Transforming growth factor-β1 involved in urotensin Ⅱ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta

Background Urotensin Ⅱ （UⅡ） is a new vasoconstrictive peptide that may activate the adventitial fibroblasts.Transforming growth factor-β1 （TGF-β1） is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UⅡ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.Methods Adventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression of α-smooth nuscle actin （α-SM-actin）, the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR （real-time RT-PCR） and Western blotting, respectively.Results UⅡ stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% （P ＜0.01）, than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10-8 mol/L of UⅡ （P ＜0.01）, which was increased by 59.9%,compared with in the control group （P ＜0.01）. The secretion of TGF-β1 induced by UⅡ was significantly blocked by SB-710411 （10-7 mol/L）, a specific antagonist of UⅡ receptor. In addition, both UⅡ （10-8 mol/L） and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UⅡ was inhibited by the specific neutralizing antibody （20 μg/ml） of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 （20 ng/ml）was inhibited by SB-710411 （10-7 mol/L）, the UⅡ receptor antagonist.Conclusion This study suggests that UⅡ could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UⅡ, and TGF-β1 signaling might be one of the important pathways by which UⅡ is involved in vascular fibrosis.

Urotensin Ⅱ promotes monocyte chemoattractant protein-1 expression in aortic adventitial fibroblasts of rat

Background Urotensin Ⅱ （Ull）,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 （MCP1） expression in rat aortic adventitial fibroblasts,so as to explore possible mechanisms in the development of vascular inflammation.Methods Growth-arrested adventitial fibroblasts were incubated in serum-free medium with UII （1010-10-7 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.Results 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 Ca2＋channel blocker nicardipine.Conclusion UII may stimulate MCP-1 expression in rat aortic adventitial fibroblasts through its receptor and Rho kinase,PKC,mitogen-activated protein kinase,calcineurin and Ca2＋ channel signal transduction,thus contributing to adventitial inflammation.

Globular adiponectin-mediated vascular remodeling by affecting the secretion of adventitial-derived tumor necrosis factor-αinduced by urotensinⅡ

Objectives:In this study,we explored how adiponectin mediated urotensinⅡ(UⅡ)-induced tumor necrosis factor-α(TNF-α)andα-smooth muscle actin(α-SMA)expression and ensuing intracellular signaling pathways in adventitial fibroblasts(AFs).Methods:Growth-arrested AFs and rat tunica adventitia of vessels were incubated with UⅡand inhibitors of signal transduction pathways for 1-24 h.The cells were then harvested for TNF-αreceptor(TNF-α-R)messenger RNA(mRNA)and TNF-αprotein expression determination by reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA),respectively.Adiponectin and adiponectin receptor(adipoR)expression was measured by RT-PCR,quantitative real-time PCR(qPCR),immunohistochemical analysis,and cell counting kit-8(CCK-8)cell proliferation experiments.We then quantified TNF-αandα-SMA mRNA and protein expression levels by qPCR and immunofluorescence(IF)staining.RNA interference(RNAi)was used to explore the function of the adipoR genes.To investigate the signaling pathway,we applied western blotting(WB)to examine phosphorylation of adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK).In vivo,an adiponectin(APN)-knockout(APN-KO)mouse model mimicking adventitial inflammation was generated to measure TNF-αandα-SMA expression by application of qPCR and IF,with the goal of gaining a comprehensive atlas of adiponectin in vascular remodeling.Results:In both cells and tissues,UⅡpromoted TNF-αprotein and TNF-α-R secretion in a dose-and time-dependent manner via Rho/protein kinase C(PKC)pathway.We detected marked expression of adipoR1,T-cadherin,and calreticulin as well as a moderate presence of adipoR2 in AFs,while no adiponectin was observed.Globular adiponectin(gAd)fostered the growth of AFs,and acted in concert with UⅡto induceα-SMA and TNF-αthrough the adipoR1/T-cadherin/calreticulin/AMPK pathway.In AFs,gAd and UⅡsynergistically induced AMPK phosphorylation.In the adventitial inflammation model,APN deficiency up-regulated the expression ofα-SMA,UⅡreceptor(UT),and UⅡwhile inhibiting TNF-αexpression.Conclusions:From the results of our study,we can speculate that UⅡinduces TNF-αprotein and TNF-α-R secretion in AFs and rat tunica adventitia of vessels via the Rho and PKC signal transduction pathways.Thus,it is plausible that adiponectin is a major player in adventitial progression and could serve as a novel therapeutic target for cardiovascular disease administration.