Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression.

BACKGROUND: Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins. METHODS: We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1fl/+). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTS: We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. CONCLUSIONS: Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.

Targeting Epsins to Inhibit Fibroblast Growth Factor Signaling While Potentiating Transforming Growth Factor-ß Signaling Constrains Endothelial-to-Mesenchymal Transition in Atherosclerosis.

BACKGROUND: Epsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODS: Using single-cell RNA sequencing combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knockout in Apoe-/- and lineage tracing/proprotein convertase subtilisin/kexin type 9 serine protease mutant viral-induced atherosclerotic mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTS: Single-cell RNA sequencing and lineage tracing revealed that epsins 1 and 2 promote EndoMT and that the loss of endothelial epsins inhibits EndoMT marker expression and transforming growth factor-ß signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in the Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 expression, which inhibits transforming growth factor-ß signaling and EndoMT. Epsins directly bind ubiquitinated fibroblast growth factor receptor-1 through their ubiquitin-interacting motif, which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic ubiquitin-interacting motif-containing peptide atheroma ubiquitin-interacting motif peptide inhibitor significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONS: We conclude that epsins potentiate EndoMT during atherogenesis by increasing transforming growth factor-ß signaling through fibroblast growth factor receptor-1 internalization and degradation. Inhibition of EndoMT by reducing epsin-fibroblast growth factor receptor-1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

Promoting Lymphangiogenesis and Lymphatic Growth and Remodeling to Treat Cardiovascular and Metabolic Diseases.

Lymphatic vessels are low-pressure, blind-ended tubular structures that play a crucial role in the maintenance of tissue fluid homeostasis, immune cell trafficking, and dietary lipid uptake and transport. Emerging research has indicated that the promotion of lymphatic vascular growth, remodeling, and function can reduce inflammation and diminish disease severity in several pathophysiologic conditions. In particular, recent groundbreaking studies have shown that lymphangiogenesis, which describes the formation of new lymphatic vessels from the existing lymphatic vasculature, can be beneficial for the alleviation and resolution of metabolic and cardiovascular diseases. Therefore, promoting lymphangiogenesis represents a promising therapeutic approach. This brief review summarizes the most recent findings related to the modulation of lymphatic function to treat metabolic and cardiovascular diseases such as obesity, myocardial infarction, atherosclerosis, and hypertension. We also discuss experimental and therapeutic approaches to enforce lymphatic growth and remodeling as well as efforts to define the molecular and cellular mechanisms underlying these processes.

A preferred sequence for organelle inheritance during polarized cell growth.

Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitantly with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether disrupting the cell cycle alters organelle inheritance order. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs when DNA replication is blocked, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division and does not require completion of S-phase.

Two sides of the same coin: Non-alcoholic fatty liver disease and atherosclerosis.

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.

Interplay Between FoxM1 and Dab2 Promotes Endothelial Cell Responses in Diabetic Wound Healing.

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 uncertain and could reveal new therapeutic targets. In our study, the molecular underpinnings of endothelial dysfunction in diabetes were investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell (EC) function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high concentrations glucose treated primary mouse skin ECs, simulating hyperglycemic conditions in diabetes mellitus. In diabetic mice with a genetic EC deficiency of Dab2 angiogenesis was reduced in vivo and in vitro when compared with wild-type mice. Restoration of Dab2 expression by injected mRNA-containing lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. At the same time, FoxM1 was downregulated in skin ECs subjected to 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. These findings indicate that restoring Dab2 expression through targeted therapies can enhance angiogenesis and wound repair in diabetes. To explore this therapeutic potential, we tested LyP-1-conjugated lipid nanoparticles (LNPs) containing Dab2 or control mRNAs to target ECs and found the former significantly improved wound healing and angiogenesis in diabetic mice. This 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.

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis.

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-ß 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-ß 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 TGFß signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.

Epsins oversee smooth muscle cell reprograming by influencing master regulators KLF4 and OCT4.

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA-seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

Targeting Liver Epsins Ameliorates Dyslipidemia in Atherosclerosis.

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.

PCBP1 regulates alternative splicing of AARS2 in congenital cardiomyopathy.

Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its splicing, are linked to infantile cardiomyopathy in humans. However, how Aars2 regulates heart development, and the underlying molecular mechanism of heart disease remains unknown. Here, we found that poly(rC) binding protein 1 (PCBP1) interacts with the Aars2 transcript to mediate its alternative splicing and is critical for the expression and function of Aars2. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these date provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.

Defective efferocytosis of vascular cells in heart disease.

The efficient phagocytic clearance of dying cells and apoptotic cells is one of the processes that is essential for the maintenance of physiologic tissue function and homeostasis, which is termed "efferocytosis." Under normal conditions, "find me" and "eat me" signals are released by apoptotic cells to stimulate the engulfment and efferocytosis of apoptotic cells. In contrast, abnormal efferocytosis is related to chronic and non-resolving inflammatory diseases such as atherosclerosis. In the initial steps of atherosclerotic lesion development, monocyte-derived macrophages display efficient efferocytosis that restricts plaque progression; however, this capacity is reduced in more advanced lesions. Macrophage reprogramming as a result of the accumulation of apoptotic cells and augmented inflammation accounts for this diminishment of efferocytosis. Furthermore, defective efferocytosis plays an important role in necrotic core formation, which triggers plaque rupture and acute thrombotic cardiovascular events. Recent publications have focused on the essential role of macrophage efferocytosis in cardiac pathophysiology and have pointed toward new therapeutic strategies to modulate macrophage efferocytosis for cardiac tissue repair. In this review, we discuss the molecular and cellular mechanisms that regulate efferocytosis in vascular cells, including macrophages and other phagocytic cells and detail how efferocytosis-related molecules contribute to the maintenance of vascular hemostasis and how defective efferocytosis leads to the formation and progression of atherosclerotic plaques.

The Role of Endothelial-to-Mesenchymal Transition in Cardiovascular Disease.

Endothelial-to-mesenchymal transition (EndoMT) is the process of endothelial cells progressively losing endothelial-specific markers and gaining mesenchymal phenotypes. In the normal physiological condition, EndoMT plays a fundamental role in forming the cardiac valves of the developing heart. However, EndoMT contributes to the development of various cardiovascular diseases (CVD), such as atherosclerosis, valve diseases, fibrosis, and pulmonary arterial hypertension (PAH). Therefore, a deeper understanding of the cellular and molecular mechanisms underlying EndoMT in CVD should provide urgently needed insights into reversing this condition. This review summarizes a 30-year span of relevant literature, delineating the EndoMT process in particular, key signaling pathways, and the underlying regulatory networks involved in CVD.

Non-alcoholic Steatohepatitis Pathogenesis, Diagnosis, and Treatment.

There has been a rise in the prevalence of non-alcohol fatty liver disease (NAFLD) due to the popularity of western diets and sedentary lifestyles. One quarter of NAFLD patients is diagnosed with non-alcoholic steatohepatitis (NASH), with histological evidence not only of fat accumulation in hepatocytes but also of liver cell injury and death due to long-term inflammation. Severe NASH patients have increased risks of cirrhosis and liver cancer. In this review, we discuss the pathogenesis and current methods of diagnosis for NASH, and current status of drug development for this life-threatening liver disease.

Intercalated disc protein Xinß is required for Hippo-YAP signaling in the heart.

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinß, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinß in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinß mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinß knock-out mice-indicating a functional and genetic interaction between Xinß and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinß modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1-10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.