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Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN−/− mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN−/− mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, 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. Osteopontin is a critical mediator of post-ischemic neovascularization. Humans express three primary OPN isoforms: OPNa, OPNb, and OPNc. This study demonstrates that OPN isoforms differentially promote functional post-ischemic arteriogenesis by promoting increased macrophage migration and macrophage accumulation and survival. Future studies will determine the molecular mechanisms underlying these divergent effects.

Background: Coronary and peripheral artery diseases result in vessel occlusion and ischemia, initiating neovascularization to restore blood flow and preserve function. We previously established that osteopontin (OPN), a matricellular 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. Methods and Results: Using a murine model of hindlimb ischemia in OPN -/- mice and 1.5x10 6 lentivirus particles expressing OPNa, OPNb or OPNc delivered IM, we found that OPN isoforms have different effects on functional perfusion recovery in vivo . OPNa increased limb perfusion 30.4%±0.8 and OPNc by 70.9%±6.3, as measured by laser Doppler perfusion imaging (d14; p2 ; 47.2%±6.1; 55.9%±6.7) and large artery (1000 - 2500 μm 2 ; 54.2%±6.1; 76.5%±10.9) ranges in vivo (n=9; p Conclusion: In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage migration and survival. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in obstructive artery disease patients.

Coronary and peripheral artery diseases lead to ischemia, initiating processes that promote neovascularization to restore blood flow and preserve tissue function. We demonstrated previously that osteopontin (OPN), a matricellular 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 assess how human OPN isoforms affect neovascularization, OPN -/- mice underwent hind limb ischemia surgery. At the time of surgery, 1.5x10 6 lentivirus particles expressing human OPNa, OPNb or OPNc were delivered by intramuscular injection. While OPNa improved limb perfusion 30.4%±0.8 in OPN -/- mice, OPNc improved perfusion by 70.9%±6.3 (d14; p

Background: WNK kinase is a serine/threonine kinase that plays an important role in normal blood pressure homeostasis. WNK3 was previously found to enhance the activity of sodium chloride cotransporter (NCC) in Xenopus oocyte. However, the mechanism through which it works remains unclear. Methods: Using overexpression and siRNA knock-down techniques, the effects of WNK3 on NCC in both Cos-7 and mouse distal convoluted cells were analyzed by Western blot. Results: We found that WNK3 significantly increased NCC protein expression in a dose-dependent manner. NCC protein expression in Cos-7 cells was markedly decreased after 2 h treatment with protease inhibitor, cycloheximide (CHX) in the NCC alone group, but was significantly decreased after 8 h treatment of CHX in the WNK3 + NCC group. WNK3 significantly increased NCC protein expression in both NCC alone and WNK3 + NCC groups regardless the overnight treatments of bafilomycin A1, a proton pump inhibitor, suggesting that WNK3-mediated increased NCC expression is not dependent on the lysosomal pathway. We further found that WNK3 group had a quicker NCC recovery than the control group using CHX pulse assay, suggesting that WNK3 increases NCC protein synthesis. WNK3 enhanced NCC protein level while reducing ERK 1/2 phosphorylation. In addition, knock-down of ERK 1/2 expression reversed WNK3-mediated increase of NCC expression. Conclusion: These results suggest that WNK3 enhances NCC protein expression by increasing NCC synthesis via an ERK 1/2-dependent signaling pathway.