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.

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.

Introduction: Vascular calcification is a detrimental ageing-related pathology that is markedly accelerated in metabolic disorders. It is driven by osteogenic differentiation of vascular smooth muscle cells (VSMCs), however epigenetic regulatory pathways activated early in this transition remain poorly defined. Methods: An in vitro calcification model was developed using primary human aortic VSMCs cultured with or without mineral stress. Epigenetic changes were assessed using targeted PCR arrays and CUT&RUN sequencing. Key findings were validated in vivo using single-cell sequencing datasets from human large arteries and spatial transcriptomic analysis in atherosclerotic carotid plaques. Transcriptomic and CUT&RUN analyses identified gene targets altered by epigenetic remodelling, and molecular tools were applied to study effects on metabolism, inflammation, apoptosis, and calcification. Results: During early calcification in response to mineral stress, SWI/SNF chromatin remodelling complexes shift toward ncBAF enrichment in pre-osteogenic VSMCs. ncBAF complexes activated transcriptional programs involved in inflammation, apoptosis, and glycolysis-all hallmarks of calcifying VSMCs. The transcription factor ETS2 was identified as a novel component of ncBAF complexes. Disruption of ncBAF or ETS2 impaired osteogenic differentiation and calcification. Notably, ETS2 expression was regulated by ncBAF, forming a positive feedback loop that reinforced VSMC phenotypic switching. Co-activation of ETS2 and ncBAF and the resulting transcriptional shifts were confirmed in human arterial single-cell datasets, with osteogenic/inflammatory clusters showing NFkB and RUNX2 activation. Spatial transcriptomics further suggested that a macrophage-rich microenvironment may promote the differentiation of smooth muscle cells toward an overt osteogenic/inflammatory phenotype. Immunohistochemistry showed that ETS2 levels correlated with calcification severity in human vessels supporting the potential clinical relevance of ETS2. Conclusions: Our findings identify a novel epigenetic mechanism in vascular calcification, where ncBAF and ETS2 cooperate to drive VSMC phenotypic switching. This ncBAF-ETS2 axis represents a potential therapeutic target to modulate VSMC plasticity and intervene early in the progression of cardiovascular calcification.

Background and aimsAtherosclerotic plaques form preferentially at vascular sites exposed to disturbed blood flow, yet the protein changes underlying this site-specific plaque development remain unclear. Mouse models are widely used to study atherosclerosis but yield only limited amounts of tissue, previously restricting proteomic studies. However, recent advances in mass spectrometry now enable proteomic profiling of very small tissue samples. We aimed to utilise this to uncover site-specific protein changes in aortic regions prone or resistant to plaque formation. MethodsAortic arches from apolipoprotein E-deficient (ApoE-/-) mice fed a Western diet (WD) for 16 weeks were dissected into plaques from the major branches and inner curvature and visibly healthy regions. Proteins were extracted, enzymatically digested, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsMore than 4000 proteins were identified per sample despite their small size (< 1 mg tissue). Principal component analysis showed clustering by both disease status and anatomical location within the aortic arch, indicating distinct proteomes. Proteins known to drive atherosclerosis - including vascular cell adhesion molecule 1 (Vcam1), apolipoprotein B (Apob), lipoprotein lipase (Lpl), and galectin 3 (Lgals3) - were most abundant in advanced plaques and decreased progressively across anatomical regions, reaching their lowest levels in healthy regions furthest from the plaques. Enrichment analysis highlighted pathways related to the extracellular matrix, immune system, hemostasis, and lipoprotein transport as central to disease progression. ConclusionsThis study demonstrates the feasibility of region-resolved proteomics in individual murine aortas and provide new molecular insights into the site-specific nature of atherosclerotic plaque development.

