Arteriosclerosis, Thrombosis, and Vascular Biology

Background: APOE4, the variant of apolipoprotein E carried by 25% of individuals, is a common genetic risk factor for cardiovascular disease (CVD). Although ApoE classically participates in lipid transport, APOE4-associated risk goes beyond impact on circulating lipids. Life-threatening CVD events including myocardial infarction and stroke are driven by atherogenesis and thrombosis. In mice ApoE4 increases atherosclerosis severity, but whether other major drivers of CVD events are influenced by ApoE4 is unknown. Methods: GWAS data for venous thromboembolism (VTE) were analyzed. In humanized APOE3 (hE3) and APOE4 (hE4) mice, thrombosis was assessed by intravital microscopy (IVM) in the mesenteric microcirculation and by inferior vena cava (IVC) partial ligation. Actions of ApoE3 versus ApoE4 on endothelial cells (EC) and their underpinnings were studied in cultured human and mouse aortic EC, interrogating interactomes with immunoprecipitation-mass spectrometry and quantifying the secretion of Von Willebrand Factor (vWF), a critical initiator of thrombosis. Single cell transcriptomics datasets were queried do localize endothelial cell gene expression. Results: GWAS showed that APOE4 is associated with increased VTE risk, and whereas plasma lipids were similar, both microvascular and venous thrombosis were markedly increased in hE4 compared to hE3 mice. In cultured EC, whereas ApoE3 attenuated vWF secretion, it was enhanced by ApoE4, and both processes were mediated by ApoE receptor 2 (ApoER2). ApoE4, but not ApoE3, suppressed VEGF eNOS activation and NO production by causing the recruitment of the protein phosphatase 2A (PP2A) catalytic subunit to ApoER2 and the activation of PP2A. PP2A deletion prevented ApoE4-induced eNOS antagonism and vWF secretion by preserving Akt activation, and the NO donor spermine NONOate negated apoE4 stimulation of vWF secretion. PP2A activity was increased in hE4 aortas and IVC, and EC ApoER2 deletion or pharmacologic PP2A inhibition fully prevented exaggerated thrombosis in hE4 mice. In human great saphenous vein ApoER2 is primarily expressed in valvular endothelium. Conclusions: APOE4 is a risk allele for thrombosis, and ApoE4 is prothrombotic in microvasculature and veins in mice. Mechanistically, the ApoE4-EC ApoER2 tandem enhances vWF secretion by recruiting and activating PP2A and antagonizing eNOS, resulting in exaggerated thrombosis. In human veins ApoER2 is expressed in valvular endothelium, which is the most common site of initiation of venous thrombosis. Targeting these processes may afford protection from both primary thrombotic disorders like VTE and acute CVD events such as myocardial infarction and stroke in 25% of the population.

IntroductionsAortic dissection (AD) is a life-threatening vascular disease with limited therapeutic targets. Apolipoprotein H (APOH), a circulating glycoprotein implicated in lipid metabolism, has not been studied in AD. MethodsPlasma APOH levels and aortic deposition were examined in AD patients. A {beta}-aminopropionitrile (BAPN) and angiotensin II (Ang-II)-induced mouse AD model with AAV-mediated Apoh knockdown was used to evaluate survival, aortic dilation, and extracellular matrix remodeling. Transcriptomic profiling, chromatin immunoprecipitation, and gene silencing in human aortic vascular smooth muscle cells (HAVSMC) were performed to dissect the mechanism. PPAR{gamma} agonist rescue was conducted in vivo. ResultsAPOH was elevated in plasma and deposited in AD aortas. Apoh knockdown improved survival, reduced AD incidence and ascending aortic dilation, and attenuated elastic fiber disruption and collagen deposition. Transcriptomics revealed enrichment of the PPAR pathway. APOH promoted HAVSMC phenotypic switching from a contractile to a synthetic state, decreasing ACTA2/TAGLN and increasing OPN/MMP9. Mechanistically, APOH upregulated NR5A1, which directly bound the PPAR{gamma} promoter to enhance PPAR{gamma} and FABP4 expression. Silencing NR5A1 or PPAR{gamma} reversed APOH-induced phenotypic switching and inflammation. In vivo, PPAR{gamma} agonist diminished the protective effects of Apoh silencing. ConclusionAPOH promotes AD progression through the NR5A1-PPAR{gamma} axis, driving vascular smooth muscle cell phenotypic switching and inflammation, and represents a potential therapeutic target. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/739043v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@323110org.highwire.dtl.DTLVardef@133260dorg.highwire.dtl.DTLVardef@1075c9eorg.highwire.dtl.DTLVardef@51a965_HPS_FORMAT_FIGEXP M_FIG C_FIG Circulating APOH promotes aortic dissection through the NR5A1-PPAR{gamma} axis in human aortic vascular smooth muscle cells. Clinical observations showed that plasma APOH levels were elevated in patients with aortic dissection. Circulating APOH acts on human aortic vascular smooth muscle cells (HAVSMC) and upregulates NR5A1, which binds to the PPARG promoter and enhances PPAR{gamma} transcription. Activation of the NR5A1-PPAR{gamma} signaling axis promotes the phenotypic transition of HAVSMCs from a contractile phenotype to a synthetic phenotype, as indicated by decreased ACTA2 and TAGLN expression and increased OPN and MMP9 expression, accompanied by enhanced production of the inflammatory mediators IL-6, MCP-1, and TNF-. Silencing NR5A1 or PPAR{gamma} reverses APOH-induced phenotypic switching and inflammatory responses, supporting the critical role of the NR5A1-PPAR{gamma} axis in APOH-mediated vascular injury.

