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Complications of chronic liver diseases - particularly hepatocellular carcinoma (HCC) - are a major cause of mortality worldwide. Several studies have shown that high or increasing levels of serum Squamous Cell Carcinoma Antigen-Immunoglobulin M complex (SCCA-IgM) are associated with development of HCC in patients with advanced liver disease and worse survival in patients with liver cancer. The aim of the present study was to assess, in patients with advanced liver disease, differences in long-term clinical outcomes in relation to baseline levels of serum SCCA-IgM. Ninety one consecutive outpatients with liver cirrhosis of different etiologies, without hepatocellular carcinoma at presentation, were enrolled from April 2007 to October 2012 in a prospective study. For a median time of 127 months, patients were bi-annually re-evaluated. SCCA-IgM complex levels were determined with a validated enzyme-linked immunosorbent assay. The results provided evidence that serum SCCA-IgM is a predictor of overall survival. The best cut-off to discriminate both HCC-free and overall survival rates was 120 AU/mL. Patients with baseline values higher than this threshold showed a substantial increase in both HCC incidence rate and all-cause mortality rate. In conclusion, a single measurement of serum SCCA-IgM helps to identify those patients with liver cirrhosis with increased risks of HCC development and mortality.

Squamous cell carcinoma antigen-1 (SCCA1) overexpression is associated with poor prognosis and chemoresistance in several tumor types, however, the underlying mechanisms remain elusive. Here, we report SCCA1 in relation to the immune and peritumoral adipose tissue microenvironment in early and advanced esophageal adenocarcinoma (EAC). In our series of patients with EAC, free SCCA1 serum levels were associated with significantly worse overall survival, and SCCA1-IgM serum levels showed a trend to a worse overall survival. Serum SCCA1 and intratumoral SCCA1 were inversely correlated with immune activation markers. In agreement with these findings, SCCA1 induced the expression of the immune checkpoint molecule programmed death ligand-1 on monocytes and a direct correlation of these 2 molecules was observed in sequential tumor sections. Furthermore, SCCA1 mRNA expression within the tumor was inversely correlated with stem cell marker expression both within the tumor and in the peritumoral adipose tissue. In vitro, in EAC cell lines treated with different chemotherapeutic drugs, cell viability was significantly modified by SCCA1 presence, as cells overexpressing SCCA1 were significantly more resistant to cell death. In conclusion, poor prognosis in EAC overexpressing SCCA1 is due to reduced tumor chemosensitivity as well as intratumoral immunity impairment, likely induced by this molecule., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objective: To explore the effects of SERPINB3 administration in murine lupus models with a focus on lupus-like nephritis. Methods: 40 NZB/W F1 mice were subdivided into 4 groups and intraperitoneally injected with recombinant SERPINB3 (7.5 μg/0.1 mL or 15 μg/0.1 mL) or PBS (0.1 mL) before (group 1 and 2) or after (group 3 and 4) the development of proteinuria (≥100 mg/dl). Two additional mice groups were provided by including 20 MRL/ lpr mice which were prophylactically injected with SERPINB3 (10 mice, group 5) or PBS (10 mice, group 6). Time of occurrence and levels of anti-dsDNA and anti-C1q antibodies, proteinuria and serum creatinine, overall- and proteinuria-free survival were assessed in mice followed up to natural death. Histological analysis was performed in kidneys of both lupus models. The Th17:Treg cell ratio was assessed by flow-cytometry in splenocytes of treated and untreated MRL/ lpr mice. Statistical analysis was performed using non parametric tests and Kaplan-Meier curves, when indicated. Results: Autoantibody levels and proteinuria were significantly decreased and time of occurrence significantly delayed in SERPINB3-treated mice vs. controls. In agreement with these findings, proteinuria-free and overall survival were significantly improved in SERPINB3-treated groups vs. controls. Histological analysis demonstrated a lower prevalence of severe tubular lesions in kidneys of group 5 vs. group 6. SERPINB3-treated mice showed an overall trend toward a reduced prevalence of severe lesions in both strains. Th17:Treg ratio was significantly decreased in splenocytes of MRL/ lpr mice treated with SERPINB3, compared to untreated control mice. Conclusions: SERPINB3 significantly improves disease course and delays the onset of severe glomerulonephritis in lupus-prone mice, possibly inducing a more tolerogenic immune phenotype.

