Protease-activated Receptor 1 And 2 Contribute To Angiotensin ?-induced Activation Of Adventitial Fibroblasts And Adventitial Remodeling

Background and Objective:Adventitial fibroblasts?AFs?can be activated by angiotensin ??Ang ??and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling.However,the detailed mechanisms underlying how Ang ? induces AF activation remain largely unknown.Protease-activated receptor?PAR?1 and 2 play a significant role in fibrogenic and inflammatory diseases.In the present study,we evaluated the mechanisms underlying PAR1-and PAR2-induced activation of rat aortic adventitial fibroblasts,and whether PAR1 and PAR2 play a role in Ang ?-induced AF activation and contribute to adventitial remodeling.Methods:Adventitial fibroblasts were isolated from thoracic aortas of 6-8 week-old male Sprague–Dawley rats.PAR1-AP?TFLLR-NH₂?and PAR2-AP?2-furoyl-LIGRLO-NH₂?were used to stimulate AFs.A cell counting kit-8?CCK-8?assay was performed to detect cell viability after various treatments.A wound scratch assay was performed to detect the ability of AF migration.Western blot was used to detect fibrogenic protein levels of?-SMA,collagen I,fibronectin,and TGF-?,and real-time PCR was used to detect mRNA levels of IL-6 and MCP-1.After pretreatment with PAR1 antagonist SCH79797 and PAR2 antagonist ENMD-1068,AFs were treated with Ang ?,and all above parameters were detected again.After pretreatment with AT1receptor antagonist losartan or AT2 receptor antagonist PD123319,Ang ? was used to stimulate AFs.Western blot was used to detect PAR1 and PAR2 expression and immunofluorescence was used to detect PAR1 and PAR2 internalization.Immunofluorescence was used to detect the co-localization of PAR1 and PAR2 with?-SMA,MCP-1 in the aorta of sham and Ang ?-treated groups.Results:We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation,including proliferation and differentiation of AFs,extracellular matrix synthesis,as well as production of pro-fibrotic cytokine TGF-?and pro-inflammatory cytokines IL-6 and MCP-1.ERK1/2-dependent signaling pathway was involved in PAR1-AP-and PAR2-AP-induced AF proliferation,and TGF-?/p-Smad2signaling pathway was involved in PAR1-AP-and PAR2-AP-induced fibrogenic proteins synthesis.Furthermore,PAR1 and PAR2 mediated Ang ?-induced AF activation,since both PAR1 and PAR2 antagonists inhibited Ang ?-induced proliferation,migration,differentiation,extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs.Further mechanistic study showed that Ang ?,via Ang ? type I receptor?AT1R?,upregulated both PAR1 and PAR2 expression,and transactivated PAR1 and PAR2,as denoted by internalization of both proteins.Finally,we found that Ang ?-treated group showed more intensive PAR1 or PAR2 staining in the adventitia,correlated with increased?-SMA and MCP-1 expression in comparison with sham group.Conclusion:The present study shows that PAR1 and PAR2 activation can directly induce AF activation,and PAR1/PAR2 transactivation is involved in Ang ?-induced AF activation.These findings suggest that PAR1 and PAR2 play a crucial role in adventitial remodeling and blockade of them might be a new therapeutic target for vascular remodeling diseases.