Antiangiogenic VEGF ₁₆₅ b Regulates Macrophage Polarization via S100A8/S100A9 in Peripheral Artery Disease

Abstract

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.

A novel voluntary weightlifting model in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway

“…RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNAseq library construction and analysis were performed as previously described . Semi‐quantitative PCR was performed as described previously .…”

Early Post-stroke Activation of Vascular Endothelial Growth Factor Receptor 2 Hinders the Receptor 1-Dependent Neuroprotection Afforded by the Endogenous Ligand

“…However, not much is known about the involvement of VEGF, or its receptors, in the polarization of macrophages/microglia under inflammation. Interestingly, very recent evidence indicates that the antiangiogenic VEGF isoform VEGF 164 b is capable of blocking VEGFR1 prompting the M1 polarization of peripheral macrophages (Ganta et al, 2019), although it is known that this form of VEGF has neuroprotective effects in the CNS (Beazley-Long et al, 2013). Also, M2-polarized peripheral macrophages in culture are known to upregulate their VEGFR1 mRNA expression (Melton et al, 2015), which in microglial cells could lead to the inhibition of the expression of the scavenger receptor A following stroke (Xu et al, 2017).…”

The complex TIE between macrophages and angiogenesis

“…Further studies revealed that in vitro generated CD11b low macrophages inhibit angiogenesis by releasing endostatin and by expressing lower levels of VEGFA compared to control macrophages (Michaeli et al, 2018). A recent study by Ganta, Choi, Farber, and Annex (2019) in a mouse hindlimb ischaemia model showed that macrophages can adopt an anti-angiogenic phenotype after VEGF 165 b binding on the macrophage VEGF receptor 1 (VEGFR1 or Flt1), which inhibited ischemic muscle neovascularization in a paracrine manner (Ganta et al, 2019). In this study, the macrophages were classified as M1 macrophages (Cd80 high Arg1 low ).…”