Epithelial Angiogenic Signaling in Biliary Pathophysiology and in Polycystic Dise

描述（由申请人提供）：我们研究的长期目标是了解胆管上皮在损伤后肝脏修复和再生中的作用。遗传性胆管病与确定的遗传缺陷作为模型疾病，以阐明基本的病理生理机制。先前资助的提案重点关注与成人显性多囊肾病（PLD-ADPKD）相关的多囊肝病，作为血管生成信号在胆道疾病中作用的范例。我们发现了可能与其他先天性和获得性肝病发病机制相关的机制。事实上，我们利用多囊蛋白诱导缺陷小鼠模型发现：1）囊上皮产生VEGF并表达其同源受体VEGFR 2; 2）VEGF介导的VEGFR 2刺激导致囊性上皮细胞的ERK 1/2依赖性增殖增加，3）PC 2缺陷的囊性胆管细胞，改变的细胞Ca 2+稳态和PKA/Ras/Raf/ERK信号传导中cAMP依赖性的增加导致mTOR/HIF-1 β介导的VEGF产生刺激，4）响应于能够耗尽ER Ca 2+储存的刺激，PC 2参与钙池操纵的钙离子内流（SOCE）; 5）如果PC 2有缺陷，则激活替代途径（钙池操纵的cAMP产生- SOcAMP），导致cAMP的不适当的过量产生; 6）VEGF/VEGFR 2通过对囊周血管细胞的旁分泌作用和对囊性上皮增殖的自分泌刺激在囊肿生长和扩张中发挥关键作用。这些发现是这一新提议的基础，其主要假设是在PLD-ADPKD中鉴定的将PC 2与VEGF分泌和VEGFR 2表达联系起来的机制在胆道病理生理学中具有普遍相关性。我们将通过三个具体的目标来解决这个假设：1）更好地理解PC 2功能库操作的Ca 2+内流和cAMP的不适当产生之间的相互作用，2）研究WT胆管细胞中的PC 2表达是否可以被细胞应激物调节，从而再现在PC 2缺陷细胞中观察到的变化; 3）研究VEGFR 2在肝损伤后胆管上皮细胞中表达的机制，阐明VEGF在肝损伤后胆管上皮细胞分支形态发生中的作用。这些研究将提出新的观点，即PC 2在胆管细胞对获得性胆管病的胆管损伤的反应的调节中起关键作用，并且反应性胆管细胞分泌的VEGF是肝脏修复的主要因素。此外，我们的研究将增加对上皮细胞中VEGF/VEGFR 2信号传导的理解，并将解决先天性和获得性胆管病的基本机制。了解胆管疾病的病理生理是保护肝功能和延长患者生存期的基本步骤

DESCRIPTION (provided by applicant): The long-term goal of our studies is to understand the role of biliary epithelium in the repair and regeneration of the liver after damage. Inherited cholangiopathies with identified genetic defects serve as model diseases to elucidate the fundamental pathophysiological mechanisms. The previously funded proposal focused on polycystic liver disease associated to Adult Dominant Polycystic Kidney Disease (PLD-ADPKD) as a paradigm for the role of angiogenic signaling in biliary diseases. We uncovered mechanisms that may be relevant for the pathogenesis of other congenital and acquired liver diseases. In fact, using mice models with inducible defects of polycystins, we found that: 1) the cystic epithelium produces VEGF and expresses its cognate receptor VEGFR2; 2) VEGF-mediated stimulation of VEGFR2 results in increased ERK1/2-dependent proliferation of the cystic epithelium, 3) PC2-defective cystic cholangiocytes, altered cellular Ca2+ homeostasis and a cAMP- dependent increase in PKA/Ras/Raf/ERK signaling results in mTOR/HIF-1�-mediated stimulation of VEGF production, 4) in response to stimuli able to deplete ER Ca2+ stores, PC2 participates in store-operated Ca2+ entry (SOCE); 5) if PC2 is defective, an alternative pathway is activated (store-operated cAMP production - SOcAMP), leading to an inappropriate overproduction of cAMP; 6) VEGF/VEGFR2 play a key role on cyst growth and expansion through paracrine effects on pericystic vascular cells, and autocrine stimulation of the cystic epithelium proliferation. These findings are the basis of this new proposal which main hypothesis is that the mechanism linking PC2 to VEGF secretion and VEGFR2 expression identified in PLD-ADPKD is of general relevance in biliary pathophysiology. We will address this hypothesis through three specific aims: 1) to better understand the interactions between PC2 function store-operated Ca2+ entry and inappropriate production of cAMP, 2) to study if PC2 expression in WT cholangiocytes can be modulated by cell stressors, thereby reproducing the changes seen in PC2-defective cells; 3) to study the mechanisms leading to VEGFR2 expression in cystic and reactive cholangiocytes, and to elucidate the role of VEGF in the branching morphogenesis of the biliary epithelium during liver repair. These studies will address the novel idea that PC2 play a pivotal role in the regulation of cholangiocyte response to biliary damage acquired cholangiopathies, and that VEGF secreted by reactive cholangiocytes is a major factor in liver repair. Furthermore, our studies will increase understanding of VEGF/VEGFR2 signaling in epithelia and will address a fundamental mechanism in congenital and acquired cholangiopathies. Understanding the pathophysiology of cholangiopathies is a fundamental step for preserving liver function and prolonging the survival of patients