Vascular adhesion protein 1 in nonalcoholic steatohepatitis: A novel biomarker?
Authors: Natalie J. Torok
Journal: Hepatology (2015)

Nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH) is a spectrum of disorders ranging from simple steatosis to progressive steatohepatitis characterized by necroinflammation, leukocyte infiltration, and lipoapoptosis leading to fibrosis and cirrhosis. A major obstacle for diagnosing NASH is that there currently are no acceptable serum biomarkers to distinguish simple steatosis from steatohepatitis.1 In the article by Weston et al.,2 vascular adhesion protein 1 (VAP-1) is described as an important chemoattractant for leukocytes both in soluble (sVAP-1) and receptor forms in three different animal models of steatohepatitis. Furthermore, they show that VAP-1 is directly involved in stellate cell activation and is a strong profibrogenic stimulus. Thus, targeting VAP-1 may result in a decrease in leukocyte recruitment and reduction of inflammation and fibrosis. According to current concepts, during NASH, pathogenic stimuli, such as oxidative stress, dysregulation of the unfolded protein response, autophagy, or inflammasome activation, result in the infiltration of leukocytes triggering immune responses. The key homing signals that are induced in the liver to attract these cells have not been well described. Unlike in other tissues, in the liver, leukocytes are known to tether directly to sinusoidal endothelial cells (SECs) without the help of selectins and instead use a different array of adhesion molecules, including hyaluronan CD44 or VAP-1 (AOC3).3 VAP-1, also known as semicarbazide-sensitive amine oxidase (SSAO), is a multifunctional ectoenzyme expressed by a variety of cells4 and catalyzes the deamination of primary amines producing aldehyde, ammonia, and H2O2. It plays a role in lymphocyte adhesion and transmigration in chronic, but not acute, inflammatory liver diseases.5 Past studies showed that VAP-1-deficient mice exhibited only mild alterations in gut mucosal immunity and did not have an increased risk for infection,6 suggesting that targeting VAP-1 could be a feasible approach to decrease leukocyte adhesion/transmigration during chronic inflammatory injury. Because elevated VAP-1 levels were also described in obesity and diabetes,7 it was plausible that it could have a significant role in the pathogenesis of NASH. Weston et al. demonstrated that, in a cohort of 74 patients with histologically confirmed NASH, serum sVAP-1 was significantly increased, compared to body mass index and metabolic phenotype-matched control patients; and in a univariate analysis, it was correlated to homeostasis model assessment-estimated insulin resistance and diabetes status. After multivariate analysis, serum sVAP-1 was independently associated with fibrosis stage. The area under the receiver operating curve (AUROC) for sVAP-1 to predict histological NASH was 0.82 (95% confidence interval [CI]: 0.74-0.9) with estimated sensitivity of 67.7% and specificity of 89.7%. The AUROC to predict F≥2 fibrosis was 0.71 (95% CI: 0.62-0.79), and with a cutoff of 1,018 ng/mL, sensitivity was estimated to be 45.2% and specificity 91.5%. Interestingly, VAP-1 was present not only in endothelial cells, but also in fibrotic septae and colocalized with alpha-smooth muscle actin–positive myofibroblasts. Mechanistic studies demonstrated that both in vitro activated hepatic stellate cells (HSCs) and isolated, active myofibroblasts express VAP-1 (AOC3 messenger RNA) and possess amine oxidase activity that is comparable to that of VAP-1 in SECs. Activated HSCs induced amine-oxidase–dependent lymphocyte migration in vitro, and this was reduced either by removal of H2O2 or by inhibiting GPCR signaling. The role of VAP-1 in vivo was first studied in the CCl4 model in the total VAP-1−/− (AOC3−/−) mice or in wild-type mice receiving anti-VAP-1 antibody. VAP-1 inhibition or deficiency has attenuated fibrosis and leukocyte infiltration. There was a marked reduction in the number of infiltrating CD8+, CD4+ T cells, and CD19+ B cells at earlier phases (2 weeks), whereas reduction in macrophage/monocytic cells was observed at later stages. To examine VAP-1 in steatohepatitis, experiments were conducted in the methionine-choline–deficient (MCD), high-fat or western lifestyle model (WLM) using trans-fats and fructose. In AOC3−/− mice, similar to the CCl4 model, reduced CD4+ and NK1.1+T-cell populations were observed. CD11b/F4/80-positive