Cardiovascular Research

One major aftermath of COVID-19 pandemic is cardiovascular consequences. SARS-CoV-2 binds to ACE2 and downregulates vasodilation. Dengue favors hypotension by weakening endothelial glycocalyx leading to plasma leakage. C1q levels, immune complexes (ICs), and proteomic profiles in serum samples from 52 COVID-19 and 19 pre-pandemic Dengue cases were studied. Unlike Dengue, COVID-19 serums showed elevated coagulation proteins promoting vaso-occlusion and peripheral artery diseases. The stress-induced chaperone and atherosclerosis marker, GRP78 (gene/ protein) was found upregulated upon SARS-CoV-2 spike expression in cardiac/ lung cell lines. Elevated GRP78 levels were also observed in serum samples from COVID-19-diagnosed individuals and subjects with myocardial infarction (MI) in post COVID-era. Surprisingly, spike antibodies (Abs) showed cross-binding to GRP78 and possibly contributed to the observed higher-level ICs in COVID-19 serums (cardiovascular embolism?). Co-localization studies showed that spike Abs (analogous to pro-atherosclerotic GRP78 auto-Abs) could directly bind to upregulated cellular GRP78 (type II hypersensitivity?). Both pathways could worsen vascular injury and atherosclerosis, leading to cardiac complications in COVID-19 cases with narrowed vessels.

Introduction: Calcific aortic stenosis (CAS) is a progressive valvular disease characterized by lipid accumulation, inflammation, and osteogenic remodeling. Emerging evidence implicates gut microbiota-derived metabolites in cardiovascular pathology, yet their contribution to valvular disease remains poorly defined. The aim of this study was to investigate gut microbiota and metabolite signatures in patients with CAS and explore causal relationships using Mendelian randomization (MR). Methods: In a prospective cohort of 54 patients with CAS and 41 age, sex, BMI-balanced non-CAS controls, we performed integrated microbiome and metabolomic profiling. Gut microbial composition was assessed by 16S rRNA sequencing, and circulating levels of tryptophan derivatives, short-chain fatty acids, bile acids, and TMA/TMAO-related metabolites were quantified. MR analyses were performed to assess causal contributions of key metabolic and inflammatory markers to CAS. Results: Baseline characteristics were comparable between groups. CAS patients exhibited a distinct tryptophan metabolic profile, characterized by higher concentrations of inflammatory kynurenine-pathway metabolites and lower indole-3-sulfate. With consistent effect sizes despite modest statistical significance after multiple testing correction. Pathway-level analyses supported preferential routing of tryptophan toward inflammatory host metabolism. In contrast, global microbiota diversity and overall community structure were preserved. However, CAS was associated with depletion of specific Firmicutes taxa, including Eubacterium coprostanoligenes, a key cholesterol-converting bacterium mediating intestinal cholesterol-to-coprostanol transformation. MR analyses suggested LDL cholesterol and lipoprotein(a) as upstream triggers of CAS, whereas ALPL and tryptophan/kynurenine metabolites appear downstream and might reflect systemic inflammation and local metabolic consumption. Sex-stratified analyses revealed enhanced kynurenine pathway activation in males, whereas females exhibited relatively higher TMAO and indole-related metabolites. Conclusion: CAS is characterized by a focused gut-host metabolic reprogramming defined by inflammatory tryptophan catabolism and loss of cholesterol-transforming microbial functions, rather than global dysbiosis. These findings identify a potential gut, valve metabolic axis contributing to valvular calcification, with potential sex-specific effects.

BackgroundCalcific aortic valve disease (CAVD) is the most common valvular heart disease in developed countries, yet no pharmacological therapy is available to slow or halt its progression. CAVD is driven by progressive calcification of aortic valve leaflets, in which myeloid cells play a central role. While macrophages have been implicated in CAVD pathogenesis, the contribution of their precursors, monocytes, remains poorly understood. We hypothesized that circulating monocytes acquire a pro-calcific and pro-inflammatory phenotype contributing to valve remodelling and CAVD progression. MethodsWe profiled circulating CD14+ monocytes from healthy volunteers (Vol), patients with CAVD, and without CAVD (NCAVD). Peripheral blood mononuclear cells (PBMCs) were isolated, and monocyte subpopulations were phenotyped by flow cytometry. Transcriptome profiling by RNA sequencing identified disease-associated gene signatures, which were validated by RT-qPCR. The CD14+ monocyte secretome was analysed using multiplex assays. Functional ability of CAVD-derived CD14+ monocytes to induce myofibroblastic transdifferentiation (MT) and osteoblastic differentiation (OD) of human valvular interstitial cells (VICS) was evaluated by immunocytochemistry and quantitative o-cresolphthalein complexone assays. ResultsIn PBMCs, CAVD monocytes displayed a subpopulation shift, with an increased proportion of CD14CD16- classical monocytes and a reduced CD14CD16 non-classical monocyte levels. In CD14+ monocytes, transcriptomic analysis revealed upregulation of inflammation-related (PDK4) and calcification-related (ATP2B1) genes, alongside downregulation of immunomodulatory genes (DDR1, IKBKE). Secretome analysis showed reduced production of immunomodulatory and anti-osteoblastogenic cytokines (IL-4, CCL3) while promoting gene expression of factors promoting MT and OD in VICS. These alterations were associated with a marked monocyte-induced increase in SMA and OPN expression in VICS and a two-fold increase in calcification. ConclusionWe demonstrate for the first time that circulating monocytes from patients with CAVD exhibit enhanced pro-inflammatory and pro-calcific properties that may contribute to CAVD progression. Additionally, we identify dysregulated gene sets within these monocytes that represent potential novel therapeutic targets for CAVD.

