Cardiovascular Research

Background: Platelets are critical drivers of thrombo-inflammatory responses in different cardiovascular diseases. Abdominal aortic aneurysm (AAA) is a progressive, life-threatening vascular disorder mainly characterised by chronic inflammation, extracellular matrix degradation, and the formation of a platelet-rich intraluminal thrombus (ILT). Experimental and clinical evidence identified platelets as main players in AAA pathology as evidenced by elevated platelet activation and procoagulant activity that critically contribute to AAA progression. Methods: The present study investigated the contribution of glycoprotein (GP)Ib alpha, the von Willebrand factor (VWF)-binding subunit of the platelet GPIb-IX-V complex, to AAA initiation and progression in experimental AAA using the ePPE mouse model and in patients. Results: Genetic ablation of platelet GPIb alpha significantly attenuated early aneurysm expansion in experimental AAA, indicating a critical role for GPIb alpha during the initial stages of aneurysm development. This initial effect was compensated at later time points showing no differences in aneurysm progression between groups. Notably, genetic deletion of GPIb alpha induced a constitutively hyperactive platelet phenotype already in naive mice that was further amplified during experimental AAA. This elevated platelet hyperactivity was mainly due to increased GPVI activation of platelets 28 days post-surgery. To assess the clinical relevance, spatial profiles of human ILT specimens from patients with AAA were analysed. In the ILT, we detected a highly compartmentalised distribution of GPIb alpha and VWF with pronounced enrichment within the luminal layer. In parallel, circulating VWF activity as well as platelet surface expression of GPIb alpha were significantly increased in patients with AAA. Conclusion: Collectively, these findings identify a dysregulated GPIb alpha-VWF axis in human AAA pathology, mainly characterised by enhanced platelet GPIb alpha surface expression and increased activity of circulating VWF.

BackgroundThe progression of abdominal aortic aneurysm (AAA) is closely associated with the phenotypic switch of vascular smooth muscle cells (VSMC). While members of the Serine/Arginine-rich splicing factor (SRs) superfamily are increasingly acknowledged for their pivotal role in modulating cell phenotype, the specific contribution of these factors to AAA development has remained underexplored. MethodsThrough integrated analyses the whole transcriptome of human and murine AAA tissues derived from clinical samples and animal models, we identified serine-arginine repeat matrix 4 (SRRM4) as a candidate gene regulating AAA formation. To investigate the role of SRRM4 in AAA formation, we used global and VSMC-specific SRRM4-knockout mice to construct AAA mouse models in two different ways. The impact of SRRM4 on AAA was further investigated in in vivo and in vitro models ResultsElevated levels of SRRM4 mRNA and protein were observed in VSMC from both human and murine AAA tissues. Targeted silencing of SRRM4 in VSMC (but not in endothelial or myeloid cells) significantly attenuated AAA formation in both AngII- and CaCl2-induced mouse models. Our results identify the RE1 silencing transcription factor (REST) as a critical target gene of SRRM4 in AAA, a master regulator of cellular phenotypic stability. We show that SRRM4 modulates VSMC phenotype through alternative splicing of the REST gene, resulting in a non-functional REST4 isoform. Furthermore, our study shows that REST directly represses STAT1, a mechanism by which SRRM4 influences VSMC phenotypic transition. Regarding clinical significance, inhibition of both STAT1 expression and function was shown to be beneficial in preventing AAA formation and slowing disease progression in mouse models. ConclusionsThis investigation uncovers a previously unrecognized role of SRRM4-mediated splicing of the REST gene in the progression of AAA and identifies a novel REST/STAT1 axis critical in the phenotypic modulation of VSMC. The findings of this study may provide insights for the development of innovative therapeutic strategies for AAA. What is New?O_LIOur study demonstrates that the RNA splicing factor SRRM4 (serine/arginine repetitive matrix 4) expression is increased in human and murine abdominal aortic aneurysm (AAA) tissues and unveils a previously undescribed role of SRRM4 in regulating AAA development. C_LIO_LISRRM4 promotes AAA development and progression through inducing splicing of REST (RE1 silencing transcription factor) gene and upregulating STAT1 (signal transducer and activator of transcription 1) expression, which result in extracellular matrix degradation, inflammation, and VSMC phenotypic switching. C_LIO_LIOur study provides proof-of-concept evidence indicating the therapeutic value of STAT1 inhibition in AAA prevention and treatment. C_LI What Are the Clinical Implications?O_LIOur study defining a novel mechanism underlying the SRRM4/REST/STAT1 axis is crucial for developing new therapeutic strategies for AAA. C_LIO_LIBased on these findings, we have initiated a prospective study (REST4-AAA; ClinicalTrials.gov Identifier: NCT07056491) to evaluate REST4 expression levels in serum of AAA patients and explore its potential as a serum biomarker for AAA prediction C_LIO_LIOur findings indicate that fludarabine, a clinically available agent reported to selectively suppress STAT1, effectively attenuates AAA progression in mice, suggesting its potential for repurposing as a therapeutic strategy for AAA. C_LI

