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Development of portosystemic collateral vessels and gastroesophageal varices is responsible for the most serious clinical consequences of portal hypertension, but effective clinical therapies are limited. Here we developed and investigated the therapeutic potential of an innovative liposomally-formulated short-interfering RNA (siRNA) technology based on clinical stage components, capable to attenuate production of the endothelial kinase insert domain receptor (KDR), which controls portosystemic collateralization and contributes to disease progression and aggravation. These siRNAs were first validated in vitro, and then, their therapeutic potential on portosystemic collateralization and pathological angiogenesis was tested in vivo in mouse models of portal hypertension (portal vein-ligation). siRNA KDR -lipoplexes efficiently transported siRNA KDR to vascular endothelial cells in mesenteric microvenules and portal vein of portal hypertensive mice, where collaterogenesis and angiogenesis take place. This systemic treatment significantly downregulated pathological KDR overexpression, without causing complete KDR knockout, preserving homeostatic baseline KDR levels and thus limiting adverse effects. siRNA KDR -lipoplex-induced endothelial-specific KDR knockdown drastically reduced by 73% the portosystemic collateralization, and impaired the pathologic angiogenic potential of vascular endothelial cells at different levels (cell proliferation, sprouting and remodeling). Targeting endothelial KDR with therapeutic siRNA KDR -lipoplexes could be a promising and plausible treatment modality for attenuating the formation of portosystemic collaterals in a clinical setting.

Objective: Pathological neovascularisation is intimately involved in portal hypertension (PH). Here, we determined the contribution of vascular stem/progenitor cells (VSPCs) to neovessel growth in PH and whether the RNA-binding protein cytoplasmic polyadenylation element binding protein-4 (CPEB4) was behind the mechanism controlling VSPC function., Design: To identify and monitor VSPCs in PH rats (portal vein-ligated), we used a combinatorial approach, including sphere-forming assay, assessment of self-renewal, 5-bromo-2'-desoxyuridine label retention technique, in vitro and in vivo stem/progenitor cell (SPC) differentiation and vasculogenic capability, cell sorting, as well as immunohistochemistry, immunofluorescence and confocal microscopy expression analysis. We also determined the role of CPEB4 on VSPC proliferation using genetically engineered mouse models., Results: We demonstrated the existence in the mesenteric vascular bed of VSPCs displaying capability to form cellular spheres in suspension culture, self-renewal ability, expression of molecules commonly found in SPCs, slow-cycling features, in addition to other cardinal properties exhibited by SPCs, like capacity to differentiate into endothelial cells and pericytes with remarkable vasculogenic activity. Such VSPCs showed, after PH induction, an early switch in proliferation, and differentiated in vivo into endothelial cells and pericytes, contributing, structurally and functionally, to abnormal neovessel formation. Quantification of VSPC-dependent neovessel formation in PH further illustrated the key role played by VSPCs. We also demonstrated that CPEB4 regulates the proliferation of the activated VSPC progeny upon PH induction., Conclusions: These findings demonstrate that VSPC-derived neovessel growth (ie, vasculogenesis) and angiogenesis cooperatively stimulate mesenteric neovascularisation in PH and identify VSPC and CPEB4 as potential therapeutic targets., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

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., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Antiangiogenic strategies have been proposed as a promising new approach for the therapy of portal hypertension and chronic liver disease. Pigment epithelium-derived factor (PEDF) is a powerful endogenous angiogenesis inhibitor whose role in portal hypertension remains unknown. Therefore, we aimed at determining the involvement of PEDF in cirrhotic portal hypertension and the therapeutic efficacy of its supplementation., Design: PEDF expression profiling and its relationship with vascular endothelial growth factor (VEGF), neovascularisation and fibrogenesis was determined in bile duct-ligated (BDL) rats and human cirrhotic livers. The ability of exogenous PEDF overexpression by adenovirus-mediated gene transfer (AdPEDF) to inhibit angiogenesis, fibrogenesis and portal pressure was also evaluated in BDL rats, following prevention and intervention trials., Results: PEDF was upregulated in cirrhotic human and BDL rat livers. PEDF and VEGF protein expression and localisation in mesentery and liver increased in parallel with portal hypertension progression, being closely linked in time and space with mesenteric neovascularisation and liver fibrogenesis in BDL rats. Furthermore, AdPEDF increased PEDF bioavailability in BDL rats, shifting the net balance in the local abundance of positive (VEGF) and negative (PEDF) angiogenesis drivers in favour of attenuation of portal hypertension-associated pathological neovascularisation. The antiangiogenic effects of AdPEDF targeted only pathological angiogenesis, without affecting normal vasculature, and were observed during early stages of disease. AdPEDF also significantly decreased liver fibrogenesis (through metalloproteinase upregulation), portosystemic collateralisation and portal pressure in BDL rats., Conclusions: This study provides compelling experimental evidence indicating that PEDF could be a novel therapeutic agent worthy of assessment in portal hypertension and cirrhosis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background & Aims: Splenomegaly is a frequent hallmark of portal hypertension that, in some cases, can be very prominent and cause symptoms like abdominal pain, splenic infarction, and cytopenia. This study characterizes the pathogenetic mechanisms leading to spleen enlargement in portal hypertensive rats and focuses on mTOR pathway as a potential modulator of splenomegaly in portal hypertension., Methods: Characterization of splenomegaly was performed by histological, hematological, immunohistochemical and Western blot analyses in rats with portal hypertension induced by portal vein ligation, and compared with sham-operated animals. The contribution of the mTOR signaling pathway to splenomegaly was determined in rats with fully developed portal hypertension and control rats by treatment with rapamycin or vehicle., Results: Our results illustrate that splenomegaly in portal hypertensive rats arises as a consequence of the interplay of several factors, including not only spleen congestion, as traditionally thought, but also enlargement and hyperactivation of the splenic lymphoid tissue, as well as increased angiogenesis and fibrogenesis. Since mTOR signaling plays a central role in immunological processes, angiogenesis and fibrogenesis, we next determined the involvement of mTOR in splenomegaly. Interestingly, mTOR signaling was overactivated in the spleen of portal hypertensive rats, and mTOR blockade by rapamycin profoundly ameliorated splenomegaly, causing a 44% decrease in spleen size. This effect was most likely accounted for the inhibitory action of rapamycin on lymphocyte proliferation, neovascularization and fibrosis., Conclusions: These findings shed light on the pathogenesis of splenomegaly in portal hypertension, and identify mTOR signaling as a potential target for therapeutic intervention in this disease.

