Processing of angiocrine alarmin IL-1α in endothelial cells promotes lung and liver fibrosis.

BACKGROUND: Fibrosis results from excessive scar formation after tissue injury. Injured cells release alarmins such as interleukin 1 (IL-1) α and ß as primary mediators initiating tissue repair. However, how alarmins from different cell types differentially regulate fibrosis remains to be explored. METHODS: Here, we used tissue specific knockout strategy to illustrate a unique contribution of endothelial cell-derived IL-1α to lung and liver fibrosis. The two fibrotic animal model triggered by bleomycin and CCl4 were used to study the effects of endothelial paracrine/angiocrine IL-1α in fibrotic progression. Human umbilical vein endothelial cells (HUVEC) were performed to explore the production of angiocrine IL-1α at both transcriptional and post-transcriptional levels in vitro. RESULTS: We found that endothelial paracrine/angiocrine IL-1α primarily promotes lung and liver fibrosis during the early phase of organ repair. By contrast, myeloid cell-specific ablation of IL-1α in mice resulted in little influence on fibrosis, suggesting the specific pro-fibrotic role of IL-1α from endothelial cell but not macrophage. In vitro study revealed a coordinated regulation of IL-1α production in human primary endothelial cells at both transcriptional and post-transcriptional levels. Specifically, the transcription of IL-1α is regulated by RIPK1, and after caspase-8 (CASP8) cleaves the precursor form of IL-1α, its secretion is triggered by ion channel Pannexin 1 upon CASP8 cleavage. CONCLUSIONS: Endothelial cell-produced IL-1α plays a unique role in promoting organ fibrosis. Furthermore, the release of this angiocrine alarmin relies on a unique molecular mechanism involving RIPK1, CASP8, and ion channel Pannexin 1.

Lung regeneration: diverse cell types and the therapeutic potential.

Lung tissue has a certain regenerative ability and triggers repair procedures after injury. Under controllable conditions, lung tissue can restore normal structure and function. Disruptions in this process can lead to respiratory system failure and even death, causing substantial medical burden. The main types of respiratory diseases are chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and acute respiratory distress syndrome (ARDS). Multiple cells, such as lung epithelial cells, endothelial cells, fibroblasts, and immune cells, are involved in regulating the repair process after lung injury. Although the mechanism that regulates the process of lung repair has not been fully elucidated, clinical trials targeting different cells and signaling pathways have achieved some therapeutic effects in different respiratory diseases. In this review, we provide an overview of the cell type involved in the process of lung regeneration and repair, research models, and summarize molecular mechanisms involved in the regulation of lung regeneration and fibrosis. Moreover, we discuss the current clinical trials of stem cell therapy and pharmacological strategies for COPD, IPF, and ARDS treatment. This review provides a reference for further research on the molecular and cellular mechanisms of lung regeneration, drug development, and clinical trials.

Endothelial H2S-AMPK dysfunction upregulates the angiocrine factor PAI-1 and contributes to lung fibrosis.

Dysfunction of the vascular angiocrine system is critically involved in regenerative defects and fibrosis of injured organs. Previous studies have identified various angiocrine factors and found that risk factors such as aging and metabolic disorders can disturb the vascular angiocrine system in fibrotic organs. One existing key gap is what sense the fibrotic risk to modulate the vascular angiocrine system in organ fibrosis. Here, using human and mouse data, we discovered that the metabolic pathway hydrogen sulfide (H2S)-AMP-activated protein kinase (AMPK) is a sensor of fibrotic stress and serves as a key mechanism upregulating the angiocrine factor plasminogen activator inhibitor-1 (PAI-1) in endothelial cells to participate in lung fibrosis. Activation of the metabolic sensor AMPK was inhibited in endothelial cells of fibrotic lungs, and AMPK inactivation was correlated with enriched fibrotic signature and reduced lung functions in humans. The inactivation of endothelial AMPK accelerated lung fibrosis in mice, while the activation of endothelial AMPK with metformin alleviated lung fibrosis. In fibrotic lungs, endothelial AMPK inactivation led to YAP activation and overexpression of the angiocrine factor PAI-1, which was positively correlated with the fibrotic signature in human fibrotic lungs and inhibition of PAI-1 with Tiplaxtinin mitigated lung fibrosis. Further study identified that the deficiency of the antioxidative gas metabolite H2S accounted for the inactivation of AMPK and activation of YAP-PAI-1 signaling in endothelial cells of fibrotic lungs. H2S deficiency was involved in human lung fibrosis and H2S supplement reversed mouse lung fibrosis in an endothelial AMPK-dependent manner. These findings provide new insight into the mechanism underlying the deregulation of the vascular angiocrine system in fibrotic organs.

