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Critical Limb Ischemia Is Associated With Increased Cryptic Collagen Regulatory Site Exposure In Human Skeletal Muscle

Mark J Hobeika, Rachel Edlin, Luis Chiriboga, Nikita Tihonov, Adam Baghban, Bart Muhs, Patrick Lamparello, Mark Adelman, Glenn Jacobowitz, Caron Rockman, Neal Cayne, Thomas Maldonado, Peter Brooks, Paul J Gagne ∙ New York University, New York, NY

Objectives: Critical limb ischemia (CLI) results in gangrene and amputation. In mouse models of CLI, matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) remodeling exposes cryptic collagen regulatory sites (e.g. HU177). HU177 exposure has been associated with successful murine limb reperfusion and new blood vessel growth in models of angiogenesis. Based on these animal studies we hypothesized that ischemic human skeletal muscle would exhibit increased HU177 exposure and increased MMP-2 and -9 levels compared to non-ischemic muscle.
Methods: Intraoperative muscle biopsies were obtained from two groups of patients with CLI: Femoral-Distal Bypass patients (non-ischemic sartorius muscle and ischemic calf muscle), and Below-Knee Amputation (BKA) patients (ischemic calf muscle). Sartorius muscle from patients without limb ischemia undergoing Endovascular Abdominal Aortic Aneurysm Repair (EVAR) served as age and co-morbidity matched controls. Immunohistochemical staining for HU177 was quantified from digital photographs (4 fields/slide). Total MMP-2 and -9 were quantified by ELISA. Results were analyzed by student t-test. P<0.05 considered significant.
Results: HU177 exposure and MMP-2 and -9 levels were markedly increased in ischemic muscle samples (Bypass/calf and BKA) compared to non-ischemic EVAR/control muscle samples (Fig 1-3)(p<0.05). Furthermore, HU177 exposure was significantly increased in the ischemic calf muscle of bypass patients compared to non-ischemic sartorius muscle from the same patients (p=0.0004.) Interestingly, significant increases in HU177 and MMP-2 and -9 were observed in the non-ischemic sartorius muscle of bypass patients compared to the non-ischemic sartorius muscle of EVAR control patients (Fig 1-3), suggesting that patients with distal limb ischemia have altered ECM remodeling in proximal, non-ischemic muscle.
Conclusions: CLI is associated with increased HU177 exposure and MMP-2 and -9 levels in ischemic skeletal muscle. Previous animal studies suggest that these ECM changes are highly favorable for angiogenesis and spontaneous limb reperfusion. Thus, despite adequate ECM remodeling, failure of spontaneous reperfusion in patients with CLI may be attributable to other dysfunctional pathways such as ECM-endothelial cell signaling.