BackgroundPeripheral artery disease is a major manifestation of atherosclerotic cardiovascular disease (ASCVD) that affects both men and women. In women, menopause increases the ASCVD risk. However, preclinical ASCVD research has historically been conducted predominantly in males, with relatively few studies focused on females and even fewer incorporating menopause models that more closely reflect human ASCVD pathobiology. Herein, we tested whether the chemical 4-vinylcyclohexene diepoxide (4-VCD)-induced ovarian failure or ovariectomy (OVX) would drive atherosclerotic development and worsen ischemic limb pathophysiology. MethodsFemale C57BL/6J mice were injected with adeno-associated virus-mediated encoding a gain-of-function mutant PCSK9 and fed an atherogenic diet for 23 weeks. Based on the baseline body weight, mice were randomly assigned to normally cycling controls (CON), 4-VCD, or OVX groups. Three weeks after the conformation of ovarian failure (4-VCD) or surgical ovarian removal (OVX), hindlimb ischemia (HLI) was induced via femoral artery ligation, and limb perfusion recovery and limb muscle performance were assessed. ResultsBoth 4-VCD treatment and OVX reduced uterus mass, without impacting body weight or composition, or circulating cholesterol levels compared to CON mice. Despite the similar metabolic and cholesterol profiles, atherosclerotic lesion areas were 1.5-1.7-fold greater in 4-VCD and OVX mice than CON mice. Perfusion recovery following HLI and plantar flexor muscle function in the ischemic limb were similar across groups, though muscle oxygenation was reduced in 4-VCD and OVX groups. ConclusionsOvarian failure and removal exacerbated atherosclerotic development but had minimal impacts on perfusion recovery and limb function following HLI. These findings confirm the inclusion of menopausal models, whether through ovarian failure or OVX, should be carefully considered to improve translatability of preclinical ASCVD studies, especially for womens health. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSWe demonstrate that both gradual ovarian failure (4-VCD) and surgical ovariectomy exacerbate atherosclerotic plaque development in a clinically relevant AAV-PCSK9 model, despite similar circulating lipid levels. In contrast, loss of ovarian function did not impair limb perfusion recovery or muscle functional outcomes following hindlimb ischemia, revealing a dissociation between atherosclerotic burden and limb functional recovery in experimental peripheral artery disease (PAD). What are the Clinical Implications?These findings provide new insight into why menopause increases atherosclerotic cardiovascular disease (ASCVD) risk while not necessarily demonstrating proportional impairments in limb recovery following ischemia. The data suggest that menopause-associated factors accelerate large-vessel atherosclerosis independent of circulating lipids, highlighting the need for targeted therapies beyond lipid lowering in postmenopausal women. Moreover, the dissociation between plaque burden and ischemic limb function underscores the importance of assessing functional outcomes in PAD independently of vascular imaging. Finally, these findings suggest that the incorporation of menopause-relevant models in preclinical research should be considered within the context of the specific biological endpoints and translational goals being evaluated.

Pulmonary arterial hypertension is a progressive, fatal disease driven by pathologic vascular remodeling including arterial medial hypertrophy, occlusive neointimal lesion formation, and venous muscularization. Current vasodilatory therapies improve hemodynamics but do not reverse established remodeling. Imatinib mesylate, a tyrosine kinase inhibitor targeting the PDGF-PDGFR signaling axis, has been proposed as an anti-remodeling therapy for pulmonary arterial hypertension and has demonstrated hemodynamic benefit in both preclinical models and clinical trials. However, prior preclinical models lack the neointimal lesions characteristic of human disease, effects on venous remodeling have not been examined, and direct histologic assessment in human trials is precluded by the invasiveness of serial lung biopsy. Here, leveraging the house dust mite mouse model of pulmonary hypertension, which recapitulates medial thickening, neointimal lesion formation, and venous muscularization, we rigorously evaluate the anti-remodeling and hemodynamic effects of imatinib during two defined remodeling stages: neointimal lesion growth and neointimal lesion maintenance. Imatinib treatment significantly reduced right ventricular systolic pressure at both stages. Despite this hemodynamic improvement, quantitative vessel-level analysis of over 1,700 arteries and 1,200 veins revealed no significant effect of imatinib on arterial medial thickness, neointimal lesion growth, neointimal lesion maintenance, or venous muscularization across any vessel size class. These findings dissociate imatinibs hemodynamic benefit from structural vascular remodeling and suggest that imatinib functions primarily as a pulmonary vasodilator rather than an anti-remodeling agent.

