Human Osteopontin Isoforms Differentially Promote Post‐Ischemic Neo‐vascularization via Divergent Effects on Macrophages in vivo and in vitro

Abstract

Coronary and peripheral artery diseases result in vessel occlusion and ischemia, initiating neovascularization to restore blood flow and preserve function. We previously demonstrated that osteopontin (OPN), a secreted cytokine, is critical to ischemia‐induced neovascularization. Unlike rodents, humans express 3 OPN isoforms (a, b, and c); however, the roles of these isoforms in neovascularization and cell migration remain undefined. To investigate if human OPN isoforms have differential effects on functional perfusion recovery in vivo, OPN−/− mice underwent hind limb ischemia (HLI) and received 1.5×106 lentivirus particles expressing OPNa, OPNb or OPNc at the time of surgery by intramuscular (IM) injection of the adductor muscle using a microsyringe. By utilizing this experimental approach, the only OPN in the system is what we add back, thus allowing us to determine how each human OPN isoform functions to influence neovascularization in vivo. OPNa and OPNc significantly improved limb perfusion, as measured by laser Doppler perfusion imaging, by 30.4%±0.8 and 70.9%±6.3, respectively (d14; p<0.001 vs. LVGFP, p<0.05 vs. hOPNb), and we confirmed by ELISA that these effects were not due to different protein expression levels (n=6, p=ns). OPNa and OPNc rescued limb function compared to control and OPNb‐treated animals (61.1%±8.2; 76.2%±9.7; p<0.05), as assessed by voluntary running wheel use. Additionally, OPNa and OPNc‐treated animals exhibit significant increases in arteriogenesis and macrophage infiltration in vivo (n=4, p<0.05 vs. GFP, OPNb). Therefore, we further hypothesized that divergent effects of OPN isoforms on arteriogenesis are due to differential effects on macrophage migration, survival, and/or polarization. Although OPN isoforms had no differential effects on macrophage polarization in vitro, OPN isoforms did differentially promote microphage migration in vitro, assessed using a modified Boyden chamber assay. While OPNa and OPNc both increased macrophage migration in vitro, OPNc was a more potent migratory stimulus than OPNa and OPNb in vitro (n=4, p<0.001 vs. no trx, OPNa, OPNb, and MCP‐1), consistent with what was observed in vivo. To determine if OPN isoforms have divergent effects on macrophage survival in vitro, OPN−/− macrophages were serum‐deprived to induce apoptosis and treated with recombinant OPNa, OPNb or OPNc proteins in vitro. Survival was assessed by FACS using FITC‐annexin V and propidium iodide staining. All isoforms significantly increased macrophage survival compared to no treatment, with OPNc increasing survival significantly greater than OPNa and OPNb (n=4, p<0.05 vs. OPNb, p<0.001 vs. no trx, and OPNa). In conclusion, human OPN isoforms exhert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage infiltration in vivo and differential direct effects on Macrophage migration and survival, but not polarization, in vitro. Taken together, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.Support or Funding Information1. T32 Training Grant #T32HL007745, NIH/NHLBI, W. Robert Taylor (PI), 01/03/2016 to present: Research Training in Academic Cardiology (Role: Post‐doctoral trainee) The goal of this training grant is to support for the highly successful postdoctoral training program in cardiovascular research in the Division of Cardiology at Emory University School of Medicine. The training program is structured around a mentor‐based environment supplemented with an extensive array of didactic educational opportunities for multidisciplinary training in basic and clinical vascular biology. Successful trainees will be well‐equipped to initiate a research career in academic cardiology. 2. K99/R00 HL119567, NIH/NHLBI, Alicia N.Lyle (PI), 04/2014–06/2019: The Role of Human Osteopontin Isoforms in Collateral Formation This project uses a novel translational therapeutic strategy to determine how the different human osteopontin (OPN) isoforms influence collateral formation.