Background: Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-laden foam cells and plaques within the arterial wall. Dysfunctional vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages contribute to disease progression. Here, we report that macrophage-specific expression of epsins, highly conserved endocytic adaptor proteins involved in clathrin-mediated endocytosis, accelerates atherosclerosis in Western diet (WD)-fed mice.

Methods: Apoe-deficient (WT/Apoe^-/-) mice and littermates with a myeloid-specific deletion of epsin 1/2 on an Apoe^-/- background (LysM-DKO/Apoe^-/-) were generated and fed a WD for 16 weeks. Single-cell RNA sequencing (scRNA-seq) was conducted to investigate the cellular and molecular mechanisms regulated by macrophage epsins during atherosclerosis. Findings from scRNA-seq were validated through metabolic profiling, qRT-PCR, immunostaining, and co-culture experiments to assess associated phenotypic changes.

Results: LysM-DKO/Apoe^-/- mice exhibited significantly reduced atherosclerotic foam cell formation compared to WT/Apoe^-/- controls. scRNA-seq analysis identified 19 major cell types, including six VSMC and five macrophage subpopulations. Modulated VSMC1 and VSMC2 subtypes were associated with inflammation, migration, and VSMC-to-macrophage transition. These populations, along with foamy-Trem2 and inflammatory macrophages, were markedly reduced in LysM-DKO/Apoe^-/- mice. Transition of modulated VSMC2 subtype into macrophages was significantly inhibited, as confirmed by both computational analysis and experimental validation. Additionally, macrophage epsin deletion reversed endothelial dysfunction, suppressed cholesterol- and glucose-mediated signaling, and reduced expression of pro-inflammatory ligands IL-1β and TNF-⍺.

Conclusion: Macrophage epsin deletion limits foam cell formation and preserves VSMC and endothelial cell phenotypes and functions. These findings reveal a potential therapeutic strategy targeting macrophage epsins to combat atherosclerosis.

Despite the development of potent drugs for modifiable risk factors, such as statins, and advances in mechanistic biomedical research, vascular disease remains the No.1 killer globally and represents a huge cost to public health. The underlying mechanisms remain incompletely understood and effective new therapies are needed. Such major challenges have promoted technological innovations and their implementations in vascular research. Unmet clinical needs and exponential technological development have synergistically advanced vascular medicine. Addressing the challenges associated with the complexity in treating cardiovascular disease requires integration of cross-disciplinary approaches and knowledge. This Research Topic thus reports new trends in a wide range of vascular medicine research from fundamental basic science to translational medicine to clinical studies.

Deciphering cell identity genes is pivotal to understanding cell differentiation, development, and cell identity dysregulation involving diseases. Here, we introduce SCIG, a machine-learning method to uncover cell identity genes in single cells. In alignment with recent reports that cell identity genes (CIGs) are regulated with unique epigenetic signatures, we found CIGs exhibit distinctive genetic sequence signatures, e.g. unique enrichment patterns of cis-regulatory elements. Using these genetic sequence signatures, along with gene expression information from single-cell RNA-seq data, SCIG uncovers the identity genes of a cell without a need for comparison to other cells. CIG score defined by SCIG surpassed expression value in network analysis to reveal the master transcription factors (TFs) regulating cell identity. Applying SCIG to the human endothelial cell atlas revealed that the tissue microenvironment is a critical supplement to master TFs for cell identity refinement. SCIG is publicly available at https://doi.org/10.5281/zenodo.14726426  , offering a valuable tool for advancing cell differentiation, development, and regenerative medicine research.

The lymphatic system maintains tissue fluid balance, and its dysfunction can result in lymphedema. Although cholesterol is essential for cellular function, its role in lymphatic development has remained unknown. Here, we identify APOA1 binding protein (AIBP) as a key regulator that promotes lymphatic endothelial cell fate specification and lymphangiogenesis. Mechanistically, AIBP reduces plasma membrane cholesterol content, thereby enhancing VEGFR3 signaling by disrupting caveolae—small plasma membrane invaginations formed by the scaffolding protein caveolin-1 (CAV-1)—and relieving CAV-1–mediated inhibition. In zebrafish and mice, AIBP loss impairs VEGFR3 signaling and lymphatic development, defects that can be rescued by CAV-1 deletion or by a VEGFR3 mutant (VEGFR3^AAA) lacking CAV-1 binding. Administration of recombinant AIBP augments VEGFC-induced lymphangiogenesis and accelerates the resolution of secondary lymphedema in adult mice. These findings define the AIBP–CAV-1 axis as a regulator of VEGFR3 signaling and lymphatic growth, offering potential therapeutic opportunities for treating lymphatic dysfunction.

Efficient lymph flow is ensured by lymphatic valves (LVs). The mechanisms that regulate LV development are incompletely understood. Here, we show that the deletion of the GPCR sphingosine 1-phosphate receptor-1 (S1PR1) from lymphatic endothelial cells (LECs) results in fewer LVs. Interestingly, LVs that remained in the terminal ileum-draining lymphatic vessels were specifically dysfunctional. Furthermore, tertiary lymphoid organs (TLOs) formed in the terminal ileum of the mutant mice. TLOs in this location are associated with ileitis in humans and mice. However, mice lacking S1PR1 did not develop obvious characteristics of ileitis. Mechanistically, S1PR1 regulates shear stress signaling and the expression of the valve-regulatory molecules FOXC2 and connexin-37. Importantly, Foxc2^+/− mice, a model for lymphedema-distichiasis syndrome, also develop TLOs in the terminal ileum. Thus, we have discovered S1PR1 as a previously unknown regulator of LV and TLO development. We also suggest that TLOs are a sign of subclinical inflammation that can form due to lymphatic disorders in the absence of ileitis.

