BACKGROUND:Individuals with JAK2V617F-mutant myeloproliferative neoplasms or clonal hematopoiesis of indeterminate potential have a markedly increased risk of cardiovascular disease, yet the mechanisms by which mutant blood cells drive vascular and cardiac dysfunction remain incompletely understood. Although the thrombopoietin receptor MPL is central to hematopoiesis and is expressed in vascular endothelial cells (ECs), its role in JAK2V617F-associated cardiovascular complications is unknown. METHODS:We generated chimeric mice with JAK2V617F-mutant blood cells and wild-type endothelium by bone marrow transplantation and challenged them with a high-fat/high-cholesterol diet to model cardiometabolic stress. RESULTS:Mice with JAK2V617F-mutant blood cells developed a distinct cardiovascular phenotype characterized by microvascular disease, increased left ventricular mass, and relatively preserved left ventricular ejection fraction. Histopathologic analysis revealed coronary arteriole stenosis, perivascular fibrosis, reduced microvascular density, and endocardial injury, without evidence of epicardial coronary stenosis or myocardial infarction. Single-cell RNA sequencing revealed activation of inflammatory, stress-response, and endothelial-to-mesenchymal transition gene signatures in ECs, most prominently within the endocardial ECs. Immunohistochemistry identified MPL expression predominantly in endocardial ECs. Thrombopoietin/MPL signaling was upregulated in endocardial ECs in mice with JAK2V617F-mutant hematopoiesis, and treatment with an anti-MPL neutralizing antibody markedly improved cardiovascular pathology, restored endocardial integrity, and increased coronary microvascular density. CONCLUSIONS:JAK2V617F-mutant hematopoiesis induces cardiac microvascular dysfunction under cardiometabolic stress. Endocardial ECs play a critical role in this pathological process, and endocardial MPL signaling constitutes a potential targetable pathway in JAK2V617F-associated cardiovascular disease.
BACKGROUND:Atherosclerosis is driven by metabolic-immune crosstalk, in which trained immunity sustains vascular inflammation. MAP17 (membrane-associated protein 17), a redox- and metabolism-regulating adaptor protein, functions as a potential upstream driver of SGLT2 (sodium-glucose cotransporter 2). We aimed to determine whether MAP17 links hyperglycemia to glycolytic activation, inflammatory polarization, and plaque progression in atherosclerosis. METHODS:MAP17 expression and its correlations with clinical risk factors were analyzed in serum from 30 patients with atherosclerosis. A trained immunity model was established in bone marrow-derived macrophages via sustained high glucose and IFN-γ (interferon-γ)/lipopolysaccharide stimulation. Functional assays were performed after MAP17 overexpression/knockdown, SGLT2 silencing, or glycolysis inhibition. RESULTS:MAP17 was significantly coupregulated in patients with atherosclerosis, with the highest levels observed in those with concomitant diabetes or metabolic syndrome, and closely associated with elevated proinflammatory M1-like cytokines. Immunohistochemistry of carotid plaques confirmed its colocalization with SGLT2 within CD68+ macrophage-rich, lipid-laden, and inflamed regions. In bone marrow-derived macrophages, high glucose robustly induced MAP17 expression, which unidirectionally upregulated SGLT2, enhanced glycolytic flux, increased lactate production, and promoted M1-like polarization and foam cell formation. MAP17 knockdown markedly suppressed SGLT2 expression, glycolysis, and TNF-α (tumor necrosis factor-α)/IL (interleukin)-1β secretion, whereas MAP17 overexpression restored glycolytic activity, proinflammatory phenotype, and foam cell generation even in SGLT2-deficient cells. In diabetic chimeric Apoe-/- mice, MAP17 activation correlated with increased glycolytic marker expression, higher proinflammatory M1-like macrophage ratios, aggravated vascular inflammation, and greater plaque burden; these effects were mitigated by MAP17 or SGLT2 silencing, or by glycolysis inhibition. CONCLUSIONS:MAP17 is a key upstream controller of the SGLT2-glycolysis axis that promotes trained immunity and accelerates atherosclerosis. Targeting MAP17 may disrupt the metabolic-inflammatory feedback loop and represents a promising therapeutic strategy for diabetic atherosclerosis.
