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.

Circulating vascular endothelial growth factor (VEGF) ligands and receptors are central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis. In response to VEGF ligand binding, VEGF receptor tyrosine kinases initiate the chain of events that transduce extracellular signals into endothelial cell responses such as survival, proliferation, and migration. These events are controlled by intricate cellular processes that include the regulation of gene expression at multiple levels, interactions of numerous proteins, and intracellular trafficking of receptor-ligand complexes. Endocytic uptake and transport of macromolecular complexes through the endosome-lysosome system helps fine-tune endothelial cell responses to VEGF signals. Clathrin-dependent endocytosis remains the best understood means of macromolecular entry into cells, although the importance of non-clathrin-dependent pathways is increasingly recognized. Many of these endocytic events rely on adaptor proteins that coordinate internalization of activated cell-surface receptors. In the endothelium of both blood and lymphatic vessels, epsins 1 and 2 are functionally redundant adaptors involved in receptor endocytosis and intracellular sorting. These proteins are capable of binding both lipids and proteins and are important for promoting curvature of the plasma membrane as well as binding ubiquitinated cargo. Here, we discuss the role of epsin proteins and other endocytic adaptors in governing VEGF signaling in angiogenesis and lymphangiogenesis and discuss their therapeutic potential as molecular targets.

BACKGROUND: The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation.

METHODS: We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed.

RESULTS: We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes.

CONCLUSIONS: These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

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.

Endothelialization of engineered vascular grafts for replacement of small-diameter coronary arteries remains a critical challenge. The ability for an acellular vascular graft to promote endothelial cell (EC) recruitment in the body would be very beneficial. This study investigated epsins as a target since they are involved in internalization of vascular endothelial growth factor receptor 2. Specifically, epsin-mimetic UPI peptides are delivered locally from vascular grafts to block epsin activity and promote endothelialization. The peptide delivery from fibrin coatings allowed for controlled loading and provided a significant improvement in EC attachment, migration, and growth in vitro. The peptides have even more important impacts after grafting into rat abdominal aortae. The peptides prevented graft thrombosis and failure that is observed with a fibrin coating alone. They also modulated the in vivo remodeling. The grafts are able to remodel without the formation of a thick fibrous capsule on the adventitia with the 100 µg mL-1 peptide-loaded condition, and this condition enabled the formation of a functional EC monolayer in the graft lumen after only 1 week. Overall, this study demonstrated that the local delivery of UPI peptides is a promising strategy to improve the performance of vascular grafts.