oa Ace2 Gene Therapy Inhibits Chronic Biliary Fibrosis in Mice

Background

The cholangiopathies or cholestatic liver diseases comprise a large group of conditions in which injury is primarily focussed on the biliary system. They include both congenital diseases such as biliary atresia and cystic fibrosis, and acquired diseases including primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC), and secondary damage to the biliary tree from obstruction, cholangitis or ischaemia. These conditions cause a specific pattern of chronic liver injury centred around damaged bile ducts which drives the development of peribiliary fibrosis and eventually biliary cirrhosis and liver failure. For most, there is no established treatment and they remain one of the most important indications for liver transplantation. As a result, there is a major need to develop new therapies which can prevent the development of chronic biliary injury and fibrosis in these diseases.

Our group made a major contribution in being the first to establish that angiotensin II (Ang II), the key effector peptide of the classic arm of the renin angiotensin system (RAS), plays a central role in the pathogenesis of hepatic fibrosis and portal hypertension (1, 2). Angiotensin converting enzyme (ACE), a key enzyme of this classic RAS, converts angiotensin I (Ang I) to the potent vasoconstrictor and profibrotic cytokine Ang II, which acts via the Ang II type 1 receptor (AT1R). Moreover, we were the first group to discover that there is an alternate ACE2 dependent arm of the RAS, which is activated in both experimental and human chronic liver disease (3). Thus, ACE2, a homologue of ACE, degrades Ang II and generates angiotensin-(1–7) (Ang-(1–7)), which in contrast to Ang II, has vasodilatory, antifibrotic, antigrowth and antiproliferative actions (4–6). These effects of Ang-(1–7) are mediated by the Mas receptor (MasR) (7). Therefore, this ACE2/Ang-(1–7)/MasR arm is thought to intrinsically regulate the RAS system by reducing Ang II levels and producing Ang (1–7), thus counterbalancing the potentially harmful effects of Ang II (Fig. 1). We have recently shown that by modulating the activity of the alternate arm of the RAS we could markedly reduce liver injury and fibrosis in short-term animal models of hepatic fibrosis (8).

Aims

Therefore, in the present study, we investigated long-term effects of ACE2 gene therapy in chronic biliary disease using multiple drug resistant gene 2 knockout (Mdr2-KO) mice, which develop progressive biliary fibrosis over 6 months.

Methods

A recombinant AAV2-LSP1 vector, constructed using the pAM backbone, was used for producing rAAV2-LSP1-ACE2 vector. The sequence of mouse ACE2, with optimized Kozak sequence, was inserted as an EcoRI/EcoRV fragment under the transcriptional control of the human antitrypsin promoter downstream of hepatic control region of the human ApoE. Control vector (rAAV-LSP1-HSA) was constructed containing human serum albumin (HSA) gene in place of the mouse ACE2 gene. Both vectors were pseudo-serotyped with the liver-specific AAV8 capsid using p5E18-VD2/8 plasmid. To investigate the efficacy of therapy early in disease progression a single injection of either ACE2 or control HSA vector was administered intra-peritoneally to 3-months-old Mdr2-KO mice with established biliary fibrosis and sacrificed them 3 months and 6 months post-treatment. Similarly, to investigate the efficacy of therapy in advanced biliary fibrosis and cirrhosis, a single injection of either ACE2 or control HSA vector was administered intra-peritoneally to 7-months-old Mdr2-KO mice with established cirrhosis and sacrificed them 2 months post-treatment. After sacrifice, blood was collected to determine liver function test and liver tissues were collected for fibrosis, liver histology, gene and protein expression analysis, and angiotensin peptide measurements. To elucidate the possible therapeutic mechanisms, Ang-(1–7), the major antifibrotic peptide produced by ACE2-induced cleavage of Ang II, was infused into 3-month-old Mdr2-KO mice for 1 month using osmotic minipump and the same end points were measured.

Results

ACE2 gene therapy increased ACE2 gene expression by more than 60-fold and ACE2 protein activity by more than 2-fold in Mdr2-KO mice compared with HSA-treated control mice. As expected, increased ACE2 expression led to a major decrease in hepatic levels of the potent profibrotic peptide Ang II with a concomitant increase in Ang-(1–7) levels. Liver injury was associated with increased release of proinflammatory cytokines such as interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP1), which recruit inflammatory molecules exacerbating injury, leading to fibrosis. We found that ACE2 gene therapy significantly (p