Gene therapy holds great promise for curing Duchenne muscular dystrophy (DMD), the most common fatal inherited childhood muscle disease. Success of DMD gene therapy depends upon functional improvement in both skeletal and cardiac muscle. Numerous gene transfer studies have been performed to correct skeletal muscle pathology, yet little is known about cardiomyopathy gene therapy. Since complete transduction of the entire heart is an impractical goal, it becomes critical to determine the minimal level of correction needed for successful DMD cardiomyopathy gene therapy. To address this question, we generated heterozygous mice that persistently expressed the full-length dystrophin gene in 50% of the cardiomyocytes of mdx mice, a model for DMD. We questioned whether dystrophin expression in half of the heart cells was sufficient to prevent stress-induced cardiomyopathy. Heart function of mdx mouse is normal in the absence of external stress. To determine the therapeutic effect, we challenged 3-month-old mice with beta-isoproterenol. Cardiomyocyte sarcolemma integrity was significantly impaired in mdx but not in heterozygous and C57Bl/10 mice. Importantly, in vivo closed-chest hemodynamic assays revealed normal left ventricular function in beta-isoproterenol-stimulated heterozygous mice. Since the expression profile in the heterozygous mice mimicked viral transduction, we conclude that gene therapy correction in 50% of the heart cells may be sufficient to treat cardiomyopathy in mdx mice. This finding may also apply to the gene therapy of other inherited cardiomyopathies.

Objective-We examined the impact of C-reactive protein (CRP) on vascular smooth muscle cell (VSMC) expression of tissue factor (TF) and TF pathway inhibitor (TFPI

Objective Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 down-regulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin (VN)-dependent cooperative binding interaction between VEGFR-2 and αVβ3. Approach and Results We studied PAI-1's effects on VEGF signaling in human umbilical vein endothelial cells (HUVECs). PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in HUVECs grown on VN, but not on fibronectin or collagen. PAI-1 inhibited binding of VEGFR-2 to β3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to VN and another requiring binding to very-low-density-lipoprotein receptor (VLDLR). PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. Conclusions PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a VN-dependent, pro-angiogenic binding interaction involving αVβ3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, VN, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.