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF)-dependent angiogenesis remain unclear. In our study, the molecular underpinnings of endothelial dysfunction in diabetes are investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FOXM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high glucose treated primary mouse skin endothelial cells. In diabetic mice with endothelial deficiency of Dab2, in vivo and in vitro angiogenesis and wound healing were reduced when compared with wild-type diabetic mice. Restoration of Dab2 expression by injected mRNA-containing LyP-1-conjugated lipid nanoparticles (LNPs) rescued impaired angiogenesis and wound healing in diabetic mice. Furthermore, FOXM1 was downregulated in skin endothelial cells under high glucose conditions as determined by RNA-sequencing analysis. FOXM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FOXM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. Our study provides evidence of the crucial roles of Dab2 and FOXM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

Cell-cell communication (CCC) is crucial for cellular function and tissue homeostasis. Existing methods for protein-oriented CCC detection often overlook metabolite-mediated CCC (mCCC), and adapting them to mCCC analysis is challenging due to fundamental differences in the underlying biological mechanisms. To fill this gap, we developed MEBOCOST, an algorithm built on scRNA-seq and metabolic flux balance analysis to detect mCCC among single cells. Comprehensive benchmarking analyses based on simulation, spatial, CRISPR screen, and clinical patient data demonstrated the robustness of MEBOCOST in detecting biologically significant mCCC events. We applied MEBOCOST to scRNA-seq datasets of human white adipose tissues and unraveled macrophages were the predominant source of mCCC reprogramming in obese patients. Moreover, analysis in mice brown adipose tissue successfully recapitulated known and further uncovered new mCCC events, including a glutamine-mediated endothelial-to-adipocyte communication, which was experimentally verified to regulate adipocyte differentiation. Therefore, MEBOCOST is a valuable tool for researchers investigating mCCC in diverse biological contexts and disease samples. MEBOCOST is freely available at https://github.com/kaifuchenlab/MEBOCOST.

Lymphatic vessels function throughout the body to drain interstitial fluids. Efficient lymph flow is ensured by lymphatic valves (LVs). However, the mechanisms that regulate LV development are incompletely understood. Here, we show that the deletion of the GPCR sphingosine 1-phosphate receptor-1 (S1PR1) from lymphatic endothelial cells (LECs) results in fewer LVs. Interestingly, LVs that remained in the terminal-ileum draining lymphatic vessels were specifically dysfunctional, and tertiary lymphoid organs (TLOs) formed in this location. TLOs in the terminal ileum are associated with ileitis in humans and mice. However, mice lacking S1PR1 did not develop obvious characteristics of ileitis. Sphingosine kinases 1 and 2 (SPHK1/2) are required for the synthesis of S1P, the ligand of S1PR1. Mice that lack Sphk1/2 in LECs recapitulate the LV and TLO phenotypes of mice that lack S1PR1. Mechanistically, S1PR1 regulates shear stress signaling and the expression of the valve-regulatory molecules FOXC2 and connexin-37. Importantly, Foxc2^+/- mice, a model for lymphedema-distichiasis syndrome, also develop TLOs in the terminal ileum. Thus, we have discovered S1PR1 as a previously unknown regulator of LV and TLO development. We also suggest that TLOs are a sign of subclinical inflammation that can form due to lymphatic disorders in the absence of ileitis.

Atherosclerosis is a chronic inflammatory condition characterized by the excessive accumulation of fat and lipid molecules, leading to the formation of foam cells and plaques in arterial walls. Dysfunction of vascular smooth muscle cells (VSMCs), fibroblast, endothelial cells, and macrophages is often associated with this pathology. We found that epsins accelerate atherosclerosis progression in individuals on a Western diet (WD). Using ApoE-deficient (ApoE^-/-) and macrophage-specific epsin deletion in ApoE^-/- backgrounds (LysM-DKO/ApoE^-/-) mice fed a WD for 16 weeks, we observed significantly reduced foam cell formation in LysM-DKO/ApoE^-/- mice compared to ApoE^-/- mice. Single-cell RNA sequencing identified 20 major cell types, including seven VSMC and five macrophage subtypes. Among the VSMC subtypes, modulating VSMC1 was involved in inflammation and migration, while modulating VSMC2 was associated with VSMC phenotype switching. In atherosclerotic mice, populations of modulating VSMC1, VSMC2, foamy-Trem2, and inflammatory macrophages increased, but significantly decreased in epsin-deficient mice. Modulating VSMC2 transition into macrophages occurred with a probability of 0.57 in ApoE^-/- mice, compared to 0.01 in LysM-DKO/ApoE^-/- mice. Epsin deletion also reversed endothelial dysfunction and downregulated cholesterol and glucose-mediated signals, as well as inflammatory ligands Il1b and C1qa. Our findings suggest that epsin deletion reduces foam cell formation and rewires VSMC and endothelial functions, offering a novel therapeutic strategy for atherosclerosis.