Chronic inflammation drives persistent systemic cytokine signaling that contributes to vascular dysfunction and secondary vasculitis, yet mechanistic studies are limited by models that fail to capture the multicellular architecture and dynamics of human arteries. In contrast, perfusing intact vessels ex vivo has limited tractability because of material availability and difficulty of genetic or biochemical manipulation. We developed a modular, perfused artery-on-a-chip platform by tri-axially bioprinting primary human vascular cells to recapitulate the concentric organization of the intimal, medial, and adventitial layers. The engineered vessels are viable longer than 21 days, with functional endothelial barriers, contractile smooth muscle behavior, and actively remodeled extracellular matrices bearing hallmarks of native vascular tissue. Addition of tumor necrosis factor alpha (TNF) induces altered transcript levels of proinflammatory mediators and secretion of cytokines and matrix-remodeling enzymes without compromising vessel viability. Importantly, this secretory response is effectively attenuated by both a small-molecule JAK1 inhibitor (ABT-317) and anti-TNF antibody (Infliximab), demonstrating the models utility for therapeutic evaluation.

BackgroundOverweight and obese individuals often exhibit lower mortality rates or better prognoses than lean or normal-weight individuals with stroke and other diseases, a phenomenon called the "obesity paradox". Carotid atherosclerosis is the primary cause of ischemic stroke, and body weight serves as a reliable surrogate for adiposity in mice. MethodsPhenotypic and genetic connections of carotid atherosclerosis with body weight were evaluated in 299 F2 mice derived from BALB/cJ and LP/J Apoe knockout (Apoe-/-) mice. F2 mice were fed a Western diet for 12 weeks. Atherosclerotic lesion sizes in left carotid arteries, body weight, coat color, plasma lipids, glucose, small dense LDL ApoB, and malondialdehyde were measured, and 11,000 single nucleotide polymorphism (SNP) markers were genotyped. ResultsCarotid lesion sizes inversely correlated with body weight in both sexes. Genome-wide scans identified two significant quantitative trait loci (QTLs) for carotid atherosclerosis on chromosomes (Chr) 6 and 15 in an additive sex model, and five QTLs on Chr 6, 7, 12, 13, and 15 in an interactive sex model. Adjusting for body weight variation downgraded Chr 15 QTL (Cath5) in both models, whereas other QTLs upgraded in the additive sex model and downgraded in the interactive sex model. Human syntenic region of Cath5 associated with carotid intima-medial thickness (cIMT) and waist-to-hip ratio (WHR). ConclusionsThese findings indicate that the obesity paradox in carotid atherosclerosis is partially driven by shared genetic components that exert opposing effects on adiposity and plaque development and act through sex-dependent mechanisms.

Background: Phenotyping of atherosclerotic plaque vulnerability has largely relied on histopathology that captures structural features, but does not fully account for clinical presentation. Proteomic profiling could uncover molecular readouts of vulnerability that refine plaque phenotyping and provide mechanistic insights. Yet, the proteomic signatures associated with plaque rupture and symptomatic presentation are poorly characterized. Methods: We profiled paired carotid plaque tissue and preoperative plasma from 88 patients undergoing carotid endarterectomy (51 symptomatic, 37 asymptomatic) using the Olink Explore 3072 platform. We related plaque protein abundance to symptomatic presentation and quantitative histopathological features, and compared the performance of histopathology- vs. proteomics-based models for discriminating symptomatic disease. Next, we developed proteomic signatures of cellular abundance and explored their associations with plaque phenotypes by using plaque single-cell RNA-sequencing (scRNA-seq) data. Finally, we assessed plaque-plasma concordance across 2,837 shared proteins. Results: Across 2,837 plaque proteins, 19 were differentially expressed in symptomatic plaques related to distinct clinical events, highlighting pathways related to neutrophil degranulation and innate immune system. FGFBP1 showed the strongest association with symptomatic presentation (log2 fold change = 1.14; P = 1.82 x 10^-6). Proteins associated with a composite vulnerability index based on histopathology were enriched for inflammatory pathways, including TNF signaling through NF{kappa}B, complement activation, and IL6-JAK-STAT3 signaling. Individual proteins also mapped to specific histopathological features, including CXCL8 associated with macrophage burden and lipid core size, and EPHB4 and PKN3 with neovascularization. A proteomics-based model discriminated symptomatic from asymptomatic plaques substantially better than a histopathology-based model (AUC 0.83 vs. 0.66; P = 0.026). Integration with scRNA-seq data enabled the development of cell-class signatures that correlated with histopathology readouts, including macrophage burden, smooth muscle cell content, and neovascularization. Plaque and plasma protein levels showed limited overall correspondence (median {rho}=0.11), although selected proteins, including FGFBP1, demonstrated concordant associations in plasma. Conclusions: Deep proteomic profiling of human carotid plaques identifies molecular signatures of symptomatic atherosclerosis that extend beyond conventional histopathology. These signatures implicate neutrophil activation and inflammatory signaling pathways as key determinants of plaque vulnerability. Although plaque and plasma proteomes are largely distinct, selected proteins may represent promising circulating biomarkers for future risk stratification.

