This project aims to clarify some aspects that would help to define a common molecular profile of vascular fibrosis, that in the penis causes Peyronie's disease (PD) and aging-related vasculogenic erectile dysfunction, and that in the arteries leads to aging-related arteriosclerosis and arterial stiffness, in order to detect cellular and molecular targets of antifibrotic therapy. We will investigate: a) the cell specificity of the alterations of gene expression occurring in penile and vascular fibrosis, clarifying the role of smooth muscle cells (SMC) and myofibroblasts in these processes; b) whether the SMC in the penile trabecular tissue are responsible for excessive collagen synthesis; c) the ontogenic relationship of myofibroblasts and SMC, and d) the effect of cell to cell interactions on their differentiation leading to excessive collagen deposition. In Aim 1, we will determine gene expression patterns in SMC and myofibroblasts in penile and vascular fibrosis. Male rats in groups: 1) "Young"; 2) "Aged"; 3) "PD-like plaque"; and 4) "Normal tunica", will receive in the corpora cavernosa or tunica albuginea an adenoviral collagen I promoter-green fluorescent protein (ColP-gfp) construct. Gfp be detected by dual fluorescence at 6 days in: a) SMC, and possibly myofibroblasts in the corpora and PDA, and: b) fibroblasts and myofibroblasts in the PDA adventitia or the tunica. Some of these cells will be dissected by laser capture microdissection (LCM). In Aim 2 we will determine the in vivo differentiation of penile fibroblasts and stem cells into SMC and myofibroblasts, and of myofibroblasts into SMC, active in collagen synthesis. Penile cultures from the rat tunica (fibroblasts) and the PD plaque (myofibroblasts) will be used as such, or after Sca1+ selection for stem cells, transfected with the ColP-gfp construct, and labeled with DAPI. Implantation will be for 1 week into: A: corpora cavernosa of old rats, with tunical: 1) fibroblasts; or 2) stem cells; and B: TGF1-induced PD-like plaque in the tunica, with: 3) tunical fibroblasts; 4) PD-myofibroblasts; or 5) tunical stem cells. End-points will be: A) LCM in tissue sections, with immunofluorescence for gfp, fibroblasts (vimentin), myofibroblasts/SMC (ASMA) and SMC (smoothelin), followed by DNA microarrays for gene expression profiles; B) fibrosis by immunohistochemistry/QIA (quantitative image analysis), RT/real time PCR, and western blots, and assays for hydroxyproline; B) for implanted DAPI+ cells: by QIA fluorescence/dual confocal microscopy for cell markers. Tissue fibrosis is an excessive deposition of collagen fibers, often accompanied by the loss of cellular mass, that occurs in most organs with aging, diabetes, injury, toxic insults, and other conditions, and that severely impairs the function of those organs and is a key factor in various diseases. Using rat animal models and cell cultures, we aim to clarify aspects that would help to define whether there is a common molecular and cellular profile of fibrosis, that in penile tissues causes Peyronie's disease (PD) and erectile dysfunction, and that in the arterial wall leads to arteriosclerosis. We will focus on studying the role of cells named fibroblasts, smooth muscle cells, and myofibroblasts, as well as adult stem cells, in these processes, and whether some of these cells can interconvert into each other. We expect that our studies would lead to the detection of novel targets of antifibrotic therapy for penile and vascular diseases of considerable public health impact.

R21DK070003

2007-08-07

2010-07-31