AimsHeart failure with preserved ejection fraction (HFpEF) afflicts millions annually and current treatments provide symptomatic relief. Here, we investigate the therapeutic potential of synthetic human Relaxin-2 (RLX) at reversing diastolic dysfunction (DD) and reducing arrhythmia vulnerability. Methods and ResultsMale ZSF1 rats were placed on a normal diet (ND, N=10 controls) or a high-fat diet (HFD, N=11), resulting in the development of DD in 11-weeks, based on serial echocardiograms (enlarged left atrium (LA), wall thickness, doppler flow: E/e). Once HFpEF was confirmed, control and HFpEF rats were randomly treated with Relaxin (400{micro}g/kg/day RLX, N=6) or the vehicle (N=5) for 2-weeks using implanted minipumps. Echocardiograms were repeated at weeks 1 and 2, then hearts were isolated, optically mapped, subjected to programmed electrical stimulation (PES) and tissues dissected for immuno-fluorescence (IF), and qPCR analysis. Circulating levels of glucose, RLX and NT-pro-ANP were measured, pre- and post-treatment. Echocardiograms indicated that RLX reversed DD by reducing LA dimensions and E/e. Optical mapping revealed that 1/3 of HFpEF hearts exhibited sustained atrial and ventricular arrhythmia which were blocked by RLX as it tended to increase conduction velocity (CV). Based on IF, RLX increased Nav1.5, Connexin-43, {beta}-catenin and Wnt1 expression. There were no significant changes in fibrosis in this HFpEF model. NT-pro-ANP was elevated in HFpEF and reduced towards control values by RLX. qPCR analysis showed that RLX decreased DKK1 and MMP1A and increased SCN5A expression compared to Vehicle treatment (N=6 and 5, respectively). ConclusionsThe ZSF1 model showed clear signs of HFpEF, including DD, enlargement of the LA, enhanced hemodynamic stress, increased vulnerability to sustained AF and VF, and elevated glucose and blood pressure. RLX treatment largely reversed DD, hemodynamic stress, and suppressed sustained arrhythmias. RLX elicited cardiac genomic changes, most likely through Wnt/canonical signaling, demonstrating RLXs potential as a therapy for HFpEF.

Aortic dissection is life-threatening due to continued loss of medial integrity that may culminate in secondary rupture within hours to days. While pre-existing defects or hemodynamic loads compound structural deterioration of the aorta, pathological progression from symptomatic dissection channel to lethal transmural tear is poorly understood. We examined the structure of referent and acutely dissected ascending aortas by microscopy. Elastic, collagen, and cellular components of non-dissected media were intricately interconnected. Medial damage in dissection lesions was traced from ingress to central to peripheral areas. Entry tears broke cleanly through successive laminae leading to cavernous false lumens in which medial structure was destroyed. Nearby laminae with widening between flanking elastic lamellae (termed minor delaminations) were filled with blood and showed severe medial damage. Farther laminae without delamination but containing red blood cells (termed blood extravasation) displayed moderate medial damage. More distant, non-delaminated laminae with accumulation of albumin but not red blood cells (termed plasma extravasation) exhibited mild medial damage. Varying medial hemorrhage with scattered sloughing of laminae was observed along the entire false lumen. We conclude that hydraulic fracturing of residual dissected media by pressurized blood via communications from the false lumen contributes to further structural weakening of the aortic wall.

IntroductionAortic stenosis (AS) is characterised by progressive aortic valve (AV) leaflet fibrosis and calcification, yet no medical therapies exist to slow disease progression. AV interstitial cells (VICs) that differentiate into myofibroblasts are central drivers of fibrosis. The Ca2+-activated K+ channel KCa3.1 promotes pro-fibrotic signalling in several fibrotic diseases, however its role in AS remains unknown. MethodsKCa3.1 protein expression was examined in paraffin embedded tissue by Immunohistochemistry from control and AS valve tissue. VICs were isolated, cultured and phenotypically characterised as myofibroblasts from AV tissue obtained from patients with severe tricuspid AS undergoing surgical AV replacement (n=19). KCa3.1 mRNA and protein expression were assessed by qRT-PCR and immunohistochemistry, and functional channel activity confirmed using patch-clamp electrophysiology. The effects of transforming growth factor-{beta}1 (TGF{beta}1) stimulation and pharmacological inhibition with the selective KCa3.1 blocker senicapoc were examined. ResultsImmunoreactive KCa3.1 channels and smooth muscle actin were detected in both control and AS aortic valve tissue, localised to elongated, nucleated interstitial cells, with significantly higher expression observed in AS tissue compared to control. Isolated VICs exhibited an activated myofibroblast phenotype, expressing THY-1, vimentin, collagen and -smooth muscle actin (SMA) (n=9). Myofibroblasts expressed KCa3.1 mRNA and protein and demonstrated functional plasma membrane channels. TGF{beta}1 stimulation increased KCa3.1, SMA and collagen type I mRNA expression, while KCa3.1 blockade with senicapoc (100 nM) significantly attenuated TGF{beta}1-induced SMA expression, stress fibre formation and collagen gel contraction. Senicapoc had no effect on myofibroblast proliferation or migration. ConclusionsWe show for the first time that functional KCa3.1 channels are expressed in human AS tissue and AV myofibroblasts, where they regulate myofibroblast contraction, -SMA expression, and differentiation, promoting pro-fibrotic activity. These responses are attenuated by the selective KCa3.1 inhibitor senicapoc. Given its established safety in phase 3 clinical trials, KCa3.1 inhibition represents a promising and readily translatable anti-fibrotic therapeutic strategy for AS.

