Macrohage depletion using clodronate liposomes delays exercise-induced collateral artery growth.3

Abstract

Background: Collateral artery growth (arteriogenesis) depends on endothelial function and inflammatory cytokines. A vasculoprotective effect of physical exercise is widely accepted. The exact mechanisms of exercise-induced collateral artery growth are incompletely understood. Methods and results: C57Bl6 mice were treated with 0.5 mg of liposomal clodronate i.v. every four days during a three week voluntary running period before unilateral femoral artery ligation. Running distance dropped from 5.5±1.0km (placebo exercise, and 4.6±0.6km (clodronate exercise, clod-ex) before ligation to 0.9±0.4 km (plac-ex) and 0.6±0.1 km (clod-ex) afterwards. Clodronate reduced circulating monocytes to 42.3% of control (liposomes without clodronate) 24h after injection. In the reticuloendothelial system (spleen), macrophage content fell to 4.1±0.2% of control. Hindlimb perfusion was assessed by laser Doppler flowmetry (LDF) before and after ligation, as well as 3 and 7 days thereafter. Directly after ligation, perfusion (ratio ligated/non-ligated hindlimb) fell to 6±1.0% in placebo sedentary (plac-sed) and to 11±1.0% in placebo exercising mice (plac-ex). Perfusion restoration was faster and more pronounced in exercising mice (increase to 81±4% in plac-ex compared to 49±11% on day 3 (p=0.020) and to 77±9% in plac-ex compared to 45±8% in plac-sed (p=0.030) 7 days after ligation). In contrast, in clod-ex, perfusion recovery was only 57±10% after 3 (p=0.056 compared to plac-ex) and 59±13% after 7 days, which was comparable to the sedentary group (clod-sed, 46±6% after 3 and 48±13% after 7 days). Immunohistochemistry was used to quantify collateral arteries and surrounding perivascular macrophages 7 days after ligation. Similar to perfusion ratio, average smooth muscle cell area of collateral arteries increased with exercise, to a significantly lesser extent so after clodronate treatment. Perivascular macrophages increased in plac-ex with a significant decrease in clodronate treated mice. Inducible NO-synthase (iNOS) mRNA in peripheral blood mononuclear cells increased in plac-ex compared to plac-sed, displaying a systemic response to exercise. Conclusion: Exercise prior to femoral artery ligation increases the speed of perfusion restoration and increases mononuclear iNOS expression. The perfusion increase is delayed by macrophage depletion. These data suggest that, next to a beneficial effect on vascular endothelium, physical exercise relies on monocytes/macrophages as the other key player in arteriogenesis.