Background and Aims: Liver fibrosis represents a global health burden, given the paucity of approved antifibrotic therapies. Liver sinusoidal endothelial cells (LSECs) play a major gatekeeping role in hepatic homeostasis and liver disease pathophysiology. In early tumorigenesis, runt-related transcription factor 3 (RUNX3) functions as a sentinel; however, its function in liver fibrosis in LSECs remains unclear. This study aimed to investigate the role of RUNX3 as an important regulator of the gatekeeping functions of LSECs and explore novel angiocrine regulators of liver fibrosis. Approach and Results: Mice with endothelial Runx3 deficiency develop gradual and spontaneous liver fibrosis secondary to LSEC dysfunction, thereby more prone to liver injury. Mechanistic studies in human immortalized LSECs and mouse primary LSECs revealed that IL-6/JAK/STAT3 pathway activation was associated with LSEC dysfunction in the absence of RUNX3. Single-cell RNA sequencing and quantitative RT-PCR revealed that leucine-rich alpha-2-glycoprotein 1 (LRG1) was highly expressed in RUNX3-deficient and dysfunctional LSECs. In in vitro and coculture experiments, RUNX3-depleted LSECs secreted LRG1, which activated HSCs throughTGFBR1–SMAD2/3 signaling in a paracrine manner. Furthermore, circulating LRG1 levels were elevated in mouse models of liver fibrosis and in patients with fatty liver and cirrhosis. Conclusions: RUNX3 deficiency in the endothelium induces LSEC dysfunction, LRG1 secretion, and liver fibrosis progression. Therefore, endothelial RUNX3 is a crucial gatekeeping factor in LSECs, and profibrotic angiocrine LRG1 may be a novel target for combating liver fibrosis.

Tumor angiogenesis is a critical driver of cancer progression; however, current anti-angiogenic therapies remain limited by resistance and toxicity. Hypoxia within the tumor microenvironment induces hypoxia-inducible factor-1α (HIF-1α), which promotes aberrant angiogenesis by upregulating vascular endothelial growth factor (VEGF) and, subsequently, delta-like ligand 4 (DLL4) in endothelial cells. A systematic screening of flavanone derivatives was performed to identify compounds capable of dual inhibition of HIF-1α and DLL4. Among 16 natural compounds evaluated, isoxanthohumol (IXN), a prenylated flavanone, emerged as the most potent, suppressing both hypoxia-induced HIF-1α accumulation in tumor cells and VEGF-induced DLL4 expression in endothelial cells. IXN markedly inhibited endothelial proliferation, migration, and tube formation in vitro. In a Lewis lung carcinoma (LLC) mouse syngeneic model, IXN monotherapy reduced tumor growth and vessel density. Notably, combination treatment with IXN and anti-PD-1 immunotherapy produced greater anti-tumor effects than either monotherapy. This combination enhanced cytotoxic T cell infiltration into the tumor core, increased granzyme B expression, and induced widespread tumor cell apoptosis, consistent with improved vascular normalization. These findings identify IXN as a promising dual-targeting agent that inhibits both HIF-1α and DLL4 and demonstrate its potential to enhance immune checkpoint blockade. Simultaneous targeting of hypoxia-driven and VEGF-DLL4-mediated angiogenic pathways represents a compelling therapeutic strategy to overcome the limitations of current anti-angiogenic and immunotherapeutic approaches.

BACKGROUND: Transcriptional regulation is fundamental to vascular homeostasis, ensuring vessel stability and function. Disruption of this regulation underlies pathological angiogenesis in cancer and vascular malformations. While RUNX3 (Runt-related transcription factor 3), a Runt-domain transcription factor, is established as a tumor suppressor and regulator of lymphocyte development, its role in endothelial cells has remained unknown. METHODS: To define the vascular function of RUNX3, we generated endothelial-specific RUNX3 knockout mice and analyzed postnatal retinal angiogenesis and oxygen-induced retinopathy. Mechanistic insights were obtained through assay for transposase-accessible chromatin with high-throughput sequencing data analysis, coimmunoprecipitation, proximity ligation assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation followed by quantitative real-time polymerase chain reaction (ChIP-qPCR). RESULTS: Loss of endothelial RUNX3 induced hypersprouting angiogenesis with excessive tip cell formation and reduced DLL4 (delta-like ligand 4) expression. RUNX3 deficiency disrupted vascular integrity by lowering VE-cadherin (vascular endothelial cadherin) and claudin-5 levels, while increasing ICAM1 (intercellular adhesion molecule 1) expression, retinal hemorrhage, and leukocyte infiltration. In the oxygen-induced retinopathy model, endothelial RUNX3 deletion aggravated pathological neovascularization. Mechanistically, RUNX3 cooperated with ERG (ETS-related gene) at ETS:RUNX motifs to regulate genes controlling sprouting ( DLL4 ), junctional stability ( CDH5 , CLDN5 ), and leukocyte adhesion ( ICAM1 ). RUNX3 formed complexes with ERG, p300, and HDAC3 (histone deacetylase 3), and proximity ligation assays showed that these interactions were enriched in the nucleus upon VEGF-A (vascular endothelial growth factor-A) stimulation. ChIP-qPCR further demonstrated that RUNX3 was required for the recruitment of these coregulators to target loci. Through differential coregulator engagement, RUNX3 functioned bidirectionally, acting as a transcriptional activator of CDH5 , CLDN5 , and DLL4 while serving as a repressor of ICAM1 . Consistently, single-cell transcriptome analysis of human arteriovenous malformations revealed dysregulated RUNX3 and target gene expression, underscoring translational relevance. CONCLUSIONS: This study provides the first evidence that RUNX3 is a pivotal transcriptional coordinator in endothelial cells. By integrating ERG and chromatin regulators at ETS:RUNX motifs, RUNX3 safeguards the balance between angiogenic activation and vascular stability. These findings establish RUNX3 as a gatekeeper of endothelial transcriptional identity and highlight its potential as a therapeutic target in pathological angiogenesis.