Arteriosclerosis, Thrombosis, and Vascular Biology

The use of nilotinib (Tasigna(R)), a second-generation tyrosine kinase inhibitor for treating chronic myeloid leukemia, increases risks for atherosclerosis. Here, we demonstrate that in endothelial cells, nilotinib activated TLR4, triggerd expression of inflammatory molecules, and increased monocyte attachment, which were all inhibited by knockdown of TLR4 or TLR4 inhibitor, CLI-095. Orally administered nilotinib profoundly accelerated atherosclerotic lesion formation in ApoE-/- mice, while co-administration of CLI-095 effectively reduced lesion areas. Our findings reveal TLR4 activation as an underlying mechanism of the pro-atherosclerotic effect of nilotinib and suggest TLR4 inhibition as an effective therapeutic approach to address vascular safety issue of nilotinib.

OBJECTIVEEvidence from previous studies has demonstrated that NRF2 can protect cardiovascular progression,which prompted us to study if NRF2 is involved in AAA development. APPROACH AND RESULTSThe difference and function of NRF2 in AAA were verified on human aortic tissues combined with scRNA-seq samples.We then used angiotensin II (Ang II) infusion mouse model with VSMCs-specific knockout to study the role of NRF2 in AAA formation.Primary cultured VSMCs were used to study how NRF2 regulates contractile VSMCs phenotype.Patient specimens were obtained to investigate the relevance of NRF2 expression to human AAA disease. NRF2 was induced in abdominal aortic VSMCs in both mouse and human AAA tissues. VSMCs-specific NRF2 knockout increased experimental AAA formation. Mechanistically, NRF2 regulates the contractile VSMCs phenotypic through miRNA145, leading to increased classic contractile protein level to prevent the development of AAA. CONCLUSIONSNRF2 is a negative regulator of AAA development, and thus may represent a potentially new therapeutic target to inhibit AAA growth and rupture.

BACKGROUNDEndothelial cell (EC) dysfunction is both a cause and consequence of vascular inflammation and lipid dysregulation in atherosclerosis, yet the molecular drivers linking EC dysfunction to systemic metabolic derangements remain incompletely understood. We previously identified Argonaute 1 (AGO1)--a canonical component of the RNA-induced silencing complex--as a regulator of EC function in angiogenesis and metabolism. In this study, we uncover a previously unrecognized, non-canonical role of nuclear AGO1 in ECs as a transcriptional coactivator of NF-{kappa}B, and demonstrate that EC-specific AGO1 inhibition simultaneously improves lipid metabolism, liver function, and vascular inflammation, thereby attenuating atherosclerosis. METHODSEC-conditional AGO1 knockout (EC-AGO1-KO) and wildtype mice were subjected to pro-atherosclerotic models induced by AAV9-PCSK9 and a western diet, or carotid artery ligation. Metabolic and vascular phenotyping and gene expression analyses were performed. In human liver sinusoidal ECs (HLSECs) and human aortic ECs (HAECs), AGO1 was knocked down using antisense oligos (ASO), followed by assays for inflammatory responses (qPCR, RNA-seq, ELISA, and monocyte adhesion). Mechanistic studies included Cut&Tag sequencing, and chromatin immunoprecipitation assays, and EC-hepatocyte co-cultures. Therapeutic effect of AGO1 inhibition was assessed using lipid nanoparticle (LNP)-delivered ASO in mice. RESULTSEC-AGO1-KO mice exhibited significantly improved plasma lipid profiles, reduced hepatic steatosis, inflammation, and fibrosis, and decreased aortic atherosclerotic burden. AGO1 knockdown in ECs dampened inflammatory responses and monocyte recruitment and enhanced hepatocyte lipid metabolism via paracrine signaling. Mechanistically, nuclear AGO1 interacted with NF-{kappa}B p65 to enhance transcription of pro-inflammatory genes including ICAM1, THBS1. LNP-delivered AGO1-ASO improved hyperlipidemia, liver function, and atherosclerosis without evident hepatotoxicity. CONCLUSIONSEndothelial AGO1 promotes vascular inflammation and liver dysfunction through a non-canonical role as an NF-{kappa}B coactivator. Its inhibition provides dual benefits--ameliorating lipid dysregulation and suppressing vascular inflammation--highlighting EC-AGO1 as a promising therapeutic target for atherosclerosis and cardiometabolic diseases.