BACKGROUND:Comprehensive investigation of endothelial cell (EC) dysfunction during atherosclerosis with single-cell omics has resulted in the proposal that ECs undergo multiple alternative cell fate decisions during disease, but lack of lineage tracing or spatial localization complicates interpretation of these data. TWIST1, a causal gene for multiple atherosclerotic vascular diseases, is activated with low shear stress in ECs, and EC-Twist1 knockout results in reduced atherosclerosis. However, it remains unclear how Twist1 affects EC phenotype and plaque biology. METHODS:We performed EC lineage tracing, in situ analysis, and scRNA-Seq in ApoE-/- mice, both before disease and after 16 weeks of high-fat diet. We also performed these studies with 2 mouse models of EC Twist1 deletion. We overexpressed TWIST1 in human coronary artery ECs exposed to different flow conditions, followed by bulk RNA-seq. Human scRNA-Seq data were used to validate key findings in the mouse model. RESULTS:We found that EC phenotypic modulation during atherosclerosis is characterized by both proinflammatory and endothelial-to-mesenchymal transition gene programs, occurring simultaneously along a single-cell fate transition. Human scRNA-Seq data validated a similar endothelial-to-mesenchymal transition during disease. We found that the commonly used Twist1 conditional allele is hypomorphic, leading to reduced Twist1 expression in multiple cell types. Using a mouse model of EC-specific Twist1 deletion, we found reduced EC phenotypic modulation, decreased lesion size, and a more stable lesion phenotype. Integration of TWIST1 overexpression in human coronary artery ECs with the mouse scRNA-seq data identified specific TWIST1-induced targets including CXCL12 and E-selectin during EC phenotypic modulation. CONCLUSIONS:Our study revealed important aspects of EC phenotypic modulation during atherosclerosis, unifying disparate observations in the field. We identified key cellular and molecular mechanisms underlying a top risk locus for multiple vascular diseases, highlighting the promotion of inflammatory endothelial-to-mesenchymal transition by TWIST1 as a key driver of disease risk.
The blood-brain barrier (BBB) is a highly specialized interface between the central nervous system and the peripheral circulation, crucial for maintaining neuronal homeostasis and protecting the brain parenchyma from potentially harmful blood-borne substances. This review examines the molecular and cellular organization of the BBB and explores how defective cell adhesion and signaling networks lead to BBB pathologies. I discuss the intricate architecture of brain endothelial cells within the context of the larger multicellular neurovascular unit, highlighting the roles of pericytes, astrocytes, as well as ECM (extracellular matrix) proteins and growth factors in vascular basement membranes. Recent advances in understanding endothelial cell tight junction dynamics, transport mechanisms, and communication pathways within the neurovascular unit are presented, with a particular emphasis on astrocyte-endothelial communication. Furthermore, I detail how abnormal astrocyte-endothelial signaling leads to BBB breakdown and contributes to various neurological disorders. In summary, this review synthesizes current knowledge of BBB biology, with particular emphasis on recent discoveries in signaling pathways, intercellular adhesion, and dynamic regulatory mechanisms that govern barrier function. By integrating findings from functional studies across multiple models, this review provides critical insights into both fundamental BBB biology and the potential development of translational approaches for treating human cerebrovascular disorders. Understanding these complex mechanisms not only advances our knowledge of normal brain homeostasis but also illuminates promising therapeutic targets and strategies for addressing conditions ranging from stroke to neurodegenerative diseases, ultimately paving the way for more effective clinical interventions that can preserve or restore BBB integrity.