Background: A substantial proportion of coronary events originate from angiographically moderate lesions, indicating that stenosis severity alone does not reflect lesion biomechanical risk. Objectives: To test whether adding lesion-adjacent pericoronary adipose tissue (PCAT) and CTCA-derived anatomy-flow descriptors to quantitative plaque assessment improves identification of future culprit lesions, with a prespecified focus on moderate stenosis. Methods: We performed a within-patient, lesion-level case-control analysis in the GeoCAD cohort, including patients undergoing coronary revascularisation during follow-up. Culprit lesions were identified from longitudinal CTCA. Stenosis severity, quantitative plaque composition, and PCAT volume were quantified (MEDIS), and vessel centreline geometry and lesion haemodynamics derived using computational modelling. Incremental prognostic value was assessed using Cox models with drop-one and stepwise workflow analyses, including a prespecified subgroup analysis of moderate stenosis lesions (25 - 49% diameter stenosis). Results: Among 46 patients (212 lesions; 55 culprit), percent area stenosis (%AS) dominated culprit lesion discrimination (HR: 2.01; 95% CI: 1.54 - 2.62; p < 0.001). In 82 moderate-stenosis lesions (30 culprit), %AS provided minimal discrimination ({Delta}C-index: 0.01; p=0.895). Culprit lesions were characterised by greater PCAT volume (HR: 1.75; 95% CI: 1.29 - 2.37; p < 0.001), higher helical flow intensity (HR: 1.35; 95% CI: 1.16 - 1.57; p < 0.001), and lower torsion (HR: 0.50; 95% CI: 0.29 - 0.84; p=0.009). Adding anatomy-flow descriptors improved risk stratification for moderate lesions beyond CTCA stenosis and plaque/PCAT features (p=0.007). Conclusions: In moderate stenosis, lesion-adjacent PCAT and anatomy-flow descriptors provided incremental prognostic information beyond luminal narrowing and plaque composition, supporting integrated CTCA phenotyping to identify high-risk nonobstructive coronary lesions.

Background: Polygenic risk scores (PRS) for coronary artery disease (CAD) are associated with cardiovascular events, but the relationship between inherited risk and routinely reported coronary computed tomography angiography (CTA) findings has not been studied. Objectives: To evaluate associations between a genome-wide PRS for angiographic coronary disease burden and coronary CTA-derived measures of atherosclerotic severity in a real-world clinical cohort. Methods: We studied Penn Medicine BioBank participants with available genotypes and clinically obtained coronary CTA reports. A previously published PRS for angiographic CAD burden was calculated using pgsc_calc. CAD-RADS scores and coronary artery calcium (CAC) values were extracted from radiology reports using the large language model Llama 3.1 8B. Associations between PRS and CAD-RADS severity were evaluated using Bayesian cumulative ordinal logit regression, while associations with log-transformed CAC burden were assessed using Bayesian linear regression. Results: Among 630 participants, median age was 59 years (IQR 49 - 68), 53% were female, 62% were genetically similar to a European reference population, and 34% to an African reference population. LLM-extracted CAD-RADS and CAC values demonstrated near-perfect agreement with manual abstraction. Higher PRS was associated with greater coronary atherosclerotic burden on CTA. Each 1-standard deviation (SD) increase in PRS was associated with a 20% higher odds of belonging to a more severe CAD-RADS category (cumulative OR 1.20, 95% credible interval 1.06-1.44). Higher PRS was also associated with greater CAC burden ({beta} 0.38, 95% credible interval 0.15 - 0.61). Conclusions: Polygenic risk for angiographic coronary disease burden is reflected in clinically reported coronary CTA severity measures, including CAD-RADS and CAC. These findings demonstrate that inherited susceptibility to CAD manifests as greater anatomic atherosclerotic burden at the time of clinical presentation and support further investigation of genetic risk integration into imaging-based cardiovascular risk assessment.

