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.

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.

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.

Lymphatic vessels grow through active sprouting and mature into a vascular complex including lymphatic capillaries and collecting vessels that ensure fluid transport. However, the signaling cues that direct lymphatic sprouting and patterning remains unclear. In this study, we demonstrated the chemokine signaling, specifically through CXCL12/CXCR4 plays critical roles in regulating lymphatic development. We showed that LEC specific CXCR4 deficient embryos and CXCL12 mutant embryos exhibited server defects in lymphatic sprouting, migration, and lymphatic valve formation. We also discovered that CXCL12, originating from peripheral nerves, directs the migration of dermal lymphatic vessels to align with nerves in developing skin. Deletion CXCR4 or blockage of CXCL12/CXCR4 activity results in reduced VEGFR3 levels on the LEC surface. This, in turn, impairs VEGFC mediated VEGFR3 signaling and downstream PI3K/AKT activities. Taken together, these data identify previously unknown chemokine signaling originating from peripheral nerves that guides dermal lymphatic sprouting and patterning. Our work identifies for the first time a neuro-lymphatics communications during mouse development and reveals a novel mechanism by which CXCR4 modulates VEGFC/VEGFR3/AKT signaling.

Background: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown.
Method: We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis.
Results: Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls.
Conclusions: These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.

Deciphering cell identity genes is pivotal to understanding cell differentiation, development, and many diseases involving cell identity dysregulation. 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 are regulated with unique epigenetic signatures, we found cell identity genes 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, enables SCIG to uncover the identity genes of a cell without a need for comparison to other cells. Cell identity gene score defined by SCIG surpassed expression value in network analysis to uncover master transcription factors regulating cell identity. Applying SCIG to the human endothelial cell atlas revealed that the tissue microenvironment is a critical supplement to master transcription factors for cell identity refinement. SCIG is publicly available at https://github.com/kaifuchenlab/SCIG, offering a valuable tool for advancing cell differentiation, development, and regenerative medicine research.

Rationale: Low density cholesterol receptor (LDLR) in the liver is critical for the clearance of low-density lipoprotein cholesterol (LDL-C) in the blood. In atherogenic conditions, proprotein convertase subtilisin/kexin 9 (PCSK9) secreted by the liver, in a nonenzymatic fashion, binds to LDLR on the surface of hepatocytes, preventing its recycling and enhancing its degradation in lysosomes, resulting in reduced LDL-C clearance. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution of circulating LDL-C to atherosclerosis, we hypothesize that liver epsins promote atherosclerosis by controlling LDLR endocytosis and degradation. Objective: We will determine the role of liver epsins in promoting PCSK9-mediated LDLR degradation and hindering LDL-C clearance to propel atherosclerosis. Methods and Results: We generated double knockout mice in which both paralogs of epsins, namely, epsin-1 and epsin-2, are specifically deleted in the liver (Liver-DKO) on an ApoE-/- background. We discovered that western diet (WD)-induced atherogenesis was greatly inhibited, along with diminished blood cholesterol and triglyceride levels. Mechanistically, using scRNA-seq analysis on cells isolated from the livers of ApoE-/- and ApoE-/- /Liver-DKO mice on WD, we found lipogenic Alb hi hepatocytes to glycogenic HNF4α hi hepatocytes transition in ApoE-/- /Liver-DKO. Subsequently, gene ontology analysis of hepatocyte-derived data revealed elevated pathways involved in LDL particle clearance and very-low-density lipoprotein (VLDL) particle clearance under WD treatment in ApoE-/- /Liver-DKO, which was coupled with diminished plasma LDL-C levels. Further analysis using the MEBOCOST algorithm revealed enhanced communication score between LDLR and cholesterol, suggesting elevated LDL-C clearance in the ApoE-/- Liver-DKO mice. In addition, we showed that loss of epsins in the liver upregulates of LDLR protein level. We further showed that epsins bind LDLR via the ubiquitin-interacting motif (UIM), and PCSK9-triggered LDLR degradation was abolished by depletion of epsins, preventing atheroma progression. Finally, our therapeutic strategy, which involved targeting liver epsins with nanoparticle-encapsulated siRNAs, was highly efficacious at inhibiting dyslipidemia and impeding atherosclerosis. Conclusions: Liver epsins promote atherogenesis by mediating PCSK9-triggered degradation of LDLR, thus raising the circulating LDL-C levels. Targeting epsins in the liver may serve as a novel therapeutic strategy to treat atherosclerosis by suppression of PCSK9-mediated LDLR degradation.

Significance: Cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality globally. Endothelial dysfunction is closely associated with the development and progression of CVDs. Patients with diabetes mellitus (DM) especially type 2 DM (T2DM) exhibit a significant endothelial cell (EC) dysfunction with substantially increased risk for CVDs. Recent Advances: Excessive reactive oxygen species (ROS) and oxidative stress are important contributing factors to EC dysfunction and subsequent CVDs. ROS production is significantly increased in DM and is critically involved in the development of endothelial dysfunction in diabetic patients. In this review, efforts are made to discuss the role of excessive ROS and oxidative stress in the pathogenesis of endothelial dysfunction and the mechanisms for excessive ROS production and oxidative stress in T2DM. Critical Issues: Although studies with diabetic animal models have shown that targeting ROS with traditional antioxidant vitamins C and E or other antioxidant supplements provides promising beneficial effects on endothelial function, the cardiovascular outcomes of clinical studies with these antioxidant supplements have been inconsistent in diabetic patients. Future Directions: Preclinical and limited clinical data suggest that N-acetylcysteine (NAC) treatment may improve endothelial function in diabetic patients. However, well-designed clinical studies are needed to determine if NAC supplementation would effectively preserve endothelial function and improve the clinical outcomes of diabetic patients with reduced cardiovascular morbidity and mortality. With better understanding on the mechanisms of ROS generation and ROS-mediated endothelial damages/dysfunction, it is anticipated that new selective ROS-modulating agents and effective personalized strategies will be developed for the management of endothelial dysfunction in DM.

FBXW7 is one of the most well-characterized F-box proteins, serving as substrate receptor subunit of SKP1-CUL1-F-box (SCF) E3 ligase complexes. SCF^FBXW7 is responsible for the degradation of various oncogenic proteins such as cyclin E, c-MYC, c-JUN, NOTCH, and MCL1. Therefore, FBXW7 functions largely as a major tumor suppressor. In keeping with this notion, FBXW7 gene mutations or downregulations have been found and reported in many types of malignant tumors, such as endometrial, colorectal, lung, and breast cancers, which facilitate the proliferation, invasion, migration, and drug resistance of cancer cells. Therefore, it is critical to review newly identified FBXW7 regulation and tumor suppressor function under physiological and pathological conditions to develop effective strategies for the treatment of FBXW7-altered cancers. Since a growing body of evidence has revealed the tumor-suppressive activity and role of FBXW7, here, we updated FBXW7 upstream and downstream signaling including FBXW7 ubiquitin substrates, the multi-level FBXW7 regulatory mechanisms, and dysregulation of FBXW7 in cancer, and discussed promising cancer therapies targeting FBXW7 regulators and downstream effectors, to provide a comprehensive picture of FBXW7 and facilitate the study in this field.