Kostallari, Enis

BET bromodomain inhibitors demonstrate significant promise as anti-inflammatory agents targeting a variety of inflammatory disorders. However, clinical data has demonstrated that the administration of non-selective BET bromodomain inhibitors has led to significant dose-limiting toxicity in clinical settings. More selective inhibitors of the second BET bromodomains, referred to as pan-D2 inhibitors, are better tolerated, however their efficacy varies significantly depending on disease indication. Here, we use three orally bioavailable inhibitors, 1–3, that are either selective for the first N-terminal bromodomain of BRD4 (BRD4-D1 selective), or pan-D1 biased, for assessing their cellular and in vivo efficacy and safety profile in two models of inflammatory liver disease. We compare their effects to known pan-D2 and pan-BET inhibitors. Our results show that pan-D1 biased inhibitor, 3, is as efficacious as the pan-BET inhibitor I-BET151 in reducing inflammation from LPS-induced and drug-induced toxicity, whereas pan-D2 inhibitors are less effective. BRD4-D1 selective inhibitors, 1 and 2, are also efficacious; however, new inhibitors with improved cellular target engagement will be necessary to more thoroughly assess their effects. Finally, BRD4-D1 selective inhibitors are better tolerated in a preclinical model of thrombocytopenia than pan-D1 biased inhibitors, while gastrointestinal (GI) toxicity may be a BRD4-driven effect. These results highlight the importance of assessing specific BET protein and bromodomain functions due to their varying roles in diverse disease models.

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.

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.

Liver sinusoidal endothelial cells (LSECs) are key players in maintaining hepatic homeostasis. They also play crucial roles during liver injury by communicating with liver cell types as well as immune cells and promoting portal hypertension, fibrosis, and inflammation. Cutting-edge technology, such as single cell and spatial transcriptomics, have revealed the existence of distinct LSEC subpopulations with a clear zonation in the liver. The signals released by LSECs are commonly called “angiocrine signaling.” In this review, we summarize the role of angiocrine signaling in health and disease, including zonation in healthy liver, regeneration, fibrosis, portal hypertension, nonalcoholic fatty liver disease, alcohol-associated liver disease, aging, drug-induced liver injury, and ischemia/reperfusion, as well as potential therapeutic advances. In conclusion, sinusoidal endotheliopathy is recognized in liver disease and promising preclinical studies are paving the path toward LSEC-specific pharmacotherapies.

LSECs are a unique population of endothelial cells within the liver and are recognized as key regulators of liver homeostasis. LSECs also play a key role in liver disease, as dysregulation of their quiescent phenotype promotes pathological processes within the liver including inflammation, microvascular thrombosis, fibrosis, and portal hypertension. Recent technical advances in single-cell analysis have characterized distinct subpopulations of the LSECs themselves with a high resolution and defined their gene expression profile and phenotype, broadening our understanding of their mechanistic role in liver biology. This article will review 4 broad advances in our understanding of LSEC biology in general: (1) LSEC heterogeneity, (2) LSEC aging and senescence, (3) LSEC role in liver regeneration, and (4) LSEC role in liver inflammation and will then review the role of LSECs in various liver pathologies including fibrosis, DILI, alcohol-associated liver disease, NASH, viral hepatitis, liver transplant rejection, and ischemia reperfusion injury. The review will conclude with a discussion of gaps in knowledge and areas for future research.

The search for non-invasive, fast, and low-cost diagnostic tools has gained significant traction among many researchers worldwide. Dielectric properties calculated from microwave signals offer unique insights into biological tissue. Material properties, such as relative permittivity (εr) and conductivity (σ), can vary significantly between healthy and unhealthy tissue types at a given frequency. Understanding this difference in properties is key for identifying the disease state. The frequency-dependent nature of the dielectric measurements results in large datasets, which can be postprocessed using artificial intelligence (AI) methods. In this work, the dielectric properties of liver tissues in three mouse models of liver disease are characterized using dielectric spectroscopy. The measurements are grouped into four categories based on the diets or disease state of the mice, i.e., healthy mice, mice with non-alcoholic steatohepatitis (NASH) induced by choline-deficient high-fat diet, mice with NASH induced by western diet, and mice with liver fibrosis. Multi-class classification machine learning (ML) models are then explored to differentiate the liver tissue groups based on dielectric measurements. The results show that the support vector machine (SVM) model was able to differentiate the tissue groups with an accuracy up to 90%. This technology pipeline, thus, shows great potential for developing the next generation non-invasive diagnostic tools.

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.