Rationale Atherosclerotic-arterial occlusions decrease tissue perfusion causing ischemia to lower limbs in patients with peripheral arterial disease (PAD). Ischemia in muscle induces an angiogenic response but the magnitude of this response is frequently inadequate to meet tissue perfusion requirements. Alternate splicing in the exon-8 of vascular endothelial growth factor (VEGF)-A results in production of pro-angiogenic VEGFxxxa isoforms (VEGF165a, 165 for the 165 amino acid product) and anti-angiogenic VEGFxxxb (VEGF165b) isoforms. Objective The anti-angiogenic VEGFxxxb isoforms are thought to antagonize VEGFxxxa isoforms and decrease activation of VEGF-Receptor-2 (VEGFR2), hereunto considered the dominant receptor in post-natal angiogenesis in PAD. Our data will show that VEGF165b inhibits VEGFR1-Signal Transducer and Activator of Transcription (STAT)-3 signaling to decrease angiogenesis in human and experimental PAD. Methods and Results In human PAD vs. control muscle-biopsies, VEGF165b: a) is elevated, b) is bound higher (vs. VEGF165a) to VEGFR1 not VEGFR2, and c) levels correlated with decreased VEGFR1, not VEGFR2, activation. In experimental PAD, delivery of an isoform specific monoclonal antibody (Ab) to VEGF165b vs. control-Ab enhanced perfusion in animal model of severe PAD (Balb/c strain) without activating VEGFR2-signaling but with increased VEGFR1-activation. Receptor pull-down experiments demonstrate that VEGF165b-inhibition vs. control increased VEGFR1-STAT3 binding and STAT3-activation, independent of janus activated kinase (Jak1)/Jak2. Using VEGFR1+/− mice that could not increase VEGFR1 after ischemia, we confirm that VEGF165b decreases VEGFR1-STAT3 signaling to decrease perfusion. Conclusions Our results indicate that VEGF165b prevents activation of VEGFR1-STAT3 signaling by VEGF165a and hence inhibits angiogenesis and perfusion recovery in PAD muscle.

Background Currently no therapies exist for treating, and improving outcomes in patients with severe peripheral arterial disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and reduce tissue loss in genetic PAD models. However, the cell specific function, downstream mechanisms or signaling involved in miR93 mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1-activation/polarization state. In the current study, we identified a novel mechanism by which miR93 regulates macrophage-polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental-PAD. Methods In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation (HSS) conditions) and in vivo experiments in preclinical-PAD models (unilateral femoral artery ligation and resection)) were conducted to examine the role of miR93-interferon regulatory factor-9 (IRF9)-immune responsive gene-1 (IRG1)-itaconic acid pathway in macrophage-polarization, angiogenesis, arteriogenesis and perfusion recovery. Results In vivo, compared to wild type (WT) controls, miR106b-93-25 cluster deficient mice (miR106b-93-25−/−) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like-macrophages following experimental-PAD. Intra-muscular delivery of miR93 in miR106b-93-25−/− PAD mice increased angiogenesis, arteriogenesis, the extent of perfusion which correlated with more M2-like-macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like-polarization even under M1-like-polarizing conditions (HSS). Delivery of bone marrow derived macrophages from miR106b-93-25−/− to WT ischemic-muscle decreased angiogenesis, arteriogenesis and perfusion, while transfer of wild-type macrophages to miR106b-93-25−/− had the opposite effect. Systematic analysis of top-differentially upregulated genes from RNA-sequencing between miR106b-93-25−/− and WT ischemic-muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage-polarization. 3′UTR luciferase-assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not a miR93 target but IRF9 that can regulate IRG1-expression is a miR93 target. In vitro, increased expression of IRF9, IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential. Conclusion We conclude that miR93 inhibits IRF9 to decrease IRG1-itaconic acid production to induce M2-like-polarization in ischemic muscle to enhance angiogenesis, arteriogenesis and perfusion recovery in experimental-PAD.

