VSMCs were prepared by the explant culture method. TGF-¦Â1 and collagen I secretions from the cells were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of TGF-¦Â1, collagen I, Smad2 and Smad3 were determined using Real-time RT-PCR and Western blotting.
UII dose-dependently promoted TGF-¦Â1 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 12 h 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-¦Â1-specific neutralizing antibody, SB-431542 (an antagonist of the TGF-¦Â1 type II receptor) and PD98059 (all P < 0.01).
This study suggests that UII could induce collagen synthesis and secretion through upregulation of TGF-¦Â1 expression and secretion in VSMCs, and that TGF-¦Â1/Smad2/3 signaling might be one of the important pathways by which UII is involved in vascular fibrosis.