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.

Residual risk of cardiovascular events remains despite treatments that effectively lower cholesterol levels and hypertension, suggesting that there must be more variables to consider in atherosclerosis treatment. Several studies have suggested sex1,2,3 and inflammation4,5 as important variables. However, a cross-cohort analysis of sex and risk factors like inflammation and lipid-handling dysfunction is needed to strengthen their connection to atherosclerosis. By using blood transcriptomic profiles of 391 male and female participants, this study revealed that inflammation and lipid-handling dysfunction have sex-specific roles in atherosclerosis. Transcriptomics of 391 human blood samples with varying degrees of atherosclerosis were used to identify sex-specific changes in immune response and lipid-handling in circulating blood cells. Preliminary analyses of both FPKM and normalized counts datasets showed that inflammatory pathway activation and enrichment increased as atherosclerotic disease severity increased across all sexes. Analysis of sex-specific differentially expressed genes (DEGs) using IPAs Canonical Pathways showed that severely impacted females had more enriched inflammatory pathways than severely impacted males. Further cross-cohort analysis of sex-specific inflammation and lipid-handling dysfunction was performed using AtheroSpectrum, a single-sample macrophage annotation tool. AtheroSpectrum confirmed that inflammation was more critical to female atherogenesis and revealed that lipid-handling dysfunction was more critical to male atherogenesis. Our study underscored the importance of inflammation and sex as variables to consider in atherosclerosis treatment, suggesting that treatment should target inflammation and consider sex. Our findings may be used for generating a model to predict atherosclerosis risk based on key DEGs, pathways, sex, and other clinical parameters when available.

ObjectiveVascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues. Approach and ResultsWe characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function of Prdm16 in vivo, we generated Prdm16 SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency of Prdm16 does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically, Prdm16 negatively regulates the expression of Tgfb2 that encodes for an upstream ligand of TGF-{beta} signaling pathway, potentially through binding to the promoter region of Tgfb2. These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state. ConclusionsOur results suggest that the CAD risk gene PRDM16 is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis.

Vascular dysfunction and coagulopathy are hallmarks of severe COVID-19. How SARS-CoV-2 infection drives endothelial dysfunction, despite the virus not infecting or replicating in endothelial cells, remains controversial. Here, we used an in vitro co-culture model of the human pulmonary epithelial-endothelial cell barrier to investigate which inflammatory mediators drive endothelial dysfunction during SARS-CoV-2 infection. SARS-CoV-2 infection of primary human bronchial epithelial cells increased adjacent endothelial cell expression of the leukocyte adhesion marker ICAM-1, disrupted endothelial VE-cadherin junctions, promoted endothelial cell death, and promoted platelet adherence to gaps in the endothelial monolayers. Dexamethasone treatment rescued these dysregulated endothelial phenotypes in infected co-cultures, confirming that inflammatory signalling was the primary driver of SARS-CoV-2-induced endothelial dysfunction. Specifically, epithelial-derived TNF and IL-1{beta} promoted endothelial dysfunction, as inhibition of TNF or IL-1R signalling blocked SARS-CoV-2-induced endothelial dysfunction in co-cultures. SARS-CoV-2-infected wild-type mice, but not TNF, IL-1{beta}, or TNF/IL-1{beta}- deficient mice, displayed increased endothelial ICAM-1 expression, while an anti-IL-1{beta} monoclonal antibody prevented SARS-CoV-2-induced ICAM-1 expression and fibrin deposition in aged K18-ACE2 mice. Our data indicate that TNF and IL-1{beta} are the specific cytokines that drive multiple aspects of endothelial dysfunction during acute SARS-CoV-2 infection, and that inhibiting their signalling pathways may provide therapeutic benefit in preventing vascular complications of COVID-19.