Goal: To evaluate the potential role of the determination of the immunocomplexed form of squamous cell carcinoma antigen [SCCA-immunoglobulin (Ig)M] for the screening of Barrett esophagus (BE) and esophageal adenocarcinoma (EAC)., Background: The cost-effectiveness of surveillance in BE is still debated and the use of biomarkers in screening and surveillance still not recommended. No information is available regarding SCCA-IgM determination in BE., Study: SCCA-IgM levels were determined (enzyme-linked immunosorbent assay) in 231 patients prospectively recruited, 71 with BE, 53 with EAC, and 107 controls, including 42 blood donors and 65 patients with gastroesophageal reflux. SCCA-IgM cutoffs between BE/EAC and controls and for BE "at risk" versus short nondysplastic BE were calculated by receiver operating characteristic curves. Immunostaining for SCCA-IgM was obtained in a subgroup of patients., Results: Median SCCA-IgM values were significantly higher in BE and EAC than in controls (P=0.0001). Patients with SCCA-IgM levels above the cutoff had a 33 times higher relative risk of harboring BE or EAC (P=0.0001). Patients "at risk," with long or dysplastic BE had SCCA-IgM levels significantly higher than those with short nondysplastic BE (P=0.035) and patients with SCCA-IgM above the cutoff had a 8 times higher relative risk of having BE "at risk." SCCA was expressed in Barrett mucosa but not in cardiac metaplasia., Conclusions: Serum SCCA-IgM determination allows the identification of patients at risk for BE/EAC and the stratification of BE patients in subgroups with different cancer risk. Because of the still limited number of controls, large, prospective studies are required to confirm this evidence.

Non-alcoholic steato-hepatitis (NASH) is a severe disease characterised by liver inflammation and progressive hepatic fibrosis, which may progress to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests that in hepatitis C virus patients steatosis and NASH are associated with faster fibrosis progression and hepatocellular carcinoma. A safe and reliable non-invasive diagnostic method to detect NASH at its early stages is still needed to prevent progression of the disease. We prospectively enrolled 91 hepatitis C virus-positive patients with histologically proven chronic liver disease: 77 patients were included in our study; of these, 10 had NASH. For each patient, various clinical and serological variables were collected. Different algorithms combining squamous cell carcinoma antigen-immunoglobulin-M (SCCA-IgM) levels with other common clinical data were created to provide the probability of having NASH. Our analysis revealed a statistically significant correlation between the histological presence of NASH and SCCA-IgM, insulin, homeostasis model assessment, haemoglobin, high-density lipoprotein and ferritin levels, and smoke. Compared to the use of a single marker, algorithms that combined four, six or seven variables identified NASH with higher accuracy. The best diagnostic performance was obtained with the logistic regression combination, which included all seven variables correlated with NASH. The combination of SCCA-IgM with common clinical data shows promising diagnostic performance for the detection of NASH in hepatitis C virus patients.

Iron overload results in cellular toxicity, tissue injury, organ fibrosis and increased risk of neoplastic transformation. SerpinB3 is a serine protease inhibitor overexpressed in the liver in oxidative stress conditions, able to induce fibrosis and increased risk of malignant transformation. Aim of the present study was to assess the effect of iron overload on SerpinB3 expression in the liver using in vivo and in vitro models.The expression of Serpinb3 was assessed in the liver of hemojuvelin knockout mice (Hjv-/-), an established model of hereditary hemochromatosis, and of wild type control mice, following dietary or pharmacological iron manipulation. To assess the direct effect of iron in vitro, cell lines were treated with different concentration of hemin or with an iron chelator.Hepatic Serpinb3 mRNA and protein were highly expressed in Hjv-/- mice, but not in wild type controls fed with a standard diet. Serpinb3 became detectable in wild type mice fed with a high iron diet or injected with iron dextran; these treatments further induced Serpinb3 expression in Hjv-/- mice. Livers expressing Serpinb3 showed a positive staining also for HIF-2α in the same areas. Hemin promoted induction of SerpinB3 mRNA in HeLa and HA22T/VGH cells, but a mild stimulation of SerpinB3 promoter activity in HeLa and Huh7 cells. In conclusion, Serpinb3 is strongly induced by iron in the mouse liver. The molecular link between iron, ROS and SerpinB3 seems to be HIF-2α, which is induced by iron overload and was previously found capable of up-regulating SerpinB3 at the transcriptional level., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

SerpinB3 is a hypoxia- and hypoxia-inducible factor-2α-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-β1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. "In vitro" experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl 4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and αSMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.

