Browsing by Author "Yaden, Benjamin"

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    Activin B Promotes Hepatic Fibrogenesis
    (2019-08) Wang, Yan; Dai, Guoli; Berbari, Nicolas; Yaden, Benjamin; Liangpunsakul, Suthat; Skalnik, David G.
    Liver fibrosis is a common consequence of various chronic liver diseases. Although transforming growth factor β 1 (TGFβ1) expression is known to be associated with liver fibrosis, the reduced clinical efficacy of TGFβ1 inhibition or the inefficiency to completely prevent liver fibrosis in mice with liver-specific knockout of TGF receptor II suggests that other factors can mediate liver fibrogenesis. As a TGFβ superfamily ligand, activin A signaling modulates liver injury by prohibiting hepatocyte proliferation, mediating hepatocyte apoptosis, promoting Kupffer cell activation, and inducing hepatic stellate cell (HSC) activation in vitro. However, the mechanism of action and in vivo functional significance of activin A in liver fibrosis models remain uncertain. Moreover, whether activin B, another ligand structurally related to activin A, is involved in liver fibrogenesis is not yet known. This study aimed to investigate the role of activin A and B in liver fibrosis initiation and progression. The levels of hepatic and circulating activin B and A were analyzed in patients with various chronic liver diseases, including end-stage liver diseases (ESLD), non-alcoholic steatohepatitis (NASH), and alcoholic liver disease (ALD). In addition, their levels were measured in mouse carbon tetrachloride (CCl4), bile duct ligation (BDL), and ALD liver injury models. Mouse primary hepatocytes, RAW264.7 cells, and LX-2 cells were used as in vitro models of hepatocytes, macrophages, and HSCs, respectively. The specificity and potency of anti-activin B monoclonal antibody (mAb) and anti-activin A mAb were evaluated using Smad2/3 luciferase assay. Activin A, activin B, or their combination were immunologically inactivated by the neutralizing mAbs in mice with progressive or established liver fibrosis induced by CCl4 or with developing cholestatic liver fibrosis induced by BDL surgery. In patients with ESLD, NASH, and ALD, increases in hepatic and circulating activin B, but not activin A, were associated with liver fibrosis, irrespective of etiology. In mice with CCl4-, BDL-, or alcohol-induced liver injury, activin B was persistently elevated in the liver and circulation, whereas activin A showed only transient increases. Activin B was expressed and secreted mainly by the hepatocytes and other cells, including cholangiocytes, activated HSCs, and immune cells. Exogenous administration of activin B promoted hepatocyte injury, activated macrophages to release cytokines, and induced a pro-fibrotic expression profile and septa formation in HSCs. Co-treatment of activin A and B interdependently activated the chemokine (C-X-C motif) ligand 1 (CXCL1)/inducible nitric oxide synthase (iNOS) pathway in macrophages and additively upregulated connective tissue growth factor expression in HSCs. Activin B and A had redundant, unique, and interactive effects on the transcripts related to HSC activation. The neutralization of activin B attenuated the development of liver fibrosis and improved liver function in mice with CCl4- or BDL-induced liver fibrosis and largely reversed the already established liver fibrosis in the CCl4 mouse model. These effects were improved by the administration of additional anti-activin A antibody. Combination of both antibodies also inhibited hepatic and circulating inflammatory cytokine production in the BDL mouse model. In conclusion, activin B is a potential circulating biomarker and potent promotor of liver fibrosis. Its levels in the liver and circulation increase significantly in both acute and chronic states of liver injury. Activin B might additively or interdependently cooperate with activin A, which directly acts on multiple liver cell populations during liver injury and fibrosis, as the combination of both proteins increases pro-inflammatory and pro-fibrotic responses in vitro. In addition, the neutralization of both activin A and activin B in vivo enhances the preventive and reversible effects of liver injury and fibrosis compared to that when activin B alone is neutralized. Our data reveal a novel target of liver fibrosis and the mechanism of activin B-mediated initiation of this process by damaging hepatocytes and activating macrophages and HSCs. Our findings show that activin B promotes hepatic fibrogenesis, and that targeting of activin B has anti-inflammatory and anti-fibrotic effects, which ameliorate liver injury by preventing or regressing liver fibrosis. Antagonizing either activin B alone or in combination with activin A prevents and regresses liver fibrosis in multiple animal studies, paving way for future clinical studies.