Background and Aims: Calcific aortic valve disease (CAVD) is a common valvular heart condition and has no effective medical therapy. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by the acquisition of somatic mutations in hematopoietic stem cells which provides a selection advantage, has been associated with incident aortic stenosis (AS), particularly when driven by TET2 mutations. Methods: We evaluated the association of CHIP and gene-specific CHIP subtypes with AS in the UK Biobank (UKB) and with aortic valve hemodynamics in the Atherosclerosis Risk in Communities (ARIC) study to prioritize gene driver mutations for mechanistic study. We then generated human ASXL1-mutant THP-1 derived macrophage like cells and evaluated the ability of THP-1 conditioned media to promote a calcific response in human valvular interstitial cells (VICs). We additionally performed mass spectrometry proteomics to characterize the ASXL1-mutant THP-1 macrophage secretome. Therapeutically relevant inflammatory pathways were inhibited with anakinra (interleukin [IL]-1) or tocilizumab (IL-6). Results: Among 449,109 individuals in the UKB with whole exome sequencing, large CHIP clones and non-DNMT3A CHIP subtypes (e.g., TET2, ASXL1, and JAK2 CHIP) were independently associated with risk of incident AS. Among 1,963 individuals in ARIC with CHIP sequencing and echocardiographic data, only ASXL1 CHIP was associated with worse aortic valve hemodynamics. In vitro, ASXL1-mutant human THP-1-derived macrophage-like cells had increased AIM2 inflammasome activation. THP-1 media promoted VIC calcification, which was accelerated in ASXL1 mutant macrophages. Proteomic profiling identified inflammatory proteins linked to valvular calcification. Accelerated VIC calcification was reduced by IL-1 inhibition by anakinra or IL-6 inhibition by tocilizumab. Conclusions: Integrating human cohort data with experimental evidence using ASXL1-mutant human macrophage-like cells and human valve cells, these findings identify ASXL1-mutant CHIP as a distinct inflammatory subtype associated with valvular calcification and support IL-1 and IL-6 signaling as candidate therapeutic pathways for future investigation in CAVD.

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.

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.

Heart regeneration varies among vertebrates, with zebrafish serving as a reference species for efficient cardiac restoration. How this capacity diversified across teleosts is an emerging question, particularly following the recent identification of non-regenerative cardiac repair in medaka and cavefish. Here, we investigate heart restorative capacity following cryoinjury in two livebearers, platyfish and swordtails (Xiphophorus species), belonging to the Poeciliidae family. We demonstrate that their hearts lack the vascularized compact myocardium, a ventricular layer implicated in the restorative response in zebrafish. Following cryoinjury, both poeciliids failed to rapidly deposit fibrotic tissue that normally reinforces the damaged ventricle. This deficiency correlates with pronounced wound protrusion. Although the remaining myocardium displayed an initial proliferative response, subsequently deposited collagenous scar tissue permanently sealed the ventricular wall, precluding complete regeneration. Transcriptomic analysis identified several divergently regulated pathways between cryoinjured hearts of zebrafish and platyfish, most notably in immune response regulation. These differences were validated by delayed leukocyte infiltration and sustained inflammation in platyfish, contrasting with the rapid and self-resolving inflammatory response in zebrafish. Our findings demonstrate that Xiphophorus species have evolved hearts with compromised regenerative capacity, characterized by initial wound protrusion and permanent scarring. These results establish that lineage-specific evolutionary traits can profoundly shape regenerative competence across teleosts, with broad implications for understanding the mechanistic basis of cardiac repair. Highlights{middle dot} Viviparous poeciliids lack vascularized compact myocardium. {middle dot} Inflammation and fibrosis are delayed in the cryoinjured platyfish ventricle. {middle dot} Ventricular cryoinjury in Xiphophorus leads to transient bulging-type deformation. {middle dot} Failure to form a myocardial bridge results in permanent scarring.