BackgroundMacrophages are key players in the pathogenesis of atherosclerosis. They trigger immune responses through their cell-surface receptors. However, how macrophages regulate those receptors in response to pro-inflammatory stimuli is not completely understood. Endocytic membrane trafficking involving receptor internalization, followed by endosomal transport and recycling of the internalized receptors, plays essential roles in balancing cell-surface receptors to meet cellular needs. Here, we explored the role of the endocytic regulator EHD1 in immune responses in macrophages and determined its contribution to atherosclerosis progression. MethodsEHD1 expression profiles in mouse and human plaques were determined by single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining. Bone marrow transplantation (BMT) by transplanting bone marrow cells from Ehd1-/- or littermate wild-type mice to irradiated Ldlr-/- mice was performed to determine the effect of EHD1 deletion on atherosclerosis progression. In vitro mechanistic studies including inflammation signaling and endocytosis assays were performed in bone marrow-derived macrophages. ResultsEHD1 expression in macrophages is enhanced as atherosclerosis progresses in both mice and humans. Histological analysis of aortic root sections from BMT mice showed that EHD1 deletion reduces lesion size. ScRNA-seq of aortic CD45+ cells demonstrated that EHD1 deletion attenuates pro-inflammatory responses and cell-cell interactions. Mechanistic studies revealed that EHD1 accelerates the endocytic recycling of TNFR2 and activates NF-kB, leading to increased expression of inflammatory cytokines. Moreover, EHD1 interacts with retromer and stabilizes sortilin, a retrograde cargo of retromer and a risk factor for atherosclerosis. ConclusionsEHD1 promotes inflammation by enhancing TNFR2-NF-kB signaling and stabilizing sortilin, leading to accelerated atherosclerosis. Our study reveals novel roles for EHD1-mediated membrane trafficking in macrophage function and paves the way to innovative therapeutic strategies that aim to address dysregulated membrane trafficking in atherosclerosis.

ObjectiveTo elucidate the sex-specific trajectories of endothelial cell (EC) transdifferentiation and their contribution to distinct atherosclerotic plaque phenotypes in males and females. Approach and ResultsWe utilized Cdh5-CreERT2/Apoe-/-/Rosa26-mTmG lineage-tracing mice to perform single-nucleus RNA sequencing (snRNA-seq) of aortic tissues from male, female, and ovariectomized female mice following 12 weeks of high-fat diet. We mapped high-resolution EC fates, identifying distinct transdifferentiation routes into smooth muscle cell (SMC)-like, adipocyte-like, macrophage-like, and fibroblast-like cells. Females exhibited higher overall EC plasticity with a preference for SMC-like and metabolically active adipocyte-like trajectories, contributing to plaque stability. Conversely, male ECs favored inflammatory macrophage-like and stress-responsive fates. Trajectory analysis revealed a novel direct EC-to-adipocyte-like transition that bypasses the canonical endothelial-to-mesenchymal transition (EndoMT). Gene regulatory network analysis, validated by in vitro knockdown assays, identified KLF2 as a homeostatic brake on transdifferentiation, while CREB5 and ESRRG emerged as critical molecular switches governing the bifurcation between adipogenic and myogenic fates. Ovariectomy in females shifted the transcriptomic landscape toward a male-like, lipid-anabolic phenotype. ConclusionsEC plasticity is a sex-dimorphic process driving differential plaque composition. We identify a novel, direct EC-to-adipocyte trajectory and pinpoint KLF2 and CREB5 as key regulators, offering new mechanistic insights into why premenopausal women develop more stable atherosclerotic lesions than men.

