Non-invasive candidate protein signature predicts hepatic venous pressure gradient reduction in cirrhotic patients after sustained virologic response.

BACKGROUND AND AIMS: A reduction in hepatic venous pressure gradient (HVPG) is the most accurate marker for assessing the severity of portal hypertension and the effectiveness of intervention treatments. This study aimed to evaluate the prognostic potential of blood-based proteomic biomarkers in predicting HVPG response amongst cirrhotic patients with portal hypertension due to Hepatitis C virus (HCV) and had achieved sustained virologic response (SVR). METHODS: The study comprised 59 patients from two cohorts. Patients underwent paired HVPG (pretreatment and after SVR), liver stiffness (LSM), and enhanced liver fibrosis scores (ELF) measurements, as well as proteomics-based profiling on serum samples using SomaScan® at baseline (BL) and after SVR (EOS). Machine learning with feature selection (Caret, Random Forest and RPART) methods were performed to determine the proteins capable of classifying HVPG responders. Model performance was evaluated using AUROC (pROC R package). RESULTS: Patients were stratified by a change in HVPG (EOS vs. BL) into responders (greater than 20% decline in HVPG from BL, or <10 mmHg at EOS with >10 mmHg at BL) and non-responders. LSM and ELF decreased markedly after SVR but did not correlate with HVPG response. SomaScan (SomaLogic, Inc., Boulder, CO) analysis revealed a substantial shift in the peripheral proteome composition, reflected by 82 significantly differentially abundant proteins. Twelve proteins accurately distinguished responders from non-responders, with an AUROC of .86, sensitivity of 83%, specificity of 83%, accuracy of 83%, PPV of 83%, and NPV of 83%. CONCLUSIONS: A combined non-invasive soluble protein signature was identified, capable of accurately predicting HVPG response in HCV liver cirrhosis patients after achieving SVR.

Loss of Hepatic Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 4 and 5 Promotes Nonalcoholic Fatty Liver Disease.

The roof plate-specific spondin-leucine-rich repeat-containing G-protein coupled receptor 4/5 (LGR4/5)-zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) module is a master regulator of hepatic Wnt/ß-catenin signaling and metabolic zonation. However, its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. The current study investigated whether hepatic epithelial cell-specific loss of the Wnt/ß-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice fed with normal diet (ND) or high-fat diet (HFD). Six-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis but the control mice did not. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with those in control mice. An ex vivo primary hepatocyte culture assay and a comprehensive bile acid (BA) characterization in liver, plasma, bile, and feces demonstrated that ND-fed Lgr4/5dLKO mice had impaired BA secretion, predisposing them to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in the livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/ß-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD.

ADAMTSL2 protein and a soluble biomarker signature identify at-risk non-alcoholic steatohepatitis and fibrosis in adults with NAFLD.

BACKGROUND & AIMS: Identifying fibrosis in non-alcoholic fatty liver disease (NAFLD) is essential to predict liver-related outcomes and guide treatment decisions. A protein-based signature of fibrosis could serve as a valuable, non-invasive diagnostic tool. This study sought to identify circulating proteins associated with fibrosis in NAFLD. METHODS: We used aptamer-based proteomics to measure 4,783 proteins in 2 cohorts (Cohort A and B). Targeted, quantitative assays coupling aptamer-based protein pull down and mass spectrometry (SPMS) validated the profiling results in a bariatric and NAFLD cohort (Cohort C and D, respectively). Generalized linear modeling-logistic regression assessed the ability of candidate proteins to classify fibrosis. RESULTS: From the multiplex profiling, 16 proteins differed significantly by fibrosis in cohorts A (n = 62) and B (n = 98). Quantitative and robust SPMS assays were developed for 8 proteins and validated in Cohorts C (n = 71) and D (n = 84). The A disintegrin and metalloproteinase with thrombospondin motifs like 2 (ADAMTSL2) protein accurately distinguished non-alcoholic fatty liver (NAFL)/non-alcoholic steatohepatitis (NASH) with fibrosis stage 0-1 (F0-1) from at-risk NASH with fibrosis stage 2-4, with AUROCs of 0.83 and 0.86 in Cohorts C and D, respectively, and from NASH with significant fibrosis (F2-3), with AUROCs of 0.80 and 0.83 in Cohorts C and D, respectively. An 8-protein panel distinguished NAFL/NASH F0-1 from at-risk NASH (AUROCs 0.90 and 0.87 in Cohort C and D, respectively) and NASH F2-3 (AUROCs 0.89 and 0.83 in Cohorts C and D, respectively). The 8-protein panel and ADAMTSL2 protein had superior performance to the NAFLD fibrosis score and fibrosis-4 score. CONCLUSION: The ADAMTSL2 protein and an 8-protein soluble biomarker panel are highly associated with at-risk NASH and significant fibrosis; they exhibited superior diagnostic performance compared to standard of care fibrosis scores. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver disease worldwide. Diagnosing NAFLD and identifying fibrosis (scarring of the liver) currently requires a liver biopsy. Our study identified novel proteins found in the blood which may identify fibrosis without the need for a liver biopsy.

