There is a large unmet need for effective therapies for cholestatic disorders, including primary sclerosing cholangitis (PSC), a disease that commonly results in liver failure. Angiotensin (Ang) II of the renin Ang system (RAS) is a potent profibrotic peptide, and Ang converting enzyme 2 (ACE2) of the alternate RAS breaks down Ang II to antifibrotic peptide Ang‐(1‐7). In the present study, we investigated long‐term effects of ACE2 delivered by an adeno‐associated viral vector and short‐term effects of Ang‐(1‐7) peptide in multiple drug‐resistant gene 2‐knockout (Mdr2‐KO) mice. These mice develop progressive biliary fibrosis with pathologic features closely resembling those observed in PSC. A single intraperitoneal injection of ACE2 therapy markedly reduced liver injury (P < 0.05) and biliary fibrosis (P < 0.01) at both established (3‐6 months of age) and advanced (7‐9 months of age) disease compared to control vector‐injected Mdr2‐KO mice. This was accompanied by increased hepatic Ang‐(1‐7) levels (P < 0.05) with concomitant reduction in hepatic Ang II levels (P < 0.05) compared to controls. Moreover, Ang‐(1‐7) peptide infusion improved liver injury (P < 0.05) and biliary fibrosis (P < 0.0001) compared to saline‐infused disease controls. The therapeutic effects of both ACE2 therapy and Ang‐(1‐7) infusion were associated with significant (P < 0.01) reduction in hepatic stellate cell (HSC) activation and collagen expression. While ACE2 therapy prevented the loss of epithelial characteristics of hepatocytes and/or cholangiocytes in vivo, Ang‐(1‐7) prevented transdifferentiation of human cholangiocytes (H69 cells) into the collagen‐secreting myofibroblastic phenotype in vitro. We showed that an increased ratio of hepatic Ang‐(1‐7) to Ang II levels by ACE2 therapy results in the inhibition of HSC activation and biliary fibrosis. Conclusion: ACE2 therapy has the potential to treat patients with biliary diseases, such as PSC.

Portal hypertension and bleeding from gastroesophageal varices is the major cause of morbidity and mortality in patients with cirrhosis. Portal hypertension is initiated by increased intrahepatic vascular resistance and a hyperdynamic circulatory state. The latter is characterized by a high cardiac output, increased total blood volume and splanchnic vasodilatation, resulting in increased mesenteric blood flow. Pharmacological manipulation of cirrhotic portal hypertension targets both the splanchnic and hepatic vascular beds. Drugs such as angiotensin converting enzyme inhibitors and angiotensin II type receptor 1 blockers, which target the components of the classical renin angiotensin system (RAS), are expected to reduce intrahepatic vascular tone by reducing extracellular matrix deposition and vasoactivity of contractile cells and thereby improve portal hypertension. However, these drugs have been shown to produce significant off-target effects such as systemic hypotension and renal failure. Therefore, the current pharmacological mainstay in clinical practice to prevent variceal bleeding and improving patient survival by reducing portal pressure is non-selective -blockers (NSBBs). These NSBBs work by reducing cardiac output and splanchnic vasodilatation but most patients do not achieve an optimal therapeutic response and a significant proportion of patients are unable to tolerate these drugs. Although statins, used alone or in combination with NSBBs, have been shown to improve portal pressure and overall mortality in cirrhotic patients, further randomized clinical trials are warranted involving larger patient populations with clear clinical end points. On the other hand, recent findings from studies that have investigated the potential use of the blockers of the components of the alternate RAS provided compelling evidence that could lead to the development of drugs targeting the splanchnic vascular bed to inhibit splanchnic vasodilatation in portal hypertension. This review outlines the mechanisms related to the pathogenesis of portal hypertension and attempts to provide an update on currently available therapeutic approaches in the management of portal hypertension with special emphasis on how the alternate RAS could be manipulated in our search for development of safe, specific and effective novel therapies to treat portal hypertension in cirrhosis.

Portal hypertension (PHT) resulting from splanchnic vasodilatation is a major cause of morbidity and mortality in patients with cirrhosis. The renin-angiotensin system (RAS) plays an important role in splanchnic vasodilatation in cirrhosis. This study investigated whether acute blockade of the vasodilatory receptors of the alternate RAS, Mas (MasR), Mas-related G-protein coupled receptor type D (MrgD), and angiotensin II type-2 receptor (AT2R) improves PHT in cirrhotic and non-cirrhotic portal hypertensive rats and counteracts systemic hypotension associated with angiotensin II type 1 receptor (AT1R) blockade. Cirrhotic bile duct ligated (BDL) or carbon tetrachloride (CCl4) injected and non-cirrhotic partial portal vein ligated (PPVL) rats were used for measurement of portal pressure (PP) and mean arterial pressure before and after an intravenous bolus injection of the MasR, MrgD, and AT2R blockers, A779, D-Pro7-Ang-(1-7) (D-Pro) and PD123319, respectively. Separate groups of rats received a combined treatment with A779 or D-Pro given 20 min after AT1R blocker losartan. Mesenteric expression of MasR, MrgD, and AT2R and circulating levels of peptide blockers were also measured. Treatment with A779 and D-Pro significantly reduced PP in cirrhotic rat models. Despite rapid degradation of A779 and D-Pro in the rat circulation, the PP lowering effect of the blockers lasted for up to 25 min. We also found that PD123319 reduced PP in CCl4 rats, possibly by blocking the MasR and/or MrgD since AT2R expression in cirrhotic mesenteric vessels was undetectable, whereas the expression of MasR and MrgD was markedly elevated. While losartan resulted in a marked reduction in PP, its profound systemic hypotensive effect was not counteracted by the combination therapy with A779 or D-Pro. In marked contrast, none of the receptor blockers had any effect on PP in non-cirrhotic PPVL rats whose mesenteric expression of MasR and MrgD was unchanged. We conclude that in addition to MasR, MrgD, a newly discovered receptor for Angiotensin-(1-7), plays a key role in splanchnic vasodilatation in cirrhosis. This implies that both MasR and MrgD are potential therapeutic targets to treat PHT in cirrhotic patients. We also conclude that the alternate RAS may not contribute to the development of splanchnic vasodilatation in non-cirrhotic PHT.

