Cholangiocyte-Derived Endothelin Signaling Mediates Biliary Injury and Liver Fibrosis

这项应用的主要目的是阐明介导胆管衰老的信号机制。 和原发性硬化性胆管炎(PSC)时的血管生成。已知(I)内皮素(ET)-1 在梗阻性胆汁淤积的胆管结扎(BDL)模型中，信号转导增强，(Ii)它主要被上调 (I)通过激活肝星状细胞(HSC)增强肝纤维化。在肝脏之外， ET-1是一种有效的血管收缩因子，可增强转化生长因子-β-1(转化生长因子-β-1，促血管生成因子) 在血管内皮细胞中表达。总体而言，ET信令(包括ET-2和ET-2)是如何传递的尚不清楚。 3)在PSC过程中介导胆管衰老和VEC增殖/血管生成。此外，还会更改 PSC过程中血管床和血管内皮细胞的增殖情况尚不清楚。我们发现ET-1、ET-2、ET-3和ET-A和 ET-B在人PSC中的表达增加，尤其是在胆管细胞和血管内皮细胞中，与多药耐药 PSC基因敲除(mdr2-/-)小鼠模型。此外，我们有新的初步数据显示血管内皮细胞增加 人PSC和mdr2-/-小鼠体内的增殖和血管生成。血管生成的变化与 胆汁淤积期的血管减少是有争议的，但考虑到门静脉高压症的PSC患者有 随着发病率和死亡率的增加，可能会发生一些由血管内皮细胞介导的潜在变化。因此， 我们的新发现首次表明PSC过程中肝血管内皮细胞的增殖和血管生成。另外， 这些发现首次深入研究了PSC过程中胆管细胞和血管内皮细胞中的ET信号。我们提供数据 提示一个反馈环，由此ET信号促进转化生长因子-β1的表达，进而增加miR-1。 125b/HIF-1α表达，已知可增加ET的表达。这些是第一批数据表明 ET/转化生长因子-β-1/mIR-125b/缺氧诱导因子-1α之间的正反馈环可延缓胆道衰老和肝脏 以自分泌和旁分泌的方式出现纤维化。此外，我们还发现，抑制mdr2-/-中的ET-A或ET-B 使用目前FDA批准的药物(用于治疗肺动脉高压)的小鼠减少胆汁 与对照组相比，衰老和肝纤维化。我们的总体假设是胆管细胞和 肝血管内皮细胞通过内皮素ET/转化生长因子-β1信号传导增加胆汁 衰老和血管内皮细胞血管生成通过自分泌和旁分泌机制。我们的发现可能会导致 寻找治疗PSC的新的有效疗法。这项申请提案是第一个 为私家侦探发展独立性，林赛·肯尼迪博士。这项提议巧妙地将 她的导师在胆管细胞生物学、microRNA信号转导和血管生成方面的背景工作 在胆汁淤积过程中进一步研究血管生物学的技术和概念。以下是成功的 完成这项申请，肯尼迪博士将对血管生成/血管减少症，VEC有更好的了解 生物学和血管与胆管的相互作用，这可以发展成独立的研究。这把钥匙 这项研究提案的要素是：(I)开发了强大的指导团队，他们将提供顶级 对申请者进行培训，(Ii)介绍新技术(喷墨、腐蚀浇注、VEC分离、 人类原代细胞分离)和概念(胆汁淤积症中的血管生物学)，(Iii)全面的指导和 职业规划，包括一对一培训、出席会议、演讲机会和发展 (4)申请者有机会开展自己独立的、成功的研究 实验室。

The main goal of this application is to clarify the signaling mechanisms mediating biliary senescence and angiogenesis during primary sclerosing cholangitis (PSC). It is known that (i) endothelin (ET)-1 signaling is enhanced in the bile duct ligation (BDL) model of obstructive cholestasis, (ii) it is primarily upregulated in cholangiocytes and (iii) enhances liver fibrosis via hepatic stellate cell (HSC) activation. Outside of the liver, ET-1 is a potent vasoconstrictor and enhances transforming growth factor-β1 (TGF-β1, pro-angiogenic factor) expression in vascular endothelial cells (VECs). Overall, it is unknown how ET signaling (including ET-2 and ET- 3) mediate biliary senescence and VEC proliferation/angiogenesis during PSC. Additionally, changes in the vascular bed and VEC proliferation during PSC is unknown. We found that ET-1, ET-2, ET-3 and ET-A and ET-B are increased, particularly in cholangiocytes and VECs, in human PSC and the multidrug resistance-2 knockout (Mdr2-/-) mouse model of PSC. Furthermore, we have novel preliminary data showing increased VEC proliferation and angiogenesis in both human PSC and in Mdr2-/- mice. Changes in angiogenesis versus vasopenia during cholestasis is controversial, but considering PSC patients with portal hypertension have an increased morbidity and mortality, there is likely some underlying VEC-mediated changes occurring. Therefore, our novel findings are the first to indicate liver VEC proliferation and angiogenesis during PSC. Additionally, these findings are the first to delve into ET signaling in cholangiocytes and VECs during PSC. We provide data indicating a feedback loop, whereby ET signaling promotes TGF-β1 expression, which can in turn increase miR- 125b/HIF-1α expression, which is known to increase ET expression. These are the first data demonstrating a positive feedback loop between ET/TGF-β1/miR-125b/HIF-1α that can perpetuate biliary senescence and liver fibrosis in autocrine and paracrine manners. Furthermore, we found that inhibition of ET-A or ET-B in Mdr2-/- mice using currently FDA-approved drugs (for the treatment of pulmonary hypertension) reduces biliary senescence and liver fibrosis when compared to controls. Our overall hypothesis is that cholangiocytes and liver VEC communicate with one another via endothelin ET/TGF-β1 signaling that increases biliary senescence and VEC angiogenesis through autocrine and paracrine mechanisms. Our findings may lead to the identification of new, effective therapeutics for the treatment of PSC. This application proposal is the first step to developing independence for the PI, Dr. Lindsey Kennedy. This proposal elegantly marries the background work of her mentors on cholangiocyte biology, microRNA signaling and angiogenesis with new techniques and concepts that further delve into vascular biology during cholestasis. Following successful completion of this application, Dr. Kennedy will have a better understanding of angiogenesis/vasopenia, VEC biology and vascular interactions with the bile ducts, and this can develop into independent studies. The KEY ELEMENTS of this research proposal are the (i) strong mentoring team developed, who will provide top-tier training for the applicant, (ii) introduction of new techniques (ink injection, corrosion casting, VEC isolation, human primary cell isolation) and concepts (vascular biology in cholestasis), (iii) comprehensive mentoring and career plan, including one-on-one training, conference attendance, presentation opportunities and development of grantsmanship, and (iv) opportunity for the applicant to develop her own independent, successful research lab.