BackgroundWe previously performed a strain intercross between atherosclerosis resistant AKR Apoe-/-mice and atherosclerosis sensitive DBA/2 Apoe-/- mice and identified the Ath28 quantitative trait locus (QTL) on the distal end of chromosome 2. Congenic strain fine mapping identified the Ath28.1 QTL atherosclerosis modifying subregion, encompassing 217 Kb, containing for only three protein-coding genes, Zbp1, Pck1, and Pmepa1, encoding respectively, Z-DNA binding protein 1, phosphoenolpyruvate carboxykinase 1, and prostate transmembrane protein androgen induced 1. MethodsThe effect of macrophage-specific knockout of Pck1 (KO) was tested using the AAV2 transduced proprotein convertase subtilisin kexin type 9 (PCSK9) overexpression mouse model of hyperlipidemia and atherosclerosis. ResultsUnexpectedly, macrophage Pck1 deficiency lowered body weight, liver weight, and HDL-cholesterol levels in both sexes, while total and non-HDL cholesterol levels were only decreased in male mice. Aortic root lesion area and necrotic lesion area were unchanged in KO mice of both sexes. ConclusionPck1 was not confirmed as an atherosclerosis modifier gene.

Background: Hypoxia develops within growing atherosclerotic lesions, inducing nuclear translocation of hypoxia-inducible factor-1 (HIF1) and metabolic reprogramming. Its role in plaque macrophages and endothelial cells has been studied, but the hypoxic plaque interior is dominated by smooth muscle cell (SMC)-derived cells, for which the role of hypoxia signaling remains unclear. Here, we investigated how loss of Hif1a in SMC lineage cells impacts plaque progression and cell phenotype in murine atherosclerosis. Methods: Atherosclerosis was induced in mice with inducible SMC-specific deletion of Hif1a (Hif1aSMC-KO) and lineage tracing of SMC-derived plaque cells. Plaque size, necrotic core size, calcification, and SMC-derived cell phenotypes were quantified in aortic root sections and gene expression changes mapped by single-cell RNA sequencing. In parallel, a cultured SMC line with or without siRNA-mediated Hif1a knockdown was exposed to hypoxia for assessments of mitochondrial function and reactive oxygen species production. Results: Hif1aSMC-KO mice developed larger plaques, with expanded necrotic cores and increased calcification, compared with littermate controls. SMC-derived plaque cells were more abundant with a higher fraction of Col2a1+ chondromyocytes, and showed elevated markers of proliferation and apoptosis, whereas macrophage and endothelial cell numbers were unaffected. Single-cell RNA sequencing analysis revealed strong dysregulation of mitochondrial genes, including electron transport chain transcripts, along with upregulation of protein folding, proteasome, and oxidative stress response pathways. In cultured SMCs subjected to hypoxia, Hif1a silencing increased cell counts, aggravated mitochondrial proton leak, and led to the accumulation of depolarized, reactive oxygen species-generating mitochondria. Further analysis of SMC-derived cells in plaques from Hif1aSMC-KO mice confirmed increased oxidative stress by 8OHdG staining. Conclusions: HIF1 maintains mitochondrial function and restrains oxidative stress in SMC-derived plaque cells in murine atherosclerosis. Its chronic loss destabilizes redox homeostasis and promotes maladaptive SMC responses, leading to SMC-driven plaque expansion, necrosis, and calcification.

BackgroundPulmonary hypertension (PH) is characterized by pulmonary vascular remodeling and smooth muscle cell accumulation, but the progenitor-like cells that contribute to this process remain incompletely defined. MethodsWe combined analyses of human pulmonary arterial hypertension lungs and experimental PH models with bulk and single-cell RNA sequencing, lineage tracing, inducible ablation of CD133+ cells, and conditional deletion of Cxcr4 in CD133+ cells. ResultsCD133 expression was markedly increased in human and experimental PH lungs. Transcriptomic analyses identified inflammatory, metabolic, chemokine-associated, and smooth muscle cell-like programs in CD133+ cells from PH lungs. Lineage tracing showed that CD133+ cells contributed to endothelial and smooth muscle cell compartments during experimental PH. Genetic ablation of CD133+ cells attenuated hypoxia-induced PH and pulmonary vascular remodeling, whereas Cxcr4 deletion in CD133+ cells reduced PH severity. ConclusionsCD133+ progenitor cells are functional contributors to pulmonary vascular remodeling, and CXCR4 signaling mediates their pathogenic activity. Targeting pathogenic CD133+ cell states or CXCL12/CXCR4 signaling may provide a strategy to limit vascular remodeling in PH.

Following aortic dissection (AD), there is a sustained risk of vascular complications, progressive false lumen aneurysm formation, and rupture. However, no effective therapy exists to prevent these complications, highlighting the need to elucidate the pathophysiology following AD. Elastic fibers are crucial for maintaining aortic wall integrity but are thought to have limited regenerative capacity once disrupted during AD. This study defined that elastic fibers were newly generated in the false lumen wall following AD in humans and mice. In human ADs, new elastic fibers were observed in the false lumen wall 6 months after onset. In mice with descending AD induced by {beta}-aminopropionitrile (BAPN), elastin mRNA was markedly upregulated in the chronic phase following AD, accompanied by elastic fiber formation. These fibers coincided with smooth muscle cell (SMC) markers within the false lumen wall. Of note, lineage tracing studies demonstrated that these cells were not derived from resident SMCs but adventitial progenitor cells. In vitro experiments further demonstrated that adventitial progenitor cells produced elastic fibers while expressing SMC markers. Collectively, these findings suggest that adventitial progenitor cells differentiate into elastogenic SMC-like cells, contributing to false lumen remodeling through de novo elastic fiber formation following AD.

