UII were inhibited significantly by a TGF-β1-specific neutralizing antibody, SB-431542 (an antagonist of the TGF-β1 type II receptor) and PD98059 (all P<0.01).

This study suggests that UII could induce collagen synthesis and secretion through upregulation of TGF-β1 expression and secretion in VSMCs, and that TGF-β1/Smad2/3 signaling might be one of the important pathways by which UII is involved in vascular fibrosis.