Liver macrophages are central in maintaining hepatic homeostasis and mediating immune responses during liver injury, including fibrosis. Macrophages may have proinflammatory or antiinflammatory properties, but which properties influence fibrosis remains unclear. To explore the role of macrophages in liver fibrosis, we performed single-cell RNA-seq in a mouse model of liver injury and found that macrophage diversity was increased. Marco was among the most significantly upregulated genes, and a population of Marcohi macrophages increased with injury and spatially segregated to nonfibrotic areas. The macrophage receptor with collagenous structure (MARCO) protein is a scavenger receptor expressed by specific subsets of macrophages, and its role in liver fibrosis is unclear. In vitro induction of Marco in bone marrow-derived macrophages decreased proinflammatory gene expression, increased antiinflammatory and antifibrotic gene expression, and enhanced phagocytosis, indicating a restorative phenotype. Adoptive transfer of MARCO+ macrophages in a mouse model of liver fibrosis reduced the expression of extracellular matrix-associated (ECM-associated) genes in hepatic stellate cells (HSCs) and reduced collagen deposition, which did not occur with the transfer of MARCO- macrophages. Therefore, MARCO+ macrophages have a tissue restorative role in the liver and attenuate fibrogenesis through interaction with HSCs, thereby providing a potential therapeutic pathway for liver fibrosis.

BACKGROUND & AIMS:Programmed death-ligand 1 (PD-L1) on hepatic stellate cells (HSCs) is required for HSC activation and suppressing T and B lymphocytes. We tested whether targeting HSC PD-L1 influenced liver inflammation and fibrosis in a carbon tetrachloride (CCl4) injury mouse model. METHODS:HSC-specific PD-L1 knockout (PD-L1HSCKO) mice were created by crossing Cd274 floxed mice to Collagen1A1-Cre mice. CCl4 was injected into PD-L1HSCKO and PD-L1HSCWT mice twice weekly for 6 weeks. Liver fibrosis was assessed by Trichrome and Picrosirius Red staining; HSC activation was determined by immunofluorescence and Western blot for HSC activation markers; liver inflammation was studied by multiplex immunofluorescence and cytokine profiling. Multiomics was leveraged to determine how targeting PD-L1 altered HSC producing collagens and cytokines/chemokines. RESULTS:Collagen deposition was reduced in CCl4-injured PD-L1HSCKO livers compared with CCl4-injured PD-L1HSCWT livers; myofibroblast density was lower in CCl4-injured PD-L1HSCKO livers compared with CCl4-injured PD-L1HSCWT livers. CCl4-injured PD-L1HSCKO livers had higher lymphocyte densities (GranzymeB+, CD8a+, CD20+) but lower Kupffer and myeloid cell densities (F4/80+ and CD11b+) compared with CCl4-injured PD-L1HSCWT livers. Serum aspartate aminotransferase and alanine aminotransferase, however, were similarly elevated by CCl4 in both groups. Spatial and bulk-cell transcriptomics revealed a global transcriptomic change of HSCs induced by PD-L1 targeting. A targeted proteomics identified that HSC secretion of a group of cytokines/chemokines, including growth/differentiation factor 15, granulocyte-macrophage colony-stimulating factor, C-X-C motif and C-C motif chemokines, was altered upon PD-L1 targeting, highlighting the role of HSC PD-L1 in HSC/Kupffer and HSC/myeloid cell interactions during HSC activation and fibrosis development. CONCLUSIONS:Targeting HSC PD-L1 altered HSC transcriptome and liver inflammation, and suppressed liver fibrosis, representing a potential therapeutic strategy for liver fibrosis.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA Abbreviations: COX-2, cyclooxygenase 2; MASH, metabolic dysfunction–associated steatohepatitis; NET, neutrophil extracellular trap. Correspondence Sofia Jerez, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, USA. Email: [email protected]

