Mas-related G protein-coupled receptor type D antagonism improves portal hypertension in cirrhotic rats.

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-Pro7 -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.

Angiotensin Converting Enzyme-2 Therapy Improves Liver Fibrosis and Glycemic Control in Diabetic Mice With Fatty Liver.

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 ß-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.

Update on New Aspects of the Renin-Angiotensin System in Hepatic Fibrosis and Portal Hypertension: Implications for Novel Therapeutic Options.

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.

Liver-Targeted Angiotensin Converting Enzyme 2 Therapy Inhibits Chronic Biliary Fibrosis in Multiple Drug-Resistant Gene 2-Knockout Mice.

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.

Current therapies and novel approaches for biliary diseases.

Chronic liver diseases that inevitably lead to hepatic fibrosis, cirrhosis and/or hepatocellular carcinoma have become a major cause of illness and death worldwide. Among them, cholangiopathies or cholestatic liver diseases comprise a large group of conditions in which injury is primarily focused on the biliary system. These include congenital diseases (such as biliary atresia and cystic fibrosis), acquired diseases (such as primary sclerosing cholangitis and primary biliary cirrhosis), and those that arise from secondary damage to the biliary tree from obstruction, cholangitis or ischaemia. These conditions are associated with a specific pattern of chronic liver injury centered on damaged bile ducts that drive the development of peribiliary fibrosis and, ultimately, biliary cirrhosis and liver failure. For most, there is no established medical therapy and, hence, these diseases remain one of the most important indications for liver transplantation. As a result, there is a major need to develop new therapies that can prevent the development of chronic biliary injury and fibrosis. This mini-review briefly discusses the pathophysiology of liver fibrosis and its progression to cirrhosis. We make a special emphasis on biliary fibrosis and current therapeutic options, such as angiotensin converting enzyme-2 (known as ACE2) over-expression in the diseased liver as a novel potential therapy to treat this condition.

The Effects of Diabetes and High-Fat Diet on Polymodal Nociceptor and Cold Thermoreceptor Nerve Terminal Endings in the Corneal Epithelium.

Purpose: There is a substantial body of evidence indicating that corneal sensory innervation is affected by pathology in a range of diseases. However, there are no published studies that have directly assessed whether the nerve fiber density of the different subpopulations of corneal sensory neurons are differentially affected. The present study explored the possibility that the intraepithelial nerve fiber density of corneal polymodal nociceptors and cold thermoreceptors are differentially affected in mice fed with a high-fat high cholesterol (HFHC; 21% fat, 2% cholesterol) diet and in those that also have diabetes. Methods: The mice were fed the HFHC diet for the duration of the experiment (up to 40 weeks). Mice in the diabetes group had hyperglycaemia induced with streptozotocin after 15 weeks on the HFHC diet. Age-matched control animals were fed a standard diet. All corneal nerve fibers were labeled with a pan neuronal antibody (antiprotein gene product 9.5), and polymodal nociceptors and cold thermoreceptors were labeled with antibodies directed against transient receptor potential cation channel, subfamily V, member 1 and transient receptor potential cation channel subfamily M member 8, respectively. Results: The mice fed a HFHC diet and those that in addition have hyperglycemia have similar reductions in corneal nerve fiber density consistent with small fiber neuropathy. Importantly, both treatments more markedly affected the intraepithelial axons of cold thermoreceptors than those of polymodal nociceptors. Conclusions: The results provide evidence that distinct subpopulations of corneal sensory neurons can be differentially affected by pathology.

The small molecule drug diminazene aceturate inhibits liver injury and biliary fibrosis in mice.

There is no established medical therapy to treat biliary fibrosis resulting from chronic inflammation in the biliary tree. We have recently shown that liver-specific over-expression of angiotensin converting enzyme 2 (ACE2) of the renin angiotensin system (RAS) ameliorated liver fibrosis in mice. Diminazene aceturate (DIZE), a small molecule drug approved by the US Food and Drug Administration, which is used to treat human trypanosomiasis, has been shown to have antifibrotic properties by enhancing ACE2 activity. In this study we sought to determine the therapeutic potential of DIZE in biliary fibrosis using bile duct ligated and multiple drug resistant gene-2 knockout mice. Additionally, human hepatic stellate (LX-2) and mouse Kupffer (KUP5) cell lines were used to delineate intracellular pathways. DIZE treatment, both in vivo and in vitro, markedly inhibited the activation of fibroblastic stellate cells which was associated with a reduced activation of Kupffer cells. Moreover, DIZE-inhibited NOX enzyme assembly and ROS generation, activation of profibrotic transcription factors including p38, Erk1/2 and Smad2/3 proteins and proinflammatory and profibrotic cytokine release. These changes led to a major reduction in biliary fibrosis in both models without affecting liver ACE2 activity. We conclude that DIZE has a potential to treat biliary fibrosis.

The Adeno-associated Virus - A Safe and Promising Vehicle for Liverspecific Gene Therapy of Inherited and Non-inherited Disorders.

INTRODUCTION: The first human adeno-associated virus (AAV) was originally discovered in 1960s as a contaminant of adenovirus stock preparation and thus it had not been of medical interest. Throughout the last three decades AAV has gained popularity to be used in gene therapy, mainly due to its replicative defectiveness and lack of pathogenicity in human. In addition, its ability to mediate stable and long-term expression in both non-dividing and dividing cells with specific tissue tropism makes AAV one of the most promising candidates for therapeutic gene transfer to treat many inherited as well as non-inherited disorders. Moreover, the use of AAV is not only restricted to overexpression of recombinant transgene, but also to over-express short hairpin RNA and microRNA to knockdown the expression of genes in targeted tissues. DISCUSSION AND CONCLUSION: This review is organized into four parts. In the first part of the review, we discuss about the discovery and history of AAV, followed by detailed AAV biology such as virus genome, virus structure and its life cycle. In the second part of the review, the discussion is centred on the molecular mechanisms of AAV and tissue transduction, including receptor recognition and cell binding, endosomal entry, virus uncoating, nuclear entry and genome replication. Advantages and limitations of using AAV as a safe vehicle for gene delivery is also discussed. In the third part of the review, we discuss about the most commonly used AAV serotypes and variants isolated from human and non-human primates, focusing on their diverse tissue tropisms, transduction efficiency, immunological profiles and their applications in animal studies. Final part of the review focuses on the recent progress of in-vivo gene transfer using AAV for inherited and non-inherited diseases in both preclinical and clinical settings with a special emphasis on potential clinical applications of AAV in the field of liver disease.