目的:内皮祖细胞(EPCs)在获得性血管生成和内皮损伤修复中起着至关重要的作用。瞬时受体电位典型通道4(TRPC4),商店操作的钙通道的关键组成部分,对于EPC功能至关重要。虽然已经阐明了TRPCs在血管疾病中的作用,TRPC4和EPC功能之间的关系,以及潜在的分子机制,尚不清楚,需要进一步阐明。
方法:从犬骨髓中分离EPCs,并通过形态学和流式细胞术进行鉴定。使用慢病毒或阴性对照将TRPC4转染到EPC中,并使用实时聚合酶链反应(RT-PCR)评估其表达。扩散,迁移,使用细胞计数试剂盒-8(CCK-8)评估管的形成,Transwell,和Matrigel分析,分别。使用酶联免疫吸附测定(ELISA)测量血管内皮生长因子(VEGF)和基质细胞衍生因子-1(SDF-1)的水平。
结果:与正常对照(NC)-shRNA组相比,TRPC4-短发夹RNA(shRNA)转染的EPC中的TRPC4mRNA表达显着降低。TRPC4沉默后迁移和成管显著减少,而增殖没有差异。此外,TRPC4沉默后,EPCs中SDF-1和VEGF的水平显着降低。
结论:TRPC4在调节内皮祖细胞的血管生成中起着至关重要的作用。沉默TRPC4可通过抑制VEGF和SDF-1表达导致血管生成减少,提示TRPC4敲低可能是血管疾病的一种新的治疗策略。
OBJECTIVE: Endothelial progenitor cells (EPCs) play a crucial role in acquired angiogenesis and endothelial injury repair. Transient receptor potential canonical channel 4 (TRPC4), a key component of store-operated calcium channels, is essential for EPC function. While the role of TRPCs has been clarified in vascular diseases, the relationship between TRPC4 and EPC function, along with the underlying molecular mechanisms, remains unclear and requires further elucidation.
METHODS: EPCs were isolated from canine bone marrow and identified by morphology and flow cytometry. TRPC4 was transfected into EPCs using lentivirus or negative control, and its expression was assessed using real-time polymerase chain reaction (RT-PCR). Proliferation, migration, and tube formation were evaluated using Cell Counting Kit-8 (CCK-8), Transwell, and Matrigel assays, respectively. Levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were measured using enzyme-linked immunosorbent assay (ELISA).
RESULTS: TRPC4 mRNA expression was significantly reduced in TRPC4-short hairpin RNA (shRNA) transfected EPCs compared to the normal control (NC)-shRNA groups. Migration and tube formation were significantly decreased after TRPC4 silencing, while proliferation showed no difference. Additionally, levels of SDF-1 and VEGF in EPCs were markedly reduced following TRPC4 silencing.
CONCLUSIONS: TRPC4 plays a crucial role in regulating angiogenesis in EPCs. Silencing of TRPC4 can lead to decreased angiogenesis by inhibiting VEGF and SDF-1 expression, suggesting that TRPC4 knockdown might be a novel therapeutic strategy for vascular diseases.