BACKGROUND:The vascular system is the largest organ in the body and underlies most chronic diseases, yet the molecular mechanisms that govern its plasticity remain poorly defined. METHODS:We applied single-cell proteomics in vascular disease, integrating it with single-cell transcriptomics to map protein regulation in healthy and Marfan syndrome aortas. RESULTS:This approach uncovered cell type-specific and cell state-specific proteins missed at the transcriptional level. Notably, we identified a decoupling of fibrillin-1 RNA and protein abundance, suggesting altered protein regulation as a potential contributor to aortic degeneration. Single-cell proteomics further resolved modulated smooth muscle cell states enriched for matrix effectors (AEBP1 [adipocyte enhancer binding protein 1], HTRA1 [high-temperature requirement A serine peptidase 1], FN1 [fibronectin 1]) and uniquely protein-level regulators (GLIPR2 [GLI pathogenesis-related 2], ITGB2 [integrin beta-2], and CD151 [cluster of differentiation 151 antigen]). CONCLUSIONS:Together, these findings establish the human single-cell proteomic atlas of the aorta, reveal altered FBN1 (fibrillin-1) protein regulation in both human and mouse Marfan syndrome, and position single-cell proteomics as a transformative framework for decoding vascular plasticity and identifying actionable effectors in disease.
BACKGROUND: Reverse cholesterol transport by HDLs (high-density lipoproteins) is considered an antiatherogenic metabolic pathway. Hepatocytes are the main contributors to the efficacy of this pathway by the production of apoA-I (apolipoprotein A1) and its lipidation by ABCA1 (ATP-binding cassette transporter A1), selective uptake of cholesterol via SR-BI (scavenger receptor class B type 1), and uptake of entire HDL particles. The molecular determinants of the latter step are not well understood. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of HDL fluorescently labeled at its protein moiety into Huh-7 hepatocarcinoma cells. Top hit genes were validated by targeted in vitro experiments and the analysis of associations between their variants and HDL-C (HDL-cholesterol) levels in the databases of the Global Lipids Genetics Consortium and the UK Biobank, as well as inborn errors of metabolism and their respective mouse models. RESULTS: The knockdown of 128 genes significantly inhibited HDL uptake. Six of them encode components of the COPI (coat protein I) coatomer, namely, COPA , COPB1 , COPB2 , COPG1 , ARCN1 , and COPZ1 . Knocking down any of them decreased the uptake of both fluorescently labeled proteins and lipids of HDL, the cell surface abundance of SR-BI, and APOA1 expression and apoA-I secretion but increased the cell surface abundance of ABCA1. Common single-nucleotide polymorphisms of ARCN1 and COPB1 were associated with significantly higher HDL-C levels in the population, while rare COPA and COPG1 variants causing immunopathies were associated with rather lower levels of HDL-C in both affected patients and the corresponding genetically modified mice. CONCLUSIONS: In hepatocytes, the COPI coatomer regulates HDL holoparticle uptake, selective lipid uptake, apoA-I secretion, and cholesterol efflux, and thereby, it influences plasma levels of HDL-C.
BACKGROUND:Traumatic brain injury (TBI) results in the release of microparticles from injured brain cells into circulation. These microparticles induce a systemic hypercoagulable state that rapidly transitions into secondary coagulopathy and endotheliopathy. We hypothesize that removing these microparticles from circulation could mitigate the TBI-induced secondary pathologies and improve outcomes. In this study, we investigated the role of Gas-6 (growth arrest-specific 6) as a scavenging factor for microparticles. METHODS:We quantified plasma Gas-6 levels in a mouse model of TBI and administered exogenous Gas-6 either before or after TBI to evaluate its effects on endotheliopathy, coagulopathy, and outcomes. Mechanistic studies assessed Gas-6-mediated clearance of circulating microparticles in TBI mice and investigated the molecular interactions by which Gas-6 binds microparticles and macrophages to facilitate microparticle scavenging. RESULTS:We found that plasma levels of Gas-6 were significantly reduced in mice subjected to severe TBI. Exogenous Gas-6 given either preinjury or postinjury attenuated coagulopathy, protected the integrity of the cerebral and pulmonary endothelium, improved neurological recovery, and increased overall survival of TBI mice. Gas-6 increased the clearance of anionic phospholipid-expressing microparticles from circulation by coupling microparticles with macrophages and monocytes through the γ-carboxyglutamate and the LG1 (laminin G-like domain 1), respectively, to facilitate phagocytosis of microparticles in the liver. CONCLUSIONS:These findings demonstrate the therapeutic potential of Gas-6 for TBI and potentially for other acute pathologies, in which microparticles initiate and propagate coagulation dysfunction and endothelial injuries.