Atherosclerosis is initiated by circulating low-density lipoprotein (LDL) cholesterol transfer into the artery wall, which is mediated by scavenger receptor class B, type I (SR-BI) in endothelial cells(1). Employing single-cell RNA sequencing in human coronary artery disease (CAD) samples, here we show that endothelial SR-BI expression is increased in atheroma, and in endothelial cells with a transcript signature indicative of responding to disturbed blood flow. In vivo in mice hypercholesterolemia and disturbed blood flow independently upregulate endothelial SR-BI; the flow-related upregulation initiates endothelial cell LDL uptake and atherogenesis. Guided by transcription factor networks, it is revealed that HIF-1 binding to human Scarb1 Intron 1 governs endothelial SR-BI transcription, and in mice HIF-1 drives hypercholesterolemia-related SR-BI upregulation and artery LDL uptake. Thus, the two major instigators of atherosclerotic lesion formation, hypercholesterolemia and disturbed blood flow, both upregulate endothelial SR-BI to drive the LDL transport that underlies the disorder. Targeting the processes regulating endothelial SR-BI potentially represents a new therapeutic strategy against CAD.

Mechanical homeostasis plays a central role in promoting and preserving optimal structure and function in the adult aorta. Although pathogenic variants can compromise homeostatic processes, it appears that intramural cells yet attempt to compensate for some genetically induced changes. In particular, lysyl oxidase is higher in the adult Marfan aorta compared with the age-matched control aorta. Here, we block lysyl oxidase in adult Fbn1C1041G/+ Marfan syndrome mice after stimulating aortic disease progression via induced hypertension. Whereas hypertension alone increases aortic dilatation, concurrent blocking of lysyl oxidase results in a dramatic increase in disease severity, driving an otherwise mild aortic phenotype in adult male Fbn1C1041G/+ Marfan mice to aneurysmal dilatations as well as dissection and rupture, with frequent premature death. Deposition and cross-linking of fibrillar collagens, among other extracellular matrix constituents, can represent a protective compensation against severe disease in the Marfan aorta. The present study emphasizes the need clinically to avoid compromising new collagen deposition and suggests that strategies to augment collagen cross-linking could be beneficial.

The Hippo signaling pathway is increasingly recognized as a key regulator of endothelial cell (EC) proliferation, migration and vascular development. However, the roles of its upstream scaffold proteins remain poorly understood. Although WWC family proteins are widely regarded as functionally redundant activators of LATS1/2 kinases, the human genome contains a third family member, WWC3, that is absent from mice, raising the possibility of species-specific regulation of endothelial Hippo signaling. Here, we assessed the roles of WWC2 and WWC3 in human ECs using siRNA-mediated knockdown. Surprisingly, we found that WWC3 is the predominant regulator of canonical Hippo signaling, with a substantially greater effect than WWC2 on LATS1/2 phosphorylation, YAP/TAZ localization and expression of Hippo target genes. Loss of WWC3 also altered endothelial morphology and induced a partial endothelial-to-mesenchymal transition-like (EndoMT-like) phenotype. By contrast, WWC2 had a lesser effect on canonical Hippo signaling, but it was required for normal VEGF signaling dynamics. Despite these distinct molecular functions, depletion of either WWC2 or WWC3 impaired EC proliferation, migration, and cord formation in vitro. Together, our findings demonstrate that WWC family proteins perform overlapping but distinct functions in human ECs, with WWC3 acting as the predominant canonical Hippo regulator, whereas WWC2 more efficiently modulates VEGF signaling. These results reveal unexpected functional specialization among WWC proteins and suggest that regulation of Hippo signaling in human ECs differs from that inferred from mouse studies.