BackgroundThis study aimed to investigate whether combined PD-1/CTLA-4 immune checkpoint inhibition predisposes the heart to a hyperinflammatory state, thereby exacerbating cardiac injury following acute myocardial infarction (MI), a critical unresolved question in cardio-oncology. MethodsMyocardial infarction was induced in Pd1-/-Ctla4+/- mice, a genetic model mimicking combined checkpoint inhibition. Key mechanistic insights were gained through in vivodepletion of CD8+ T cells (using anti-CD8a antibody) and pharmacological inhibition of the JAK-STAT1 pathway (using Tofacitinib). Cardiac function, structural injury, and immune responses were comprehensively assessed via echocardiography, flow cytometry, immunofluorescence, and molecular analyses. ResultsCompared to wild-type controls, Pd1-/-Ctla4+/- mice exhibited significantly increased post-MI mortality, worse cardiac function, and larger infarct size. Mechanistically, the aggravated injury was driven by an amplified infiltration of activated, IFN-{gamma}-producing CD8+ T cells, which activated the JAK-STAT1 pathway in macrophages, polarizing them towards a pro-inflammatory state. Depleting CD8+ T cells or inhibiting the JAK-STAT1 pathway effectively attenuated macrophage-driven inflammation and improved all aspects of post-MI injury. ConclusionsCombined PD-1/CTLA-4 blockade exacerbates post-infarction cardiac injury by promoting CD8+ T cell-mediated activation of macrophages via the JAK-STAT1 axis. This work elucidates MI as a context-dependent immune-related adverse event in ICI therapy and identifies CD8+ T cells and the JAK-STAT1 pathway as promising therapeutic targets for cardioprotection in these patients. RESEARCH PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study identifies acute myocardial infarction (MI) as a potential, context-dependent immune-related adverse event in the setting of combined PD-1/CTLA-4 checkpoint inhibition, shifting the paradigm beyond the classic focus on myocarditis. C_LIO_LIIt elucidates a novel pathogenic axis where combined checkpoint deficiency exacerbates post-MI injury specifically through CD8+ T cell-derived IFN-{gamma}, which activates macrophages via the JAK-STAT1 pathway. C_LI What Question Should Be Addressed Next?O_LIFuture studies should employ anti-PD-1/CTLA-4 monoclonal antibodies in wild-type or humanized mouse models to validate findings and better recapitulate the pharmacokinetics of clinical ICI therapy, strengthening translational relevance. C_LIO_LIThe long-term consequences of this primed inflammatory state on chronic cardiac remodeling, heart failure development, and the potential interplay with atherosclerosis warrant further investigation. C_LI

BackgroundHypertrophic cardiomyopathy (HCM) is a heritable trait with marked variability in expression and outcomes. Our aims were to discover new genetic loci associated with HCM and to test the effect of a new polygenic risk score (PRS) on incidence, phenotype and outcomes, stratified by sarcomere genotype status. MethodsA discovery genome-wide association study (GWAS) was performed on 2,284 HCM cases and 4,525 controls. Two fixed-effects meta-analyses combined our discovery GWAS with single-trait and multi-trait results from a published study. Discovered loci underwent comprehensive bioinformatic analysis including functional and druggability annotations. A PRS using loci from the two meta-analyses was evaluated for association with: HCM diagnosis in 411,213 individuals from UK Biobank (UKBB); imaging phenotypes in individuals without HCM; a composite endpoint (including all-cause mortality and transplantation) and sudden cardiac death (SCD) in 1,756 HCM cases. PRS analyses were stratified by sarcomere genotype status. ResultsThree loci were found in the discovery GWAS (BAG3, FHOD3 and novel locus PPP1R3A). In the meta-analyses, 70 unique loci were identified, four novel (MYPN, YWHAE, NOS1AP and OBSCN). Bioinformatic analyses identified NOS1AP as a candidate HCM gene. A new PRS was significantly associated with HCM diagnosis (hazard ratio [HR] 3.19, 95% CI:2.46-4.14, for top 5% vs lower 95%; HR 1.88, 95% CI:1.72-2.06, per SD increase). Significant associations were found between PRS and greater left ventricular (LV) wall thickness and higher LV ejection fraction in UKBB participants without HCM. Sarcomere-negative HCM cases in the top 20% of the PRS distribution had an increased risk of SCD (HR 2.72, 95% CI:1.03-7.17). ConclusionsWe report novel HCM loci. A new PRS predicted the risk of HCM development and associated imaging characteristics in the UKBB and outcomes in an HCM cohort.

