CELL-SELECTIVE EXPRESSION OF FIBROTIC GENE PATHWAYS

描述（由申请人提供）：本项目旨在阐明一些方面，这些方面将有助于定义血管纤维化的共同分子特征，即阴茎中的血管纤维化导致佩洛尼氏病（PD）和衰老相关的血管源性勃起功能障碍，动脉中的血管纤维化导致衰老相关的动脉硬化和动脉僵硬，以检测抗纤维化治疗的细胞和分子靶点。我们将研究：a)阴茎和血管纤维化中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)阴茎小梁组织内的SMC是否负责过多的胶原合成；c)肌成纤维细胞与SMC的成体关系，d)细胞间相互作用对其分化导致过度胶原沉积的影响。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)“岁”;3)“pd样斑块”；4)“正常膜”，将在海绵体或白膜中接受腺病毒I型胶原启动子绿色荧光蛋白（ColP-gfp）的构建。第6天用双荧光法检测Gfp: a) SMC，体表和PDA中的肌成纤维细胞，b) PDA外膜或被膜中的成纤维细胞和肌成纤维细胞。其中一些细胞将被激光捕获显微解剖（LCM）解剖。在Aim 2中，我们将确定阴茎成纤维细胞和干细胞向SMC和肌成纤维细胞的体内分化，以及肌成纤维细胞向参与胶原合成的SMC的体内分化。从大鼠被膜（成纤维细胞）和PD斑块（肌成纤维细胞）中培养的阴茎将被使用，或者在Sca1+选择干细胞后，用ColP-gfp构建体转染，并用DAPI标记。1周植入：A：老龄大鼠海绵体，囊膜：1)成纤维细胞；2)干细胞；B: TGF¿1诱导的膜内pd样斑块，其中：3)膜成纤维细胞；4) PD-myofibroblasts;或者是囊干细胞。终点将是：A)组织切片的LCM，用免疫荧光法检测gfp、成纤维细胞（vimentin）、肌成纤维细胞/SMC （ASMA）和SMC (smoothelin)，然后用DNA微阵列检测基因表达谱；B)免疫组织化学/定量图像分析（QIA）、RT/real time PCR和western blots检测纤维化，并检测羟脯氨酸；B)对于植入的DAPI+细胞：采用QIA荧光/双共聚焦显微镜进行细胞标记。组织纤维化是指胶原纤维的过度沉积，通常伴随着细胞质量的损失，发生在大多数器官中，包括衰老、糖尿病、损伤、毒性损伤和其他情况，并严重损害这些器官的功能，是各种疾病的关键因素。利用大鼠动物模型和细胞培养，我们的目标是澄清一些方面，这些方面将有助于确定是否存在一种共同的纤维化分子和细胞特征，即在阴茎组织中导致Peyronie病（PD）和勃起功能障碍，以及在动脉壁中导致动脉硬化。我们将重点研究成纤维细胞、平滑肌细胞和肌成纤维细胞以及成体干细胞在这些过程中的作用，以及这些细胞中的一些细胞是否可以相互转化。我们期望我们的研究将导致发现对公共卫生有重大影响的阴茎和血管疾病的抗纤维化治疗的新靶点。

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.