背景：VEGF 165 a增加了microRNA-17-92簇的表达，促进了发育、视网膜和肿瘤血管生成。我们先前已经表明，VEGF 165 b，一种可选择剪接的VEGF-A异构体，在缺血内皮细胞（ECs）中抑制VEGFR-STAT3通路，降低其血管生成能力。在缺血巨噬细胞（Møs）中，VEGF 165 b抑制VEGFR1使S100A8/A9表达增加，进而推动M1型极化。我们目前的研究旨在确定VEGF 165 b抑制对MiR-17-92簇在临床前PAD中进行灌流恢复的调节是否有效。方法：采用股动脉结扎切除引起的腹股沟下肢缺血（HLI）作为临床前PAD模型。采用缺氧血清饥饿（HSS）作为体外PAD模型。采用特异VEGF 165 b抗体抑制/中和VEGF 165 b。结果：对实验性PAD模型中miR-17-92簇成员（miR-17-18a-19a-19b-20a-92）的系统分析表明，VEGF 165 b抑制诱导HSS-ECs和HSS骨髓来源巨噬细胞（BMDMs）中该簇内miRNA-17-20a表达增加，与相应的正常和/或同型IgG对照相比增强了灌流恢复。与生物信息学分析一致的是，miR-17和miR-20a的一个共同靶标为RCAN3，Argonaute-2 pull-down实验显示与相应对照相比，HSS-ECs和HSS-BMDMs的RISC复合物中miR-17-20a表达下降，RCAN3表达上升。抑制miR-17-20a导致RCAN3水平下降，减少缺血性血管生成并促进M1型极化，从而损害灌流恢复。最后，通过使用STAT3抑制剂、S100A8/A9沉默剂和VEGFR1缺陷的ECs和Møs，我们展示了VEGF 165 b抑制通过VEGFR1-STAT3或VEGFR1-S100A8/A9独立于缺血ECs和缺血Møs中激活miR-17-20a-RCAN3通路。结论：我们的数据揭示了VEGF 165 b抑制在PAD中激活缺血血管中一个迄今为止未被认识的治疗性“miR-17-20a-RCAN3”通路，该通路与VEGFR1-STAT3/S100A8/A9无关，且只在VEGF 165 b抑制时才能被激活。

Background: VEGF 165 a increases the expression of microRNA-17-92 cluster, promoting developmental, retinal, and tumor angiogenesis. We have previously shown that VEGF 165 b, an alternatively spliced VEGF-A isoform, inhibits the VEGFR-STAT3 pathway in ischemic endothelial cells (ECs) to decrease their angiogenic capacity. In ischemic macrophages (Møs), VEGF 165 b inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGF 165 b inhibition promotes perfusion recovery by regulating the miR-17-92 cluster in preclinical PAD. Methods : Hind limb ischemia (HLI) induced by femoral artery ligation and resection was used as a preclinical PAD model. Hypoxia serum starvation (HSS) was used as an in vitro PAD model. VEGF 165 b was inhibited/neutralized by an isoform-specific VEGF 165 b antibody. Results: Systematic analysis of miR-17-92 cluster members (miR-17-18a-19a-19b-20a-92) in experimental-PAD models showed that VEGF 165 b-inhibition induces miRNA-17-20a (within miR-17-92 cluster) in HSS-ECs and HSS-bone marrow derived macrophages (BMDMs) vs. respective normal and/or isotype matched IgG controls to enhance perfusion-recovery. Consistent with the bioinformatics analysis that revealed RCAN3 as a common target of miR-17 and miR-20a, Argonaute-2 pull-down assays showed decreased miR-17-20a expression and higher RCAN3 expression in the RISC complex of HSS-ECs and HSS-BMDMs vs. the respective controls. Inhibiting miR-17-20a induced RCAN3 levels to decrease ischemic angiogenesis and promoted M1-like polarization to impair perfusion recovery. Finally, using STAT3 inhibitors, S100A8/A9 silencers and VEGFR1-deficient ECs and Møs, we show that VEGF 165 b inhibition activates the miR-17-20a-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 in ischemic ECs and ischemic Møs, respectively. Conclusion: Our data revealed a hereunto unrecognized therapeutic 'miR-17-20a-RCAN3' pathway in the ischemic vasculature that is VEGFR1-STAT3/S100A8/A9 independent and is activated only upon VEGF 165 b inhibition in PAD.