Diabetes is associated with endothelial dysfunction, impaired wound healing, and increased thrombotic risk, yet the impact of diabetes on endothelial secretory organelles remains poorly understood. Weibel-Palade bodies (WPBs) are specialised endothelial granules that store and release von Willebrand factor (VWF) and other vasoactive cargo essential for haemostasis, inflammation, and vascular repair. Here, we investigated how diabetic environments influence WPB biogenesis and VWF structure under physiologically relevant flow conditions. Acute exposure of endothelial cells to constant or fluctuating high glucose concentrations, designed to model diabetic glycaemic conditions, did not alter WPB number or morphology under either static or high laminar shear stress conditions. In contrast, primary endothelial cells derived from a diabetic donor exhibited reduced Akt and eNOS signalling, significantly fewer WPBs, reduced intracellular VWF content, and shorter stimulus-evoked VWF strings compared with non-diabetic endothelial cells. Although total cellular VWF levels were reduced, high molecular weight (HMW) VWF content within endothelial lysates was not significantly altered. Plasma from diabetic patients demonstrated elevated circulating VWF levels together with marked inter-patient heterogeneity in VWF multimer composition. These findings suggest that chronic diabetes-associated endothelial dysfunction, rather than hyperglycaemia alone, alters WPB biology and VWF handling. We propose that dysregulated basal endothelial secretion may deplete endothelial VWF stores, limiting appropriate stimulus-coupled WPB release during vascular injury and contributing to defective vascular repair in diabetes.

Objective: To compare RNA-sequencing-derived transcriptomic profiles of thoracic aortic aneurysm tissue from individuals with bicuspid versus trileaflet aortic valves. Methods: Human ascending aortic tissue was collected from patients undergoing cardiac surgery at a single institution between January 2021 and December 2022 with bicuspid aortic valves (BAV) and trileaflet aortic valves (TAV) with (-A) and without (-N) thoracic aortic aneurysm. TAV-N tissue was collected from heart transplant donors. The decision to perform ascending aortic replacement was at surgeon discretion following ACC/AHA guidelines. Bulk RNA was extracted from the aortic wall, and Illumina RNA Sequencing performed. Differential gene expression analysis, enrichment analyses, network analysis, and deconvolution single cell-mapping were performed in R. Cell-type specificity of differentially expressed genes was determined using an established Aorta single cell RNA sequencing matrix. Results: Tissue samples from 60 patients were included: 4 TAV-N, 16 BAV-N, 28 BAV-A, and 12 TAV-A. Average absolute aortic diameter was 5.1 +/- 0.38 cm for BAV-A and 5.3 +/- 0.44 cm for TAV-A, as measured on pre-operative CT. Gene ontology analyses of differentially expressed genes revealed enrichment of genes associated with extracellular matrix (ECM) organization, cellular receptor interactions and vascular smooth muscle cell (VSMC) function in BAV-A and BAV-N. In contrast, analysis of TAV-A versus TAV-N showed enrichment in genes associated with immune and inflammatory processes. Cell-type specificity analysis revealed a downregulation of genes associated with ECM components, cell signaling, and ECM remodeling in mesenchymal cells, VSMCs, and matrix fibroblasts specifically in BAV-A versus BAV-N. Conclusions: The transcriptome changes observed in aneurysmal aortas of BAV and TAV patients are distinct, suggesting mechanistic differences contributing to aneurysm development and progression. The observed differences in gene expression between the non-aneurysmal aortas may signify a predisposition to aneurysm development unique to BAV aortopathy.

Bicuspid aortic valve (BAV) is one of the most common congenital heart defects but its genetic basis remains incompletely defined. Extracellular matrix components play key roles in outflow tract (OFT) and valve development, but their contribution to BAV is not fully established. Following the analysis of a cohort of BAV patients, we identified a family harbouring a rare human ELASTIN (ELN) variant (p.Gln691X). To assess its pathogenicity, we generated a zebrafish elna/b double knockout (KO) using an RNAless CRISPR Cas9 strategy to avoid genetic compensation. This mutant exhibited cardiovascular defects including OFT anomalies, reduced stroke volume and dysmorphic aortic valves, highlighting Elastins critical role in cardiac development. We then used this model to test the ELN variant identified in the BAV family. We found that wild-type ELN mRNA was able to restore normal cardiac function and morphology, whereas the variant ELN mRNA failed to do so. This study establishes a robust in vivo model to assess ELN variant pathogenicity and provides evidence linking ELASTIN to BAV, opening new avenues for uncovering the genetic mechanisms underlying BAV.

Cells deploy adaptive programs to maintain homeostasis under stress, yet mechanisms counteracting damage triggered by transmembrane signaling remain poorly defined. Using a hyperaldosteronism model, we examined how autophagy regulates aldosterone-mediated mineralocorticoid receptor (MR) activation. In human umbilical vein endothelial cells (HUVECs), aldosterone induced autophagy, as evidenced by elevated Beclin-1, an increased LC3-II/LC3-I ratio, and reduced SQSTM1/p62. Aldosterone also promoted MR translocation from the cytosol to the nucleus. Co-immunoprecipitation and immunofluorescence revealed direct interaction and colocalization between MR and Beclin-1, as well as enhanced MR-lysosome association. Domain mapping showed that the Beclin-1 middle domain (161-241 AA) binds the MR C-terminal region (601-984 AA). Bioinformatic prediction and ChIP-qPCR confirmed that MR occupies the promoters of IL-1{beta}, IL-6, and TNF- upon aldosterone stimulation. Beclin-1 overexpression attenuated MR nuclear translocation, promoter binding, and inflammatory cytokine expression, whereas Beclin-1 knockdown reversed these effects. In vivo, aldosterone-infused Beclin-1 transgenic (Becn1-tg) mice exhibited lower blood pressure, reduced aortic medial thickening, and attenuated cardiac hypertrophy relative to wild-type controls, with no difference in body weight. Our findings identify Beclin-1 as a critical negative regulator of aldosterone signaling through an autophagy-dependent negative feedback loop. By interacting with MR and directing it toward lysosomal sequestration, Beclin-1 limits MR nuclear translocation and transcriptional activity, thereby mitigating aldosterone-induced vascular inflammation and cardiovascular injury. HighlightsAldosterone activates autophagy and promotes MR-Beclin-1 interaction in HUVECs Beclin-1 binds the C-terminal MR domain and directs MR to lysosomal degradation Beclin-1 overexpression suppresses MR nuclear translocation and cytokine gene activation Beclin-1 transgenic mice are protected from aldosterone-induced cardiovascular injury