BackgroundAtherosclerosis is driven by macrophage foam cell formation resulting from excessive oxidized low-density lipoprotein (oxLDL) accumulation and chronic vascular inflammation. This study evaluated the therapeutic potential of Aegeline, Atorvastatin, and their combined in mitigating oxLDL-induced inflammatory responses, cholesterol accumulation, and oxLDL uptake in human THP-1 macrophages. MethodsTHP-1 monocytes were differentiated into macrophages using a 72-hour differentiation protocol followed by a 48-hour resting period, confirmed via CD14 surface marker characterization. Macrophages were exposed to DiI-oxLDL and treated with Aegeline, Atorvastatin, or their combination. Key inflammatory cytokines and chemokines (CRP, TNF-, IL-6, and IL-8) were measured using ELISA. Cholesterol efflux capacity and cellular oxLDL uptake were quantitatively assessed using fluorescence retention assays and immunofluorescence imaging. ResultsDifferentiation of THP-1 monocytes to macrophages resulted in marked down-regulation of CD14 expression. DiI-oxLDL exposure triggered significant pro-inflammatory mediator secretion (p<0.001) and excessive intracellular cholesterol accumulation. Single-agent treatment with Aegeline or Atorvastatin significantly attenuated oxLDL-induced elevations of CRP, TNF-, IL-6, and IL-8. Atorvastatin alone strongly suppressed CRP expression back to physiological baseline levels (p=ns vs. control). Notably, the combination of Aegeline and Atorvastatin demonstrated enhanced, broad-spectrum anti-inflammatory efficacy, achieving superior suppression of TNF- (p=ns vs. control), IL-6, and IL-8 compared to monotherapies. Furthermore, both agents promoted cholesterol efflux and suppressed oxLDL uptake, with the combination treatment producing the lowest residual intracellular cholesterol levels (p<0.001). ConclusionAegeline and Atorvastatin effectively suppress oxLDL-induced macrophage inflammatory cascades and intracellular lipid overload. While Atorvastatin monotherapy exerts robust control over CRP and oxLDL loading, combining Aegeline with Atorvastatin provides synergistic efficacy, enhancing cholesterol efflux and restoring pro-inflammatory cytokine expression toward physiological levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/744794v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d90d88org.highwire.dtl.DTLVardef@1079202org.highwire.dtl.DTLVardef@2d659org.highwire.dtl.DTLVardef@4685af_HPS_FORMAT_FIGEXP M_FIG C_FIG

NOTCH3 is a transmembrane receptor highly expressed in vascular mural cells where it contributes to blood vessel formation and homeostasis. NOTCH3 expression declines in the vasculature with aging, and dysregulated NOTCH3 signalling is implicated in pulmonary arterial hypertension, cancer progression and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). RNA-based approaches targeting NOTCH3 are emerging as potential therapeutic strategies, however, the consequences of NOTCH3 suppression in mature vascular smooth muscle cells (VSMCs) remain incompletely understood. Here, we investigated the molecular and functional effects of siRNA-mediated NOTCH3 knockdown in human aortic smooth muscle cells. Transfection with NOTCH3-targeting siRNA efficiently suppressed NOTCH3 transcript and protein levels. Quantitative proteomics revealed remodelling of extracellular matrix (ECM), cytoskeletal and metabolic pathways, with enrichment of collagen biosynthesis and inhibition of glycolytic signalling. Specifically, NOTCH3 knockdown increased ECM components, including COL3A1, elevated F-actin, and upregulated the actin regulator, CTTN. In parallel, glycolytic capacity was reduced, accompanied by decreased expression of the glycolytic enzyme ENO2. Despite reduced VEGFA and alteration in angiogenic signalling proteins, endothelial network formation in co-cultures, as well as VSMC proliferation and migration remained unaffected. Finally, NOTCH3 interactome analysis revealed key collagen and actin-regulating proteins. These findings identify NOTCH3 as an important regulator of ECM homeostasis, cytoskeletal organisation, and glycolytic metabolism. The preservation of primary cellular functions despite molecular remodelling highlights the adaptive capacity of VSMCs. These findings demonstrate that therapeutic modulation of NOTCH3 may alter vascular cell biology which warrants consideration during development of RNA-based therapeutics for CADASIL and other NOTCH3-associated diseases.

