Biology subjects

Cowan, D. B.

Publications and source records attributed to Cowan, D. B..

9 recordsLinked to original sources

bioRxiv2024

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

BackgroundThe protein tyrosine phosphatase CD45 is expressed in all nucleated cells of the hematopoietic system and in mitral valve endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndoMT). Our recent work indicated that activation of endogenous CD45 in human endothelial colony-forming cells (ECFCs) induced expression of multiple EndoMT marker genes. We hypothesized that CD45 may contribute to atherosclerosis; however, detailed molecular mechanisms underlying how CD45 may contribute to EndoMT and the impact of therapeutic manipulation of CD45 expression in atherosclerosis are unknown. MethodsWe generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE-deficient (WT/ApoE-/-) background and fed them a Western diet (WD) to produce atherosclerosis. We enriched mouse aortic ECs with anti-CD31 beads to perform single-cell RNA sequencing. Cellular, biochemical and molecular approaches were used to investigate the effect of endothelial CD45-specific deletion on EndoMT and lesion development in an ApoE-/- mouse model of atherosclerosis. ResultsEC-iCD45KO mice showed reductions in lesion development, plaque macrophage infiltration, and expression of cell adhesion molecules when compared to WT/ApoE-/- controls. Single-cell RNA sequencing revealed that loss of endothelial CD45 decreases EndoMT marker expression and TGF-{beta} signaling in atherosclerotic mice, which is associated with reduction of lesions. Mechanistically, CD45 loss increases Fibroblast Growth Factor Receptor 2 (FGFR2) expression in mouse aortic ECs and Kruppel-like Factor 2 (KLF2) expression in the aortic root. Endothelial CD45-deficiency also inhibits EndoMT and TGF{beta} signaling in atherosclerosis. ConclusionsOur findings demonstrate that genetic depletion of endothelial CD45 protects against EndoMT-driven atherosclerosis by promoting FGFR2 and KLF2 expression while inhibiting Transforming Growth Factor beta (TGF{beta}) signaling and EndoMT. Consequently, targeting endothelial CD45 may represent a novel therapeutic strategy to reduce EndoMT in atherosclerosis.

cell biology↗

bioRxiv2024

Targeting Liver Epsins Ameliorates Dyslipidemia in Atherosclerosis

BackgroundThe 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. MethodsLiver-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. ResultsWestern 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 Ldlrhi 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. ConclusionsLiver 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.

cell biology↗

bioRxiv2024

Targeted Dab2 Restoration: A Novel Approach for Enhanced Wound Healing in Diabetic Conditions

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.

cell biology↗

bioRxiv2024

Epsins promote atherosclerosis through VSMC phenotypic modulation

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.

cell biology↗

bioRxiv2023

Targeting endothelial FOXO1 protects diabetic β-cells and improves wound healing

The forkhead box O1 (FOXO1) transcription factor plays critical roles in regulating not only metabolic activity but also angiogenesis in the vascular endothelium1-4. Our previous studies show that epsin endocytic adaptors can regulate both angiogenesis and lymphangiogenesis5-7. 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 {beta}-cell function8-11. Excessive IGF2, especially elevated local IGF2 levels in islets, may represent a risk factor for developing diabetes12-15; however, the underlying molecular mechanisms by which aberrant angiogenesis and endothelium-derived factors regulate pancreatic {beta}-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 {beta}-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 IGFR215-17 and CTCF18-21 can reduce IGF2 levels and may ameliorate {beta}-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 {beta}-cell function and fate through CTCF and IGF2-IGF2R, providing a potential strategy for ameliorating diabetes and accelerating cutaneous wound healing.

cell biology↗

bioRxiv2023

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.

developmental biology↗

bioRxiv2022

Targeting Epsins to Inhibit FGF Signaling while Potentiating TGF-β Signaling Constrains Endothelial-to-Mesenchymal-Transition in Atherosclerosis

