Vascular Biology Center, Medical College of Georgia at Augusta University, 1460 Laney-Walker Blvd, Augusta, GA, 30912, USA.
Department of Medicine (Cardiology), University of Illinois at Chicago, Chicago, IL, USA.
Angiogenesis. 2023 Feb;26(1):77-96. doi: 10.1007/s10456-022-09852-7. Epub 2022 Aug 19.
VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1 mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced HO signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.
血管内皮生长因子受体 2（VEGFR2）信号在血管内皮细胞（EC）中的传递受 NADPH 氧化酶（NOXs）和线粒体产生的活性氧（ROS）调节，这在出生后血管生成中发挥着重要作用。然而，ROS 如何通过高度扩散信号增强 VEGFR2 信号和修复性血管生成仍然不清楚。蛋白二硫键异构酶 A1（PDIA1）是一种依赖于氧化还原环境的氧化还原酶。我们假设 PDIA1 作为一种氧化还原传感器来增强血管生成。在这里，我们发现 PDIA1 在人 EC 受到 VEGF 刺激时与 VEGFR2 共免疫沉淀或与 VEGFR2 或早期内体标志物 Rab5 共定位在核周区。PDIA1 沉默通过抑制 VEGFR2 信号显著降低 VEGF 诱导的 EC 迁移、增殖和球体发芽。在机制上，VEGF 刺激通过 NOX4-线粒体 ROS 轴迅速增加 PDIA1 的 Cys-OH 形成。用 Ser 替换活性四个 Cys 残基的“氧化还原无效”突变型 PDIA1 的过表达显著抑制了 VEGF 诱导的 PDIA1-CysOH 形成和血管生成反应，从而降低了 VEGFR2 磷酸化。Pdia1 小鼠在发育性视网膜和 Matrigel 塞模型以及离体主动脉环发芽模型中表现出血管生成受损。使用后肢缺血模型的研究表明，PDIA1 在缺血肌肉的血管生成 EC 中表达显著增加，并且 EC 特异性 Pdia1 条件性敲除小鼠的缺血诱导肢体灌注恢复和新血管生成受损。这些结果表明，PDIA1 可以通过 Cys-OH 形成感应 VEGF 诱导的 HO 信号，从而在 EC 中促进 VEGFR2 信号和血管生成，从而增强出生后血管生成。氧化的 PDIA1 可能是治疗缺血性血管疾病的潜在治疗靶点。
