Robert M. Berne Cardiovascular Research Center (V.C.G., M.C., B.H.A.), University of Virginia, Charlottesville.
Division Cardiovascular Medicine, Department of Medicine (V.C.G., B.H.A.), University of Virginia, Charlottesville.
Circulation. 2019 Jan 8;139(2):226-242. doi: 10.1161/CIRCULATIONAHA.118.034165.
Atherosclerotic occlusions decrease blood flow to the lower limbs, causing ischemia and tissue loss in patients with peripheral artery disease (PAD). No effective medical therapies are currently available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing vascular endothelial growth factor (VEGF)-A levels, a potent proangiogenic growth factor to induce angiogenesis, and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGFa) and VEGFxxxb (VEGFb) isoforms with existing literature focusing on VEGFb's role in inhibiting vascular endothelial growth factor receptor 2-dependent angiogenesis. However, we have recently shown that VEGFb blocks VEGF-A-induced endothelial vascular endothelial growth factor receptor 1 (VEGFR1) activation in ischemic muscle to impair perfusion recovery. Because macrophage-secreted VEGFb has been shown to decrease angiogenesis in peripheral artery disease, and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGFb in regulating macrophage function in PAD.
Femoral artery ligation and resection were used as an in vivo preclinical PAD model, and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-Doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, ELISAs, immunostainings, flow cytometry, quantitative polymerase chain reaction analysis, and RNA sequencing were performed to determine a role of VEGFb in regulating macrophage phenotype and function in PAD.
First, we found increased VEGFb expression with increased M1-like macrophages in PAD versus non-PAD (controls) muscle biopsies. Next, using in vitro hypoxia serum starvation, in vivo pre clinical PAD models, and adoptive transfer of VEGFb-expressing bone marrow-derived macrophages or VEGFR1 bone marrow-derived macrophages (M1-like phenotype), we demonstrate that VEGFb inhibits VEGFR1 activation to induce an M1-like phenotype that impairs ischemic muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage polarization by RNA-Seq analysis of hypoxia serum starvation-VEGFR1 versus hypoxia serum starvation-VEGFR1 bone marrow-derived macrophages.
In our current study, we demonstrate that increased VEGFb expression in macrophages induces an antiangiogenic M1-like phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGFb results in S100A8/S100A9-mediated calcium influx to induce an M1-like phenotype that impairs ischemic muscle revascularization and perfusion recovery.
动脉粥样硬化阻塞会减少下肢的血流量，导致外周动脉疾病（PAD）患者发生缺血和组织丧失。目前尚无有效的医学疗法可诱导严重 PAD 患者的血管生成并促进灌注恢复。旨在诱导血管内皮生长因子（VEGF）-A 水平的临床试验，VEGF-A 是一种强有力的促血管生成生长因子，可诱导血管生成和灌注恢复，但并未成功。VEGF-A 外显子-8 的选择性剪接导致 VEGFxxxa（VEGFa）和 VEGFxxxb（VEGFb）异构体的形成，现有文献侧重于 VEGFb 在抑制血管内皮生长因子受体 2 依赖性血管生成中的作用。然而，我们最近发现，VEGFb 阻断缺血肌肉中 VEGF-A 诱导的血管内皮生长因子受体 1（VEGFR1）激活，从而损害灌注恢复。由于巨噬细胞分泌的 VEGFb 已被证明可减少外周动脉疾病中的血管生成，并且众所周知巨噬细胞在调节缺血肌肉血管重塑中发挥重要作用，因此我们研究了 VEGFb 在调节 PAD 中巨噬细胞功能中的作用。
股动脉结扎和切除被用作体内 PAD 临床前模型，而低氧血清饥饿被用作 PAD 的体外模型。进行激光多普勒灌注成像、缺血肌肉的过继细胞转移、免疫印迹分析、ELISA、免疫染色、流式细胞术、定量聚合酶链反应分析和 RNA 测序实验，以确定 VEGFb 在调节 PAD 中巨噬细胞表型和功能中的作用。
首先，我们发现 PAD 肌肉活检中 VEGFb 表达增加，M1 样巨噬细胞增多。接下来，我们使用体外低氧血清饥饿、体内临床前 PAD 模型和过继转移表达 VEGFb 的骨髓衍生巨噬细胞或 VEGFR1 骨髓衍生巨噬细胞（M1 样表型），证明 VEGFb 抑制 VEGFR1 激活以诱导 M1 样表型，从而损害缺血肌肉的新血管生成。随后，我们通过低氧血清饥饿-VEGFR1 与低氧血清饥饿-VEGFR1 骨髓衍生巨噬细胞的 RNA-Seq 分析发现 S100A8/S100A9 是巨噬细胞极化的 VEGFR1 下游调节剂。
在我们目前的研究中，我们证明了巨噬细胞中 VEGFb 表达的增加诱导了一种抗血管生成的 M1 样表型，直接损害了血管生成。VEGFb 对 VEGFR1 的抑制导致 S100A8/S100A9 介导的钙内流，从而诱导 M1 样表型，损害缺血肌肉再血管化和灌注恢复。