AimMaternal iron deficiency (ID) during pregnancy induces cardiovascular adaptations, including reduced blood pressure and improved cardiac efficiency in hypertensive pregnancy. Iron is essential for mitochondrial function, particularly oxidative phosphorylation, where it serves as a cofactor within electron transfer complexes. Given the high metabolic demands of the maternal heart and irons central role in mitochondrial metabolism, we examined how maternal ID affects cardiac mitochondrial ultrastructure, respiration, dynamics, and redox status in pregnant spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Methods and ResultsFemale SHR and WKY rats were fed iron-replete or iron-restricted diets before and throughout gestation. On gestational day 21, cardiac mitochondrial ultrastructure was assessed by transmission electron microscopy (TEM), respiration by high-resolution respirometry, and the expression of proteins involved in fusion, fission, autophagy, and apoptosis markers by immunoblotting. Antioxidant gene expression was quantified by RT-qPCR. Data were analyzed by two-way ANOVA with Holm-Sidaks post hoc test. Maternal iron restriction reduced hemoglobin levels in both strains. TEM revealed enlarged, morphologically heterogeneous mitochondria with reduced and disrupted cristae architecture in ID dams of both strains. Iron restriction reduced succinate-supported respiration and tended to reduce NADH-supported respiration, in both strains. SHR dams exhibited reduced fusion signalling, reflected by a lower L-OPA1:S-OPA1 ratio. MFN1 expression was reduced by ID in both strains, whereas MFN2 expression was lower in SHR and further reduced by ID. In contrast, DRP1 phosphorylation increased selectively in ID-WKY dams. Iron restriction increased LC3-II:I ratio and BNIP3 in SHR, and increased PINK1 in both strains, while Parkin and p62 were unchanged. Antioxidant gene expression increased in ID-SHR but decreased in ID-WKY dams. Despite these alterations, markers of oxidative damage and apoptosis were unchanged by iron restriction. ConclusionMaternal ID induces marked remodeling of myocardial mitochondrial ultrastructure and selectively constrains iron-dependent respiration in hypertensive pregnancy without overt oxidative damage or apoptosis. These mitochondrial alterations occur alongside previously observed reductions in blood pressure and improved cardiac efficiency, suggesting favorable hemodynamic adaptations may coexist with underlying bioenergetic constraints in the maternal heart. Translational PerspectiveMaternal iron deficiency anemia (IDA) may alter the course of hypertensive pregnancy in ways not evident from hemodynamic indices alone. Here, IDA was associated with abnormal myocardial mitochondrial ultrastructure, selective reductions in respiratory capacity and stress response pathways, despite previously observed improvements in blood pressure and cardiac efficiency. These findings suggest that favourable hemodynamic changes may reflect reduced metabolic demand rather than enhanced bioenergetic capacity. If confirmed in human pregnancy, management of ID in women with underlying hypertension may need closer attention to cardiac metabolic health, as cardiovascular adaptions could coexist with myocardial stress and may vary with anemia severity and duration.

BackgroundPreeclampsia (PE) is a hypertensive disorder of pregnancy characterized by systemic inflammation, oxidative stress, and endothelial dysfunction. Although maternal vascular dysfunction is well established in PE, the mechanisms underlying fetal vascular injury remain poorly understood. We investigated whether inflammatory signaling activates NADPH oxidase 5 (NOX5) and contributes to oxidative stress and dysfunction in human umbilical arteries from pregnancies complicated by PE. MethodsUmbilical arteries and serum samples were obtained from normotensive pregnant women (NP) and women with PE. Vascular reactivity, nitric oxide (NO) bioavailability, reactive oxygen species (ROS) generation, cytokine levels, and NOX isoform expression were evaluated in human umbilical arteries and EA.hy926 endothelial cells. Pharmacological inhibition of NOX5, TNF- neutralization, Ca{superscript 2} channel blockade, and siRNA-mediated NOX5 silencing were used to investigate mechanisms. ResultsPE umbilical arteries exhibited increased vasoconstrictor responses, oxidative stress, and NOX5 expression, accompanied by impairment of NO bioavailability. NOX5 inhibition reversed vascular hyperreactivity in PE vessels. Exposure of normotensive umbilical arteries to PE serum reproduced the PE vascular phenotype, characterized by enhanced ROS generation, reduced NO levels, and hypercontractility. In endothelial cells, PE serum induced TNF--dependent Ca{superscript 2} influx, oxidative stress, and reduced NO production. Both pharmacological and genetic inhibition of NOX5 prevented these alterations. ConclusionsPE promotes fetal vascular dysfunction through activation of a TNF-/Ca2+/NOX5 signaling pathway that amplifies oxidative stress and impairs NO bioavailability. These findings identify NOX5 as a previously unrecognized mediator of umbilical artery dysfunction in PE and suggest the TNF-/Ca2+/NOX5 axis as a potential therapeutic target in hypertensive pregnancies.

