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.

Background: The low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins' canonical role in regulating endocytosis of cell surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis.

Methods: Liver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE^-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE^-/- mice with established atherosclerosis.

Results: Western diet (WD)-induced atherosclerosis was significantly attenuated in ApoE^-/-/Liver-DKO mice compared with ApoE^-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE^-/- and ApoE^-/-/Liver-DKO mice revealed epsin-deficient Ldlr^hi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes showed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE^-/-/Liver-DKO mice compared with ApoE^-/- controls, correlating with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE^-/-/Liver-DKO mice compared with ApoE^-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing an increased epsin1 and epsin2 expression in atherosclerotic cardiovascular disease patients.

Conclusions: Liver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance in the liver.

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.

One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long noncoding RNAs (lncRNAs), could provide new, more targeted therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome-associated lncRNA and show that its expression was induced in the heart upon pathological cardiac hypertrophy and that its deletion in mice exacerbated stress-induced cardiac hypertrophy and augmented protein translation. In contrast, overexpression of CARDINAL attenuated cardiac hypertrophy in vivo and in vitro and suppressed hypertrophy-induced protein translation. Mechanistically, CARDINAL interacted with developmentally regulated GTP-binding protein 1 (DRG1) and blocked its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 was downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.

Atherosclerosis, a chronic systemic inflammatory condition, is implicated in most cardiovascular ischemic events. The pathophysiology of atherosclerosis involves various cell types and associated processes, including endothelial cell activation, monocyte recruitment, smooth muscle cell migration, involvement of macrophages and foam cells, and instability of the extracellular matrix. The process of endothelial-to-mesenchymal transition (EndoMT) has recently emerged as a pivotal process in mediating vascular inflammation associated with atherosclerosis. This transition occurs gradually, with a significant portion of endothelial cells adopting an intermediate state, characterized by a partial loss of endothelial-specific gene expression and the acquisition of “mesenchymal” traits. Consequently, this shift disrupts endothelial cell junctions, increases vascular permeability, and exacerbates inflammation, creating a self-perpetuating cycle that drives atherosclerotic progression. While endothelial cell dysfunction initiates the development of atherosclerosis, autophagy, a cellular catabolic process designed to safeguard cells by recycling intracellular molecules, is believed to exert a significant role in plaque development. Identifying the pathological mechanisms and molecular mediators of EndoMT underpinning endothelial autophagy, may be of clinical relevance. Here, we offer new insights into the underlying biology of atherosclerosis and present potential molecular mechanisms of atherosclerotic resistance and highlight potential therapeutic targets.

This study investigates the molecular underpinnings of endothelial dysfunction in diabetes, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGFR2 signaling and endothelial cell function. Our research reveals critical insights into the downregulation of Dab2 and FoxM1 in endothelial cells (ECs) under hyperglycemic conditions that leads to impaired angiogenesis and delayed wound healing. These findings Substantiate our hypothesis that restoring Dab2 expression through targeted therapies could enhance angiogenesis and wound repair in diabetic environments. In vitro experiments involved treating primary murine brain ECs with high glucose concentrations, simulating hyperglycemic conditions in diabetes mellitus. Bulk RNA-sequencing analysis identified significant downregulation of Dab2, FoxM1, and genes involved in cell cycle progression, cell growth, survival, glycolysis, and oxidative phosphorylation. In vivo, ECs isolated from diabetic mice showed a marked decrease in Dab2 and FoxM1 compared to controls, validated by immunostaining and western blot analysis. Notably, FoxM1 was found to directly bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. Dab2 deficiency led to enhanced lysosomal degradation of VEGFR2 in high-glucose-treated ECs, reducing VEGFR2 signaling. This was further supported by in vitro experiments showing decreased proliferation and angiogenic capability in Dab2-deficient brain ECs. Correspondingly, diabetic mice lacking Dab2 exhibited slower wound healing and reduced neovascularization. To explore therapeutic potential, we employed Dab2-mRNA encapsulated in lipid nanoparticles, significantly improving wound healing and angiogenesis in diabetic mice. This study provides substantial evidence of the crucial roles of Dab2 and FoxM1 in diabetic endothelial dysfunction and proposes targeted gene delivery systems as a promising treatment for diabetic vascular complications.