There is considerable experimental evidence that the renin angiotensin system (RAS) plays a central role in both hepatic fibrogenesis and portal hypertension. Angiotensin converting enzyme (ACE), a key enzyme of the classical RAS, converts angiotensin I (Ang I) to angiotensin II (Ang II), which acts via the Ang II type 1 receptor (AT1R) to stimulate hepatic fibrosis and increase intrahepatic vascular tone and portal pressure. Inhibitors of the classical RAS, drugs which are widely used in clinical practice in patients with hypertension, have been shown to inhibit liver fibrosis in animal models but their efficacy in human liver disease is yet to be tested in adequately powered clinical trials. Small trials in cirrhotic patients have demonstrated that these drugs may lower portal pressure but produce off-target complications such as systemic hypotension and renal failure. More recently, the alternate RAS, comprising its key enzyme, ACE2, the effector peptide angiotensin-(1–7) (Ang-(1–7)) which mediates its effects via the putative receptor Mas (MasR), has also been implicated in the pathogenesis of liver fibrosis and portal hypertension. This system is activated in both preclinical animal models and human chronic liver disease and it is now well established that the alternate RAS counter-regulates many of the deleterious effects of the ACE-dependent classical RAS. Work from our laboratory has demonstrated that liver-specific ACE2 overexpression reduces hepatic fibrosis and liver perfusion pressure without producing off-target effects. In addition, recent studies suggest that the blockers of the receptors of alternate RAS, such as the MasR and Mas related G protein-coupled receptor type-D (MrgD), increase splanchnic vascular resistance in cirrhotic animals, and thus drugs targeting the alternate RAS may be useful in the treatment of portal hypertension. This review outlines the role of the RAS in liver fibrosis and portal hypertension with a special emphasis on the possible new therapeutic approaches targeting the ACE2-driven alternate RAS.

Splanchnic vasodilatation contributes to the development and aggravation of portal hypertension (PHT). We previously demonstrated that in cirrhosis, angiotensin‐ mediates splanchnic vasodilatation through the Mas receptor (MasR). In this study, we investigated whether the recently characterized second receptor for angiotensin‐(1–7), Mas‐related G protein‐coupled receptor type D (MrgD), contributes to splanchnic vasodilatation in cirrhotic and noncirrhotic PHT. Splanchnic vascular hemodynamic and portal pressure were determined in two rat models of cirrhotic PHT and a rat model with noncirrhotic PHT, treated with either MrgD blocker D‐Pro 7 ‐Ang‐(1‐7) (D‐Pro) or MasR blocker A779. Gene and protein expression of MrgD and MasR were measured in splanchnic vessels and livers of cirrhotic and healthy rats and in patients with cirrhosis and healthy subjects. Mesenteric resistance vessels isolated from cirrhotic rats were used in myographs to study their vasodilatory properties. MrgD was up‐regulated in cirrhotic splanchnic vessels but not in the liver. In cirrhotic rats, treatment with D‐Pro but not A779 completely restored splanchnic vascular resistance to a healthy level, resulting in a 33% reduction in portal pressure. Mesenteric vessels pretreated with D‐Pro but not with A779 failed to relax in response to acetylcholine. There was no splanchnic vascular MrgD or MasR up‐regulation in noncirrhotic PHT; thus, receptor blockers had no effect on splanchnic hemodynamics. Conclusion : MrgD plays a major role in the development of cirrhotic PHT and is a promising target for the development of novel therapies to treat PHT in cirrhosis. Moreover, neither MrgD nor MasR contributes to noncirrhotic PHT.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and is frequently associated with type 2 diabetes. However, there is no specific medical therapy to treat this condition. Angiotensin‐converting enzyme 2 (ACE2) of the protective renin angiotensin system generates the antifibrotic peptide angiotensin‐(1‐7) from profibrotic angiotensin II peptide. In this study, we investigated the therapeutic potential of ACE2 in diabetic NAFLD mice fed a high‐fat (20%), high‐cholesterol (2%) diet for 40 weeks. Mice were given a single intraperitoneal injection of ACE2 using an adeno‐associated viral vector at 30 weeks of high‐fat, high‐cholesterol diet (15 weeks after induction of diabetes) and sacrificed 10 weeks later. ACE2 significantly reduced liver injury and fibrosis in diabetic NAFLD mice compared with the control vector injected mice. This was accompanied by reductions in proinflammatory cytokine expressions, hepatic stellate cell activation, and collagen 1 expression. Moreover, ACE2 therapy significantly increased islet numbers, leading to an increased insulin protein content in β‐cells and plasma insulin levels with subsequent reduction in plasma glucose levels compared with controls. Conclusion: We conclude that ACE2 gene therapy reduces liver fibrosis and hyperglycemia in diabetic NAFLD mice and has potential as a therapy for patients with NAFLD with diabetes.

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