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.

Purpose: SOX10 mutations previously implicated in Waardenburg syndrome (WS), have now been linked to Kallmann Syndrome [KS], the anosmic form of idiopathic hypogonadotropic hypogonadism (IHH). We investigated whether SOX10 -associated WS and IHH represent elements of a phenotypic continuum within a unifying disorder or if they represent phenotypically distinct allelic disorders. Methods: Exome sequencing from 1309 IHH subjects (KS: 632; normosmic idiopathic hypogonadotropic hypogonadism [nIIHH:677) were reviewed for SOX10 rare sequence variants (RSVs). The genotypic and phenotypic spectrum of SOX10 -related IHH (this study & literature) and SOX10 -related WS cases (literature) were reviewed and compared with SOX10 -RSV spectrum in gnomAD population. Results: Thirty-seven SOX10 -associated IHH cases were identified: Current study:16 KS; 4 nIHH; literature:16 KS; 1 nIHH. Twenty-three IHH cases (62%; all KS), had ≥1 known WS-associated feature(s). Moreover, five previously reported SOX10- associated WS cases showed IHH-related features. Four SOX10 missense RSVs showed allelic overlap between IHH-ascertained and WS-ascertained cases. The SOX10-HMG domain showed an enrichment of RSVs in disease-states vs. gnomAD. Conclusions: SOX10 mutations contribute to both anosmic (KS) and normosmic (nIHH) forms of IHH. IHH and WS represent SOX10 -associated developmental defects that lie along a unifying phenotypic continuum. The SOX10 -HMG domain is critical for the pathogenesis of SOX10-related human disorders.

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.

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