Pathogenic variants of COL4A1 and COL4A2, which encode the 1 and 2 chains of type IV collagen in vascular basement membranes, cause cerebral small vessel disease and intracerebral hemorrhage, but the signaling mechanisms that precede vascular damage remain incompletely defined. Here, we investigated cerebral arterial function in mice heterozygous for the Col4a2+/G646D mutation. Cerebral pial arteries from aged (12-month-old), but not young (3-month-old), Col4a2+/G646D mice exhibited impaired pressure-induced myogenic tone, whereas downstream parenchymal arterioles retained normal myogenic responsiveness, revealing a segment-selective vascular defect. The frequency of subcellular Ca2+ signals (Ca2+ sparks) generated by the release of Ca2+ through ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) was increased, and large-conductance Ca2+-activated K+ (BK) channel activity was elevated, in cerebral pial artery smooth muscle cells (SMCs) from aged Col4a2+/G646D mice. SR Ca2+ store load and Ryr2 transcript abundance were unchanged in these cells, but Stimulated Emission Depletion super-resolution microscopy revealed that RyR2 protein clusters were enlarged compared with controls. Protein kinase A (PKA) activation reproduced RyR2 cluster enlargement, whereas PKA inhibition normalized RyR2 cluster organization and BK channel activity in SMCs from aged Col4a2+/G646D mice. These findings identify age-associated, PKA-dependent, nanoscale remodeling of RyR2 clusters as a previously unknown mechanism underlying cerebral arterial dysfunction in COL4A2-related disease.

Background and aimsCardiovascular-kidney-metabolic (CKM) syndrome stages confer graded CVD risk, but the underlying stage-specific molecular mechanisms remain undefined. MethodsIn 355,724 UK Biobank participants (median follow-up 13.5 years), we mapped CKM stages (0-3) to incident CVD. Using proteomics (n=37,785) and metabolomics (n=190,112), we identified stage-specific biomarkers via LASSO and XGBoost-SHAP. Mediation analyses were performed to quantify the proportion of the CKM-CVD association that was statistically accounted for by these biomarkers. The proportion of the protective association between cardiovascular health (Lifes Crucial 9 [LC9]) and incident CVD that was mediated by the same molecules was quantified. ResultsCVD risk increased across CKM stages. Beyond 11 pan-stage proteins (e.g., RTN4R,LEP) and 29 pan-stage metabolites (e.g.,GlycA), stage-specific molecular signatures emerged, whose pathway enrichment revealed a shift from metabolic/extracellular matrix dysregulation (Stage 1) to inflammation (Stage 2) to hypoxia/fibrosis (Stage 3). The proportion of the CKM-CVD risk association statistically accounted for by these molecules shifted accordingly: ADM (42.9%) in Stage 1, FABP4 (24.6%) in Stage 2, and HAVCR1 (28.0%) in Stage 3. High CVH (LC9[≥]80) was associated with approximately 80% lower CVD risk in Stages 0-2; a proportion of this protective association was statistically accounted for by the same stage-specific molecules. ConclusionsThese findings reveal a stage-ordered molecular continuum--from ECM remodeling to inflammation to fibrosis--that redefines CKM-driven CVD risk, and the strong protection of high CVH in early stages was statistically accounted for in part by these stage-specific molecules, generating the hypothesis that CVH may reduce risk through these modifiable pathways and providing a molecular framework for future stage-adapted intervention trials.

