CELL-SELECTIVE EXPRESSION OF FIBROTIC GENE PATHWAYS

描述（由申请人提供）：该项目旨在阐明有助于定义血管纤维化的常见分子特征的某些方面，即在阴茎中导致佩罗尼病（PD）和衰老相关的血管性勃起功能障碍，以及在动脉中导致衰老相关的动脉硬化和动脉硬化，以检测抗纤维化治疗的细胞和分子靶点。我们将调查：a）阴茎和血管纤维化中发生的基因表达改变的细胞特异性，阐明平滑肌细胞（SMC）和肌成纤维细胞在这些过程中的作用; B）阴茎小梁组织中的SMC是否负责过度的胶原合成; c）肌成纤维细胞和SMC的个体发生关系，和d）细胞与细胞的相互作用对其分化的影响，导致过度的胶原沉积。在目的1中，我们将确定阴茎和血管纤维化中SMC和肌成纤维细胞的基因表达模式。各组雄性大鼠：1）“年轻”; 2）“老化”; 3）“PD样斑块”;和4）“正常图尼卡”，将在海绵体或图尼卡白蛋白中接受腺病毒胶原I启动子-绿色荧光蛋白（ColP-gfp）构建体。在第6天，通过双重荧光检测GFP：a）体和PDA中的SMC和可能的肌成纤维细胞，以及：B）PDA外膜或图尼卡中的成纤维细胞和肌成纤维细胞。其中一些细胞将通过激光捕获显微切割（LCM）进行切割。在目标2中，我们将确定阴茎成纤维细胞和干细胞在体内分化为SMC和肌成纤维细胞，以及肌成纤维细胞分化为SMC，在胶原合成中具有活性。来自大鼠图尼卡（成纤维细胞）和PD斑块（肌成纤维细胞）的阴茎培养物将原样使用，或在Sca 1+选择干细胞后使用，用ColP-gfp构建体转染，并用DAPI标记。植入1周：A：老年大鼠的海绵体，具有内膜：1）成纤维细胞;或2）干细胞;和B：图尼卡中TGF β 1诱导的PD样斑块，具有：3）内膜成纤维细胞; 4）PD-肌成纤维细胞;或5）内膜干细胞。终点为：A）组织切片中的LCM，用免疫荧光检测gfp、成纤维细胞（波形蛋白）、肌成纤维细胞/SMC（ASMA）和SMC（平滑蛋白），然后用DNA微阵列检测基因表达谱; B）通过免疫组织化学/QIA（定量图像分析）、RT/真实的PCR和蛋白质印迹以及羟脯氨酸测定法检测纤维化; B）植入的DAPI+细胞：通过QIA荧光/双共聚焦显微镜检测细胞标志物。组织纤维化是胶原纤维的过度沉积，通常伴随着细胞质量的损失，其发生在具有老化、糖尿病、损伤、毒性损伤和其他病症的大多数器官中，并且严重损害那些器官的功能，并且是各种疾病的关键因素。使用大鼠动物模型和细胞培养，我们的目标是澄清方面，这将有助于确定是否有一个共同的分子和细胞的纤维化，在阴茎组织导致佩罗尼氏病（PD）和勃起功能障碍，并在动脉壁导致动脉硬化。我们将专注于研究名为成纤维细胞，平滑肌细胞和肌成纤维细胞以及成体干细胞的细胞在这些过程中的作用，以及其中一些细胞是否可以相互转化。我们希望我们的研究将导致检测到抗纤维化治疗的阴茎和血管疾病的相当大的公共卫生影响的新目标。

DESCRIPTION (provided by applicant): 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: TGF¿1-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.