BACKGROUND:The lung vasculature is comprised of a series of branching vessels extending from the main pulmonary artery to the alveolar capillaries, then back to the pulmonary veins. Lung endothelial cells (EC) exist along this continuum, exposed to gradients of shear stress, oxygen tension, and pressure. Single-cell RNA sequencing has identified lung EC subsets, but many aspects of the vascular continuum, including vessel size and capillary polarity, remain undefined from transcriptomic data. METHODS:We created an EC-enriched single-cell RNA sequencing data set from the P3 mouse lung. Using diffusion pseudotime, we developed an analytical framework to delineate transcriptomic gradients and assign vessel-size scores to categorize individual EC along the vascular continuum. We validated size-related gene expression patterns with fluorescence in situ hybridization and tested the application of this framework to transcriptomic data sets derived from diverse species and developmental stages. RESULTS:We categorized capillary 1, arterial, and venous EC along 2 gradients: arterio-venous zonation and vessel size, distinguishing large arteries from arterioles, large veins from venules, and revealing arterio-venous polarity within the capillaries. Our data recapitulated previously established zonally defined cell signaling axes, identified unique cellular communication patterns in large versus small vessels, and localized injury-induced venous EC proliferation to vessels of specific size. This analytical framework was successfully applied to categorize lung EC by size in several published mouse and human data sets across different stages of lung development. CONCLUSIONS:Our findings provide a new approach to analyze transcriptional data to map EC along the pulmonary vascular tree, enabling the assignment of individual EC to vessels of a relative size. This framework allows the inference of spatial information from gene expression alone, thus providing novel mechanistic insights into pulmonary vascular diseases affecting specific vascular segments.
BACKGROUND:Endothelial dysfunction is critical for the pathogenesis of atherosclerosis, particularly in arterial regions exposed to disturbed flow (DF). ADCY4 (adenylate cyclase 4) catalyzes the production of cAMP (cyclic adenosine monophosphate), a ubiquitous second messenger that regulates cellular and physiological processes. This study investigated ADCY4 as a shear-stress-responsive gene in endothelial cells (ECs) for the regulation of vascular endothelial inflammation during atherogenesis. METHODS:Integrated analysis of RNA sequencing data sets from human vascular ECs exposed to unidirectional flow or DF, and single-cell RNA-seq data from mouse partial ligation carotid arteries, was performed. ADCY4 expression was quantified in cultured ECs under various flow conditions. Nanoparticles carrying Cdh5 promoter-driven CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and Adcy4-specific guide RNA plasmids were used to achieve EC-specific Adcy4 knockout in ApoE-/- mice for atherogenesis studies. RESULTS:ADCY4 was upregulated in ECs under unidirectional flow but downregulated under DF. We identified that KLF2 (Krüppel-like factor 2) directly bound to the ADCY4 promoter and transcriptionally enhanced its expression in human aortic ECs. ADCY4 knockdown in ECs under unidirectional flow increased proinflammatory gene expression and monocyte adhesion, whereas ADCY4 overexpression under DF produced opposite changes. Further studies showed ADCY4 attenuated endothelial inflammation by inhibiting NF-κB (nuclear factor κB) signaling. We performed partial carotid ligation in the high-fat diet-fed ApoE-/- mouse atherosclerosis model, demonstrating that EC-specific Adcy4 knockout increased vascular endothelial inflammation and monocyte infiltration, thus promoting atherosclerotic plaque formation. Aggravated atherogenesis was also observed in EC-Adcy4 knockout high-fat diet-fed ApoE-/- mice, with increased atherosclerotic lesions in en face aortas and aortic arches. Finally, semaglutide increased ADCY4 expression in human aortic ECs under DF and reduced vascular endothelial inflammation, monocyte accumulation, and atherosclerotic plaque formation. These beneficial effects were substantially impaired when EC ADCY4 was absent. CONCLUSIONS:ADCY4 regulation of the cAMP/PKA (protein kinase A)-NF-κB pathway in the endothelium advances our understanding of vascular inflammation in atherogenesis and provides opportunities for therapeutic intervention of atherosclerotic cardiovascular disease.
