Ductular reaction promotes intrahepatic angiogenesis through Slit2-Roundabout 1 signaling

Background and aims: Ductular reaction (DR) expands in chronic liver diseases and correlates with disease severity. Besides its potential role in liver regeneration, DR plays a role in the wound-healing response of the liver, promoting periductular fibrosis and inflammatory cell recruitment. However, there is no information regarding its role in intrahepatic angiogenesis. In the current study we investigated the potential contribution of DR cells to hepatic vascular remodeling during chronic liver disease. Approach and results: In mouse models of liver injury, DR cells express genes involved in angiogenesis. Among angiogenesis-related genes, the expression of Slit2 and its receptor Roundabout 1 (Robo1) was localized in DR cells and neoangiogenic vessels, respectively. The angiogenic role of the Slit2-Robo1 pathway in chronic liver disease was confirmed in ROBO1/2-/+ mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which displayed reduced intrahepatic neovascular density compared to wild-type mice. However, ROBO1/2 deficiency did not affect angiogenesis in partial hepatectomy. In patients with advanced alcohol-associated disease, angiogenesis was associated with DR, and up-regulation of SLIT2-ROBO1 correlated with DR and disease severity. In vitro, human liver-derived organoids produced SLIT2 and induced tube formation of endothelial cells. Conclusions: Overall, our data indicate that DR expansion promotes angiogenesis through the Slit2-Robo1 pathway and recognize DR cells as key players in the liver wound-healing response.Supported by grants from Fondo de Investigación Sanitaria Carlos III (FIS), cofinanced by Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, “Una manera de hacer Europa” (FIS PI20/00765, PI17/00673, to P.S.-B; FIS 18-PI18/00862, to I.G and M.C); from the National Institute on Alcohol Abuse and Alcoholism (1U01AA026972-01 and AGAUR 2017-SGR-01456, to P.S.-B.); and from the European Foundation for Alcohol Research (EA1653, to P.S.-B.). M.C. is funded by the Ramon y Cajal program from the Ministerio de Ciencia e Innovación RYC2019-026662-I. P.G. is funded by the Agencia de Gestió d'Ajuts Universitaris i de Recerca 2014 SGR 708, Centro de Investigaciónen Red Enfermedades Hepáticas y Digestivas (CIBERehd), and Institució Catalana de Recerca i Estudis Avançats. S.A. received a grant from the Ministerio de Educación, Cultura y Deporte (FPU17/04992). B.A.-B. is funded by the Instituto de Salud Carlos III (FI16/00203

Apelin signaling modulates splanchnic angiogenesis and portosystemic collateral vessel formation in rats with portal hypertension

BACKGROUND/AIMS: Angiogenesis is a pathological hallmark of portal hypertension. Although VEGF is considered to be the most important proangiogenic factor in neoangiogenesis, this process requires the coordinated action of a variety of factors. Identification of novel molecules involved in angiogenesis is highly relevant, since they may represent potential new targets to suppress pathological neovascularization in angiogenesis-related diseases like portal hypertension. The apelin/APJ signaling pathway plays a crucial role in angiogenesis. Therefore, we determined whether the apelin system modulates angiogenesis-driven processes in portal hypertension. METHODS: Partial portal vein-ligated rats were treated with the APJ antagonist F13A for seven days. Splanchnic neovascularization and expression of angiogenesis mediators (Western blotting) was determined. Portosystemic collateral formation (microspheres), and hemodynamic parameters (flowmetry) were also assessed. RESULTS: Apelin and its receptor APJ were overexpressed in the splanchnic vasculature of portal hypertensive rats. F13A effectively decreased, by 52%, splanchnic neovascularization and expression of proangiogenic factors VEGF, PDGF and angiopoietin-2 in portal hypertensive rats. F13A also reduced, by 35%, the formation of portosystemic collateral vessels. CONCLUSIONS: This study provides the first experimental evidence showing that the apelin/APJ system contributes to portosystemic collateralization and splanchnic neovascularization in portal hypertensive rats, presenting a potential novel therapeutic target for portal hypertension

Sequential functions of CPEB1 and CPEB4 regulate pathologic expression of vascular endothelial growth factor and angiogenesis in chronic liver disease.

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease. METHODS: We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed. RESULTS: Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation. CONCLUSIONS: We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-29491 and SAF2014-55473-R to MF; BFU2011-30121, BFU2014-54122-P and Consolider RNAREG CSD2009-00080 to RM; PI13/00341 to JB; PI11/01562 and PI14/00125 to PN), Generalitat de Catalunya (SGR1436 to RM; SGR1108 to JB), Fundación Botín by Banco Santander through its Santander Universities Global Division (to RM), AECC Scientific Foundation (to RM and MF), Worldwide Cancer Research (to RM and MF) and RETIC Cancer RD12/0036/0051/FEDER to PN. GFM is funded by a Juan de la Cierva contract from MINECO. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). CIBERehd is an initiative from the Instituto de Salud Carlos III. We also thank Dr Francisco X Real for his contribution to knockout mice generation