The circadian molecular clock is a 24 hour cellular timekeeper that influences many features of cardiovascular function. Disruption of the circadian clock via misalignment with the light dark cycle is associated with a higher incidence of cardiovascular disease and raises cardiovascular risk factors in humans. Nonetheless, the cell type specific molecular basis for how misalignment affects the vasculature remains poorly understood. To address this, we performed single cell RNA sequencing from whole mouse aorta at ZT0, ZT6, ZT12, and ZT18 under aligned and acutely misaligned (6 hour phase advance) light dark cycles in both male and female mice. Leveraging Bayesian variational inference, we estimated posterior waveforms for 141,752 cells across four major cell types and identified hundreds of cycling genes in vascular smooth muscle cells (SMCs) and fibroblasts. Pathway and transcription factor enrichment analyses revealed coordinated circadian activity in cholesterol biosynthesis, smooth muscle contraction, and extracellular matrix organization. Notably, SMC genes implicated in phenotypic switching showed coordinated temporal patterns, with genes promoting switching peaking at dusk and genes restraining switching peaking at dawn. Comparing males and females, we found that female SMCs are broadly more rhythmic, with higher amplitudes and nearly twice as many cycling genes after controlling for cell counts and library sizes, a sex difference that was cell type specific and not observed in fibroblasts. After acute misalignment, cycling genes showed reduced amplitudes, and peak times showed limited adaptation to the new light dark cycle. Given that the central clock is known to adapt near completely during this timeframe, these observations suggest internal misalignment between central and peripheral rhythms. Moreover, altered relative timing of core clock genes within cells indicates that misalignment is created at the intracellular level as well. In SMCs, gene expression patterns were consistent with proteostatic stress, including broad downregulation of protein chaperones and stress response genes, which cells appear to cope with by upregulating protein degradation pathways. In parallel, in vivo vascular phenotyping showed increased vascular permeability in both sexes, reduced urinary nitrate in males, and increased microvascular thrombus formation in males following acute misalignment. This atlas provides a resource for understanding how circadian misalignment disrupts vascular homeostasis and may contribute to cardiovascular disease risk.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease characterized by progressive medial degeneration, impaired mechanical integrity, and extracellular matrix (ECM) degradation. However, no pharmacological therapy has been proven to halt aneurysm progression or prevent dissection or rupture. Vascular smooth muscle cells (VSMCs) are vital for maintaining medial architecture by sensing and remodeling the surrounding ECM; however, the mechanism by which abnormal ECM mechanics are transmitted to nuclear transcriptional programs that disrupt aortic wall matrix homeostasis remains incompletely understood. Integrative transcriptomic screening of Lysyl oxidase (LOX)-deficient and ?-aminopropionitrile (BAPN)-induced TAAD models identified vestigial-like family member 4 (VGLL4) as a mechanosensitive transcriptional regulator of TAAD. VGLL4 was enriched in VSMCs and markedly increased in aortas from patients with TAAD and BAPN-induced TAAD mice. VSMC specific deletion of Vgll4 protected mice from BAPN-induced aortic dilation, dissection, rupture-associated mortality, vascular stiffening, ECM degradation, and medial destruction. Mechanistically, pathological matrix remodeling and mechanical stress induced VGLL4 expression in VSMCs, where VGLL4 cooperated with specificity protein 1 (SP1) to activate Wisp1 transcription. In vivo, VSMC-enriched Wnt-inducible signaling pathway protein (WISP1) overexpression exacerbated TAAD progression, whereas Wisp1 knockdown protected against BAPN-induced TAAD and mitigated the severe aortic phenotype driven by VGLL4 overexpression. Secreted WISP1 bound Tissue Inhibitor of Metalloproteinases 3 (TIMP3) through its C-terminal domain and impaired TIMP3-mediated MMP9 inhibition, thereby increasing MMP9 proteolytic activity and accelerating ECM degradation. Consistently, in vivo Wisp1 knockdown protected against BAPN-induced TAAD. Together, these findings define the VGLL4-WISP1-TIMP3/MMP9 axis, which couples pathological ECM mechanics to nuclear transcriptional activation and protease-dependent matrix degradation in VSMCs. This pathway promotes medial structural failure, aortic mechanical stability loss, and TAAD progression, identifying WISP1 as a potential therapeutic target for preserving aortic wall matrix homeostasis.