Background: Squamous cell carcinoma antigen (SCCA)-IgM complex has been described as a promising tool to identify patients with progressive liver disease at higher risk of hepatocellular carcinoma (HCC) development in retrospective studies., Aim: To assess the clinical value of this biomarker in patients with cirrhosis in a prospective study., Methods: Patients with overt cirrhosis were prospectively evaluated at 6-month intervals for HCC development and decompensation with clinical examination, liver ultrasound, α-fetoprotein measurement. SCCA-IgM was measured in serum by immunoenzymatic assay. Median follow-up duration was 52 months (range 12-68 months)., Results: 70 patients (26% male; mean age 56±10 years) were enrolled. The main aetiological factors were alcohol (44%) and hepatitis C (34%). Baseline values of SCCA-IgM were significantly higher in patients who developed HCC. Positivity of the biomarker at baseline was associated with a significantly shorter HCC-free survival, while α-fetoprotein (cut off >20 ng/ml) was not significant. SCCA-IgM positivity and hepatitis C were significant prognostic factors for HCC development. The biomarker was not associated with the development of clinical complications of cirrhosis., Conclusion: This prospective study demonstrates that in patients with cirrhosis SCCA-IgM is associated with HCC development and may be useful for clinical management of cirrhotic patients at higher risk of HCC development., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

SerpinB3 has been recently described as an early marker of liver carcinogenesis, but the potential mechanistic role of this serpin in tumor development is still poorly understood. Overexpression of Myc often correlates with more aggressive tumour forms, supporting its involvement in carcinogenesis. Yes-associated protein (Yap), the main effector of the Hippo pathway, is a central regulator of proliferation and it has been found up-regulated in hepatocellular carcinomas. The study has been designed to investigate and characterize the interplay and functional modulation of Myc by SerpinB3 in liver cancer. Results from this study indicate that Myc was up-regulated by SerpinB3 through calpain and Hippo-dependent molecular mechanisms in transgenic mice and hepatoma cells overexpressing human SerpinB3, and also in human hepatocellular carcinomas. Human recombinant SerpinB3 was capable to inhibit the activity of Calpain in vitro, likely reducing its ability to cleave Myc in its non oncogenic Myc-nick cytoplasmic form. SerpinB3 indirectly increased the transcription of Myc through the induction of Yap pathway. These findings provide for the first time evidence that SerpinB3 can improve the production of Myc through direct and indirect mechanisms that include the inhibition of generation of its cytoplasmic form and the activation of Yap pathway.

SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells. Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively. Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.

Aims/hypothesis: Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited., Methods: Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out., Results: Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement., Conclusions/interpretation: We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.

The physiological roles of the protease inhibitor SERPINB3 (SB3) are still largely unknown. The study was addressed to assess the biological effects of this serpin in vivo using a SB3 transgenic mouse model. Two colonies of mice (123 transgenic for SB3 and 148 C57BL/6J controls) have been studied. Transgenic (TG) mice showed longer survival than controls and the difference was more remarkable in males than in females (18.5% vs 12.7% life span increase). In TG mice decreased IL-6 in serum and lower p66shc in the liver were observed. In addition, TG males showed higher expression of mTOR in the liver. Liver histology showed age-dependent increase of steatosis and decrease of glycogen storage in both groups and none of the animals developed neoplastic lesions. In conclusion, the gain in life span observed in SB3-transgenic mice could be determined by multiple mechanisms, including the decrease of circulating IL-6 and the modulation of ageing genes in the liver.