Rupture of the fibrous cap is the primary cause of clinical complications from atherosclerosis. Smooth muscle cells (SMCs) are a major contributor to fibrous cap development and stability through de-differentiation to extracellular matrix-producing cells. We previously showed that the antioxidant enzyme cytoglobin (CYGB) is expressed in vascular SMCs and regulates SMC dependent vascular remodeling and gene expression. In the present study, we investigated the function of SMC-CYGB in atherosclerosis. To this end, we generated a mouse line with SMC-specific deletion of Cygb and simultaneous SMC-lineage tracing. We found that SMC specific deletion of CYGB increased fibrous cap thickness in a 17-week PCSK9-AAV8 gain of function combined with Western diet mouse model of atherosclerosis. SMC specific deletion of CYGB increased collagen deposition and SMC cellularity of the fibrous cap in the absence of changes in total plaque and necrotic core sizes. CYGB expression in SMCs was associated with transdifferentiation towards a fibroblast-like, matrix remodeling phenotype. Finally, CYGB was expressed in the fibrous cap of human coronary atherosclerotic lesions and was associated with ACTA2 positive cells. These results provide first-time evidence that SMC-CYGB reduces plaque stability by decreasing cap thickness, collagen deposition, and SMC cellularity.

Elevated levels of lipoprotein(a) (Lp(a)) are an independent risk factor for the development of atherothrombotic diseases. However, it is unknown if Lp(a) directly promotes thrombus formation, inhibits thrombus clearance, or merely accelerates the underlying atherosclerotic processes that culminate in plaque rupture. While numerous studies indicate that the apolipoprotein(a) (apo(a)) component of Lp(a) can inhibit plasminogen activation and fibrinolysis, recent evidence suggests that these effects may not be retained in Lp(a). An alternative mechanism through which Lp(a) may promote atherothrombotic events is by impacting platelet function. However, the effects of Lp(a) on platelet function and thrombosis have never been directly assessed in blood clots formed from flowing whole blood. Using a transgenic mouse model expressing high plasma concentrations of apo(a), we showed using a laser-induced mesenteric vessel injury model employing intravital microscopy that apo(a) increased platelet and fibrin volumes in the thrombi without affecting fibrinolysis. In a ferric chloride-induced mouse carotid artery thrombosis model, we found that apo(a) substantially reduced occlusion times and led to more stable thrombi; importantly, we also demonstrated that the effects of Lp(a) could be mitigated by low-dose aspirin therapy. We evaluated the prothrombotic potential of Lp(a) in human blood clots formed under arterial flow conditions using a Chandler loop apparatus. In these studies, we showed that the presence of Lp(a) during thrombogenesis inhibited lysis of the thrombi, without directly impacting fibrinolysis. Lp(a) promoted platelet accumulation in the Chandler thrombi and facilitated the development of fibrin networks that displayed features of fibrinolysis resistance. In an analysis of the UK Biobank, participants with Lp(a) [≥]125 nmol/L had a higher risk for arterial thrombosis of non-atherosclerotic etiology but not a higher risk for venous thromboembolism. Collectively, these findings demonstrate that Lp(a) is inherently prothrombotic and likely promotes arterial thrombosis in vivo in part through promoting platelet activation. These findings explain how elevated Lp(a) is an important risk factor for arterial thrombosis either with or without an atherosclerotic etiology as well as observational primary prevention data suggesting that aspirin reduces atherothrombotic risk specifically in patients with elevated Lp(a).

BackgroundLymphatic valves are specialized structures within lymphatic vessels that ensure unidirectional lymph transport. Defective lymphatic valve formation is associated with lymphedema, a chronic disease characterized by impaired lymph drainage and the accumulation of protein-rich interstitial fluid. Lymphatic valves form during late embryonic stages in mice, with oscillatory shear stress implicated in regulating the molecular mechanisms which control lymphatic valve formation. However, the molecular mechanisms governing lymphatic valve formation remain incompletely understood. Although CXCR4 is regulated by shear stress in blood vessels, whether CXCL12/CXCR4 signaling regulates lymphatic valve formation and the underlying molecular mechanisms remain unknown. MethodsTo investigate the roles of CXCR4 in the regulation of lymphatic valve development, we utilized lymphatic endothelial cell (LEC)-specific Cxcr4 knockout (Flt4CreERT2, Cxcr4f/f) mice. To determine the source of CXCL12, major ligand for CXCR4 in the mesentery, we used global Cxcl12-/-, and Cxcl12-DsRed knock-in/knockout (KIKO) reporter mice, as well as conditional Cxcl12 knockout mouse lines. To determine the molecular mechanisms by which CXCL12/CXCR4 regulates lymphatic valve development, primary human dermal LECs were exposed to oscillatory shear stress (OSS) to mimic valve-associated flow, followed by analysis of downstream signaling pathways and valve related gene expression. ResultsLEC-specific loss of CXCR4 displayed impaired lymphatic valve development. Flt4CreERT2, Cxcr4f/f mice showed a significant reduction in valve numbers in embryonic mesenteric lymphatic vessels. Similarly, reduced valve numbers were observed in Cxcl12-/- embryos, indicating CXCL12/CXCR4 is required for embryonic mesentery collecting lymphatic valve formation. Cxcl12-DsRed KIKO mice revealed that blood vessels, opposed to nerves, were the major source of CXCL12 in the embryonic mesentery. In align with this finding, EC-specific Cxcl12 deletion recapitulated defective valve phenotypes observed in Cxcl12-/- embryos. Mechanistically, CXCR4 knockdown in primary human dermal LECs attenuated OSS induced phosphorylation of AKT and FOXO1, leading to increased nuclear localization of FOXO1 and reduced expression of FOXC2, an essential transcription factor governing lymphatic valve development. Consistent with these findings, lymphatic valves of LEC-Cxcr4 deficient mice exhibited increased FOXO1 nuclear localization. Importantly, pharmacological activation of AKT reduced FOXO1 nuclear accumulation and restored lymphatic valve numbers in LEC-Cxcr4 deficient mesenteric lymphatic vessels. ConclusionsOur findings reveal CXCL12/CXCR4 signaling acts as a critical regulator of lymphatic valve development. CXCL12/CXCR4 signaling integrates into the flow dependent AKT/FOXO1/FOXC2 signaling axis to coordinate lymphatic valve development and morphogenesis. Taken together, our study uncovers a previously unknown role of CXCL12/CXCR4 signaling pathway in the regulation of lymphatic valve development. Targeting CXCL12/CXCR4/AKT/FOXO1 axis may represent a promising therapeutic strategy for improving lymphatic valve development to improve lymphatic function for the treatment of lymphedema.