BACKGROUND: Hereditary hemorrhagic telangiectasia is a genetic disorder caused by loss-of-function mutations in components of the bone morphogenetic protein signaling pathway, leading to arteriovenous malformations. Most prior work has treated BMP (bone morphogenetic protein) component depletion as mechanistically interchangeable, yet whether distinct genes converge on a shared mechanism remains unclear. We aimed to understand the molecular relationship between BMP signaling and endothelial flow response that leads to arteriovenous malformation formation. METHODS: We expose human endothelial monolayers treated with small interfering RNA against SMAD4 or ALK1 to laminar flow and analyze flow-responsive transcriptomics, flow-responsive BMP signaling activation dynamics, cell polarity, and morphology. We analyze the cell-autonomous and noncell-autonomous migration dynamics of endothelial cells treated with siSMAD4 or siALK1 . Using the postnatal mouse retina model, we study endothelial cell distribution changes over time in mosaic settings, and assess the remodeling capabilities of Smad4 iECKO or Alk1 iECKO , relative to littermate controls. RESULTS: This study shows that depletion of SMAD4 or ALK1 leads to fundamentally distinct mechanisms of vascular malformation. SMAD4 deficiency enhances endothelial responses to blood flow, including transcriptional activation and migration against flow, causing excessive capillary pruning and the development of single large shunts. In contrast, ALK1 deficiency disrupts flow sensing, impairs cell polarization and migration, and promotes a persistent angiogenic state, resulting in dense, hypervascularized networks. RNA sequencing across static and flow conditions identifies both flow-dependent and flow-independent transcriptional changes, suggesting early defects in endothelial fate specification. Mosaic in vitro models show that mutant cells co-opt neighboring wild-type cells, while in vivo tracking confirms mutation-specific migration behavior. CONCLUSIONS: These findings reveal divergent cellular programs driving arteriovenous malformations and underscore the need for gene-specific diagnostic and therapeutic strategies.
BACKGROUND:Calcific aortic valve (AV) disease (CAVD) is recognized as an active pathological process involving extracellular matrix remodeling. This study investigates extracellular matrix remodeling through proteomic analysis and a novel mouse model of aortic stenosis. METHODS:Proteomic and glycoproteomic analyses were conducted on AV leaflets from heart transplant donors (n=29) and patients with CAVD (n=17). Each CAVD sample was subdivided into noncalcified and calcified regions. To investigate the functional impact of extracellular matrix remodeling on aortic stenosis, we crossed apolipoprotein E-deficient mice (ApoE-/-) with mice lacking the catalytic domain of ADAMTS5 (Adamts5Δcat) to generate a mouse model combining hyalectan accumulation with hypercholesterolemia. RESULTS:Proteomic and glycoproteomic analyses revealed hyalectan accumulation in CAVD compared with control valves. Versican predominated in noncalcified regions, while aggrecan was enriched in calcified regions. The shift in hyalectan composition correlated with changes in AV pressure gradient, elevated osteoblast-like cell markers, and inflammatory proteins, most notably pentraxin 3. Both versican and aggrecan are characterized by their ability to bind hyaluronan and serve as substrates of ADAMTS5. In Adamts5Δcat/ApoE-/- mice, hyalectan accumulation was associated with narrowed aortic cusp separation and increased post-AV velocity. Proteomic analysis of AVs from Adamts5Δcat/ApoE-/- mice revealed elevated versican, aggrecan, and pentraxin 3, recapitulating key features of human CAVD. Single-cell RNA sequencing and in vitro experiments linked versican to activated valve interstitial cells, while aggrecan colocalized with calcification markers in osteoblast-like cells. Pentraxin 3 was bound to hyaluronan and accumulated in calcified AVs. ADAMTS5 deficiency was sufficient to cause intact versican accumulation and promote valve interstitial cell activation, accompanied by increased expression of osteopontin. Osteopontin is a ligand of CD44, a principle hyaluronan receptor. CONCLUSIONS:This study highlights hyaluronan remodeling during aortic stenosis pathogenesis. A shift from versican to aggrecan in human CAVD correlates with changes in AV pressure gradient. In Adamts5Δcat/ApoE-/- mice, impaired hyalectan catabolism promotes aortic stenosis.
