H. Albadawi1,2, L. M. Crowley1, M. W. Koulopoulos1, H. Yoo1, M. T. Watkins1,2 2Harvard School Of Medicine,Brookline, MA, USA 1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA
Introduction: Claudication, a form of demand ischemia (DI) in patients with peripheral arterial disease (PAD) has been associated with altered skeletal muscle fiber morphology and fatty degeneration. Metabolic syndrome exacerbates exercise intolerance in PAD due to impaired circulation and metabolism. The diet-induced obese (DIO) mouse provides a robust model of humans at risk for PAD and diabetes. This study evaluated the expression of growth factors, cytokine signaling pathways, and regulators of adipocyte differentiation in DIO mice in the setting of DI.
Methods: DIO C57BL6 mice underwent unilateral femoral artery ligation followed by two weeks recovery. Mice were then randomized into two groups. The first group (n=8) was subjected to 1hr of daily treadmill exercise (12m/min, 10° incline) for 4 weeks to induce DI, and a parallel group (n=7) remained sedentary (SI) for 4 weeks. Hindlimb muscles were examined histologically for capillary density (CD31 immunohistochemistry) and muscle cross sectional area (CSA). Hindlimb skeletal muscles were analyzed for the levels of the pro-angiogenic cytokines G-CSF and VEGF using multiplex assay while the phosphorylation of the signaling transduction molecules, pSTAT3 and pERK1/2 and the brown adipocyte marker uncoupling protein-1 (UCP-1) levels were measured by western blotting. Real-time qPCR analysis was used to assess gene expression of the energy metabolism transcription factor PGC1-α. Statistical analysis was performed with student t-test.
Results: (see table) Exercise significantly enhanced capillary density (p=0.0086) and markedly lowered CSA (p=0.0012) in DI muscle compared to SI. These findings were paralleled by a significant increase in G-CSF (p= 0.0006) and pSTAT3 (p=0.0114) protein levels as well as enhanced PGC1-α (p= 0.014) gene expression. There was no difference in the pERK1/2 (p=0.23) or VEGF (p=0.2) protein levels between the two groups. Furthermore, DI muscle had significantly diminished UCP-1 protein levels compared to SI muscle.
Conclusions: These data suggest that DI enhances capillary density but lowers muscle CSA. Since VEGF and ERK1/2 levels were not different between DI and SI muscles, the enhanced capillary density may be primarily dependent on G-CSF and STAT3 signaling pathway after 4 weeks of DI. Exercise also appears to modulate adipogenic differentiation and muscle energy metabolism, evident by the decrease in UCP-1 protein and increase in PGC1-α gene expression. Ongoing regular exercise in patients with PAD is important to augment microvascular perfusion and decrease fatty degeneration, thereby enhancing muscle adaptation to ameliorate claudication and improve limb function.
