Gao, Jinhang

Cellular and molecular heterogeneity in the liver has been increasingly recognized to drive liver fibrosis progression, but the particular events that occur initially in response to liver injury and trigger immune cell recruitment remain unclear. Here, we identify epigenetically aberrant liver sinusoidal endothelial cells (LSECs) as key players in this process. Mechanistically, the epigenetic readers like bromodomain-containing protein 4–dependent (BRD4-dependent) super enhancers (SEs) activate proinflammatory genes, including promyelocytic leukemia (PML). The PML protein, in turn, binds BRD4 and amplifies proinflammatory angiocrine signaling through phase separation–dependent SE activation via PML/BRD4 condensate formation. In mouse models, LSEC-specific depletion of the PML/BRD4 complex mitigates liver inflammation and fibrosis. Single-cell RNA-seq reveals that epigenetically aberrant LSECs exhibit a reprogrammed proinflammatory angiocrine landscape in mouse fibrotic livers. TIMP1 + LSECs promote the recruitment of CD63 + monocyte–derived macrophages (MoMFs) during liver fibrosis progression. Thereby, PML/BRD4 in LSECs governs inflammatory immune cell recruitment in liver fibrosis. Pharmacological BRD4 inhibition or epigenetic PML-SE repression alleviates liver inflammation and fibrosis. In conclusion, PML/BRD4-mediated SE activation via phase separation drives proinflammatory angiocrine signaling in LSECs, initiating the inflammatory cascade and subsequent immune cell recruitment during liver fibrosis.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

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.