BACKGROUNDThoracic aortic dissection (TAD) is a life-threatening vascular disease that requires effective drug treatment to prevent progression and rupture. Because arachidonic acid metabolism is involved in inflammation and vascular homeostasis, we investigated the roles of arachidonic acid metabolites in TAD pathogenesis and their utility as therapeutic targets. METHODSSerum metabolomics analysis was performed to characterize arachidonic acid metabolites in TAD patients and a TAD mouse model. 12/15-LOX expression was profiled in the aortic tissues of TAD patients and the TAD mouse model. Four-week-old male Alox15 knockout mice (Alox15-/-), 12-HETE-treated mice, ML351 (12/15-LOX inhibitor)-treated mice, and LY255283 (leukotriene B 4 receptor 2 [BLT2] antagonist)-treated mice received {beta}-aminopropionitrile monofumarate (BAPN, 1 g/kg/day) for 4 weeks to model TAD, then underwent assessment of TAD progression. Interaction of 12-HETE produced by macrophages with BLT2 receptor-expressing cells was detected by molecular docking and immunoblotting. RESULTSSerum levels of 12-HETE and the expression of 12/15-LOX in aortic tissue were significantly increased in TAD patients and BAPN-treated TAD mice. BAPN-induced TAD progression was significantly ameliorated in Alox15-deficient or -suppressed mice. 12-HETE directly interacted with BLT2 receptors on macrophages, activating the downstream NOX-1/ROS/NF-{kappa}B signaling pathway to induce inflammatory cytokine release. This initiated inflammatory cell recruitment and exacerbated extracellular matrix degradation, leading to phenotype switching in vascular smooth muscle cells (VSMCs). Additionally, treatment with ML351 and LY255283 significantly reduced the rates of dissection rupture and combined treatment could maximize the curative effect. CONCLUSIONS12-HETE may amplify the inflammatory cascade and trigger aberrant phenotype switching in VSMCs during TAD development. The reduction of circulating 12-HETE or antagonism of its receptor may be new targets for TAD prevention and treatment. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThe expression levels of 12/15-LOX and its metabolite 12-HETE were elevated in TAD patients and TAD mice. C_LIO_LIIncreased levels of 12-HETE directly bind to BLT2 receptors in macrophages, thereby initiating inflammatory cascades that downregulate VSMC differentiation markers through the suppression of IL-6. C_LIO_LIDeletion or pharmacologic inhibition of 12/15-LOX and suppression of BLT2 mitigated TAD development by alleviating inflammation and VSMC phenotype switching. C_LI What Are the Clinical Implications?O_LIThe inhibition of 12-HETE-related pathways, through mechanisms such as reducing the plasma 12-HETE content or blocking its receptor, may represent a novel therapeutic strategy for TAD. C_LIO_LIFurther studies are needed to explore the diagnostic value of serum 12-HETE as a novel biomarker for TAD. C_LI

Pathological retention of LDL in the intima is involved in atherosclerosis, although the retention mechanisms are not well-understood. Previously, we reported Sterile Alpha Motif Domain Containing 1 (SAMD1), a protein secreted by intimal smooth muscle cells in atherosclerotic lesions, appears to bind LDL in extracellular matrix around intimal cells. Fab-fragment inhibitors of apparently irreversible SAMD1/LDL binding reduced LDL retention in carotid injury models, but did not have a significant effect on early spontaneous lesion development. Our histology of mouse atherosclerosis models revealed extensive SAMD1 expression, possibly related to cell phenotype modulation and antigen presentation. Although the normal function of SAMD1 is unknown, it may have multiple epigenetic roles. For this report, we generated SAMD1-/-, SAMD1-/+, and SAMD1-/+ apoE-/- mice to further explore SAMD1s role in atherosclerosis. SAMD1 was found in tissues throughout the SAMD1+/+ and SAMD1-/+ embryos. Homozygous loss of SAMD1 was embryonic lethal: at embryonic day 14, organs were partially developed and/or degraded; heads and brains were malformed; no blood vessels were observed; red blood cells were scattered and pooled, primarily near the embryo surface; and cell death was occurring. Development appeared normal in heterozygous SAMD1 embryos, but postnatal genotyping showed a reduced ability to thrive. Growth of atherosclerotic lesions in SAMD1-/+ apoE-/- after 35 weeks was not significantly less than in mice SAMD1+/+ apoE-/- mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