BACKGROUNDEpsin 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. METHODSUsing single cell RNA sequencing (scRNA-seq), combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knock-out mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTSScRNA-seq and lineage tracing revealed that epsins 1 and 2 promote EndoMT, and the loss of endothelial epsins inhibits EndoMT marker expression as well as transforming growth factor-beta signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 (FGFR1) expression that inhibits TGF-{beta} signaling and EndoMT. Epsins directly bind ubiquitinated FGFR1 through their ubiquitin-interacting motif (UIM), which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic UIM-containing peptide API significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONSWe conclude that epsins potentiate EndoMT during atherogenesis by increasing TGF-{beta} signaling through FGFR1 internalization and degradation. Inhibition of EndoMT by reducing epsin-FGFR1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

cell biology↗

bioRxiv2022

Targeting Epsins by nanotherapy regulates lipid metabolism and promotes 1 ABCG1-mediated cholesterol efflux to fortify atheroma regression

BACKGROUNDExcess 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 (NPs), which encapsulate small interfering RNAs to suppress Epsins. METHODSWe used single cell RNA sequencing (scRNA-seq) with our newly developed algorithm MEBOCOST 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 (LysM-DKO-ABCG1fl/+). The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTSWe 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 NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly 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. CONCLUSIONSOur 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. Novelty and SignificanceO_ST_ABSWHAT IS KNOWN?C_ST_ABSO_LIEpsin endocytic adaptor proteins are upregulated in human and mouse atherosclerotic lesions C_LIO_LILesional macrophages internalize lipids primarily through scavenger receptor-mediated endocytosis such as CD36 and SR-A C_LIO_LIMacrophage-mediated cholesterol efflux and reverse cholesterol transport is crucial to atheroma resolution C_LI WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE?O_LIScRNA-seq combined with the newly-developed algorithm MEBOCOST reveals that Epsins are involved in macrophage-mediated lipid metabolic regulation C_LIO_LIMacrophage epsins promote lipid uptake by targeting CD36 endocytosis and membrane recycling via the Epsin ENTH domain C_LIO_LIEpsins bind ubiquitinated ABCG1--resulting in the endocytosis and lysosomal degradation of this cholesterol transporter, which reduces cholesterol efflux. C_LIO_LIMacrophage-specific, nanoparticle-mediated RNAi delivery exhibits a therapeutic benefit for the treatment of atherosclerosis C_LIO_LIAtherosclerotic plaque regression using this nanoparticle delivery platform represents a clinically-relevant approach for the treatment of advance atherosclerosis C_LI BRIEF SUMMARYDespite the development of new cholesterol-lowering therapies, including the recently approved PCSK9 small interfering RNA (siRNA) antagonists, patients still face a tremendous risk of developing major acute cardiovascular events resulting from chronic inflammation in the plaque. We employed a novel nanomedicine platform containing a stabilin-2 targeting peptide (S2P) to deliver Epsin-specific siRNAs to lesional macrophages. We discovered that inhibition of these adaptor proteins in lesional macrophages significantly diminished plaque size and necrotic core area, increased fibrous cap thickness, and promoted plaque regression.

cell biology↗

bioRxiv2017

Transit and integration of extracellular mitochondria in human heart cells

Tissue ischemia adversely affects the function of mitochondria, which results in impairment of oxidative phosphorylation and compromised recovery of the affected organ. The impact of ischemia on mitochondrial function has been most extensively studied in the heart because of the morbidity and mortality associated with injury to this organ. Because conventional methods to preserve cell viability and function following an ischemic injury are limited in their efficacy, we developed a unique approach to protect the heart by transplanting respiration-competent mitochondria isolated from a non-ischemic tissue to the ischemic region. Our experiments in animals have shown that transplantation of isolated mitochondria to injured heart tissue leads to decreases in cell death, increases in energy production, and improvements in contractile function. We also discovered that exogenously-derived mitochondria injected or perfused into ischemic hearts were readily internalized by cardiac cells through actin-dependent endocytosis. Here, we describe the use of three-dimensional super-resolution microscopy and transmission electron microscopy to determine the intracellular fate of exogenous mitochondria in non-dividing human iPS-derived cardiomyocytes and dividing primary human cardiac fibroblasts. We show isolated mitochondria are internalised in human cardiac cells within minutes and then transported to endosomes and lysosomes. The majority of exogenous mitochondria escape from these compartments and fuse with the endogenous mitochondrial network, while some organelles are degraded through hydrolysis. Understanding this process may guide the development of treatments directed at replacing or augmenting impaired mitochondria in ischemic tissues and provide new options to rejuvenate dysfunctional mitochondria in a wide range of human diseases and disorders.

cell biology↗