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

Purpose Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts which have become myofibroblasts (α-smooth muscle actin positive cells) and migrate to the neointima. There is increased expression of hypoxia inducible factor-1 alpha (HIF-1α in venous neointimal hyperplasia formation in experimental animal model and clinical samples. We hypothesized that under hypoxic stimulus (HIF-1α fibroblasts will convert to myofibroblasts through a matrix metalloproteinase-2 (MMP-2) mediated pathway. Materials and methods Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1α, α-smooth muscle actin, MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for α-smooth muscle actin, collagen, and fibronectin was performed. Results At all time points, there was significantly increased expression of HIF-1α in the hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was significantly increased expression α-smooth muscle actin at all time points which peaked by 48 hours in hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) by 48-72 hours by hypoxic fibroblasts (P<0.05). By 72 hours, there was significant increase in TIMP-2 expression (P<0.05). Immunohistochemical analysis demonstrated increased expression for α-smooth muscle actin, collagen, and fibronectin as the length of hypoxia increased. Conclusions Under hypoxia, fibroblasts will convert to myofibroblasts through a MMP-2 mediated pathway which may provide insight into the mechanism of venous neointimal hyperplasia.

Matrix metalloproteinases and their inhibitors in pulmonary hypertension

“…In particularly, a significant increase in active MMP-2, TIMP-1 and TIMP-2 production was also observed under hypoxic stimulus when compared to normoxic fibroblasts [44]. It has been shown that the reactive oxygen species (ROS)-induced oxidative stress increases MMP activity and potentially modulates fibroblast proliferation and collagen synthesis [49].…”

“…Different cell types that constitute the cellular component of the vasculature, including endothelium, SMCs, fibroblasts and infiltrating innate immune cells which infiltrate the vessel wall, are described as the major cellular sources of MMPs in adult vasculature [41][42][43][44] (table S1). The diverse functions of MMPs in vasculature mainly depend on its expression and activity.…”

“…With regard to fibroblasts, the homeostatic relationship between resident fibroblasts and the collagen matrix keeps them in a quiescent, undifferentiated state [3]. However, normal fibroblasts subjected to hypoxia display hypoxia-induced phenotypic switching to myofibroblasts, through the MMP-2/TIMP mediated pathway [44]. Specifically, PDGF, TGF-b1, tenascin-C (TN-C), fibronectin and ET-1 exhibit mitogenic activity and induce the myofibroblast phenotype both in vitro and in vivo [85], probably by regulating the MMP/TIMP balance.…”

Cardioprotection via adaptation to hypoxia: expanding the timeline and targets?

“…For example, there is evidence that cultured murine fibroblasts exposed to hypoxia can change their phenotype to myofibroblasts [13]. Although such cells produce collagen, it is the contractile ability of a-smooth muscle actin which may be of particular relevance as myofibroblasts are known to produce scar contracture [5].…”

“…Hypoxia has been shown to stimulate collagen synthesis by cultured human vascular-derived myofibroblasts [24]. In contrast, the effects on matrix metalloproteinases have been mixed: several studies reported increases in MMP-2 synthesis in cultured rodent fibroblasts [1,13], whereas exposure of cultured human myofibroblasts to chronic hypoxia attenuated MMP-2 activation [20]. The overall effect of hypoxia on collagen deposition is, however, best assessed in the intact heart.…”