Targeting Endothelial Necroptosis Disrupts Profibrotic Endothelial-Hepatic Stellate Cells Crosstalk to Alleviate Liver Fibrosis in Nonalcoholic Steatohepatitis.

Chronic liver diseases affect over a billion people worldwide and often lead to fibrosis. Nonalcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes, is characterized by liver fibrosis, and its pathogenesis remains largely unknown, with no effective treatment available. Necroptosis has been implicated in liver fibrosis pathogenesis. However, there is a lack of research on necroptosis specific to certain cell types, particularly the vascular system, in the context of liver fibrosis and NASH. Here, we employed a mouse model of NASH in combination with inducible gene knockout mice to investigate the role of endothelial necroptosis in NASH progression. We found that endothelial cell (EC)-specific knockout of mixed lineage kinase domain-like protein (MLKL), a critical executioner involved in the disruption of cell membranes during necroptosis, alleviated liver fibrosis in the mouse NASH model. Mechanistically, EC-specific deletion of Mlkl mitigated the activation of TGFß/Smad 2/3 pathway, disrupting the pro-fibrotic crosstalk between endothelial cells and hepatic stellate cells (HSCs). Our findings highlight endothelial MLKL as a promising molecular target for developing therapeutic interventions for NASH.

Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3 + monocytes, Isg15 + macrophages, Ifit3 + neutrophils, and Il12b + DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3 + monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15 + macrophages; Il12b and Ccl22 by Il12b + DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3 + neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.

Deficiency of endothelial FGFR1 signaling via upregulation of ROCK2 activity aggravated ALI/ARDS.

Vascular leakage and inflammation are pathological hallmarks of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Endothelial cells (ECs) serve as a semipermeable barrier and play a key role in disease progression. It is well known that fibroblast growth factor receptor 1 (FGFR1) is required for maintaining vascular integrity. However, how endothelial FGFR1 functions in ALI/ARDS remains obscure. Here, we revealed that conditional deletion of endothelial FGFR1 aggravated LPS-induced lung injury, including inflammation and vascular leakage. Inhibition of its downstream Rho-associated coiled-coil-forming protein kinase 2 (ROCK2) by AAV Vec-tie-shROCK2 or its selective inhibitor TDI01 effectively attenuated inflammation and vascular leakage in a mouse model. In vitro, TNFα-stimulated human umbilical vein endothelial cells (HUVECs) showed decreased FGFR1 expression and increased ROCK2 activity. Furthermore, knockdown of FGFR1 activated ROCK2 and thus promoted higher adhesive properties to inflammatory cells and higher permeability in HUVECs. TDI01 effectively suppressed ROCK2 activity and rescued the endothelial dysfunction. These data demonstrated that the loss of endothelial FGFR1 signaling mediated an increase in ROCK2 activity, which led to an inflammatory response and vascular leakage in vivo and in vitro. Moreover, inhibition of ROCK2 activity by TDI01 provided great value and shed light on clinical translation.

Endothelial anthrax toxin receptor 2 plays a protective role in liver fibrosis.

Hepatocellular carcinoma is one of the leading cancers worldwide and is a potential consequence of fibrosis. Therefore, the identification of key cellular and molecular mechanisms involved in liver fibrosis is an important goal for the development of new strategies to control liver-related diseases. Here, single-cell RNA sequencing data (GSE136103 and GES181483) of clinical liver non-parenchymal cells were analyzed to identify cellular and molecular mechanisms of liver fibrosis. The proportion of endothelial subpopulations in cirrhotic livers was significantly higher than that in healthy livers. Gene ontology and gene set enrichment analysis of differentially expressed genes in the endothelial subgroups revealed that extracellular matrix (ECM)-related pathways were significantly enriched. Since anthrax toxin receptor 2 (ANTXR2) interacts with the ECM, the expression of ANTXR2 in the liver endothelium was analyzed. ANTXR2 expression in the liver endothelium of wild-type (WT) mice significantly decreased after a 4-time sequential injection of carbon tetrachloride (CCl4) to induce liver fibrosis. Next, conditional knockout mice selectively lacking Antxr2 in endothelial cells were generated. After endothelial-specific Antxr2 knockout mice were subjected to the CCl4 model, the degree of liver fibrosis in the knockout group was significantly more severe than that in the control group. In addition, ANTXR2 in human umbilical vein endothelial cells promoted matrix metalloproteinase 2 (MMP2) activation to degrade the ECM in vitro. Finally, endothelial-specific overexpression of Antxr2 alleviated the development of liver fibrosis following adeno-associated virus treatment. Collectively, these results suggested that endothelial ANTXR2 plays a protective role in liver fibrosis. This function of ANTXR2 may be achieved by promoting MMP2 activation to degrade the ECM.

Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Comprehensive Review of the Vascular Niche in Regulating Organ Regeneration and Fibrosis.

The vasculature occupies a large area of the body, and none of the physiological activities can be carried out without blood vessels. Blood vessels are not just passive conduits and barriers for delivering blood and nutrients. Meanwhile, endothelial cells covering the vascular lumen establish vascular niches by deploying some growth factors, known as angiocrine factors, and actively participate in the regulation of a variety of physiological processes, such as organ regeneration and fibrosis and the occurrence and development of cancer. After organ injury, vascular endothelial cells regulate the repair process by secreting various angiocrine factors, triggering the proliferation and differentiation process of stem cells. Therefore, analyzing the vascular niche and exploring the factors that maintain vascular homeostasis can provide strong theoretical support for clinical treatment targeting blood vessels. Here we mainly discuss the regulatory mechanisms of the vascular niche in organ regeneration and fibrosis.

Trimethylamine-N-oxide (TMAO) mediates the crosstalk between the gut microbiota and hepatic vascular niche to alleviate liver fibrosis in nonalcoholic steatohepatitis.

Liver fibrosis is one main histological characteristic of nonalcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes with no approved therapies. The role of the gut microbiota in NASH pathogenesis has not been thoroughly illustrated, especially how the gut microbiota derives metabolites to influence the distal liver in NASH. Here, we performed 16S rDNA amplicon sequencing analysis of feces from a mouse NASH model induced by a Western diet and CCl4 injury and found genera under Streptococcaceae, Alcaligenaceae, Oscillibacter, and Pseudochrobactrum, which are related metabolites of TMAO. Injection of the gut microbial metabolite TMAO reduced the progression of liver fibrosis in the mouse NASH model. Further analysis revealed that the anti-fibrotic TMAO normalized gut microbiota diversity and preserved liver sinusoidal endothelial cell integrity by inhibiting endothelial beta 1-subunit of Na (+), K (+)-ATPase (ATP1B1) expression. Collectively, our findings suggest TMAO-mediated crosstalk between microbiota metabolites and hepatic vasculature, and perturbation of this crosstalk disrupts sinusoidal vasculature to promote liver fibrosis in NASH.

Dopamine receptor D2 antagonism normalizes profibrotic macrophage-endothelial crosstalk in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Currently there is no effective treatment for liver fibrosis, which is one of the main histological determinants of non-alcoholic steatohepatitis (NASH). While Hippo/YAP (Yes-associated protein) signaling is essential for liver regeneration, its aberrant activation frequently leads to fibrosis and tumorigenesis. Unravelling "context-specific" contributions of YAP in liver repair might help selectively bypass fibrosis and preserve the pro-regenerative YAP function in hepatic diseases. METHODS: We used murine liver fibrosis and minipig NASH models, and liver biopsies from patients with cirrhosis. Single-cell RNA-sequencing (scRNA-Seq) was performed, and a G-protein-coupled receptor (GPCR) ligand screening system was used to identify cell-selective YAP inhibitors. RESULTS: YAP levels in macrophages are increased in the livers of humans and mice with liver fibrosis. The increase in type I interferon and attenuation of hepatic fibrosis observed in mice specifically lacking Yap1 in myeloid cells provided further evidence for the fibrogenic role of macrophage YAP. ScRNA-Seq further showed that defective YAP pathway signaling in macrophages diminished a fibrogenic vascular endothelial cell subset that exhibited profibrotic molecular signatures such as angiocrine CTGF and VCAM1 expression. To specifically target fibrogenic YAP in macrophages, we utilized a GPCR ligand screening system and identified a dopamine receptor D2 (DRD2) antagonist that selectively blocked YAP in macrophages but not hepatocytes. Genetic and pharmacological targeting of macrophage DRD2 attenuated liver fibrosis. In a large animal (minipig) NASH model recapitulating human pathology, the DRD2 antagonist blocked fibrosis and restored hepatic architecture. CONCLUSIONS: DRD2 antagonism selectively targets YAP-dependent fibrogenic crosstalk between macrophages and CTGF+VCAM1+ vascular niche, promoting liver regeneration over fibrosis in both rodent and large animal models. LAY SUMMARY: Fibrosis in the liver is one of the main histological determinants of non-alcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes. Our study demonstrates that a macrophage-specific deficiency in Yes-associated protein (YAP) attenuates liver fibrosis. Dopamine receptor D2 (DRD2) antagonism selectively blocks YAP in macrophages and thwarts liver fibrosis in both rodent and large animal models, and thus holds potential for the treatment of NASH.