IgM antibodies bound to different cancer antigens have shown recently a higher diagnostic value, compared with the corresponding free molecule, giving rise to a new family of biomarkers. High or increasing levels of Squamous Cell Carcinoma Antigen (SCCA)-IgM immune complexes were associated with more advanced liver disease and increased risk of development of HCC. Rheumatoid factor (RF) represents a long-standing problem of interference for immunometric assays. The aim of the present study was to examine the specificity of SCCA-IgM in relation to the presence of RF reactivity in patients infected with hepatitis C virus (HCV). Sera of 73 patients with cirrhosis, infected with HCV, (mean age ± SD: 66 ± 13 years; M/F: 45/28), including 21 patients with HCC, were studied. SCCA-IgM immune complexes levels were measured by a commercial ELISA. To evaluate the possible interfering effect of RF, the standard calibrator, positive for SCCA-IgM, was spiked with serial dilutions of a RF positive or negative serum. SCCA-IgM immune complexes were positive in 35 out of 73 (48%) patients, while RF activity was found in 10 out of 73 (14%) patients. Patients with cirrhosis with RF activity had significantly higher levels of SCCA-IgM, compared to RF negative cases; however, no significant correlation between SCCA-IgM and RF values was observed. In samples created artificially the same results in terms of reactivity for SCCA-IgM were obtained, regardless of the presence of RF activity. These findings support the lack of correlation between the two parameters found in sera of patients infected with HCV., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development., Aim: To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels., Methodology and Findings: 79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140-0.427) vs. 0.140 (IQR 0.140-0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079)., Conclusions: IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels.

SERPINB3 has been found in chronic liver damage and hepatocellular carcinoma, but not in normal liver. By direct mRNA sequencing, a new SERPINB3 polymorphism (SCCA-PD) has been identified, presenting the substitution Gly351Ala in the reactive center loop of the protein. The prevalence of the SCCA-PD isoform has been found to be significantly higher in patients with cirrhosis than in patients with chronic liver disease and in normal subjects. The aim of this study was to investigate the biological and functional activity of SERPINB3 isoforms using in vitro models. HepG2 and Huh7 cells lines were transfected with plasmid vectors containing wild-type SERPINB3 or its polymorphic variant SCCA-PD and their expression at transcriptional and protein level was determined. To assess the functional activity, both recombinant proteins were produced and kinetic analysis was carried out using papain and cathepsin-L as target proteases. In addition, the inhibition of JNK kinase activity by SERPINB3 isoforms was assessed. The crystal structure of wild-type SERPINB3 at 2.7 Å resolution was used for preparation of refined 3D models of the two isoforms. The results showed that transcriptional activity and protein expression of the two isoforms were similar in both transfected cell lines. Both SERPINB3 preparations exerted a dose-dependent protease inhibitory activity, but the effect of SCCA-PD was higher than that of the wild-type isoform. This result was supported by 3D modelling, where increased hydrophobic profile of the SCCA-PD isoform, introduced by the G351A mutation, was detected. In addition, at high protein concentration, SCCA-PD revealed a 16% higher inhibitory effect on c-Jun phosphorylation by JNK1, compared with wild-type SERPINB3. In conclusion, the single amino acid substitution in the SERPINB3 reactive site loop improves the functional activity of SCCA-PD isoform. This different antiprotease activity might favor disease progression in patients carrying this polymorphism.

Transforming growth factor-beta1 (TGF-beta1) is the master cytokine in the pathogenesis of liver fibrosis. TGF-beta1 and extent of fibrosis were correlated recently to the serpin SERPINB3 in idiopathic pulmonary fibrosis, a chronic disease recalling liver cirrhosis. The aim of this study was to assess the relation between SERPINB3, TGF-beta1 and fibrosis in chronic liver diseases and to determine the effect of this serpin on TGF-beta1 expression using in vitro models. SERPINB3 and TGF-beta1 were evaluated in liver biopsies of 94 patients with chronic liver disease. The effect of SERPINB3 on TGF-beta1 expression was determined in primary human hepatocytes, HepG2 and Huh7 cells transfected with intact SERPINB3 human gene or with reactive site loop deleted mutants. A significant correlation between TGF-beta1 and SERPINB3 at the protein level was observed in liver biopsies, confirmed by a positive correlation at mRNA level. Both proteins were correlated to the extent of liver fibrosis. All transfected cells showed increased TGF-beta1 mRNA and protein production and the integrity of the reactive site loop of the serpin was crucial to achieve this effect. In conclusion, chronically damaged hepatocytes produce SERPINB3 and TGF-beta, and the anti-protease activity of this serpin might be implicated in TGF-beta1 induction.