Vascular endothelial cell (EC) dysfunction is a pathological mediator of he development, progression, and clinical manifestations of atherosclerotic disease. Inflammation is associated with EC dysfunction, but the responsible mechanisms are not well characterized. There is substantial evidence that serum proprotein convertase subtilisin/kexin type 9 (PCSK9) is increased in pro-inflammatory states and that elevated PCSK9 levels are associated with adverse cardiovascular outcomes after controlling for traditional risk factors, including low-density lipoprotein (LDL) cholesterol. Here we investigate PCSK9 as a novel link between inflammation and vascular EC dysfunction, as assessed by nitric oxide (NO) bioavailability. Tumor necrosis factor alpha (TNF-), a pro-inflammatory cytokine, increased PCSK9 mRNA expression and PCSK9 protein levels in isolated human aortic ECs, which were accompanied by reduced total and phosphorylated endothelial nitric oxide synthase (eNOS) protein levels and NO bioavailability. Finally, genetic PCSK9 reduction utilizing a PCSK9 specific siRNA in human aortic ECs resulted in the rescue of phosphorylated eNOS protein levels and NO bioavailability. Our results demonstrate that PCSK9 is increased in human aortic ECs exposed to a pro-inflammatory stimulus and that this increase is associated with EC dysfunction. Silencing of TNF-mediated augmentation of PCSK9 expression utilizing a small interfering RNA against PCSK9 rescued the inflammation-induced EC dysfunction. These results indicate that PCSK9 is a causal link between inflammation and EC dysfunction, a potent driver of atherosclerotic cardiovascular disease.

BACKGROUNDNeointimal hyperplasia (NH) is a common pathological response to vascular injury and mediated primarily by vascular smooth muscle cell (VSMC) migration and proliferation. The COP9 signalosome (CSN) is formed by 8 canonical subunits (CSN1 through CSN8) with its deneddylation activity residing in CSN5. Each or some of CSN subunits may have deneddylation-independent function. Despite strong evidence linking the CSN to cell cycle regulation in cancer cells, the role of the CSN in vascular biology remains obscure. METHODSNeointimal CSN5 expression in the lung tissue of pulmonary hypertension (PAH) patients was assessed with immunohistochemistry. Adult mice with smooth muscle cell-restricted CSN5 knockout (CSN5-SMKO) or CSN8 hypomorphism (CSN8-hypo) and cultured mouse VSMCs were studied to determine the role and governing mechanisms of the CSN in NH. NH was induced by ligation of the left common carotid artery (LCCA) and PDGF-BB stimulation was used to mimic the vascular injury in cell cultures. RESULTSRemarkably higher CSN5 levels were detected in the neointimal VSMCs of the pulmonary arteries of human PAH. LCCA ligation induced NH and significantly increased the mRNA and protein levels of CSN subunits in the LCCA wall of adult wild type mice. CSN5-SMKO impaired Cullin deneddylation and the nuclear export of p27 in vessel walls and markedly inhibited VSMC proliferation in mice. On the contrary, CSN8-hypo significantly exacerbated NH and VSMC proliferation in vivo and in cellulo. Cytoplasmic CSN5 mini-complexes and the nuclear export of p27 were significantly increased in CSN8-hypo mouse vessels and cultured CSN8-hypo VSMCs. Nuclear export inhibition with leptomycin attenuated the PDGF-BB-induced increases in VSMC proliferation in both CSN8-hypo and control VSMCs. Further, genetically disabling CSN5 nuclear export but not disabling CSN5 deneddylase activity suppressed the hyperproliferation and restored p27 nuclear localization in CSN8 hypomorphic VSMCs. Interestingly, CSN deneddylase inhibition by CSN5i-3 did not alter the hyperproliferation of cultured CSN8-hypo VSMCs but suppressed wild type VSMC proliferation in cellulo and in vivo and blocked neointimal formation in wild type mice. CONCLUSIONThe CSN promotes VSMC proliferation and NH in injured vessels through deneddylation activity and CSN5-mediated nuclear export.

