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Hypoxia Induces Upregulation Of MMP-9 In Human Skeletal Muscle Myoblasts

Mikel Sadek, Sofia Bajwa, Lorenzo Zanotti, Xiaolu Li, Mark Hobeika, Peter Brooks, Paul Gagne ∙ New York University Medical Center, New York, NY

Introduction: Limb ischemia leads to gangrene and limb loss. In a mouse ischemic limb model we have shown that matrix metalloproteinase-9 (MMP-9) mediated extracellular matrix (ECM) remodeling exposes cryptic collagen regulatory sites associated with angiogenesis and reperfusion. MMP-9 has been attributed to infiltrating inflammatory cells (e.g. neutrophils, macrophages). It has been shown that ischemic skeletal muscle supports angiogenesis in part through VEGF production. Realizing MMP-9’s importance in ECM remodeling and angiogenesis, we hypothesized that ischemic skeletal muscle would also produce MMP-9. Consequently, we studied whether cultured human skeletal muscle myoblasts (HSMMs) exposed to hypoxic conditions would produce MMP-9. Methods: Cell lines of passage 5 HSMMs were cultured under normoxic or hypoxic (1%pO2) conditions for 48h. RNA and protein were harvested from the cell extracts. The RNA was analyzed with quantitative real-time PCR for MMP-9 expression. RNA polymerase II was the normalization control. Western blot analysis was performed using anti-MMP-9 antibody, with appropriate positive and negative controls. Actin served as the loading control. Results were analyzed by densitometry using GelExpert 3.5. All experiments were performed in triplicate. Immunohistochemical staining for MMP-9 was conducted on HSMMs in culture. All results were analyzed by student t-test, where appropriate. P < 0.05 is significant. Results: HSMMs produce MMP-9 based on PCR and Western Blot analysis. Futhermore, hypoxia upregulates MMP-9 mRNA expression at 48h in HSMMs with a fold change of 3.29 ± 0.72, p < 0.05 (Fig. 1). Hypoxia also upregulates MMP-9 protein levels at 48h with a fold change of 1.68 ± 0.27, p < 0.05 (Figs. 1 and 2). Immunohistochemistry demonstrates intracellular MMP-9 staining in hypoxic HSMMs (Fig. 3). Conclusion: In response to hypoxia, HSMMs exhibit increased MMP-9 transcription and translation compared to normoxic cells. Furthermore, MMP-9 is demonstrable in the intracellular space. Thus, skeletal muscle myoblasts are capable of producing the MMP-9 required for the ECM remodeling and cryptic collagen site exposure associated with subsequent angiogenesis. This represents a novel finding by which skeletal muscle tissue may promote angiogenesis and ischemic limb reperfusion.