BackgroundVein graft failure due to intimal hyperplasia and maladaptive remodeling remains a major limitation of peripheral bypass surgery. Although vascular remodeling is recognized as a multilayered process, layer-specific molecular mechanisms that distinguish adaptive from negative remodeling remain incompletely understood. We aimed to investigate the vascular microenvironment of patent and stenotic grafts using spatial transcriptomics. MethodsVein specimens were obtained from three patients undergoing revision surgery. For each patient, a matched set of three samples was collected: unused saphenous vein (Denovo), normally healed vein graft (Non-stenosed), and stenosed vein graft (Stenosed) (n = 3 patients). GeoMx Digital Spatial Profiling with the Human Whole Transcriptome Atlas was used to map gene expression across intima, medial, and adventitial layers. Differential expression, gene ontology, spatial deconvolution, and immunohistochemistry were integrated for analysis. ResultsNon-stenosed and Stenosed grafts shared transcriptional features distinct from Denovo veins, particularly in pathways related to cell proliferation. Non-stenosed grafts showed increased expression of CDKN1A across all vascular layers, whereas Stenosed grafts exhibited enhanced mitogen-activated protein kinase (MAPK) pathway activity, reduced DUSP1-mediated regulation, and increased macrophage accumulation. ECM remodeling showed layer-specific organization, with VCAN and ACAN enriched in the intima and DCN in the adventitia, while Stenosed grafts demonstrated a trend toward collagen-dominant remodeling. Cell deconvolution suggested a shift toward vascular smooth muscle cell (VSMC)-dominant architecture after arterialization, with modest enrichment of synthetic VSMC signatures in stenotic regions. ConclusionsVein graft stenosis appears to be associated with layer-specific alterations in cell cycle regulation, inflammatory signaling, extracellular matrix remodeling, and VSMC phenotype. Spatial transcriptomic analysis reveals molecular heterogeneity not captured by bulk approaches and provides preliminary insight into graft remodeling. These findings may inform future studies to improve long-term graft patency.

BACKGROUNDLysine methyltransferase SET7 activates gene expression through mono- methylation of histone H3 at lysine 4 (H3K4me1) and modulates protein function via mono- methylation of non-histone proteins (Kme1). However, its role and molecular targets in endothelial dysfunction associated with cardiometabolic disorders remain unknown. METHODSEndothelial-specific Setd7 knockout (Setd7EC-KO) mice were generated and endothelial function assessed in WT and Setd7EC-KO mice after high-fat diet (HFD). Human aortic endothelial cells (HAECs) were used to investigate SET7 expression and function under metabolic stress. SET7-dependent histone and non-histone targets were identified by proteomic and ChIP analyses. Insights from these datasets guided the design of bioinformatic, molecular, and functional studies to define their regulatory mechanisms. Clinical relevance was evaluated in human arteries. RESULTSHFD selectively increased endothelial SET7 expression in WT mouse aortas. Despite comparable metabolic abnormalities, Setd7EC-KO mice were protected from HFD- induced endothelial dysfunction, oxidative stress, and inflammation. In HAECs, high glucose emerged as the strongest inducer of SET7 expression, promoting pro-inflammatory and pro- oxidant gene expression, monocyte adhesion, and ROS generation. These effects were reproduced by overexpression of catalytically active SET7 and reversed by its inhibition or silencing. Proteomic and ChIP analyses revealed that SET7-dependent H3K4me1 activates transcription of spliceosome components, linking aberrant mRNA splicing to endothelial inflammation and oxidative stress. Moreover, Kme1-proteomics identified endothelial nitric oxide synthase (eNOS) as a direct SET7 substrate. Bioinformatic analyses and mutagenesis experiments demonstrated that SET7-mediated mono-methylation of eNOS at K494 disrupts calmodulin (CaM) binding and impairs NO synthesis. These molecular signatures were also observed in internal mammary arteries from patients with vascular disease and hyperglycemia. CONCLUSIONSSET7 drives endothelial dysfunction through a dual mechanism: 1) H3K4me1-dependent activation of splicing machinery triggering inflammation and oxidative stress, and 2) eNOS mono-methylation at K494 reducing NO bioavailability. Targeting SET7 may therefore represent a promising avenue to safeguard endothelial homeostasis in cardiometabolic disease. GRAPHIC ABSTRACTA graphic abstract is available for this article.