The earliest events in calcific aortic valve disease (CAVD) occur at the valve endothelium, where the cells lining the leaflet undergo NF-kappaB (Nuclear Factor kappa B)-driven inflammatory activation and endothelial-to-mesenchymal transition (EndMT). The endothelial glycocalyx (EG) sets the threshold at which endothelial cells (ECs) respond to such cues, yet its composition and function in the aortic valve remain almost entirely uncharacterized. Here we define the cell-surface proteoglycan (CPG) landscape of human aortic valve endothelial cells (HAVECs) under inflammatory stimulation and identify syndecan-3 (SDC3) as a regulator of valve endothelial activation. Among the proteoglycans examined, SDC3 was highly expressed and inflammation-responsive. Silencing SDC3 in unstimulated HAVECs was sufficient to upregulate NF-kappaB pathway components and the leukocyte adhesion molecules vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin, and it amplified their induction upon TNF-alpha challenge. Pharmacological inhibition of IKKbeta; abolished this difference, placing SDC3 upstream of NF-kappaB, while SDC3 overexpression attenuated tumor necrosis factor-alpha (TNF-alpha)-induced VCAM1, the inverse of the knockdown effect. Loss of SDC3 further primed HAVECs toward a mesenchymal, EndMT-prone phenotype under transforming growth factor-beta 2 (TGF-beta2) stimulation, marked by increased smooth muscle protein 22-alpha (SM22alpha) and reduced platelet endothelial cell adhesion molecule (PECAM1/CD31). Together, these findings identify SDC3 as an endogenous restraint on the inflammatory and mesenchymal programs that characterize the endothelial phase of CAVD and establish the valve endothelial glycocalyx as a previously unappreciated yet potent regulator of aortic valve endothelial dysfunction. Preserving or augmenting SDC3 may warrant investigation as a means of holding valve endothelial cells in a quiescent state.

Background: Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome associated with ageing, obesity and impaired nitric oxide signalling. Preclinical models often do not capture the sex-specific and cardiometabolic features observed in patients. We hypothesised that biological sex and the degree of nitric oxide synthase (NOS) inhibition would influence development of HFpEF-like versus HFrEF-like phenotypes. Methods and Results: Male and female C57BL/6J mice (>24 weeks) were exposed to a high-fat diet (HFD) combined with low-dose (0.3 g/L; female only) or high-dose (0.5 g/L; male and female) NOS inhibition using N({omega})-nitro-L-arginine methyl ester (L-NAME) for 15 weeks. Female mice receiving low-dose L-NAME+HFD developed a HFpEF-like phenotype characterised by impaired diastolic function, exercise intolerance and preserved systolic function. Increasing NOS inhibition did not worsen diastolic dysfunction but induced inflammatory and stress-associated transcriptional pathways in female hearts. In contrast, male mice given high-dose L-NAME+HFD developed hypertension, elevated ventricular pressures and impaired systolic function, consistent with a HFrEF-like phenotype. Despite different cardiac phenotypes, circulating lipidomic profiling revealed broadly conserved sphingolipid and phospholipid remodelling, with sex-specific regulation of phosphatidylinositol and lysophosphatidylcholine species. Transcriptomic analyses identified shared regulation of extracellular matrix, calcium-handling and metabolic pathways, whereas greater NOS inhibition was associated with transcriptional signatures related to inflammatory signalling, cellular stress responses and mitochondrial homeostasis. Conclusions: Cardiometabolic stress does not produce a uniform HF phenotype. Instead, biological sex and the degree of NOS inhibition direct distinct functional and molecular remodelling trajectories, identifying HFpEF-like dysfunction as one of several potential cardiac responses to cardiometabolic injury.