Marfan syndrome (MFS) is an inherited connective tissue disorder caused by pathogenic variants in FBN1, encoding fibrillin-1, with life-threatening aortic complications arising in part from endothelial cell (EC) dysfunction. To study this in a human model, we generated hiPSC-derived ECs from three MFS patients (iMFS-ECs). We show that iMFS-ECs recapitulate known disease phenotypes, including impaired alignment in the direction of flow. Moreover, we found that iMFS-ECs do not recover from TNF--induced loss of barrier integrity, due to sustained EC contractility. iMFS-ECs exhibited TNF--induced ICAM1 upregulation and NF-{kappa}B activation comparable to healthy donor-derived hiPSC-ECs by bulk RNA-seq, while expression of genes linked to cytoskeletal arrangements, cell signaling and ECM remodeling were dysregulated. In conclusion, we show that hiPSC derived ECs can serve as a model to investigate MFS pathology. These findings establish a human iPSC platform for MFS endothelial research and suggest impaired inflammatory resolution as a novel therapeutic target.

BackgroundAdipose tissue surrounding the heart and vasculature plays critical roles in cardiovascular homeostasis and disease, yet the cellular and molecular milieu of these depots at single-cell resolution remains incompletely characterized. Understanding how regional adipocytes differ transcriptionally and communicate with neighboring cardiovascular cells is essential for developing targeted therapeutic strategies. MethodsWe performed single-nucleus RNA sequencing (snRNA-seq) on human adipose tissue from four anatomically distinct depots: ascending aorta, left atrium, right coronary artery, and subcutaneous fat. We characterized cellular composition, adipocyte and progenitor heterogeneity, depot-specific transcriptional programs, and intercellular communication networks. We further examined signaling remodeling in disease contexts, including atrial fibrillation and aortic aneurysm. ResultsWe identified six transcriptionally distinct adipocyte subpopulations and six adipocyte stromal and progenitor cell (ASPC) subpopulations were shared across depots but showed marked differences in abundance and gene expression reflecting developmental imprinting, including HOX family genes and anterior-posterior patterning programs. Intercellular communication analysis revealed depot-specific ligand-receptor interactions, with EPHA signaling identified as selectively enriched in the left atrial adipose depot. Disease-state analyses demonstrated extensive change in cell-cell communication in atrial fibrillation and aortic aneurysm, with differential regulation of FN1, EGF, SLIT, NOTCH, and CD46 signaling pathways. ConclusionsOur study reveals that cardiac and vascular adipose depots harbor transcriptionally specialized adipocytes and progenitors with distinct intercellular communication programs that are remodeled in atrial fibrillation and aortic aneurysm.

Cardiovascular calcification is present in practically all cardiac diseases, which are the top killers in the world today1, and is particularly associated with atherosclerosis2, aortic stenosis3 and rheumatic fever4. If not the direct cause of death, calcification contributes considerably to complications that can lead to heart failure5. Nonetheless, the origins and mechanisms of cardiovascular calcification are still strongly debated3,6-12. Just over a decade ago, it has been reported that nano and micron-sized calcified spherical particles, formed from a single crystal of magnesium-containing calcium phosphate, were the first calcified structure that could be detected in cardiovascular tissue13. These particles were found even before any sign of cardiac disease was present and were present in all stages of cardiac diseases13. The ubiquity of these particles suggests their importance for the origins and development of cardiovascular calcific diseases. Here, we show that these particles originate from platelet {delta}-granules and are present in mammals, birds and lizards. Based on our results, we suggest a new mechanism for the origins of these particles, complementing existing models of cardiovascular calcification7,14, and bringing a new, early, and hitherto unaccounted key event in the process of cardiovascular calcification. This new mechanism model, along with a better understanding of the early stages of cardiovascular calcification, could open the path for the development of pharmacological prevention and treatment solutions for several cardiac diseases.