BackgroundProtease-activated receptor 4 (PAR4) is the only functional thrombin receptor on mouse platelets. Expression and activation of platelet PAR4 was shown to influence hemostatic plug stability and contributes to thrombosis in different murine arterial and venous thrombosis models. PAR4 activation by thrombin and other serine proteases occurs at the canonical activation site at Arginine (Arg) 59 in mice and Arg47 in humans. If murine PAR4 has functional non-canonical activation sites as shown for other PARs is unknown. ObjectiveTo investigate canonical and potentially non-canonical PAR4 signaling in mice, we generated a mouse model expressing a functional, thrombin-cleavage resistant PAR4 by changing Arg59 to Alanine (Ala) 59 in murine PAR4 (PAR4R59A). PAR4R59A mice were used to assess the impact of impaired canonical (thrombin)-dependent PAR4 signaling on hemostasis and thrombosis in mice. MethodsWe analyzed platelet aggregation, platelet integrin activation and -granule release ex vivo. Hemostasis and thrombosis in PAR4R59A and their control mice was compared using the jugular vein needle puncture injury-induced hemostasis model, and the ferric chloride-induced carotid artery and electrolytic injury-induced femoral vein thrombosis models. ResultsPlatelets of PAR4R59A mice did not response to thrombin but responded normally to PAR4 agonist peptide (PAR4AP) stimulation in aggregation assays. Platelets of PAR4R59A and their control mice exhibited comparable responses to ADP, convulxin or PAR4AP regarding integrin activation and -granular release. PAR4R59A mice exhibited impaired hemostasis in the jugular vein needle puncture model, and were protected from ferric chloride-induced arterial thrombosis and from electrolytic injury induced thrombosis in the femoral vein. ConclusionThe novel PAR4R59A mouse expresses a thrombin-insensitive but still functional PAR4. We propose that the new mouse line will increase the in vivo investigation of canonical PAR4 signaling pathways and may reveal unknown non-canonical PAR4 signaling in different pathologies.

BackgroundAtherosclerosis is a chronic inflammatory vascular disorder with persistent residual inflammation even after standard lipid-lowering therapy. Mounting evidence from bench to bedside suggests that diabetes and obesity accelerate atherosclerosis development. Tirzepatide (TZP), a dual Glucagon-Like Peptide-1 Receptor/Glucose-Dependent Insulinotropic Polypeptide Receptor (GLP-1R/GIPR) agonist approved for treating diabetes and obesity, has demonstrated proven cardiometabolic efficacy in large cardiovascular outcome trials. However, it remains largely uncertain whether TZP attenuates atherosclerosis independent of its anti-diabetic and anti-obese effects through direct actions on the vasculature. MethodsWe established atherosclerotic mouse models under diabetic, obese, and non-diabetic/non-obese conditions. Analysis of covariance (ANCOVA) and pair-feeding experiments were applied to experimentally decouple weight-dependent metabolic improvement from intrinsic vasculoprotection. Molecular and cell biological assays in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were performed to dissect the underlying signaling mechanisms. ResultsTZP markedly reduced aortic plaque burden and inflammation, restrained necrotic core enlargement, and improved plaque stability across all experimental mouse models. Both ANCOVA and pair-feeding experiments confirmed that these atheroprotective effects were independent of food intake and body weight loss. Furthermore, TZP attenuated systemic and vascular inflammation in Tumor Necrosis Factor- (TNF)-treated C57BL/6J mice, and this protection occurred without changes in body weight or blood glucose levels. Mechanistically, TZP directly targeted endothelial cells and activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/endothelial nitric oxide synthase (eNOS) pathway, increased eNOS phosphorylation and nitric oxide bioavailability, consequently downregulating the expression of the pro-inflammatory adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). ConclusionsTZP arrests atherosclerosis progression through weight loss-independent anti-inflammatory mechanisms. These findings implicate TZP as a promising therapeutic drug for mitigating residual vascular inflammation in patients with atherosclerotic cardiovascular disease (ASCVD), irrespective of glycemic status or obesity. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LITirzepatide exerts direct anti-atherosclerotic effects in preclinical mouse models of atherosclerosis under diabetic, obese, and non-obese conditions. C_LIO_LITirzepatide directly targets endothelial GLP-1R/GIPR and downstream cAMP/PKA/eNOS signaling pathway to suppress NF-{kappa}B-driven vascular inflammation, thereby uncovering a previously unrecognized vasculoprotective mechanism underlying its cardiovascular benefits C_LI What Are the Clinical Implications?O_LITirzepatide exerts direct vascular protective effects independent of body weight reduction, suggesting that its cardiovascular benefits may extend beyond glycemic control and obesity management. C_LIO_LITirzepatide may represent a promising therapeutic drug for addressing residual vascular inflammation in ASCVD patients, including those without overt diabetes or obesity C_LI