BACKGROUNDSmooth muscle cells (SMCs) substantially contribute to the development of atherosclerosis through a process called "phenotypic switching." Our previous work identified an SMC-derived intermediate cell type, termed "SEM" cells, which plays a crucial role in SMC transition to other cell types and in lesion development. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuates atherosclerosis in mice coincident with suppression of SEM cell formation. However, the effect of RA signaling on advanced disease and the underlying molecular mechanisms by which RA modulates SMC transition to SEM cells are largely unknown. METHODSWe applied SMC lineage tracing atheroprone mice and biochemistry and cell and molecular biology techniques (e.g., RNA sequencing, quantitative reverse transcription PCR, co-immunoprecipitation, and chromatin immunoprecipitation-quantitative PCR) to reveal the regulatory mechanisms of RA signaling in SMC transition to SEM cells. RESULTSActivation of RA signaling with ATRA in established atherosclerosis significantly reduced SEM cells and lesion size while increasing fibrous cap thickness. Mechanistically, retinoic acid receptor alpha (RAR) directly targets the promoters of Ly6a and Ly6c1 in mouse SMCs, and activation of RA signaling recruits EZH2 to the regulatory elements triggering local H3K27me3. Distinct from a molecular model that reported for RA recruitment of HDAC1 during embryogenesis, RAR/EZH2 complex recruits SIRT1 and SIRT6, rather than classical HDACs, to the regulatory regions of key SEM cell marker genes. This subsequently reduces multiple acetylated histone modifications (e.g., H3K27ac, H3K4ac, H3K9ac, H3K14ac, H3K56ac) with recruitment of the transcription corepressor, NCOR1, to repress downstream SEM cell marker genes. CONCLUSIONSOur findings provide novel mechanistic insights into RA modulating SMC phenotypic switching in atherosclerosis, suggesting molecular targets for preventive and therapeutic interventions for atherosclerosis and its clinical complications.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs limits their clinical utility. Combinatorial strategies enhance their antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E2, formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE2 synthesis /response pathway - global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4 - on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr-/-) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE2 biosynthesis, reflected by its major urinary metabolite, PGE2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4-/- mice was impaired. Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.

BACKGROUNDEndothelial cells lining the vasculature form a polarised secretory organ that releases proteins apically into the circulation and basally into the subendothelial matrix. While this secretion plays many critical roles in homeostasis, the potential roles for endothelial secretion in vascular disease remain under-studied. METHODSWe treated polarised monolayers of primary human endothelial cells with native and oxidised LDL and identified the proteins secreted from the apical and basal endothelial surfaces by tandem mass tag mass spectrometry. RESULTSTreatment with either native or oxidised LDL led to increased secretion of a cohort of 21 mostly pro-fibrotic proteins. We focussed on fibronectin, which was secreted apically or basally, depending on the direction of LDL treatment. LDL-stimulated fibronectin secretion did not require well-characterised endothelial LDL receptors but was instead mediated by the poorly characterised scavenger receptor Scarf1 and the cholesterol-sensitive SNARE proteins syntaxin 4 and 6. This LDL-stimulated secretion did not involve increased fibronectin expression but instead appeared to result from a decrease in fibronectin turnover and a re-routing of intracellular sorting. CONCLUSIONSThis novel endothelial secretory pathway links circulating LDL to the release of pro-fibrotic proteins from the endothelium, supporting a role for endothelial cell secretion in the progression of lipid-induced fibrotic disease.

Pericytes (PCs) play crucial roles in capillary maturation, stability, and homeostasis. Impaired PC coverage and function are implicated in various diseases, including pulmonary arterial hypertension (PAH). Challenges investigating PC biology are largely due to the lack of a concise marker, resulting in difficulty distinguishing PCs from other mural cell populations, including smooth muscle cells (SMCs) and fibroblasts (FBs). Utilizing bioinformatic analysis and RNAscope, we identified HIG hypoxia-inducible domain family member 1B (Higd1b) as a unique and conserved gene marker for PCs and generated a novel knockin mouse line, Higd1b-CreERT2, which precisely labels PCs in the lung and heart. Human lung single-cell RNAseq suggested the presence of two HIGD1B+ PC subtypes with different functions. By lineage tracing pulmonary Higd1b+ cells exposed to hypoxia in vivo, we identified Type 1 PCs remained in the capillary network, while Type 2 PCs accumulated in the arterioles and coexpressed SMC markers and increased levels of Vimentin, associated with focal adhesion pathways. These results suggest that Type 1 PCs are specialized for supporting capillary EC homeostasis and quiescent, while Type 2 PCs are lineage active and located close to the border zone of the arterioles and capillaries, which may be motile and transition to SMC-like cells in hypoxia-induced pulmonary hypertension. The discovery of PC-type specialization in capillaries transforms our understanding of the structure, function and regulation of pulmonary capillary circulation and their contribution to vascular remodeling.