Trained immunity and clonal hematopoiesis are two newly identified immunological phenomena that contribute to the pathophysiology of atherosclerotic cardiovascular disease. These two phenomena share some convergent molecular mechanisms, such as IL-1{beta} being a central regulator and involvement of epigenetic enzymes. Therefore, we hypothesize that presence of clonal hematopoiesis driver mutations (CHDMs) can predispose to an increased capacity to build trained immunity. We previously characterized how the presence of CHDMs relates to immune cell function and vasculometabolic complications in a cohort of older individuals with overweight and obesity. From this cohort we now selected 17 individuals with CH due to DNMT3A mutations and 15 without any known CHDMs. We performed in depth immune characterization via flow cytometry, functional assays with monocytes and neutrophils, and we measured the capacity to build trained immunity using {beta}-glucan and oxLDL as stimuli. We corroborated our previous findings of lower ex vivo cytokine production capacity of PBMCs from individuals with DNMT3A mutations. Importantly, presence of DNMT3A CHDMs associated with higher trained immunity response. Moreover, we demonstrated that individuals with DNMT3A mutations were characterized with higher CD10+ mature neutrophils and a lower neutrophil MPO release upon TLR2 stimulation. In conclusion, presence of DNMT3A CHDMs is associated with increased susceptibility to build a hyperresponsive trained monocyte phenotype. The exact molecular mechanisms behind this phenomena requires further investigation.

BackgroundHydrogen sulfide (H2S) is an important endothelial-derived vasodilator, but the signaling mechanism remains incompletely understood. We previously demonstrated that H2S-mediated vasodilation requires transient receptor potential vanilloid type 4 (TRPV4) channels. Because H2S has been reported to enhance heme oxygenase (HO) activity and HO-derived carbon monoxide (CO) regulates endothelial signaling, we hypothesized that H2S-mediated vasodilation requires HO-2-derived CO. MethodsPressure myography was performed in isolated rat mesenteric arteries to determine the contribution of HO, TRPV4, eBK, and SK/IK channels to H2S-mediated vasodilation. HO-2 sulfhydration was assessed using a maleimide assay, and spatial association among HO-2 and TRPV4 was examined using proximity ligation assays in human aortic endothelial cells. ResultsH2S Selicited concentration-dependent vasodilation that was abolished by HO inhibition. Repletion of CO restored H2S-mediated vasodilation in the presence of HO inhibition. CO-mediated vasodilation was abolished by TRPV4 and SK/IK inhibition but was unaffected by eBK inhibition. H2S increased HO-2 sulfhydration and enhanced HO activity. In endothelial cells, HO-2 and TRPV4 exhibited close spatial association. ConclusionsThese findings support a model in which H2S stimulates HO-2-derived CO production, leading to TRPV4-dependent endothelial signaling, SK/IK activation, and vasodilation. Together, the data support the existence of an endothelial HO-2/TRPV4/SK/IK signaling domain that contributes to H2S-mediated vascular reactivity.

