To address our goals, we used: (a) micro-CT analysis of long bones and skulls to assess phenotypic differences, (b) histomorphometric analyses to assess parameters of endochondral bone formation, and (c) ex vivo studies to assess bone cell function. occurs. Connective tissue growth factor CTGF/CCN2, which is overexpressed in muscular dystrophies, correlates with the degree and severity of fibrosis in many diseases. However, the role of CTGF/CCN2 in skeletal muscle fibrosis characteristic of Duchenne Muscular Dystrophy (DMD) remains unknown. To test the hypothesis that CTGF might not only contribute to conversion of already damaged muscle into scar tissue, but that it could by itself also directly contribute to skeletal muscle deterioration, we used two experimental approaches: 1) Determine the effect of loss of CTGF function in the progression of fibrosis in mdx mice, a murine model of DMD, in a genetically CTGF-reduced model (mdx-CTGF+/mice) and a decrease of CTGF activity model (blocking antibodies). 2) Evaluate the effect of gain of CTGF levels through overexpression in the tibialis anterior muscle of wild-type mice using an adenovirus containing the CTGF mouse sequence (Ad-mCTGF). CTGF protein levels were significantly elevated in the muscles of dystrophic mdx mice compared to wild-type mice. CTGF levels were significantly reduced in mdx-CTGF+/mice and this correlated with a significant decrease of fibrosis, as determined by fibronectin and collagen levels. We also detected less muscle damage in mdx-CTGF+/mice compared to mdx mice, as evidenced by more normal muscle tissue structure and reduced levels of myogenic precursors such as myogenin and embryonic myosin. Impressively, the decrease of CTGF in mdx-CTGF+/mice caused GSK3368715 an improvement of the specific GLI1 isometric contractile force despite the absence of dystrophin. FG-3019 is an antibody inhibitor of CTGF that GSK3368715 is currently in clinical trials for treatment of pulmonary and liver fibrosis and pancreatic cancer. Administration of FG-3019 to mdx mice for 1 month produced a similar effect as genetic depletion, resulting in decreased fibrosis and increased isometric contractile force. In contrast, CTGF overexpression induced extensive skeletal muscle damage, which was followed by a massive regeneration of the damaged muscle, as indicated by increased embryonic myosin and fibers with centrally located nuclei. It also induced strong fibrosis with increased levels of fibronectin, collagen, decorin and -smooth muscle actin (-SMA). Moreover, CTGF overexpression caused a decrease of the specific isometric contractile force. Strikingly, when CTGF overexpression stopped, the entire phenotype proved to be reversible, in parallel with normalization of CTGF levels. Altogether these results provide strong evidence for a critical role of CTGF in skeletal muscle fibrosis in vivo. CTGF not only acts downstream of muscle injury, but contributes directly to deterioration of skeletal muscle phenotype and function. These observations underscore the importance of CTGF in the patho-physiology of muscular dystrophies and suggest that targeting CTGF might have significant potential in development of novel therapies for Duchenne muscular dystrophy and related diseases. (Financial support provided by CARE PFB 12/2007, MDA 89419 and Fondecyt 11080212, Conicyt AT-24100047. Material support (antibodies) provided by FibroGen, Inc.) NORMALIZATION OF ANGIOGENESIS AND VASCULOGENESIS BY CCN1: IMPLICATIONS IN OCCULAR NEOVASCULAR DISEASES Adeel Hasan1, Nataliya Pokeza1, Douglas Lazzaro2, Lynn Shaw3, Maria B. Grant3andBrahim Chaqour1,2 Department of Cell Biology1, Department of Ophthalmology2, SUNY Downstate Medical Center, Brooklyn, NY;3Dept of Pharmacology and Therapeutics, University of Florida, Gainesville, Fl The formation and maturation of blood vessels by angiogenesis (sprouting from preexisting blood vessels) or vasculogenesis GSK3368715 (de novo formation of vessels from angioblasts or stem cells) is orchestrated by a constellation of physical and chemical factors whose spacio-temporal patterns of expression and concentration are tightly regulated. Functional redundancy among some angiogenic and vasculogenic factors is generally assumed but dysregulation of some key factors may result in either vascular regression or the formation of an abnormal vasculature, a hallmark feature of ischemic retinopathies including retinopathy of prematurity. ROP is characterized by a vaso-obliteration phase caused by a disrupted oxygen environment in the retina, and a subsequent ischemia-induced neovessel formation phase characterized by the growth of abnormal leaky blood vessels which rupture and cause blindness. At present, the contribution of local and systemic regulation of normal and pathologic vessel growth is unclear. The CCN1 protein also known as cysteine-rich protein 61 is a dynamically expressed and locally produced protein required for proper angiogenesis and vasculogenesis during development. Interestingly, the expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia..
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