Background: Autophagy is a homeostatic pathway supporting stress adaptation and is dysregulated in atherosclerosis. Its potential as a biomarker or therapeutic target in atherosclerotic vascular disease (ASVD) remains incompletely defined. We measured autophagic flux in peripheral blood mononuclear cells (PBMCs) from patients with peripheral arterial disease (PAD) or carotid stenosis (CS), compared with healthy controls, and explored clinical outcome associations. Methods: Ninety-four patients with PAD or CS and 19 healthy controls were studied. Autophagic flux was quantified from fresh blood using a validated ex vivo chloroquine inhibition ELISA measuring LC3BII accumulation. Major adverse cardiovascular events (MACE) and major adverse limb events (MALE) were ascertained over a median follow up of 828 days. Results: The ASVD cohort comprised claudication (n = 16), chronic limb threatening ischemia (CLTI; n = 49), and CS (n = 29). Autophagic flux was higher in ASVD than controls (mean 281.4 vs. 182.3 ng LC3BII/mg protein/h; p < 0.0001) and remained independently associated after multivariable adjustment. Within CLTI, concurrent infection was associated with lower flux (p = 0.001), approaching control levels (p = 0.327). In CLTI, higher flux quartiles were associated with lower MACE risk, most strongly for quartile 3 (hazard ratio 0.07 vs. quartile 1, 95% CI 0.01 to 0.50; p = 0.009). Conclusion: Autophagic flux is elevated in PBMCs from ASVD patients, independent of age and sex. Attenuated flux in CLTI with concurrent infection may indicate autophagic exhaustion in advanced disease. The association between higher flux and lower MACE in CLTI suggests prognostic utility, warranting evaluation in larger prospective studies.

Background: Calcific aortic valve disease (CAVD) is the most common valvular heart disease in older adults and currently has no approved pharmacological therapy to prevent or slow disease progression. Aortic valve replacement, either surgically or by transcatheter aortic valve implantation (TAVI), therefore remains the only effective treatment for severe CAVD. Monocytes play an important role in driving inflammation and disease progression in CAVD. As the disease progresses and the aortic valve becomes increasingly stenotic, circulating monocytes passing through the narrowed valve are repeatedly exposed to pathological haemodynamic forces, including markedly elevated shear stress, before their recruitment into the valve tissue. However, how exposure to these mechanical forces reprograms circulating monocytes influences their subsequent responses within the valve remain poorly understood. Methods and Results: In this study, we used an engineered model of aortic valve stenosis combined with phosphoproteomic profiling to investigate how prolonged exposure to pathological shear stress reprograms circulating monocytes and influences their subsequent phenotype and function following recruitment into the valve matrix. We further used TAVI as a unique clinical model to determine whether restoration of physiological haemodynamics in patients with severe aortic stenosis reverses the shear-induced monocyte phenotype observed in our experimental model. We found that prolonged exposure of monocytes to pathological shear stress induced extensive remodelling of the proteomic and phosphoproteomic landscape, with enrichment of pathways associated with cellular metabolism, cytoskeletal organisation, and inflammatory signalling. Kinase activity analysis further identified increased protein kinase C (PKC) activity accompanied by suppression of AKT signalling. Consistent with these molecular changes, pathological shear stress increased actin polymerisation, mitochondrial membrane potential, intracellular calcium signalling, and PKC activity. Using a bioengineered extracellular matrix model that recapitulates the glycosaminoglycan-rich microenvironment of the stenotic aortic valve we found that prior exposure to pathological shear stress altered monocyte-to-macrophage differentiation, reducing oxidised LDL uptake and foam-cell formation while promoting co-expression of CD80 and CD163, indicative of a mixed macrophage phenotype. Finally, using a myeloid-specific Piezo1 knockout mouse model, we identified Piezo1 as a key mechanosensor linking pathological shear stress to PKC activation, mitochondrial responses, and persistent changes in monocyte phenotype following migration into the extracellular matrix. Conclusions: These findings establish a mechanistic link between abnormal valve haemodynamics, metabolic and inflammatory reprogramming of circulating monocytes, extracellular matrix remodelling, and immune dysregulation during CAVD progression, highlighting monocyte mechanotransduction and downstream PKC/Piezo1 signalling as potential therapeutic targets.