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    The Roles of Activin A and B in Liver Inflammation and Fibrosis
    (2019-05) Hamang, Matthew J.; Dai, Guoli; Marrs, James; Yaden, Benjamin
    Liver fibrosis is the result of different types of chronic liver diseases, such as cholestatic liver disease and nonalcoholic steatohepatitis, among others. Fibrosis, if left unchecked, may progress to the point of cirrhosis – permanently affecting liver function detrimentally and potentially leading to development of hepatocellular carcinoma. Inflammatory response following tissue injury is vital for the initiation of fibrosis; chronic inflammation results in abnormal tissue healing and promotes a pro-fibrogenic response. Activins are cytokines that have been identified as members of the TGFβ superfamily of growth and differentiation factors. Activin A and B, in particular, have been identified as having roles in the pathophysiology of liver disease, but have not been investigated thoroughly. We treated mice with concanavalin A, a potent T-cell mitogen with liver specificity when administered intravenously, and characterized the resulting response to liver injury and how activin A and B are modulated during this acute inflammatory phase. We showed that activin B is highly increased in circulation following inflammation, as well as locally in the liver as well as the spleen. We then neutralized activin A and B via neutralizing antibodies in our concanavalin A-induced liver injury model to determine if inhibition of these ligands may confer protective effects during the acute inflammatory response in liver. Neutralization of either activin A or activin B protected hepatocytes, improved liver function, and significantly reduced circulating cytokines following concanavalin A administration. Finally, we determined whether inhibition of activin A or B might prevent or reverse the development of liver fibrosis after disease has been established. We induced liver fibrosis in mice via the hepatotoxin carbon tetrachloride, and then treated with neutralizing antibodies while still maintaining carbon tetrachloride administration. Neutralization of activin A and B markedly reduced liver fibrosis, protected hepatocytes, and improved liver function. Our findings implicate both activin A and B as major players in the acute inflammatory response to liver injury, as well as during chronic injury and fibrogenesis, and demonstrate the therapeutic potential of targeting these ligands for the treatment of fibrosis in chronic liver diseases.
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    TGF-beta signaling in an in vivo model of NASH
    (2016) Culver, Alexander; Dai, Guoli; Yaden, Benjamin; Marrs, James
    A burgeoning area of focus within liver disease research is centered on the concomitant muscle atrophy present in end stage liver disease patients which shows a correlation to severity of hepatic fibrosis and transplant survival outcomes. Of particular interest, nonalcoholic steatohepatitis (NASH) is a form of liver disease that is characterized as the hepatic manifestation of metabolic syndrome. If left untreated, the disease can progress to the state of cirrhosis and hepatocellular carcinoma requiring transplant. Concordant with increasing global prevalence of obesity, NASH is projected to become the leading cause for liver transplants by 2020. Due to a lack of therapeutic options, these patients represent a large unmet medical need in the western world. A major hurdle to therapeutic research is the lack of a quick, reproducible, and cost effective in vivo model that recapitulates the plethora of pathologies and their molecular underpinnings manifested by this disorder. Our studies attempted to validate and expand upon a two-hit model of NASH, which incorporated both the integral comorbidities associated with metabolic challenges of obesity along with liver injury. The two-hit model manifests not only the hepatic morphohistological characteristics of the disease, but also incorporates the obligatory muscle atrophy. To further elaborate on the potential direct link between liver and skeletal muscle and remove any confounding issues associated with the model, in vitro administration of hepatotoxins representing various pathologies associated with liver disease, were used to recapitulate the liver-muscle endocrine signaling that exists in vivo. Our data shows that a variety of hepatoxins can elicit hepatocellular damage which releases factors that inhibits myotube size in vitro. The two hit model also preserves many of conserved molecular underpinnings observed in clinical hepatic fibrosis. Of particular interest, the TGFβ superfamily has been demonstrated to play an important regulatory role in the progression of fibrosis in NASH patients. TGFβ, Activin A, and Follistatin are members of the highly conserved family that are increased in NASH patients. Furthermore, these proteins have a well-studied role in muscle health, regeneration, and mass that has been hypothesized to be conserved between liver and muscle tissues. Surprisingly, novel expression of the myokine and negative regulator of muscle mass Gdf8 (myostatin) was increased in our in vivo model as well. Our studies focused on the molecular interactions of these TGFβ superfamily members and their role on liver disease progression. Through specific inhibition of these proteins (Activin A and Gdf8), we demonstrated that they appear to play key individual roles in the progression of the concomitant muscle atrophy observed in NASH patients. Interestingly, superior efficacy was gained with the treatment of a pan inhibitor of these proteins (Activin A, B, Gdf8 etc.) via a soluble decoy receptor (ActRIIB-Fc), suggesting an additional unaccounted for ligand. Activin B, was found to be increased in two separate in vivo models of liver fibrosis (two-hit model and BDL), has been implicated in regulating muscle mass. Our data suggest a pivotal role for several members of the TGFβ superfamily in NASH associated muscle atrophy. Therapies designed to treat liver fibrosis and the resultant decrements in muscle mass and force must account for these agents which will require pan inhibition of TGFβ superfamily ligands that signal through the ActRIIB receptor.