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.

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.

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.

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.

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.

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.

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Gene therapy is a novel promising approach for treating a spectrum of inherited and non-inherited disorders by delivering therapeutic genes to specific organs or tissues. Of the viral vectors that have been used to date to deliver the genes of interest, the adenoassociated viral (AAV) vector appears to be the most safe and effective vehicle and has the ability to maintain long-term gene and protein expression following a single injection of the vector. Gene therapy studies using AAV vector have shown significant progress not only in animal models but also in human gene therapy with no known pathogenicity. While success has been achieved in gene therapy using AAV vector to deliver the target genes for inherited disorders, however, clinical trials are yet to begin to see whether gene therapy has promise for treatment of non-inherited diseases. This chapter describes AAV biology, viral structure, and cell entry mechanisms, with special emphasis on AAV tissue tropism achieved by manipulating different serotypes and capsid engineering. This chapter also discusses successful application of the AAV vector for non-inherited disorders in animal models with particular reference to liver fibrosis, outlining advantages, disadvantages, and future challenges that this therapy may face.