BET bromodomain inhibitors regulate keratinocyte plasticity.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Lack of the pH-sensing Receptor TDAG8 [GPR65] in Macrophages Plays a Detrimental Role in Murine Models of Inflammatory Bowel Disease.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. METHODS: The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. RESULTS: mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naïve T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. CONCLUSIONS: TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.

Identification of Small Molecules Which Induce Skeletal Muscle Differentiation in Embryonic Stem Cells via Activation of the Wnt and Inhibition of Smad2/3 and Sonic Hedgehog Pathways.

The multilineage differentiation capacity of mouse and human embryonic stem (ES) cells offers a testing platform for small molecules that mediate mammalian lineage determination and cellular specialization. Here we report the identification of two small molecules which drives mouse 129 ES cell differentiation to skeletal muscle with high efficiency without any genetic modification. Mouse embryoid bodies (EBs) were used to screen a library of 1,000 small molecules to identify compounds capable of inducing high levels of Pax3 mRNA. Stimulation of EBs with SMIs (skeletal muscle inducer, SMI1 and SMI2) from the screen resulted in a high percentage of intensively twitching skeletal muscle fibers 3 weeks after induction. Gene expression profiling studies that were carried out for mode of actions analysis showed that SMIs activated genes regulated by the Wnt pathway and inhibited expression of Smad2/3 and Sonic Hedgehog (Shh) target genes. A combination of three small molecules known to modulate these three pathways acted similarly to the SMIs found here, driving ES cells from 129 as well as Balb/c and C57Bl/6 to skeletal muscle. Taken together, these data demonstrate that the SMI drives ES cells to skeletal muscle via concerted activation of the Wnt pathway, and inhibition of Smad2/3 signaling and Shh pathways. This provides important developmental biological information about skeletal muscle differentiation from embryonic stem cells and may lead to the development of new therapeutics for muscle disease.

DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.

Many patients suffering from developmental disorders harbor submicroscopic deletions or duplications that, by affecting the copy number of dosage-sensitive genes or disrupting normal gene expression, lead to disease. However, many aberrations are novel or extremely rare, making clinical interpretation problematic and genotype-phenotype correlations uncertain. Identification of patients sharing a genomic rearrangement and having phenotypic features in common leads to greater certainty in the pathogenic nature of the rearrangement and enables new syndromes to be defined. To facilitate the analysis of these rare events, we have developed an interactive web-based database called DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources) which incorporates a suite of tools designed to aid the interpretation of submicroscopic chromosomal imbalance, inversions, and translocations. DECIPHER catalogs common copy-number changes in normal populations and thus, by exclusion, enables changes that are novel and potentially pathogenic to be identified. DECIPHER enhances genetic counseling by retrieving relevant information from a variety of bioinformatics resources. Known and predicted genes within an aberration are listed in the DECIPHER patient report, and genes of recognized clinical importance are highlighted and prioritized. DECIPHER enables clinical scientists worldwide to maintain records of phenotype and chromosome rearrangement for their patients and, with informed consent, share this information with the wider clinical research community through display in the genome browser Ensembl. By sharing cases worldwide, clusters of rare cases having phenotype and structural rearrangement in common can be identified, leading to the delineation of new syndromes and furthering understanding of gene function.