Background: Atherosclerotic occlusions decrease blood flow to the lower limbs causing ischemia and tissue loss in patients with peripheral artery disease (PAD). Currently, no effective medical therapies are available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing VEGF-A levels, a potent pro-angiogenic 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 (VEGF165a) and VEGFxxxb (VEGF165b) isoforms with existing literature focusing on VEGF165b’s role in inhibiting VEGFR2 dependent angiogenesis. However, we have recently shown that VEGF165b blocks VEGF-A induced endothelial VEGFR1 activation in ischemic muscle to impair perfusion recovery. Since macrophage secreted VEGF165b 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 VEGF165b in regulating macrophage function in PAD. Methods: Femoral artery ligation and resection was 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, enzyme-linked immunosorbent assays, Immunostainings, flow cytometry, qPCR analysis and RNA-Seq analysis were performed to determine a role of VEGF165b in regulating macrophage phenotype and function in PAD. Results: First, we found increased VEGF165b-expression with increased M1-like-macrophages in PAD vs. non-PAD (controls) muscle-biopsies. Next, using in vitro hypoxia serum starvation (HSS), in vivo pre-clinical PAD models and adoptive-transfer of VEGF165b-expressing bone marrow-derived macrophages (BMDM) or VEGFR1+/− BMDM (M1-like-phenotype), we demonstrate that VEGF165b 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 HSS-VEGFR1+/+ vs. HSS-VEGFR1+/− BMDM. Conclusion: In our current study, we demonstrate that increased VEGF165b-expression in macrophages induces an anti-angiogenic M1-like-phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF165b results in S100A8/S100A9 mediated calcium influx to induce an M1-like-phenotype that impairs ischemic-muscle revascularization and perfusion recovery.

Angiogenesis is the growth of new blood vessels from pre-existing microvessels. Peripheral arterial disease (PAD) is caused by atherosclerosis that results in ischemia mostly in the lower extremities. Clinical trials including VEGF-A administration for therapeutic angiogenesis have not been successful. The existence of anti-angiogenic isoform (VEGF165b) in PAD muscle tissues is a potential cause for the failure of therapeutic angiogenesis. Experimental measurements show that in PAD human muscle biopsies the VEGF165b isoform is at least as abundant if not greater than the VEGF165a isoform. We constructed three-compartment models describing VEGF isoforms and receptors, in human and mouse, to make predictions on the secretion rate of VEGF165b and the distribution of various isoforms throughout the body based on the experimental data. The computational results are consistent with the data showing that in PAD calf muscles secrete mostly VEGF165b over total VEGF. In the PAD calf compartment of human and mouse models, most VEGF165a and VEGF165b are bound to the extracellular matrix. VEGF receptors VEGFR1, VEGFR2 and Neuropilin-1 (NRP1) are mostly in ‘Free State’. This study provides a computational model of VEGF165b in PAD supported by experimental measurements of VEGF165b in human and mouse, which gives insight of VEGF165b in therapeutic angiogenesis and VEGF distribution in human and mouse PAD model.

Introduction: Classic VEGF-A induced angiogenesis involves VEGFR2 (VR2)-PI3K-AKT-eNos activation. Ischemia induces VEGF165b (V165b, an anti-angiogenic VEGF-A isoform) levels in Peripheral Arterial Disease (PAD) muscle. Since V165b competes with pro-angiogenic VEGF-A isoforms to bind and block VEGF-A dependent activation of VR2 and angiogenesis, we hypothesized that “V165b inhibition removes the anti-angiogenic brakes on VR2-PI3K-Akt signaling to promote therapeutic angiogenesis and perfusion recovery in PAD”. Methods and Results: Hind limb ischemia (HLI), an experimental PAD model was performed by femoral artery ligation and resection in worse recovery animal strains (Balbc and type 2-Diabetes (T2D), n=10/group). Mice were treated i.m. with isoform specific V165b blocking antibody or IgG immediately post-surgery. V165b inhibition significantly increased perfusion recovery (Balbc: V165b-Ab=75.35±4.7 vs. IgG=50.13±2.4%, T2D: 65.70±5.8 vs. 44.04±6.08%) assessed by laser Doppler. Furthermore, V165b inhibition increased vascular density by ~2 fold in ischemic muscle and significantly decreased necrosis scores in both animal strains compared to IgG. In vitro, HUVECs treated with V165b-Ab showed significantly higher capillary like tube formation on matrigel suggesting that endogenous V165b inhibition is sufficient to promote angiogenesis. In stark contrast to our hypothesis, V165b inhibition significantly induced VR1 activation but not VR2, along with Stat3 activation, decreased Akt, Erk1/2 activation and decreased P53 levels (in d3 post HLI tissue samples) compared to IgG. In vitro, HUVECs treated with V165b-Ab also showed significant increase in VR1 and Stat3 activation compared to IgG. Moreover, ligand-receptor binding experiments showed that V165b inhibition significantly increased the binding of not only VEGF-A but also VEGF-B and PlGF to VR1 in ischemic muscle as well as in HUVECs in vitro (P<0.05 is considered significant). Conclusion: We conclude that V165b not only competes with VEGF-A but also with VEGF-B and PlGF to bind and block VR1 activation in ischemic muscle. Our data point towards a novel VR1 signaling that activates Stat3 and inhibits P53 to promote perfusion recovery in ischemic muscle post V165b inhibition.