Therapeutic siRNA targeting endothelial KDR decreases portosystemic collateralization in portal hypertension

Abstract

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). siRNAKDR-lipoplexes efficiently transported siRNAKDR 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. siRNAKDR-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 siRNAKDR-lipoplexes could be a promising and plausible treatment modality for attenuating the formation of portosystemic collaterals in a clinical setting.

Targeted Transfection Using PEGylated Cationic Liposomes Directed Towards P-Selectin Increases siRNA Delivery into Activated Endothelial Cells

“…Certainly, progress towards the clinical use of siRNA therapeutics depends on the development of suitable siRNA carriers [42,43]. There were several attempts to develop proper nanocarriers, such as liposomes for specific and efficient siRNA delivery to endothelium in vitro and in vivo [44]. There are many challenges in the development of efficacious lipid-based siRNA nanocarriers such as the appropriate composition of liposomes, the ratio of cationic lipid to nucleic acid used for lipoplexes formation, protection of siRNA against degradation, optimal concentration of either siRNA and liposomes, cytotoxicity of lipoplexes, efficient cellular uptake and effective endosomal release.…”

Recent Advances in Practical Methods for Liver Cell Biology: A Short Overview

“…As opposed to in vitro assays, using in vivo assays, these hypotheses can be validated by immunohistochemical, histological and molecular biology procedures. Recently, the use of angiogenesis inhibitors in vivo has shown a potential effect in chronic liver disease, such as inhibiting the VEGF signaling pathway, combining treatments with VEGF and PDGF inhibitors directed against endothelial cells and pericytes, or gene therapy with cell-targeted molecule-targeted liposomal small interference RNAs (Table 1) [53,88,89]. Endogenous angiogenesis inhibitors using adenovirus-mediated gene transfer are also applied (Table 1) [56,90].…”

Interplay Between Macrophages and Angiogenesis: A Double-Edged Sword in Liver Disease

“…Fat accumulation in visceral fat depots, including the mesentery, induces a state of chronic inflammation that may reach the liver through the portal venous system, contributing to activate HSCs and increase fibrogenesis (74). Pathological angiogenesis during chronic liver disease also takes place extrahepatically, playing a major role in the formation of portosystemic collaterals (1,(75)(76)(77)(78)(79)(80) and the development and maintenance of splanchnic hyperdynamic circulation and portal hypertension (81)(82)(83)(84)(85).…”

“…Due to the relevance of VEGF as a key angiogenic regulator, it is one of the main targets. Approaches used to inhibit angiogenesis in chronic liver disease and portal hypertension include neutralizing monoclonal antibodies (78), tyrosin kinase inhibitors (24,(83)(84)(85), or therapeutic small interference RNAs targeting VEGF receptor-2 (76). Attenuation of oxidative stress and inflammation has also been shown to reduce pathological angiogenesis in liver disease and portal hypertension (86)(87)(88).…”