Background: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation., Methods: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody., Results: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests., Conclusions: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis.

Squamous Cell Carcinoma Antigen-1 (SCCA1) overexpression has been observed in tumours of epithelial origin and in hepatocellular carcinoma. Previous data indicate that this serpin inhibits apoptosis, while its proliferative activity was only recently proposed. The aim of this study was to evaluate the effect of SCCA1 on liver cells in a transgenic mouse model after partial hepatectomy. Twenty-one C57BL/6J mice (11 transgenic for human SCCA1, 10 wild-type) underwent partial hepatectomy and were sacrified after one week. Apoptosis and proliferation markers were determined in the liver at sacrifice, while a cytokine panel was measured in serum. Transgenic mice showed a relative liver weight significantly higher than wild-type mice at sacrifice (mean +/- SD, 5.38+/-0.50% vs 4.84+/-0.29%, p=0.0221), while no difference (p=0.2403) was observed in two untreated control groups (6 transgenic, 6 wild-type mice). Active caspase-3 was significantly lower in transgenic mice than in wild-type mice (p=0.0047). The transgenic mouse group showed overall higher proliferative activity at sacrifice, compared to wild-type mice, with increased proliferation parameters. Cytokine analysis revealed a remarkable and opposite sex-dependent behaviour of interleukin (IL)-6 after hepatectomy. At variance with wild-type mice, a significant IL-6 increase was documented only in transgenic females (p=0.0313), even more relevant than that observed in wild-type males. In conclusion, transgenic mice expressing SCCA1 showed higher liver regenerative potential compared to wild-type mice, supporting the dual role of this serpin as an anti-apoptotic and pro-proliferative stimulus for liver cells in vivo.

The serine protease inhibitor SerpinB3 has been described as critical mediator of liver fibrosis and it has been recently proposed as an additional hepatokine involved in NASH development and insulin resistance. Protease Activated Receptor 2 has been identified as a novel regulator of hepatic metabolism. A targeted therapeutic strategy for NASH has been investigated, using 1-Piperidine Propionic Acid (1-PPA), since this compound has been recently proposed as both Protease Activated Receptor 2 and SerpinB3 inhibitor. The effect of SerpinB3 on inflammation and fibrosis genes was assessed in human macrophage and stellate cell lines. Transgenic mice, either overexpressing SerpinB3 or carrying Serpinb3 deletion and their relative wild type strains, were used in experimental NASH models. Subgroups of SerpinB3 transgenic mice and their controls were also injected with 1-PPA to assess the efficacy of this compound in NASH inhibition. 1-PPA did not present significant cell and organ toxicity and was able to inhibit SerpinB3 and PAR2 in a dose-dependent manner. This effect was associated to a parallel reduction of the synthesis of the molecules induced by endogenous SerpinB3 or by its paracrine effects both in vitro and in vivo , leading to inhibition of lipid accumulation, inflammation and fibrosis in experimental NASH. At mechanistic level, the antiprotease activity of SerpinB3 was found essential for PAR2 activation, determining upregulation of the CCAAT Enhancer Binding Protein beta (C/EBP-β), another pivotal regulator of metabolism, inflammation and fibrosis, which in turn determined SerpinB3 synthesis. 1-PPA treatment was able to inhibit the PAR2 - C/EBP-β - SerpinB3 axis and to protect from NASH development and progression, supporting the potential use of a similar approach for a targeted therapy of NASH. [Display omitted] • SerpinB3 is a mediator of liver inflammation, steatosis and fibrosis. • The Protease Activated Receptor 2 (PAR2) is activated by SerpinB3. • The CCAAT Enhancer Binding Protein-beta is essential for SerpinB3 synthesis. • 1-Piperidine Propionic Acid inhibits PAR2 and protects from non-alcoholic steatohepatitis. [ABSTRACT FROM AUTHOR]