VEGF induces stress fiber formation in fibroblasts isolated from dystrophic muscle

Abstract

Treatment with vascular endothelial growth factor (VEGF) to reduce ischemia and enhance both endogenous muscle repair and regenerative cell therapy in Duchenne muscular dystrophy (DMD) has been widely proposed in recent years. However, the interaction between angiogenesis and fibrosis, a hallmark feature of DMD, remains unclear. To date, it has not been determined whether VEGF exerts a pro-fibrotic effect on DMD-derived fibroblasts, which may contribute to further disease progression. Thus, the purpose of this study was to investigate the effect of exogenous VEGF on fibroblast cultures established from a murine model of DMD. Primary fibroblast cultures were established from gastrocnemius and diaphragm muscles of 10 week-old mdx/utrn+/- mice. Quantitative polymerase chain reaction (qPCR) was employed to assess changes in transcript expression of alpha-smooth muscle actin (Acta2), type-1 collagen (Col1a1), connective tissue growth factor (Ctgf/ccn2) and fibronectin (Fn1). Immunofluorescence and Western blot analysis was further employed to visualize changes in protein expression of alpha-smooth muscle actin (α-SMA), CTGF/CCN2 and fibronectin. mRNA levels of Col1a1, Ctgf/ccn2, and FN did not increase following treatment with VEGF in fibroblasts derived from either diaphragm or gastrocnemius muscles. Acta2 expression increased significantly in diaphragm-derived fibroblasts following treatment with VEGF. Morphological assessment revealed increased stress fiber formation in VEGF-treated fibroblasts compared to the untreated control fibroblasts. The findings from this study suggest that further investigation into the effect of VEGF on fibroblast function is required prior to the utilization of the growth factor as a treatment for DMD.

Hypoxic Signaling in Skeletal Muscle Maintenance and Regeneration: A Systematic Review

“…In skeletal muscle, both hypoxia-inducible transcription factors have a pro-angiogenetic function by inducing VEGF expression and the development of capillaries that express CD31 in vivo (Niemi et al, 2014 ). VEGF is not only essential for angiogenesis but also for skeletal muscle regeneration (Germani et al, 2003 ), even though it could have pro-fibrotic effects by inducing stress fiber formation in muscle-specific fibroblasts of dystrophic muscles (Gutpell and Hoffman, 2015 ).…”

Targeting angiogenesis in Duchenne muscular dystrophy

“…Gutpell et al [ 107 ] have investigated the effect of VEGF administration on fibrosis markers using heterozygous mdx /utrn +/− mice lacking dystrophin and partially utrophin. Interestingly, the pro-fibrotic response of skeletal muscle fibroblasts isolated from 10-week-old mdx /utrn +/− mice to VEGF administration, with increased Acta2 expression encoding alpha-smooth muscle actin and enhanced stress-fiber formation, in comparison to untreated control fibroblasts was detected [ 108 ]. Moreover, also Gutpell et al [ 86 ] in another study have shown that although expression of VEGF is significantly decreased in the diaphragm (but not in GM) of mdx /utrn +/− mice, intramuscular, short-term delivery of low dose VEGF does not improve blood flow, as evidenced by applying dynamic contrast-enhanced computed tomography (DCE-CT).…”

HIF-hypoxia signaling in skeletal muscle physiology and fibrosis

“…Furthermore, although VEGF plays a pro-angiogenic role as a downstream effector of the HIF-mediated hypoxic signaling, it could have pro-fibrotic effects. VEGF induces stress fiber formation in fibroblasts isolated from dystrophic muscle, a marker of differentiation to myofibroblasts, which are the main producers of ECM (Gutpell and Hoffman 2015). Therefore, sustained/chronic hypoxic signaling activation in dystrophic muscle due to vasculature damage could be one of the mechanisms contributing to fibroblast-overactivation, as a result of VEGF overexpression, which finally elicits fibrosis.…”