Perivascular adipose tissue (PVAT), an intriguing layer of fat surrounding blood vessels, regulates vascular tone and mediates vascular dysfunction through mechanisms that are not well understood. Here we show with single nucleus RNA-sequencing of thoracic aortic PVAT from Dahl SS rats that a high-fat (HF) hypertensive diet induces coordinated changes in gene expression across the diverse cell types within PVAT. HF diet produced sex-specific alterations in cell-type proportions and genes related to remodeling of extracellular matrix dynamics and vascular integrity and stiffness, as well as changes in cell-cell communication pathways involved in angiogenesis, vascular remodeling, and mechanotransduction. Gene regulatory network analysis with virtual transcription factor knockout in adipocytes identified specific nuclear receptors that could be targeted for suppression or potential reversal of HF diet-induced changes. Interestingly, generative deep learning models were able to predict cross-cell-type perturbations in gene expression, indicating a hypertensive disease signature that characterizes HF-diet-induced perturbations in PVAT.

BackgroundAtherosclerosis occurs preferentially in the arteries exposed to disturbed flow (d-flow), while the stable flow (s-flow) regions are protected even under hypercholesterolemic conditions. We recently showed that d-flow alone initiates flow-induced reprogramming of endothelial cells (FIRE), including the novel concept of partial endothelial-to-immune-cell-like transition (partial EndIT), but was not validated using a genetic lineage-tracing model. Here, we tested and validated the two-hit hypothesis that d-flow is an initial instigator of partial FIRE but requires hypercholesterolemia to induce a full-blown FIRE and atherosclerotic plaque development. MethodsMice were treated with adeno-associated virus expressing proprotein convertase subtilisin/kexin type 9 and a Western diet to induce hypercholesterolemia and/or partial carotid ligation (PCL) surgery to expose the left common carotid artery (LCA) to d-flow. Single-cell RNA sequencing (scRNA-seq) analysis was performed using cells obtained from the intima and leftover LCAs and the control right common carotid arteries at 2 and 4 weeks post-PCL. Comprehensive immunohistochemical staining was performed on EC-specific confetti mice treated with PCL and hypercholesterolemic conditions at 4 weeks post-PCL to validate endothelial reprogramming. ResultsAtherosclerotic plaques developed by d-flow under hypercholesterolemia at 2 and 4 weeks post-PCL, but not by d-flow or hypercholesterolemia alone, as expected. The scRNA-seq results of 98,553 single cells from 95 mice revealed 25 cell clusters; 5 EC, 3 vascular smooth muscle cell (SMC), 5 macrophage (M{Phi}), and additional fibroblast, T cell, natural killer cell, dendritic cell, neutrophil, and B cell clusters. Our scRNA-seq analyses showed that d-flow under hypercholesterolemia transitioned healthy ECs to full immune-like (EndIT) and, more surprisingly, foam cells (EndFT), in addition to inflammatory and mesenchymal cells (EndMT). Further, EC-derived foam cells shared remarkably similar transcriptomic profiles with foam cells derived from SMCs and M{Phi}s. Comprehensive lineage-tracing studies using immunohistochemical staining of canonical protein and lipid markers in the EC-specific confetti mice clearly demonstrated direct evidence supporting the novel FIRE hypothesis, including EndIT and EndFT, when d-flow was combined with hypercholesterolemia. Further, reanalysis of the publicly available human carotid plaque scRNA-seq and Perturb-seq datasets supported the FIRE hypothesis and a potential mechanistic link between the genes and FIRE. ConclusionWe provide evidence supporting the two-hit hypothesis: ECs in d-flow regions, such as the branching points, are partially reprogrammed, while hypercholesterolemia alone has minimal endothelial reprogramming effects. Under hypercholesterolemia, d-flow fully reprograms arterial ECs, including the novel EndIT and EndFT, in addition to inflammation and EndMT, during atherogenesis. This single-cell atlas provides a crucial roadmap for developing novel mechanistic understanding and therapeutics targeting flow-sensitive genes, proteins, and pathways of atherosclerosis.