BackgroundEndothelial cell (EC) injury induced by disturbed flow drives neointimal hyperplasia in arteriovenous fistulas (AVFs), where CD34+ cell-mediated repair may be involved. PIEZO1 and VEGFR2 are important mechanosensors with critical role in maintaining endothelial function. However, whether PIEZO1 interacts with VEGFR2 during CD34+ cell differentiation to orchestrate the vascular repair remains unknown. MethodsAVF model was established in several mouse strains. Single cell RNA sequencing was performed for human and mouse samples. Cd34-CreERT2; R26-tdTomato; Piezo1flox/flox mice were used to investigate the effect of Piezo1 deletion on endothelial repair in AVFs. CD34-high human umbilical vein ECs (CD34high HUVECs) was sorted and exposed to different flow patterns to determine the role of shear stress in CD34high cell differentiation. Co-immunoprecipitation, proximal ligation assay and complementary approaches were performed to delineate mechanotransduction initiated by PIEZO1-VEGFR2 interaction. ResultsSingle cell RNA sequencing and immunostaining showed abundant CD34high cells in the vessel wall of AVFs in humans and animal models. Exposure of CD34high HUVECs to different flow patterns showed that laminar shear stress downregulated CD34 while upregulating VE-cadherin and claudin-5 expression. In contrast, oscillatory flow produced the opposite effects, indicating impaired endothelial maturation. PIEZO1 knockdown in CD34high HUVECs attenuated shear stress-induced endothelial marker expression. In Cd34 conditional Piezo1 knockout mouse model of AVF, we observed decreased number of CD34-derived cells, more compact cellular arrangement, and attenuated neointimal hyperplasia. Mechanistically, we found PIEZO1 interacts with VEGFR2, thereby mediating the distinct effects of laminar and oscillatory shear stress on AKT-FoxO1 axis, which critically regulates endothelial marker expression. Furthermore, pharmacological activation of AKT signaling in AVF mouse model enhanced CD34+ cell-mediated endothelial repair and attenuated neointimal hyperplasia. ConclusionPIEZO1-VEGFR2 complex-mediated mechanotransduction plays a key role in regulating CD34+ cell-derived endothelial repair in AVFs via AKT-FoxO1 axis. AKT activation enhances endothelial maturation, thereby attenuating neointimal hyperplasia in AVFs. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIn arteriovenous fistulas (AVFs), abnormal shear stress induces endothelial cell injury, and the resulting neointimal hyperplasia is a major cause of anastomotic stenosis. C_LIO_LICD34 cells actively participate in vascular endothelial repair. C_LIO_LIPIEZO1 is a mechanoreceptor mediating endothelial sensing of hemodynamic shear stress, contributing to the maintenance of atheroprotective endothelial phenotype under laminar shear stress, whereas its activation induces pro-inflammatory effects under disturbed shear stress. C_LI What New Information Does This Article Contribute?O_LICD34 cells participate in repairing endothelial injury induced by abnormal shear stress in AVFs. PIEZO1 knockout in CD34+ cells improve endothelial repair and attenuates neointimal hyperplasia in AVF. C_LIO_LILaminar shear stress induces CD34 downregulation and upregulates VE-cadherin and claudin-5 expression in CD34-high human umbilical vein endothelial cells, whereas oscillatory shear stress upregulates CD34 expression and suppresses VE-cadherin and claudin-5 expression. C_LIO_LIMechano-stimuli lead to PIEZO1-VEGFR2 complex formation regulating CD34 cell-mediated endothelial repair through the downstream AKT-FoxO1 axis. C_LI Abnormal hemodynamic shear stress-induced endothelial injury initiates neointimal hyperplasia in AVFs. The present study identifies PIEZO1 as a key mechanosensor that regulates CD34+ cell-derived endothelial repair in response to distinct blood flow patterns. PIEZO1 promotes CD34+ cell differentiation into mature ECs for endothelial repair under laminar shear stress, whereas it disrupts the differentiation of CD34+ cells into mature endothelium under oscillatory shear stress. Mechanistically, a novel shear stress-sensing complex comprising PIEZO1 and VEGFR2 was identified in regulating flow-induced differentiation of CD34+ cells into mature ECs via the AKT-FoxO1 signaling axis, thereby controlling the expression of endothelial maturation markers VE-Cadherin and Claudin-5. These findings define a novel PIEZO1-VEGFR2 mechanotransduction axis in CD34+ cell-mediated endothelial repair and support AKT pathway activation as a potential therapeutic strategy against neointimal hyperplasia in AVFs.

Background: Abdominal aortic aneurysms (AAA) are characterized by dilation of the aorta that can lead to aortic rupture and death. The transcriptional co-activators Yes-Associated Protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) are mechanosensitive effectors of the highly conserved Hippo signaling pathway. It is hypothesized that cell-specific YAP/TAZ signaling in endothelial cells (EC) plays a pivotal role in mediating AAA formation and rupture. Methods: Single-cell RNA-sequencing in human AAAs was performed and differentially expressed genes (DEGs) were identified in the endothelial cell cluster. YAP/TAZ mRNA and protein expression were also assessed in human AAA and control aortic tissue. Two established murine AAA models were used with male C57BL/6 and EC-CreERT2-YAPfl/fl/TAZfl/fl mice with/without Verteporfin (VPF, YAP/TAZ inhibitor) and XMU-MP-1 (YAP/TAZ activator) treatments. On postoperative days 14 and 28, aortic diameter, histology, cytokine, and MMP2 expressions were evaluated. Results: A significant alteration in EC-specific differentially expressed YAP/TAZ-related genes was observed in which 242 genes were upregulated and 71 genes were downregulated in AAA compared to controls. Human AAA tissue showed a significant increase in YAP and TAZ protein expressions compared to controls. Elastase-treated EC-YAP/TAZ-/- mice showed a significant decrease in AAA diameter compared to littermate controls. Histological quantification revealed preservation of -smooth muscle actin, reduced elastin fiber breaks, and decreased macrophage infiltration in EC-YAP/TAZ-/- mice compared to littermate controls. Importantly, pharmacological inhibition of YAP/TAZ using VPF significantly attenuated AAAs in two experimental murine models. In vitro data demonstrates that VPF inhibits endothelial cell YAP expression, downregulating pathways associated with pathogenic angiogenesis and vascular inflammation. Conclusions: These data suggest that EC-specific YAP/TAZ signaling mediates AAA formation. Pharmacological inhibition of the Hippo pathway can significantly mitigate aortic inflammation and vascular remodeling to decrease the progression of AAAs and prevent aortic rupture.