cells were unchanged in this dietary model, but there were less CD11b+/Gr+ monocytic cells. Fibrosis was reduced both in AOC3−/− mice and those treated with the anti-VAP1 antibody. Because the MCD model does not recapitulate the metabolic features of human NASH, studies were performed in the high-fat diet and WLM. Surprisingly, serum alanine aminotransferase levels did not decrease in AOC3−/− mice in these models, although there was a significant reduction in CD4+ and NK1.1+ cells after 9 months of steatogenic diet, and this was comparable to the data from the MCD model. The investigators next postulated that VAP-1 SSAO activity was required for development of steatohepatitis and generated a mouse model where VAP-1 was rendered catalytically inactive. When these mice were placed on the MCD diet, similar reductions in CD4+ and NK1.1CD3+ cells were observed as in AOC3−/− mice, suggesting that intact SSAO activity is required for leukocyte homing and progression of steatohepatitis. This is of particular importance, given that the direct signals for leukocyte recruitment have not previously been established. The reduction of inflammatory infiltrate was in parallel with decreasing fibrogenic markers. Given that VAP-1 expression was induced in fibrotic stroma, the next question was whether it had direct profibrogenic effects. Short-term treatment with recombinant VAP-1 did not induce HSC activation or proliferation; however, it enhanced cell spreading and wound closure. LX2 cells, a human activated HSC cell line when transfected with the catalytically active form of VAP-1, exhibited a significant induction of COL1a1 and LOXL2. The fact that VAP-1 antibody (that does not bind to the active site) in vivo also blocked fibrosis suggests that VAP-1 has both enzyme-dependent and -independent profibrogenic effects that would need to be addressed in future studies. Taken together, the investigators have demonstrated complex proinflammatory and -fibrogenic effects of VAP-1 during steatohepatitis involving either recruitment of leukocyte populations and/or a direct effect on HSC activation (Fig. 1). Moreover, serum sVAP-1 levels correlated to histological findings in progressive NASH in a well-characterized cohort of patients. Therefore, sVAP-1 either alone or combined with other potential markers of fibrosis could become a biomarker of disease progression in NASH. Proposed actions of VAP-1 in steatohepatitis. 1. In HSCs, there is a VAP-1-mediated induction of fibrogenic transcripts that is SSAO dependent. 2. Cleaved sVAP-1 in serum correlates to fibrogenic activity in the liver of NASH patients. 3. VAP-1 expressed in SECs or active HSCs enhance recruitment of CD4+, CD8+, and NK1.1+CD3+ cells likely involving SSAO activity/H2O2 production. An important question to address in the future is the mechanism of sVAP-1 cleavage. Dysregulated insulin responses are thought to be linked to VAP-1 shedding; however, the main cellular sources (SECs vs. stromal cells or myofibroblasts) or the mechanisms of cleavage in the liver are not known. Previously, tumor necrosis factor alpha (TNF-α) was shown to regulate VAP-1 ectodomain shedding by matrix metalloproteinases in adipose tissue8; thus, it is plausible that rising serum sVAP-1 reflects increasing leukocytic/monocytic infiltration, TNF-α activity, and sVAP-1 cleavage, leading to a vicious cycle. Interestingly, steatosis was also reduced by deletion of AOC3 or its inhibition by antibodies. As in these studies, no cell-specific approaches were used where there could be multiple explanations (e.g., the role of adipose tissue-derived adipokines could be considered). The concept that VAP-1 plays a role in HSC activation is novel. Given that alternative splicing is described to result in multiple VAP-1 transcripts with some encoding proteins, it would be important to know which form was responsible for the observed profibrogenic effects in NASH. In previous studies, VAP-1 induction was also observed in the fibrotic stroma of biopsy samples with primary biliary cirrhosis, primary sclerosing cholangitis, and alcoholic liver disease. This means that VAP-1 may have inherent profibrogenic roles independent of the metabolic phenotype. Further studies are needed to clarify the role of VAP-1 in stellate cells, or whether its decreased expression/shedding could be correlated to fibrosis reversal. Natalie J. Torok, M.D. University of California Davis and VA Northern California Health System, Sacramento, CA