Citations

Background: Immune cell–driven inflammation is a key mediator of metabolic dysfunction–associated steatohepatitis (MASH) progression. We have previously demonstrated that pharmacological sphingosine 1-phosphate (S1P) receptor modulation ameliorates MASH and is associated with attenuated accumulation of intrahepatic macrophage and T-cell subsets. Although S1P receptors are expressed on several immune cell types, given the prominent role of monocyte-derived recruited macrophages in the sterile inflammation of MASH, we hypothesized that deletion of S1P receptor 1 (S1P 1 ) on myeloid cells may ameliorate MASH by reducing the accumulation of proinflammatory monocyte-derived macrophages in the liver. Methods: The LyzMCre approach was used to generate myeloid cell–specific knockout mice, termed S1pr1 MKO . Littermate S1pr1 loxp/loxp mice were used as wild-type controls. MASH was established by feeding mice a high-fat, -fructose, and -cholesterol (FFC) diet for 24 weeks, which led to the development of steatohepatitis and MASH-defining cardiometabolic risk factors. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by mass cytometry and immunohistochemistry. Results: Histological examination demonstrated a reduction in liver inflammatory infiltrates and fibrosis in high-fat, -fructose, and -cholesterol–fed S1pr1 MKO compared to wild-type. There was a corresponding reduction in alanine aminotransferase, a sensitive marker for liver injury. As determined by mass cytometry, a significant decrease in recruited macrophages was noted in the livers of high-fat, -fructose, and -cholesterol–fed S1pr1 MKO mice compared to wild-type. Gene ontology pathway analysis revealed significant suppression of the peroxisome proliferator–activated receptor gamma and mitogen-activated protein kinase pathways in S1pr1 MKO consistent with attenuated MASH in mice. Conclusions: Deletion of S1P 1 in myeloid cells is sufficient to attenuate intrahepatic accumulation of monocyte-derived macrophages and ameliorate murine MASH.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB–dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

Objective: Circulating exosome-enriched extracellular vesicles (EVs) have drawn considerable importance in obesity-related insulin-resistance (IR). We sought to compare the proteomics profile of serum exosomes from normal individuals and those with obesity and IR. Methods: We isolated serum exosomes from male subjects with obesity and insulin resistance (Ob-IR, HOMA-IR > 2.0) and lean/overweight insulin-sensitive (Normal (N), HOMA-IR < 2.0) individuals. The differential protein expression between the two groups was detected by a label-free quantitative mass spectrometry analysis followed by GO annotation and ingenuity pathway analysis (IPA). Results: We identified 23 upregulated and 46 downregulated proteins between Ob-IR and N groups. Some of these proteins are involved in altering insulin signaling (VPS13C, TBC1D32, TTR, and ADIPOQ), inflammation (NFκB and CRP), and B-cell proliferation/activation (IGLV4-69, IGKV1D-13, and IGHV4-28). GO analysis revealed that the differentially expressed proteins (DEPs) are mainly involved in regulating immune cell activation and are located in extracellular space. IPA analysis showed that top molecules mediating IR, inflammation and B-cell activation were upregulated in Ob-IR subjects compared to N subjects. Conclusions: Serum exosomal proteins can be used as biomarkers to identify the future risk of diabetes and a therapeutic target to prevent or slow down the progression of diabetes in high-risk individuals.

Background and Aims Acute kidney injury (AKI) has a poor prognosis in cirrhosis. Given the variability of creatinine, the prediction of AKI and dialysis by other markers is needed. The aim of this study is to determine the role of serum and urine metabolomics in the prediction of AKI and dialysis in an inpatient cirrhosis cohort. Approach and Results Inpatients with cirrhosis from 11 North American Consortium of End‐stage Liver Disease centers who provided admission serum/urine when they were AKI and dialysis‐free were included. Analysis of covariance adjusted for demographics, infection, and cirrhosis severity was performed to identify metabolites that differed among patients (1) who developed AKI or not; (2) required dialysis or not; and/pr (3) within AKI subgroups who needed dialysis or not. We performed random forest and AUC analyses to identify specific metabolite(s) associated with outcomes. Logistic regression with clinical variables with/without metabolites was performed. A total of 602 patients gave serum (218 developed AKI, 80 needed dialysis) and 435 gave urine (164 developed AKI, 61 needed dialysis). For AKI prediction, clinical factor–adjusted AUC was 0.91 for serum and 0.88 for urine. Major metabolites such as uremic toxins (2,3‐dihydroxy‐5‐methylthio‐4‐pentenoic acid [DMTPA], N2N2dimethylguanosine, uridine/pseudouridine) and tryptophan/tyrosine metabolites (kynunerate, 8‐methoxykyunerate, quinolinate) were higher in patients who developed AKI. For dialysis prediction, clinical factor–adjusted AUC was 0.93 for serum and 0.91 for urine. Similar metabolites as AKI were altered here. For dialysis prediction in those with AKI, the AUC was 0.81 and 0.79 for serum/urine. Lower branched‐chain amino‐acid (BCAA) metabolites but higher cysteine, tryptophan, glutamate, and DMTPA were seen in patients with AKI needing dialysis. Serum/urine metabolites were additive to clinical variables for all outcomes. Conclusions Specific admission urinary and serum metabolites were significantly additive to clinical variables to predict AKI development and dialysis initiation in inpatients with cirrhosis. These observations can potentially facilitate earlier initiation of renoprotective measures.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.