Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo.

Therapeutic angiogenesis using vascular endothelial growth factor (VEGF) is considered a promising new therapy for patients with arterial obstructive disease. Clinical improvements observed consist of improved muscle function and regression of rest pain or angina. However, direct evidence for improved vascularization, as evaluated by angiography, is weak. In this study, we report an angiogenesis-independent effect of VEGF on ischemic skeletal muscle, ie, upregulation of myoglobin after VEGF treatment. Mice received intramuscular injection with adenoviral VEGF-A or either adenoviral LacZ or PBS as control, followed by surgical induction of acute hindlimb ischemia at day 3. At day 6, capillary density was increased in calf muscle of Ad.VEGF-treated versus control mice (P<0.01). However, angiographic score of collateral arteries was unchanged between Ad.VEGF-treated and control mice. More interestingly, an increase in myoglobin was observed in Ad.VEGF-treated mice. Active myoglobin was 1.5-fold increased in calf muscle of Ad.VEGF-treated mice (P< or =0.01). In addition, the number of myoglobin-stained myofibers was 2.6-fold increased in Ad.VEGF-treated mice (P=0.001). Furthermore, in ischemic muscle of 15 limb amputation patients, VEGF and myoglobin were coexpressed. Finally, in cultured C2C12 myotubes treated with rhVEGF, myoglobin mRNA was 2.8-fold raised as compared with PBS-treated cells (P=0.02). This effect could be blocked with the VEGF receptor tyrosine kinase inhibitor SU5416. In conclusion, we show that VEGF upregulates myoglobin in ischemic muscle both in vitro and in vivo. Increased myoglobin expression in VEGF-treated muscle implies an improved muscle oxygenation, which may, at least partly, explain observed clinical improvements in VEGF-treated patients, in the absence of improved vascularization.

Adenoviral activin a expression prevents intimal hyperplasia in human and murine blood vessels by maintaining the contractile smooth muscle cell phenotype.

Activin A alters the characteristics of human arterial smooth muscle cells (SMCs) toward a contractile, quiescent phenotype. We hypothesize that activin A may prevent SMC-rich neointimal hyperplasia. Here, we study the effect of adenovirus-mediated expression of activin A on neointima formation in vitro and in vivo. Human saphenous vein organ cultures, in which a neointima is formed spontaneously, were infected either with activin A- or lacZ-adenovirus. Activin A-overexpression reduces neointima formation by 78%, whereas no significant reduction was observed after control infection. In addition, the effect of activin A on neointima formation was assessed in vivo in mice with cuffed femoral arteries. In activin A adenovirus-infected mice (IV injection), neointimal hyperplasia is reduced by 77% compared with the SMC-rich neointima in mock-infected or in noninfected mice. Cultured human saphenous vein SMCs and murine aorta SMCs were incubated with activin A and an increased expression of SM22alpha and SM alpha-actin mRNA, and SM alpha-actin protein was demonstrated. Laser-capture microdissection on sections of cuffed murine arteries and subsequent real-time RT-PCR established in vivo induction of SM alpha-actin mRNA in the media of activin A-treated mice. In summary, activin A inhibits neointima formation in vitro and in vivo by preventing SMC dedifferentiation.