BACKGROUND: MGP (matrix Gla protein) serves as an inhibitor of vascular calcification by limiting elastin degradation and regulating BMP (bone morphogenetic protein) activity. Mutation of the conserved proline residue 64 to glycine in MGP abolishes BMP binding in vitro. We hypothesized that selective loss of BMP binding would elucidate the contribution of BMP to cell differentiation in Mgp -deficient mice. METHODS: Computational analyses using AlphaFold3 and molecular dynamics simulations were performed to determine the structural effects of γ-carboxylation and the proline residue 64 to glycine mutation. The vascular phenotype of Mgp -knockin mice expressing the proline residue 64 to glycine mutation was compared with wild-type (WT) and global Mgp -knockout mice. Proximity ligation assay was performed to assess MGP-BMP4 in aortic cells. Single-cell RNA-sequencing was used to identify cellular alterations in the vascular media. RESULTS: Molecular dynamics simulation revealed 5 Ca 2+ ions coordinated by γ-carboxylated Glu residues in the MGP dimer. The proline residue 64 to glycine mutation did not disrupt Ca 2+ binding but likely suppressed conformational changes required for BMP4 binding. Unlike Mgp - KO mice, Mgp -knockin mice did not develop vascular calcification, elastic lamina proteolysis, and endothelial-mesenchymal transition, but exhibited vascular fibrosis. MGP-BMP4 interaction observed in WT aortic cells was barely detectable in Mgp -knockin aortic cells. Single-cell RNA-sequencing revealed increased fractions of smooth muscle cells and enhanced myofibroblast differentiation in the Mgp -knockin aortas compared with WT. In Mgp -knockin aortas, SMAD2 expression was significantly increased throughout the vessel wall compared with WT aortas. In contrast, SMAD1/5/9 activation in the Mgp -knockout aortas was more confined to the endothelium, whereas it localized to the media-adventitia transition in WT and Mgp -knockin aortas. Both Mgp -knockin and Mgp -knockout mice developed arteriovenous malformations in the lungs, kidneys, and brain. CONCLUSIONS: Our findings suggest that MGP plays a versatile role in preserving vascular integrity, in part serving as an important BMP-trap aimed at directing vascular cell differentiation.
BACKGROUND:Atherosclerosis is a chronic vascular disease with a complex pathophysiology involving smooth muscle cell (SMC) dysfunction, and Olfm2 (olfactomedin 2) plays a role in SMC phenotype modulation. However, it is unclear if Olfm2 is involved in the development of atherosclerosis. METHODS:Olfm2 global and SMC-specific knockout models were used to study its role in the development of atherosclerosis. RESULTS:We found that Olfm2 was upregulated in atherosclerotic plaques of high-fat diet-fed ApoE-/- (apolipoprotein E) mice in vivo and in oxLDL (oxidized low-density lipoprotein)-treated SMCs in vitro, suggesting the involvement of Olfm2 in SMC foam cell formation (SFCF). Indeed, knockdown of Olfm2 inhibited, while overexpression of Olfm2 promoted oxLDL-induced SFCF. Moreover, lipid accumulation and atherosclerotic lesion areas were significantly reduced in the aortas of high-fat diet-fed Olfm2-/- ApoE-/- mice compared with those in ApoE-/- mice, demonstrating that Olfm2 is a novel regulator promoting atherosclerosis. Mechanistically, Olfm2 physically interacted with IκBα (inhibitor of nuclear factor κB) and facilitated the release of NF-κB (nuclear factor κB) from the NF-κB/IκBα complex, leading to activation of NF-κB signaling, which, in turn, promoted SFCF and subsequent atherosclerosis development. Olfm2 functions in atherosclerosis and SFCF were further confirmed by using an inducible SMC-specific Olfm2-deficient mouse model. CONCLUSIONS:Olfm2 is a novel regulator that promotes SFCF and atherosclerosis by activating NF-κB signaling. Targeting Olfm2 may be a novel potential therapeutic strategy against atherosclerosis.
