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 biolo...