BACKGROUND Endothelial cells (ECs) exhibit organ-specific functional diversity that shapes vascular homeostasis, disease susceptibility, and therapeutic accessibility. Although single-cell transcriptomic studies have defined endothelial heterogeneity at the RNA level, the in vivo cell-surface protein landscape that directly mediates vascular signaling and drug targeting remains incompletely characterized. Here, we mapped organ-specific endothelial surface proteomes atlas in vivo to define tissue-enriched vascular protein candidates relevant to organ-selective therapeutic design. METHODS We generated Cdh5-CreERT; Cre-iPEEL mice, referred to here as CHRP mice, in which membrane-tethered horseradish peroxidase is induced selectively in ECs after tamoxifen treatment, and compared CHRP labeling with non-selective NHS-Biotin vascular labeling. Following in vivo biotin-phenol perfusion, endothelial surface proteins were enriched by streptavidin affinity purification and analyzed by mass spectrometry across six organs. Proteomic profiles were used to resolve tissue- and subtype-associated endothelial surface signatures, compare protein and transcript detection patterns, and nominate tissue-selective endothelial membrane candidates, which were annotated using ChEMBL compound-target information. RESULTS Compared with non-selective NHS-Biotin labeling, CHRP improved endothelial specificity and produced clearer separation of tissue-resolved endothelial surface proteomes across brain, white adipose tissue, small intestine, kidney, lung, and skeletal muscle. CHRP proteomics revealed pronounced organ-specific heterogeneity and resolved canonical arterial, venous, and capillary programs, as well as specialized endothelial signatures including blood-brain barrier and glomerular endothelial features. Comparison with single-cell endothelial references revealed systematic differences between transcriptomic and proteomic detection of endothelial membrane proteins. Further analysis identified tissue-selective endothelial membrane candidates, and ChEMBL annotation linked a subset of these candidates to existing compound-target records, supporting the candidate atlas as a resource for future tissue-selective vascular targeting studies. CONCLUSIONS CHRP-based in vivo proximity labeling enables systematic, protein-level mapping of organ-specific endothelial surface proteomes. Together with transcriptomic comparison and compound-target annotation, this study provides a tissue-resolved endothelial surfaceome resource for vascular biology and future organ-selective therapeutic target evaluation.

Abdominal aortic aneurysms (AAAs) occur predominantly in the elderly population and currently there is no effective pharmacological therapy for mitigating AAA growth and preventing impending rupture. Proprotein subtilisin kexin type 9 (PCSK9) gene has been identified as a specific risk-locus for AAA development. However, the mechanistic and clinical role of PCSK9-mediated signaling in AAAs has not been delineated. We demonstrate that treatment with PCSK9 inhibitors, such as Evolocumab, mitigates vascular inflammation and remodeling, resulting in attenuated aneurysm growth in clinical datasets as well as experimental models of AAA and aortic rupture. Mechanistically, Evolocumab immunomodulates macrophage reprogramming to enhance clearance of apoptotic smooth muscle cells via MerTK-dependent efferocytosis that ameliorates aortic inflammation and vascular remodeling. Furthermore, Evolocumab increases the expression of oxidized phosphatidylserine species and decreases expression of lysophospholipids, succinate, and glycolytic intermediates within the aortic wall compared to untreated controls, further enhancing the pro-resolving functions of macrophages. Collectively, our data demonstrates the ability of PCSK9 inhibition to regulate macrophage-specific efferocytosis that limits AAA progression and prevents aortic rupture.

BACKGROUNDEndothelial cell senescence induces endothelial dysfunction, thereby contributing to atherosclerosis progression. Circular RNAs (circRNAs) play diverse roles in multiple physiological and pathological processes. N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic RNAs and dynamically regulates RNA fate and function. However, the functions and therapeutic potential of m6A-modified circRNAs in endothelial cell senescence remain unknown. METHODSm6A-modified circRNAs associated with endothelial cell senescence were screened by circRNA expression and m6A-circRNA microarray profiling of endothelial cells and mouse aortic intima. circEZH2 expression was validated in endothelial cells, vascular tissues, and human atherosclerotic plaques by RT-qPCR, and RNA fluorescence in situ hybridization. The role of circEZH2 in endothelial senescence and atherosclerosis was assessed in vitro and in vivo. RNA pull-down, mass spectrometry, RNA immunoprecipitation, co-immunoprecipitation, ubiquitination assays, and rescue experiments were used to define the underlying mechanism. RESULTSWe identified A novel m6A-modified circRNA, circEZH2, that was downregulated in the aged aortic intima and advanced plaques. CircEZH2 was stabilized by m6A reader IGF2BP2. Endothelial cell-specific overexpression of circEZH2 delayed senescence and suppressed atherosclerosis progression. At the cellular level, circEZH2 overexpression delayed senescence, decreased p53/p21 levels and increased angiogenic activity of endothelial cells, while circEZH2 knockdown exhibited the opposite effect. Mechanistically, circEZH2 functions as a scaffold to promote USP37-mediated deubiquitination, thereby stabilizing ZNF326. Moreover, endothelial cell-specific knockdown of ZNF326 counteracts the anti-senescent and anti-atherosclerotic effects mediated by circEZH2 overexpression. CONCLUSIONSIn summary, the present study identifies circEZH2 as a novel suppressor of endothelial cell senescence, highlighting its potential as a therapeutic target for age-related atherosclerosis. GRAPHIC ABSTRACTA graphic abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/730538v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1bce806org.highwire.dtl.DTLVardef@124ff87org.highwire.dtl.DTLVardef@40911org.highwire.dtl.DTLVardef@ef3d26_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract.C_FLOATNO Schematic model of m6A-modified circEZH2 regulation in endothelial cell senescence and atherosclerosis. In young endothelial cells, IGF2BP2 is highly expressed and recognizes m6A-modified circEZH2, thereby maintaining its RNA stability. CircEZH2 stabilizes ZNF326 protein through USP37-mediated deubiquitination, which leads to suppression of p21 and p53, delays endothelial cell senescence, ameliorates endothelial dysfunction, and ultimately suppresses the progression of atherosclerosis. C_FIG What Are the Clinical Implications?This study identifies circEZH2 as a novel m6A-modified circular RNA that is reduced in the aged aortic intima and in endothelial cells within advanced atherosclerotic plaques. Endothelial circEZH2 overexpression delays endothelial cell senescence, preserves endothelial function, and suppresses atherosclerotic lesion formation, supporting an important role for circEZH2 in vascular aging-associated atherosclerosis. Mechanistically, circEZH2 acts as a scaffold to enhance USP37-mediated deubiquitination and stabilization of ZNF326, while endothelial ZNF326 knockdown counteracts the anti-senescent and anti-atherosclerotic effects of circEZH2. These findings reveal the circEZH2-ZNF326 axis as a previously unrecognized mechanism regulating endothelial senescence and atherosclerosis progression. Our work supports the potential of circEZH2-based gene therapy as a novel therapeutic approach for atherosclerosis.