Histone variant H3.3 maintains adult haematopoietic stem cell homeostasis by enforcing chromatin adaptability.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability.

Targeting epigenetically maladapted vascular niche alleviates liver fibrosis in nonalcoholic steatohepatitis.

Chronic hepatic diseases such as nonalcoholic steatohepatitis (NASH) suppress liver regeneration and lead to fibrosis and cirrhosis. Decoding the cellular and molecular network underlying this fibrotic maladaptation might aid in combatting NASH, a growing health challenge with no approved therapies. Here, we used multiomics analysis of human cirrhotic liver, a Western diet­ and carbon tetrachloride (CCl4)­induced minipig NASH model, and genetically modified mice to unravel the landscape of the vascular adaptome at the single-cell level, in which endothelial cells (ECs) and TH17 cells jointly contribute to liver cirrhosis. We found that epigenetics-dependent hepatic vascular maladaptation enriches fibrogenic TH17 cells to promote liver fibrosis in mice, minipigs, and human patients with cirrhosis. Further analysis of humans, minipigs, and mice suggested that cross-talk between histone deacetylase 2 (HDAC2) and DNA methyltransferase 1 (DNMT1) promoted liver EC maladaptation to promote production of angiocrine IGFBP7 and ADAMTS1 in extracellular vesicles, recruiting fibrogenic TH17 cells to the liver. Pharmacological targeting of HDAC2 and DNMT1 alleviated fibrosis in a minipig NASH model. We conclude that epigenetically reprogrammed vascular adaptation contributes to liver fibrosis. Targeting of a vascular adaptation node might block maladaptive vascularization to promote liver regeneration in NASH.

Reversal of emphysema by restoration of pulmonary endothelial cells.

Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.

Selective Targeting of Vascular Endothelial YAP Activity Blocks EndMT and Ameliorates Unilateral Ureteral Obstruction-Induced Kidney Fibrosis.

Kidney fibrosis is accompanied by vascular dysfunction. Discovering new ways to ameliorate dysfunctional angiogenesis may bypass kidney fibrosis. YAP (Yes-associated protein) plays a multifaceted role during angiogenesis. Here, we found that selectively targeting YAP signaling in the endothelium ameliorates unilateral ureteral obstruction (UUO)-induced kidney fibrosis. Genetic deletion of Yap1, encoding YAP protein, in VE-cadherin+ endothelial cells inhibited endothelial-to-mesenchymal transition (EndMT) and dysfunctional angiogenesis and improved obstructive nephropathy and kidney fibrosis. Treatment with the systemic YAP inhibitor verteporfin worsened kidney fibrosis symptoms because of its lack of cell specificity. In an attempt to identify endothelial-specific YAP modulators, we found that G-protein-coupled receptor coagulation factor II receptor-like 1 (F2RL1) was highly expressed in vessels after UUO-induced kidney fibrosis. The F2RL1 peptide antagonist FSLLRY-NH2 selectively blocked YAP activity in endothelial cells and ameliorated kidney fibrosis. Thus, selective antagonization of endothelial YAP activity might bypass kidney fibrosis and provide new avenues for the design of antifibrotic therapies.

An Epigenetic Mechanism Underlying Chromosome 17p Deletion-Driven Tumorigenesis.

Chromosome copy-number variations are a hallmark of cancer. Among them, the prevalent chromosome 17p deletions are associated with poor prognosis and can promote tumorigenesis more than TP53 loss. Here, we use multiple functional genetic strategies and identify a new 17p tumor suppressor gene (TSG), plant homeodomain finger protein 23 (PHF23). Its deficiency impairs B-cell differentiation and promotes immature B-lymphoblastic malignancy. Mechanistically, we demonstrate that PHF23, an H3K4me3 reader, directly binds the SIN3-HDAC complex through its N-terminus and represses its deacetylation activity on H3K27ac. Thus, the PHF23-SIN3-HDAC (PSH) complex coordinates these two major active histone markers for the activation of downstream TSGs and differentiation-related genes. Furthermore, dysregulation of the PSH complex is essential for the development and maintenance of PHF23-deficient and 17p-deleted tumors. Hence, our study reveals a novel epigenetic regulatory mechanism that contributes to the pathology of 17p-deleted cancers and suggests a susceptibility in this disease. SIGNIFICANCE: We identify PHF23, encoding an H3K4me3 reader, as a new TSG on chromosome 17p, which is frequently deleted in human cancers. Mechanistically, PHF23 forms a previously unreported histone-modifying complex, the PSH complex, which regulates gene activation through a synergistic link between H3K4me3 and H3K27ac.This article is highlighted in the In This Issue feature, p. 1.