Hypoxia-induced Phenotypic Switch of Fibroblasts to Myofibroblasts through a Matrix Metalloproteinase 2/Tissue Inhibitor of Metalloproteinase–mediated Pathway: Implications for Venous Neointimal Hyperplasia in Hemodialysis Access

Abstract

Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts that have become myofibroblasts (ie, alpha-smooth muscle actin [SMA]-positive cells) and migrate to the neointima. There is increased expression of hypoxia-inducible factor (HIF)-1alpha in venous neointimal hyperplasia formation in experimental animal models and clinical samples. It was hypothesized that, under hypoxic stimulus (ie, HIF-1alpha), fibroblasts will convert to myofibroblasts through a matrix metalloproteinase (MMP)-2-mediated pathway. Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1alpha, alpha-SMA, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for alpha-SMA, collagen, and fibronectin was performed. At all time points, there was significantly increased expression of HIF-1alpha in the hypoxic fibroblasts compared with normoxic fibroblasts (P < .05). There was significantly increased expression of alpha-SMA at all time points, which peaked by 48 hours in hypoxic fibroblasts compared with normoxic fibroblasts (P < .05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in TIMP-1 by 48-72 hours by hypoxic fibroblasts (P < .05). By 72 hours, there was significant increase in TIMP-2 expression (P < .05). Immunohistochemical analysis demonstrated increased expression of alpha-SMA, collagen, and fibronectin as the duration of hypoxia increased. Under hypoxic conditions, fibroblasts will convert to myofibroblasts through an MMP-2-mediated pathway, which may provide insight into the mechanism of venous neointimal hyperplasia.