Background: Platelets play a critical role in thrombo-inflammation following acute myocardial infarction (AMI). Objectives: Their impact on hepatic thrombopoietin (TPO) regulation after AMI remains poorly understood to date. Methods: Wildtype, thrombocytopenic or GPVI deficient mice underwent ischemia/reperfusion (I/R) injury. Platelet activation and turnover, and hepatic expression of different receptors involved in TPO regulation were analyzed. Results: Here, we identify a heart-liver axis that dynamically regulates platelet production post AMI. An elevated platelet turnover with increased reticulated as well as desialylated platelets early after AMI was detected. This was accompanied by the upregulation of specific receptors such as Asgr1/2 and IL-6R and increased phosphorylation of STAT3 in the liver and elevated numbers of megakaryocytes in spleen tissue. Consequently, increased TPO plasma levels at 24h post AMI were detected and platelet counts were rapidly restored after AMI. Platelet depletion induces a compensatory increase in hepatic TPO expression and plasma TPO levels, accompanied by dysregulated STAT3 signaling. This response contrasts with GPVI-deficient (Gp6-/-) mice, which exhibit no major alterations in TPO regulation, suggesting distinct mechanisms between acute thrombocytopenia and chronic platelet receptor deficiency in platelet homeostasis after AMI. These findings provide the first evidence of a direct heart-liver axis regulating platelet homeostasis after AMI, driven by inflammatory and hepatic signaling pathways. Conclusions: Our study highlights a previously unrecognized role of the heart-liver axis in controlling platelet turnover, with hepatic STAT3 signaling as a key mediator. These insights enhance our understanding of post AMI platelet homeostasis and may be critical for future therapeutic strategies.

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.

BackgroundNeutrophil extracellular traps (NETs) contribute to immunothrombosis and arterial thrombosis. Mechanisms underlying myocardial infarction after SARS-CoV-2 vaccination remain poorly understood. ObjectivesTo investigate histopathologic and immunothrombotic features of coronary thrombi in patients with ST-elevation myocardial infarction (STEMI) after SARS-CoV-2 vaccination. MethodsWe performed a retrospective matched cohort study including patients with STEMI undergoing primary percutaneous coronary intervention between January 2021 and March 2023. Coronary thrombi obtained by aspiration were analyzed by histopathology, immunohistochemistry, and confocal microscopy for NET detection. Vaccinated and unvaccinated patients were matched by age and sex. Associations between vaccination status and thrombus characteristics were assessed after adjustment for SARS-CoV-2 serologic status. ResultsAmong 44 matched patients (23 vaccinated and 21 unvaccinated), NETs were identified in 14 vaccinated patients (61%) and 5 unvaccinated patients (24%; P = .01). Vaccination was associated with increased odds of NET-positive thrombi after adjustment for SARS-CoV-2 serology (odds ratio, 5.1; 95% CI, 1.36-19.45; P = .02). No associations were observed between vaccination and polymorphonuclear cell density, fibrin deposits, plaque fragments, or anti-platelet factor 4 staining. Among patients vaccinated within 100 days before STEMI, NET-positive thrombi were associated with shorter intervals between vaccination and myocardial infarction (median [IQR], 25 [11-64] vs 57 [40-84] days; P = .02). ConclusionsSARS-CoV-2 vaccination was associated with increased NET presence in coronary thrombi from patients with STEMI, suggesting a potential NET-mediated immunothrombotic mechanism independent of classical vaccine-induced immune thrombotic thrombocytopenia.