Innate immunity is a critical contributor to graft rejection and cardiovascular complications after organ transplantation, with increasing evidence indicating that innate immune memory significantly influences graft outcomes. The most extensively studied form, trained immunity, involves epigenetic and metabolic reprogramming of innate immune cells, altering their inflammatory responsiveness. Numerous transplantation-related factors, including metabolic disturbances such as hypercholesterolemia and hyperglycemia, can induce trained immunity, and both experimental and clinical studies have linked it to graft survival. Although trained immunity reflects responses to nonspecific stimuli, innate allogeneic memory shows that innate immune cells can recognize nonself and mount donor-specific memory responses. This review covers the current knowledge on both forms of innate immune memory in the context of solid organ transplantation.
BACKGROUND:Monocyte-derived macrophages play a significant role in the initiation and progression of atherosclerosis by transforming into lipid-laden foam cells and regulating vascular inflammation. However, the molecular mechanisms that regulate macrophage lipid accumulation, phenotype, efferocytic capacity, and atherosclerosis are incompletely understood. Our preliminary studies revealed increased expression of LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) in human atherosclerotic arteries. Additional experiments demonstrated increased LGR4 levels in atherogenic oxidized low-density lipoprotein-treated macrophages. However, the macrophage-specific role of LGR4 in atherogenesis has never been investigated. METHODS:To investigate the role of myeloid cell Lgr4 in atherosclerosis development, myeloid cell-specific Lgr4 knockout (Lgr4f/f LysM Cre+/-, Lgr4ΔM) and littermate control Lgr4f/f (Lgr4WT) mice were injected intraperitoneally with adeno-associated viral vector (serotype 8) for hPCSK9 and fed a Western diet for 16 weeks. Various in vitro cell-based assays, molecular biology techniques, and immunohistological approaches were used to evaluate the functional roles of macrophage Lgr4 and underlying signaling mechanisms. RESULTS:Oil red O staining of whole aortas and aortic root sections demonstrated reduced atherosclerosis in Lgr4ΔM mice compared with sex-matched Lgr4WT mice. However, no changes in circulating monocyte frequencies were detected. Histochemical staining performed on aortic root sections revealed smaller necrotic cores and higher collagen content in Lgr4ΔM mice. Additionally, Lgr4ΔM mice exhibited lower fat mass and blood glucose levels, while plasma total cholesterol was comparable to that of control mice. Further in vitro and ex vivo studies demonstrated reduced lipid accumulation, enhanced efferocytic capacity, a suppressed proinflammatory phenotype, and attenuated expression of different low-density lipoprotein uptake genes in Lgr4-deficient macrophages. Moreover, Lgr4 knockout macrophages displayed increased cholesterol efflux capacity and reduced activation of oxidized low-density lipoprotein-induced Wnt (Wingless-related integration site)/β-catenin signaling. CONCLUSIONS:These findings suggest that myeloid cell Lgr4 contributes to atherosclerotic lesion formation via stimulating macrophage lipid accumulation, impairing efferocytic capacity, and promoting a proinflammatory phenotype. Collectively, these results identify macrophage LGR4 as a novel therapeutic target for atherosclerosis.
