Abstract：
BACKGROUND: Vascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-β) activation is critical. Understanding TGF-β activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-β activation and vascular fibrosis development.
METHODS: In TAK patients, PCSK5 and TGF-β expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-β in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients.
RESULTS: Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-β and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-β (pro-TGF-β) to the mature form by binding the pro-TGF-β cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-β binding, decreasing TGF-β activation and ECM expression, which was also partially validated in leflunomide-treated patients.
CONCLUSIONS: The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-β and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-β binding, presenting a new TAK treatment approach.
METHODS: In TAK patients, PCSK5 and TGF-β expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-β in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients.
RESULTS: Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-β and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-β (pro-TGF-β) to the mature form by binding the pro-TGF-β cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-β binding, decreasing TGF-β activation and ECM expression, which was also partially validated in leflunomide-treated patients.
CONCLUSIONS: The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-β and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-β binding, presenting a new TAK treatment approach.
摘要：
背景：血管纤维化直接导致大动脉炎（TAK）的血管增厚，其中持续的转化生长因子β（TGF-β）激活至关重要。了解TGF-β活化调节并阻断它可能在TAK中产生治疗作用。枯草杆菌蛋白酶/kexin5型前卵白转化酶(PCSK5)rs6560480(T/C)与TAK发育有关。在这项研究中,我们评估了TAK中PCSK5rs6560480基因型与PCSK5表达之间的关联,并探讨了其在TGF-β活化和血管纤维化发展中的分子作用.
方法：在TAK患者中，ELISA和免疫组化染色检测血浆和主动脉组织中PCSK5和TGF-β的表达,并对PCSK5rs6560480进行基因分型。通过蛋白质印迹（WB）和免疫组织化学染色检查PCSK5与细胞外基质（ECM）表达之间的相关性。通过免疫共沉淀进行检测以检测外膜成纤维细胞（AAF）中PCSK5和TGF-β之间的相互作用。通过WB检测下游信号通路并用适当的抑制剂进行验证。在细胞培养和TAK患者中探索了抑制PCSK5作用的潜在免疫抑制剂。
结果：与PCSK5rs6560480CT患者相比，PCSK5rs6560480TT患者的PCSK5水平明显更高，血管病变增厚更多。在TAK血管病变中，α-平滑肌肌动蛋白（α-SMA）阳性肌成纤维细胞中PCSK5的表达显着增加。在体外培养中，过表达PCSK5促进了AAF和主动脉中TGF-β和下游SMAD2/3的激活和ECM的表达。机制研究支持PCSK5通过结合pro-TGF-β切割位点将前体TGF-β(pro-TGF-β)激活为成熟形式。来氟米特抑制PCSK5和pro-TGF-β结合，降低TGF-β活化和ECM表达，这在来氟米特治疗的患者中也得到了部分验证。
结论：研究结果揭示了PCSK5通过TGF-β和下游SMAD2/3通路激活在TAK血管纤维化中的新型促纤维化机制。来氟米特可能通过破坏PCSK5和pro-TGF-β的结合而具有抗纤维化作用，提出了一种新的TAK治疗方法。
方法：在TAK患者中，ELISA和免疫组化染色检测血浆和主动脉组织中PCSK5和TGF-β的表达,并对PCSK5rs6560480进行基因分型。通过蛋白质印迹（WB）和免疫组织化学染色检查PCSK5与细胞外基质（ECM）表达之间的相关性。通过免疫共沉淀进行检测以检测外膜成纤维细胞（AAF）中PCSK5和TGF-β之间的相互作用。通过WB检测下游信号通路并用适当的抑制剂进行验证。在细胞培养和TAK患者中探索了抑制PCSK5作用的潜在免疫抑制剂。
结果：与PCSK5rs6560480CT患者相比，PCSK5rs6560480TT患者的PCSK5水平明显更高，血管病变增厚更多。在TAK血管病变中，α-平滑肌肌动蛋白（α-SMA）阳性肌成纤维细胞中PCSK5的表达显着增加。在体外培养中，过表达PCSK5促进了AAF和主动脉中TGF-β和下游SMAD2/3的激活和ECM的表达。机制研究支持PCSK5通过结合pro-TGF-β切割位点将前体TGF-β(pro-TGF-β)激活为成熟形式。来氟米特抑制PCSK5和pro-TGF-β结合，降低TGF-β活化和ECM表达，这在来氟米特治疗的患者中也得到了部分验证。
结论：研究结果揭示了PCSK5通过TGF-β和下游SMAD2/3通路激活在TAK血管纤维化中的新型促纤维化机制。来氟米特可能通过破坏PCSK5和pro-TGF-β的结合而具有抗纤维化作用，提出了一种新的TAK治疗方法。
