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.

Lymphatics perform essential transport and immune cell regulatory functions to maintain homeostasis in the gastrointestinal (GI) system. Although blood and lymphatic vessels function as parallel and integrated systems, our understanding of lymphatic structure, regulation and functioning lags far behind that of the blood vascular system. This chapter reviews lymphatic flow, differences in lymphangiogenic and hemangiogenic factors, lymphatic fate determinants and structural features, and examines how altered molecular signaling influences lymphatic function in organs of the GI system. Innate errors in lymphatic development frequently disturb GI functioning and physiology. Expansion of lymphatics, a prominent feature of GI inflammation, may also play an important role in tissue restitution following injury. Destruction or dysregulation of lymphatics, following injury, surgery or chronic inflammation also appears to exacerbate GI disease activity and morbidity. Understanding the physiological roles played by GI lymphatics is essential to elucidating their underlying contributions to forms of congenital and acquired forms of GI pathology, and will provide novel approaches for treatment of these conditions.

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.

The pathophysiology of inflammatory bowel disease (IBD) includes leukocyte infiltration, blood and lymphatic remodeling, weight loss and protein enteropathy. The roles of angiopoietin-2 (Ang-2) in initiating gut inflammation, leukocyte infiltration and angiogenesis are not well understood. Several important differences were seen in the development of experimental IBD in Ang-2-/- mice. Although weight change and disease activity differ only slightly in WT and Ang-2-/- + DSS treated mice, leukocyte infiltration, inflammation and blood and lymphatic vessel density is significantly attenuated compared to WT+ DSS mice. Gut capillary fragility and water export (stool blood and form) appear significantly earlier in Ang-2-/- + DSS mice vs. WT. Colon lengths were also significantly reduced in Ang-2-/- and gut histopathology was less severe in Ang-2-/-compared to WT + DSS. Lastly, the decrease in serum protein content in WT + DSS was less severe in Ang-2-/- + DSS, thus protein losing enteropathy (PLE) a feature of IBD is relieved by Ang-2-/-. These data demonstrate that in DSS colitis, Ang-2 mediates inflammatory hemangiogenesis, lymphangiogenesis and neutrophil infiltration to reduce some, but not all clinical features of IBD. The implications for Ang-2 manipulation in the development of IBD and other inflammatory diseases and treatments involving Ang-2 are discussed.

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We built a whole‐body computational model to study the role of the poorly understood vascular endothelial growth factor (VEGF)165b splice isoform in peripheral artery disease (PAD). This model was built and validated using published and new experimental data from cells, mice, and humans, and explicitly accounts for known properties of VEGF165b: lack of extracellular matrix (ECM)‐binding and weak phosphorylation of vascular endothelial growth factor receptor‐2 (VEGFR2) in vitro. The resulting model captures all known information about VEGF165b distribution and signaling in human PAD, and provides novel, nonintuitive insight into VEGF165b mechanism of action in vivo. Although VEGF165a and VEGF165b compete for VEGFR2 in vitro, simulations show that these isoforms do not compete for VEGFR2 at much lower physiological concentrations. Instead, reduced VEGF165a may drive impaired VEGFR2 signaling. The model predicts that VEGF165b does compete for binding to VEGFR1, supporting a VEGFR1‐mediated response to anti‐VEGF165b. The model predicts a key role for VEGF165b in PAD, but in a different way than previously hypothesized.

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