Background: Endothelial dysfunction is a central driver of cardiovascular and inflammatory diseases, yet the post-translational mechanisms that preserve endothelial homeostasis remain incompletely understood. Protein neddylation, the covalent conjugation of a ubiquitin-like modifier, regulates diverse cellular processes, yet its physiological role in the vascular endothelium remains unknown. This study investigated whether protein neddylation is required to preserve endothelial identity and vascular homeostasis. Methods: We generated tamoxifen-inducible endothelial-specific Nae1 knockout mice to inhibit neddylation and combined bulk RNA sequencing, single-cell and single-nucleus transcriptomics, quantitative proteomics, biochemical analyses, and gain- and loss-of-function approaches to define the role of endothelial neddylation in vascular homeostasis and inflammatory injury. Results: Endothelial-specific Nae1 deletion caused rapid mortality associated with vascular leakage, platelet accumulation, inflammation, and multi-organ injury. Multi-omics analyses demonstrated profound loss of endothelial identity, characterized by suppression of core endothelial programs and activation of inflammatory, procoagulant, and pyroptotic pathways. Single-cell analyses revealed progressive endothelial dysfunction culminating in depletion of the endothelial population and remodeling of the vascular niche. Mechanistically, endothelial neddylation deficiency activated gasdermin D (GSDMD)- and gasdermin E (GSDME)-dependent pyroptosis, whereas dual inhibition of GSDMD and GSDME markedly attenuated inflammatory transcriptomic remodeling, vascular injury, hepatocyte death, immune cell infiltration, and platelet accumulation. Translational analyses demonstrated reduced endothelial neddylation in experimental endotoxemia and decreased expression of neddylation pathway components in human atherosclerosis and COVID-19 datasets. Conversely, restoration of endothelial neddylation partially reversed inflammatory endothelial transcriptomic reprogramming in vivo. Conclusions: NAE1-dependent protein neddylation is an essential regulator of endothelial identity and vascular integrity. Loss of endothelial neddylation promotes gasdermin-dependent pyroptosis and thrombo-inflammatory vascular injury, whereas restoration of the neddylation pathway mitigates inflammatory endothelial dysfunction. These findings identify endothelial neddylation as a fundamental mechanism maintaining vascular homeostasis and a potential therapeutic target for cardiovascular and inflammatory diseases.

Background: Murine models are widely used to investigate cardiovascular disease (CVD), yet many established models rely on invasive surgery or genetic manipulation to induce hyperlipidemia and therefore may not fully capture the gradual cardiovascular consequences of mutant hematopoiesis. Tie2-Cre/-FF1/- (Tie2FF1) mice express JAK2V617F mutation in both hematopoietic and endothelial cells and develop spontaneous CVD on a standard chow diet. Methods: We applied a comprehensive, non-invasive cardiovascular phenotyping protocol to 1-year-old Tie2FF1 mice and age-matched controls. Assessments included transthoracic echocardiography, coronary flow reserve by Doppler ultrasound, quantitative analysis of coronary arteriolar structure and perivascular fibrosis, endocardial histology and subendocardial collagen deposition, and histologic evaluation of pulmonary and hepatic congestion. Results: Tie2FF1 mice demonstrated left ventricular dilation, increased cardiac mass, and mildly reduced systolic function without obstructive epicardial coronary disease. Coronary microvascular remodeling was evident by arteriolar narrowing and increased perivascular fibrosis, while coronary flow reserve was markedly reduced in an exploratory cohort. Endocardial endothelial disruption and increased subendocardial fibrosis were also observed. In addition, Tie2FF1 mice showed increased lung and liver weights, pulmonary fluid accumulation, alveolar septal thickening, pulmonary vascular fibrosis, hepatic sinusoidal dilatation, and centrilobular hepatocyte swelling, providing objective evidence of clinically relevant heart failure. Significance: These findings establish Tie2FF1 mice as a spontaneous, non-invasive model with features of coronary microvascular and endocardial endothelial dysfunction and heart failure with mildly reduced left ventricular ejection fraction. The integrated approach described here provides a practical framework for comprehensive murine cardiovascular phenotyping beyond traditional atherosclerosis- and surgery-based models.

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.