AimsPlasminogen activator inhibitor-1 (PAI-1) regulates plasmin-mediated proteolysis, thereby influencing vascular stability and tissue remodeling. Angiotensin II (AngII) induces an increase in PAI-1 during the development of ascending thoracic aortic aneurysm (ATAA). The initial purpose of this study was to determine whether deletion of PAI-1 influenced development of ATAA. Subsequently, this study aimed to define the early pathological events preceding cardiac fibrosis in PAI-1 deficiency and the structural domain responsible for its protective effect. Methods and resultsAngII was infused for 4 weeks in whole-body PAI-1 deficient (PAI-1-/-) mice and their wild-type littermates (PAI-1+/+) to examine the role of PAI-1 in ATAA. PAI-1 deficiency did not alter AngII-induced aortopathy but revealed a striking cardiac phenotype characterized by replacement fibrosis predominantly within the epicardium and posterior septum. Ferric iron, indicative of prior hemorrhage, was coincident with fibrosis. Similar phenotypes were observed in PAI-1-/- mice infused with norepinephrine for 4 weeks. To define the pathological events preceding cardiac fibrosis, either AngII or norepinephrine was infused for 1 week in PAI-1+/+ or -/- mice. Both infusions induced extensive epicardial hemorrhage and posterior septal fibrosis in PAI-1-/- mice. To explore the initiation of cardiac pathology, AngII was infused for approximately 1 day. PAI-1-/- mice developed diffuse hemorrhage and cardiomyocyte injury localized to the posterior septum, pathologic changes that preceded overt fibrosis. Finally, to determine the protective domain of PAI-1, saline or AngII was administered to mice harboring loss-of-function point mutations in the protease inhibitory (PAI-1Ala/Ala) or somatomedin B-binding domains (PAI-1AK/AK). Compared to saline infusion, 1 week of AngII induced hemorrhage and heterogeneous fibrosis in PAI-1Ala/Ala, but not PAI-1AK/AK mice. ConclusionsThese findings support that, under hemodynamic stress, PAI-1 deficiency promotes early cardiac hemorrhage and cardiomyocyte injury that lead to fibrosis. Mutational studies implicate dysregulated plasmin generation as an initiator of cardiac injury and fibrosis. TRANSLATIONAL PERSPECTIVECardiac fibrosis has been reported in a human population with PAI-1 deficiency and currently lacks targeted therapy. Our findings demonstrate that in animal models, PAI-1 deficiency confers susceptibility to cardiac injury in response to hemodynamic stress, which may accelerate fibrotic remodeling. Mutational disruption of the protease-inhibitory domain of PAI-1 induced similar pathology, supporting a protective role for this function. These observations suggest that interventions aimed at controlling hypertension, promoting endothelial integrity, or regulating plasmin activation could reduce fibrotic remodeling in this population.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening pulmonary vascular disease, characterized by persistent thrombotic obstruction and progressive pulmonary vascular remodeling, yet the molecular mechanisms linking persistent thrombosis to vascular remodeling remain incompletely understood. Clinical studies have reported elevated plasma von Willebrand factor (VWF) levels and reduced ADAMTS13 in patients with CTEPH, but whether VWF/ADAMTS13 dysregulation contributes directly to disease pathogenesis remains unclear. Here, using newly established rat models of CTEPH, we identify a causative role for dysregulation of the VWF-ADAMTS13 axis in chronic thromboembolic progression. CTEPH rats developed persistent, unresolved VWF- and fibrin-rich thrombi accompanied by markedly increased endothelial VWF deposition. In contrast, ADAMTS13 expression and activity were significantly reduced in CTEPH rats. Consistent with these findings, genetic Adamts13 deficiency further exacerbates pulmonary microvascular thrombosis and accelerated early mortality following disease induction. Mechanistically, ultra-large (UL)-VWF accumulated on the pulmonary endothelial surface, promoting robust platelet recruitment under shear. This platelet-VWF interaction stimulated the release of platelet-derived pro-remodeling mediators, including TGF-{beta}1 and PDGF-BB. Genetic ablation of Vwf markedly reduced in situ microvascular thrombosis within pulmonary arterioles, attenuated pulmonary arterial remodeling, and improved pulmonary hemodynamics. Moreover, treatment with recombinant ADAMTS13 reduced endothelial UL-VWF accumulation, suppressed platelet activation, and effectively prevented thrombosis and platelet-driven pro-remodeling signaling in CTEPH rats. Collectively, these findings identify dysregulation of the VWF-ADAMTS13 axis as a key driver of pulmonary thrombosis and vascular remodeling in CTEPH and support therapeutic targeting of this pathway as a potential disease-modifying strategy. Key PointsO_LIVWF-ADAMTS13 dysregulation promotes persistent pulmonary thrombosis and platelet-driven arterial remodeling within pulmonary arterioles. C_LIO_LIRecombinant ADAMTS13 treatment or VWF ablation abrogates pulmonary arterial thrombosis and halts vascular remodeling in CTEPH. C_LI