BACKGROUND & AIMS:Congestion alters the microenvironment of the liver sinusoid along the portal-central axis. We studied spatial changes in immune cells in the sinusoid that contribute to congestive fibrosis and portal hypertension (PHTN). METHODS:To visualize the distribution of immune cells in congestive hepatopathy (CH), we performed imaging mass cytometry (IMC) on liver tissue from patients with CH, Fontan-associated liver disease (FALD), and controls. We performed partial ligation of the inferior vena cava (pIVCL) to simulate CH in mice and isolated primary liver cells for single-cell RNA-sequencing (scRNA-seq) to study zonation of liver sinusoidal endothelial cells (LSECs). After pIVCL, mice were treated with intraperitoneal injections of AMG487, an inhibitor of the CXCL9 receptor, or a neutralizing antibody to CXCL9. RESULTS:Intra-hepatic macrophages are enriched in CH and FALD. Given the role of CXCL9 in macrophage patterning in the liver, we performed RNA in situ hybridization (RNAish) in CH and determined that CXCL9 was highly expressed in LSECs in FALD, suggesting that LSECs recruit macrophages in CH. After pIVCL, treatment with AMG487 or an antibody to CXCL9 attenuated portal pressures, fibrosis, and intra-hepatic macrophages. To study changes in LSECs that promote macrophage chemotaxis, we performed scRNA-seq after pIVCL and sham procedures. Analysis revealed 3 LSEC subpopulations according to sinusoidal location. RNAish identified peri-central LSECs as the predominant source of CXCL9 in FALD. In vitro analyses revealed that β-catenin and hypoxia inducible factor-1 α regulate CXCL9 transcription in peri-central LSECs. CONCLUSIONS:CXCL9 derived from peri-central LSECs enriches intra-hepatic macrophages in CH and FALD, contributing to congestive fibrosis and PHTN. Strategies to target LSEC-derived CXCL9 may prevent the progression of CH and FALD.

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.

Chronic livers diseases, including non-alcoholic steatohepatitis (NASH), alcohol-related liver disease (ALD) and viral hepatitis, may lead to cirrhosis, which is a leading cause of death (Xu et al., 2022; Devarbhavi et al., 2023). Efficient therapies don’t exist, defining a space of unmet needs. Metabolic pathways play an important role in the establishment of chronic liver diseases, where hepatic stellate cells, immune cells and other cell types play crucial roles (Chen et al. Kostallari and Shah, 2016; Drinane et al., 2017; Maiers et al., 2017; Du et al., 2018; Kostallari et al., 2018; Haak et al., 2019; Hilscher et al., 2019; Arab et al., 2020; Azad et al., 2020; Gao et al., 2020; Mejias et al., 2020; Yaqoob et al., 2020; Kostallari et al., 2021; Greuter et al., 2022; Kostallari et al., 2022; McConnell et al., 2023; Xiao et al., 2023). However, their study is just starting to flourish. This second volume of “Chronic Liver Disease: New Targets and New Mechanisms” Research Topic continues to present recent advances in the field of chronic liver disease which might point towards the next potential therapeutic advances.

EDITORIAL article Front. Mol. Biosci., 18 July 2023Sec. Molecular Diagnostics and Therapeutics Volume 10 - 2023 | https://doi.org/10.3389/fmolb.2023.1237824

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis.

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2 , in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C’s epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement.

Purpose We evaluated biological effects of distinct local anesthetics on human adipose-derived mesenchymal stem cells when applied to reduce periprocedural pain during mesenchymal stem cell injections. Methods and Materials Metabolic activity (MTS assay), viability (Live/Dead stain), and gene expression (quantitative real-time reverse-transcriptase polymerase chain reaction) were measured in mesenchymal stem cells incubated with various concentrations of lidocaine, ropivacaine, or bupivacaine during a 12-hr time course. Results Cell viability and metabolic activity decreased in a dose, time, and substance-specific manner after exposure to lidocaine, ropivacaine, and bupivacaine, with ropivacaine being the least cytotoxic. Cell viability decreases after brief exposure (<1.5 hrs) at clinically relevant concentrations (eg, 8 mg/ml of lidocaine, 2.5 mg/ml of ropivacaine or bupivacaine). Mesenchymal stem cells exposed to local anesthetics change their expression of mRNA biomarkers for stress response (EGR1, EGR2), proliferation (MKI67, HIST2H4A), ECM (COL1A1, COL3A1), and cell surface marker (CD105). Conclusions Local anesthetics are cytotoxic to clinical-grade human mesenchymal stem cells in a dose-, time-, and agent-dependent manner and change expression of ECM, proliferation, and cell surface markers. Lidocaine and bupivacaine are more cytotoxic than ropivacaine. Single-dose injections of local anesthetics may affect the biological properties of mesenchymal stem cells in vitro but may not affect the effective dose of MSCs in a clinical setting.