ANGPT2 is widely recognized as a critical regulator of pathological neovascularization. By analyzing scRNA-seq data from neonatal retinas, we demonstrate that Angpt2 transcripts are highly enriched in tip cells relative to other endothelial subtypes, where Angpt1/4 expression is absent. However, mechanisms underlying ANGPT2 function at angiogenic fronts remain inadequately understood. Here, we show that endothelial Angpt2 deletion severely disrupted retinal vascularization, characterized by neovascular tufts and micro-hemorrhage. Similar angiogenic defects also occurred in the brain, but were less evident in other tissues examined. Mechanistically, ANGPT2 insufficiency attenuated retinal tip cell invasion with aberrant mural cell coverage, compromising sprouting into non-vascularized tissues. Retinal RNA-seq analysis revealed that transcripts associated with endothelial migration and junction assembly were reduced in Angpt2 mutants compared to littermate controls, while upregulated genes were enriched in hypoxia-responsive pathways and mural cell development. Notably, abnormal mural-tip cell associations were detected within 48 hours post-Angpt2 deletion, displaying also a hypoxia-driven transcriptomic signature. These closely resemble the vascular pathologies observed in human retinopathy of prematurity. In contrast, Angpt1 insufficiency or Angpt4 deficiency primarily affected venous morphogenesis. Collectively, our findings imply that ANGPT2 is essential for driving tip cell invasion during sprouting angiogenesis, and that its insufficiency triggers hypoxia-driven vascular anomalies.

APOL1 risk variants are the strongest genetic determinants of HIV-associated nephropathy (HIVAN), yet the mechanisms linking inflammation to APOL1-mediated podocyte injury remain poorly understood because authentic patient-derived human disease models are lacking. Using urine-derived podocytes established from children with HIVAN and endogenous APOL1 reporter cell lines derived from these cells, we identified a previously unrecognized pathway of inflammatory, cathepsin-dependent APOL1 proteolysis. Endogenous APOL1 cleavage was detected in patient-derived podocytes, whereas reporter cell lines enabled the identification and functional characterization of N-terminal and C-terminal APOL1 fragments with distinct intracellular localization and pathogenic functions. The nuclear N-terminal fragment activated inflammatory transcriptional programs and promoted podocyte injury, whereas the membrane-associated C-terminal fragment mediated membrane toxicity and remained susceptible to pharmacologic inhibition by inaxaplin. Cathepsin S directly cleaved APOL1 in vitro, linking inflammatory signaling to APOL1 fragmentation. These findings identify inflammatory APOL1 proteolysis as a mechanism that partitions APOL1 toxicity into distinct pathogenic programs and nominate APOL1 processing as a therapeutic target for HIV-associated and other APOL1-mediated kidney diseases.

Uterine fibroids (leiomyomas) are highly prevalent benign tumors defined by excessive extracellular matrix (ECM) deposition, altered vascular structure, and progressive tissue stiffening, yet the cellular programs that coordinate these features remain poorly understood. Cellular senescence has been implicated in fibroid biology, but whether senescence represents a uniform state or distinct, functionally specialized cell identities within fibroids is unknown. Here, we identify the distinct heterogeneous populations of senescent cells ("senotypes") present in human fibroids and characterize their role in shaping the fibroid microenvironment. Using single-cell RNA sequencing (scRNA-seq) integrated with a senescence gene signature and protein-level validation, we identify senescent cells (SnC) distributed across fibroblast, mural, and endothelial compartments, each exhibiting distinct transcriptional programs. SnC endothelial cells (ECs) are enriched in fibroids relative to matched myometrium and activate TEAD4-associated mechanosensing, angiogenic, and immune signaling pathways, despite being associated with impaired vessel maturation in situ. In parallel, SnC fibroblast and mural populations in fibroids upregulated SRF-associated cytoskeletal and ECM programs, accompanied by increased COL6A3 expression and collagen VI deposition, consistent with tissue stiffening. Ligand-receptor and spatial analyses reveal that these SnC populations function as interconnected signaling hubs, coordinating immune cell recruitment and stromal remodeling. Importantly, analysis of human fibroids treated with collagenase demonstrated a reduction in both ECM density and SnC burden, supporting a reinforcing relationship between matrix mechanics and senescence. Together, these findings establish senescence in fibroids as a heterogeneous, mechanically reinforced, and network-driven process that links vascular dysfunction, immune signaling, and fibrosis, highlighting distinct SnC states as potential translational targets for non-surgical therapies.