The prevalence of non-alcoholic fatty liver disease (NAFLD) and atherosclerosis remain high, which is primarily due to widespread adoption of a western diet and sedentary lifestyle. NAFLD, together with advanced forms of this disease such as non-alcoholic steatohepatitis (NASH) and cirrhosis, are closely associated with atherosclerotic-cardiovascular disease (ASCVD). In this review, we discussed the association between NAFLD and atherosclerosis and expounded on the common molecular biomarkers underpinning the pathogenesis of both NAFLD and atherosclerosis. Furthermore, we have summarized the mode of function and potential clinical utility of existing drugs in the context of these diseases.

Hypothesis VSMC play crucial roles in atherosclerosis via phenotypic switching. The trans-differentiation of VSMC into other cell types might contribute to atherosclerotic lesion development, progression, and the subsequent diseases such as myocardial infarction or stroke. Epsins, a family of endocytic adaptors, are crucial for atherosclerosis development and progression; yet, the role of epsins in VSMC phenotypic modulation is unknown.

Methods and Results To decipher the role of VSMC epsins in regulating atheroma development and progression, we created WT and SMC-specific inducible epsin1/2 double knockout (SMC-iDKO) mice on ApoE^-/- background fed a western diet. Using single-cell RNA-seq analysis, we found VSMC and EC population significantly changed in SMC-iDKO/ApoE^-/- mice compared to ApoE^-/- mice. Using immunofluorescent and FACS analysis, we observed that both VSMC and EC marker genes expressions were up-regulated in DKO-VSMCs indicating that DKO-SMCs were maintaining their SMC phenotype and may further trans-differentiating into ECs via a process called MEndoT, mesenchymal to endothelial transition. Given the critical role ofKLF4 in regulating VSMCs phenotype during atheroma progression, we observed diminished KLF4 expression in DKO-SMCs compared to WT treated with oxLDL. Furthermore, we found that epsins interacted with KLF4 with Epsin-UIM domain and their interaction improved KLF4 stability and promoted KLF4 to transfer into nuclei. In this study, we found that epsins were highly expressed and positively correlated with the lesion severity in VSMCs in atherosclerotic. Using immunofluorescent and Oil Red O stainings, we observed reduced lipid accumulation, lesion size and macrophage infiltration but elevated VSMCs in the cap of lesions in SMC-iDKO/ApoE^-/- mice compared with ApoE^-/- mice.

Conclusions In conclusions, we demonstrated an unexpected role of epsins in regulating phenotypic switching by repressing SM-contractile and EC marker genes expression through an epigenetic regulatory mechanism. Our data suggest that epsins may be a therapeutic target for treating occlusive vascular diseases, and uncover regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

The forkhead box O1 (FOXO1) transcription factor plays critical roles in regulating not only metabolic activity but also angiogenesis in the vascular endothelium^1–4. Our previous studies show that epsin endocytic adaptors can regulate both angiogenesis and lymphangiogenesis^5–7. Endothelial cells (ECs) lining the inside of blood vessels are continuously exposed to circulating insulin and insulin-like growth factors (IGFs). Emerging evidences suggest that ECs can affect β-cell function^8–11. Excessive IGF2, especially elevated local IGF2 levels in islets, may represent a risk factor for developing diabetes^12–15; however, the underlying molecular mechanisms by which aberrant angiogenesis and endothelium-derived factors regulate pancreatic β-cell function in diabetes remain unclear. Here, we report that the pancreas of diabetic patients as well as the pancreas, skin, and plasma of streptozotocin/high fat diet (STZ/HFD)-induced diabetic mice and db/db mice contains excess IGF2, which can lead to β-cell dysfunction and apoptosis. Single-cell transcriptomics combined with mass spectrometry analysis reveal that endothelial-specific knockout of FOXO1 increases circulating soluble and cell-membrane or intracellular expression levels of IGF type 2 receptor (IGF2R) and CCCTC-binding factor (CTCF), while decreasing IGF2 levels in diabetes. Both IGFR2^15–17 and CTCF^18–21 can reduce IGF2 levels and may ameliorate β-cell decline associated with excess IGF2 in diabetes. Furthermore, depletion of FOXO1, epsins, or knockdown of ULK1 inhibits autophagy formation in ECs, preventing degradation of vascular endothelial growth factor receptor 2 (VEGFR2) to promote angiogenesis and improve wound healing in diabetes. Our findings reveal that endothelial FOXO1 regulates epsin-dependent angiogenesis and affects β-cell function and fate through CTCF and IGF2-IGF2R, providing a potential strategy for ameliorating diabetes and accelerating cutaneous wound healing.