抑制抗血管生成的VEGF165b可激活一条新的miR-17-20a-钙加压素-3通路，该通路可使外周动脉疾病中的缺血肌肉血管再生。

Inhibiting Anti-angiogenic VEGF165b Activates a Novel miR-17-20a-Calcipressin-3 Pathway that Revascularizes Ischemic Muscle in Peripheral Artery Disease.

作者信息

Batan S, Kuppuswamy S, Wood M, Reddy M, Annex B H, Ganta V C

机构信息

Vascular Biology Center, Department of Medicine, Augusta University, Augusta-GA-30912.

Medical College of Georgia, Augusta University, Augusta-GA-30912.

出版信息

Res Sq. 2023 Aug 14:rs.3.rs-3213504. doi: 10.21203/rs.3.rs-3213504/v1.

DOI:10.21203/rs.3.rs-3213504/v1

PMID:37645966

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10462251/

Abstract

BACKGROUND

VEGFa increases the expression of microRNA-17-92 cluster, promoting developmental, retinal, and tumor angiogenesis. We have previously shown that VEGFb, 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), VEGFb inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGFb 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 PAD model. VEGFb was inhibited/neutralized by an isoform-specific VEGFb antibody.

RESULTS

Systematic analysis of miR-17-92 cluster members (miR-17-18a-19a-19b-20a-92) in experimental-PAD models showed that VEGFb-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 VEGFb 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 VEGFb inhibition in PAD.

摘要

背景

血管内皮生长因子A（VEGFa）可增加微小RNA-17-92簇的表达，促进发育、视网膜及肿瘤血管生成。我们之前已经表明，血管内皮生长因子B（VEGFb）是VEGF-A的一种可变剪接异构体，可抑制缺血内皮细胞（ECs）中的VEGFR-STAT3信号通路，从而降低其血管生成能力。在缺血巨噬细胞（Møs）中，VEGFb抑制VEGFR1以诱导S100A8/A9表达，从而驱动M1样极化。我们当前的研究旨在确定在临床前外周动脉疾病（PAD）中，VEGFb抑制是否通过调节miR-17-92簇来促进灌注恢复。

方法

采用股动脉结扎和切除诱导的后肢缺血（HLI）作为临床前PAD模型。采用缺氧血清饥饿（HSS）作为PAD模型。使用异构体特异性VEGFb抗体抑制/中和VEGFb。

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，与各自的正常和/或同型匹配IgG对照相比，VEGFb抑制可诱导HSS-ECs和HSS-骨髓来源巨噬细胞（BMDMs）中miRNA-17-20a（在miR-17-92簇内）表达增加，从而增强灌注恢复。与生物信息学分析结果一致，该分析显示RCAN3是miR-17和miR-20a的共同靶点，AGO2下拉实验表明，与各自对照相比，HSS-ECs和HSS-BMDMs的RNA诱导沉默复合体（RISC）中miR-17-20a表达降低，RCAN3表达升高。抑制miR-17-20a可诱导RCAN3水平降低，减少缺血性血管生成，并促进M1样极化，从而损害灌注恢复。最后，使用STAT3抑制剂、S100A8/A9沉默剂以及VEGFR1缺陷的ECs和Møs，我们发现，在缺血ECs和缺血Møs中，VEGFb抑制分别独立于VEGFR1-STAT3或VEGFR1-S100A8/A9激活miR-17-20a-RCAN3信号通路。