Unlabelled: Portal hypertension, the most important complication in patients with cirrhosis of the liver, is a serious and life-threatening disease for which there are few therapeutic options. Because angiogenesis is a pathological hallmark of portal hypertension, the goal of this study was to determine the effects of sorafenib-a potent inhibitor of proangiogenic vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor beta (PDGFR-beta), and Raf kinases-on splanchnic, intrahepatic, systemic, and portosystemic collateral circulations in two different experimental models of portal hypertension: rats with prehepatic portal hypertension induced by partial portal vein ligation and rats with intrahepatic portal hypertension and secondary biliary cirrhosis induced by bile duct ligation. Such a comprehensive approach is necessary for any translational research directed toward defining the efficacy and potential clinical application of new therapeutic agents. Sorafenib administered orally once a day for 2 weeks in experimental models of portal hypertension and cirrhosis effectively inhibited VEGF, PDGF, and Raf signaling pathways, and produced several protective effects by inducing an approximately 80% decrease in splanchnic neovascularization and a marked attenuation of hyperdynamic splanchnic and systemic circulations, as well as a significant 18% decrease in the extent of portosystemic collaterals. In cirrhotic rats, sorafenib treatment also resulted in a 25% reduction in portal pressure, as well as a remarkable improvement in liver damage and intrahepatic fibrosis, inflammation, and angiogenesis. Notably, beneficial effects of sorafenib against tissue damage and inflammation were also observed in splanchnic organs., Conclusion: Taking into account the limitations of translating animal study results into humans, we believe that our findings will stimulate consideration of sorafenib as an effective therapeutic agent in patients suffering from advanced portal hypertension.

Background: Angiogenesis is an important determinant of the pathophysiology of portal hypertension contributing to the formation of portosystemic collateral vessels and the hyperdynamic splanchnic circulation associated to this syndrome. Somatostatin and its analogues, like octreotide, have been shown to be powerful inhibitors of experimental angiogenesis., Aim: To determine whether octreotide has angioinhibitory effects in portal hypertensive rats., Methods: Partial portal vein-ligated (PPVL) rats were treated with octreotide or vehicle during 4 or 7 days. Splanchnic neovascularization and VEGF expression were determined by histological analysis and western blotting. Expression of the somatostatin receptor subtype 2 (SSTR2), which mediates the anti-angiogenic effects of octreotide, was also analyzed. Formation of portosystemic collaterals (radioactive microspheres) and hemodynamic parameters were also measured., Results: Octreotide treatment during 4 days markedly and significantly decreased splanchnic neovascularization, VEGF expression by 63% and portal pressure by 15%, whereas portosystemic collateralization and splanchnic blood flow were not modified. After 1 week of octreotide injection, portal pressure was reduced by 20%, but inhibition of angiogenesis escaped from octreotide therapy, a phenomenon that could be related to the finding that expression of SSTR2 receptor decreased progressively (up to 78% reduction) during the evolution of portal hypertension., Conclusion: This study provides the first experimental evidence showing that octreotide may be an effective anti-angiogenic therapy early after induction of portal hypertension, but not in advanced stages most likely due to SSTR2 down-regulation during the progression of portal hypertension in rats. These findings shed light on new mechanisms of action of octreotide in portal hypertension.

Unlabelled: Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) pathways are crucial to angiogenesis, a process that contributes significantly to the pathogenesis of portal hypertension. This study determined the effects of inhibition of VEGF and/or PDGF signaling on hyperdynamic splanchnic circulation and portosystemic collateralization in rats with completely established portal hypertension, thus mimicking the situation in patients. Portal vein-ligated rats were treated with rapamycin (VEGF signaling inhibitor), Gleevec (PDGF signaling inhibitor), or both simultaneously when portal hypertension was already fully developed. Hemodynamic studies were performed by transit-time flowmetry. The extent of portosystemic collaterals was measured by radioactive microspheres. The expression of angiogenesis mediators was determined by Western blotting and immunohistochemistry. Combined inhibition of VEGF and PDGF signaling significantly reduced splanchnic neovascularization (i.e., CD31 and VEGFR-2 expression) and pericyte coverage of neovessels (that is, alpha-smooth muscle actin and PDGFR-beta expression) and translated into hemodynamic effects as marked as a 40% decrease in portal pressure, a 30% decrease in superior mesenteric artery blood flow, and a 63% increase in superior mesenteric artery resistance, yielding a significant reversal of the hemodynamic changes provoked by portal hypertension in rats. Portosystemic collateralization was reduced as well., Conclusions: Our results provide new insights into how angiogenesis regulates portal hypertension by demonstrating that the maintenance of increased portal pressure, hyperkinetic circulation, splanchnic neovascularization, and portosystemic collateralization is regulated by VEGF and PDGF in portal hypertensive rats. Importantly, these findings also suggest that an extended antiangiogenic strategy (that is, targeting VEGF/endothelium and PDGF/pericytes) may be a novel approach to the treatment of portal hypertension.