■损伤或炎症后由内皮细胞(EC)释放的微泡(MV)含有组织因子(TF),并介导与基础平滑肌细胞(SMC)的通讯。Ser253磷酸化的TF与丝状蛋白A共定位在迁移的SMC的前缘。在这项研究中,内皮源性TF-MV的影响,对人冠状动脉SMC(HCASMC)迁移进行了检查。
■来自人冠状动脉EC(HCAEC)的表达TFWt的MV加速了HCASMC迁移,但胞质结构域缺失的TF较低。此外,用TFAsp253-MV孵育,或TFAsp253在HCASMC中的表达,减少细胞迁移。阻断TF因子VIIa(TF-fVIIa)促凝血/蛋白酶活性,或抑制HCASMC上的PAR2信号,废除了加速迁移。单独用fVIIa孵育增加了HCASMC迁移,但补充TF后显着增强。既不是单独的重组TF,因子Xa,PAR2激活肽(SLIGKV)也不影响细胞迁移。在其他实验中,在用TF-fVIIa刺激之前,用对应于TF的细胞质结构域的肽转染HCASMC。仅当所述肽在Ser253的位置被磷酸化时,细胞迁移才被抑制。在HCASMC中丝状蛋白A的突变形式的表达表明TF而不是PDGF-BB增强了迁移,依赖于丝素A中repeat-24的存在。HCASMC与TFWt-MV的孵育显着降低了Smoothelin-B蛋白的水平,并上调FAK表达。
■总而言之,Ser253磷酸化的TF和fVIIa作为MV-货物被EC释放,与PAR2一起作用于SMC以促进迁移,对于正常的动脉稳态以及,在血管疾病的发展过程中。
UNASSIGNED: Microvesicles (MV) released by endothelial cells (EC) following injury or inflammation contain tissue factor (TF) and mediate communication with the underlying smooth muscle cells (SMC). Ser253-phosphorylated TF co-localizes with filamin A at the leading edge of migrating SMC. In this study, the influence of endothelial-derived TF-MV, on human coronary artery SMC (HCASMC) migration was examined.
UNASSIGNED: MV derived from human coronary artery EC (HCAEC) expressing TFWt accelerated HCASMC migration, but was lower with cytoplasmic domain-deleted TF. Furthermore, incubation with TFAsp253-MV, or expression of TFAsp253 in HCASMC, reduced cell migration. Blocking TF-factor VIIa (TF-fVIIa) procoagulant/protease activity, or inhibiting PAR2 signaling on HCASMC, abolished the accelerated migration. Incubation with fVIIa alone increased HCASMC migration, but was significantly enhanced on supplementation with TF. Neither recombinant TF alone, factor Xa, nor PAR2-activating peptide (SLIGKV) influenced cell migration. In other experiments, HCASMC were transfected with peptides corresponding to the cytoplasmic domain of TF prior to stimulation with TF-fVIIa. Cell migration was suppressed only when the peptides were phosphorylated at position of Ser253. Expression of mutant forms of filamin A in HCASMC indicated that the enhancement of migration by TF but not by PDGF-BB, was dependent on the presence of repeat-24 within filamin A. Incubation of HCASMC with TFWt-MV significantly reduced the levels of Smoothelin-B protein, and upregulated FAK expression.
UNASSIGNED: In conclusion, Ser253-phosphorylated TF and fVIIa released as MV-cargo by EC, act in conjunction with PAR2 on SMC to promote migration and may be crucial for normal arterial homeostasis as well as, during development of vascular disease.