The epidermal growth factor receptor (EGFR) family network comprises 4 receptors (EGFR, ERBB2, ERBB3, ERBB4) and numerous ligands, and is dysregulated in many cancers. Since anti-cancer drugs that target these receptors are cardiotoxic for some patients, it is important to understand the network in cardiac cells. Data from the Human Protein Atlas established that EGFR family members and their ligands are differentially expressed in cardiac cell types. Ligand expression was altered in human failing hearts and may contribute to disease. These ligands stimulated extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt in rat cardiomyocytes but to different degrees. Afatinib (at a concentration to inhibit all EGF family receptors) was used to assess the role of the network in a mouse model of cardiac hypertrophy induced by angiotensin II (AngII). Echocardiography and segmental strain analysis demonstrated that afatinib reduced AngII-induced cardiac hypertrophy and caused cardiac dysfunction. This was associated with loss of cardiomyocyte hypertrophy, enhanced cardiac fibrosis, and reduced expression of Nrg1. NRG1 binds to ERBB4 in cardiomyocytes which homodimerizes or heterodimerises with ERBB2. The role of ERBB2 in the cardiomyocyte response to NRG1 compared with EGF was dissected using tucatinib (a selective ERBB2 inhibitor) and mRNA expression profiling. Most, but not necessarily all, of the response to NRG1 required ERBB2 signalling; most, but not all, of the response to EGF did not. Thus, the EGFR family network plays an important role in the heart. Understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with EGFR family anti-cancer drugs. Clinical perspectivesO_LIAnti-cancer drugs that target the epidermal growth factor receptor (EGFR) family are cardiotoxic for some patients; it is therefore important to understand the network in cardiac cells. C_LIO_LIThe EGFR family and their ligands are differentially expressed in cardiac cells with changes in ligand expression in heart failure; inhibition of all receptors in a mouse model of hypertrophy reduces cardiac hypertrophy and causes cardiac dysfunction with attenuation of cardiomyocyte hypertrophy and enhanced cardiac fibrosis and loss of neuregulin 1 (NRG1); in rat cardiomyocytes, NRG1 signalling to gene expression is largely mediated via ERBB2. C_LIO_LIThe EGFR family network plays an important role in the heart; understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with anti-cancer drugs targeted against it. C_LI

BackgroundHypertension and preeclampsia are clinically distinct, yet biologically related conditions characterized by vascular dysfunction and elevated cardiovascular risk. Although genome-wide association studies (GWAS) have identified loci associated with blood pressure traits and preeclampsia, the functional mechanisms linking shared variants to gene regulation and clinical phenotypes remain unclear. MethodsWe integrated GWAS summary statistics for hypertension, systolic blood pressure (SBP), diastolic blood pressure (DBP), and preeclampsia to identify shared variants (p [&le;] 1x10-). Cis-expression quantitative trait loci (eQTL) analyses were performed in whole blood using RNA-seq data from 180 African American women. Significant associations (FDR [&le;] 0.05) were evaluated for replication across vascular, metabolic, and endocrine tissues in the Genotype-Tissue Expression (GTEx) project. Associations between gene expression and blood pressure traits were also assessed. ResultsWe identified 4,792 shared GWAS variants, of which 4,663 were tested in eQTL analyses, yielding 1,837 significant variant-gene associations across 78 genes. Replication in GTEx confirmed 645 associations involving 24 genes, many showing cross-tissue regulatory effects. Three genes (C4B, HLA-C, and HLA-DQB1) demonstrated convergent evidence across GWAS, gene regulation, and expression-trait analyses. C4B expression was positively associated with hypertension and SBP, while HLA-C showed consistent negative associations with hypertension, SBP, and DBP. HLA-DQB1 expression was specifically associated with DBP, suggesting trait-specific effects. ConclusionsThese findings highlight immune-related pathways as key mediators linking hypertension and preeclampsia. Integrating genetic, transcriptomic, and phenotypic data provides a framework for identifying functionally relevant loci and advancing mechanistic insights into cardiometabolic and pregnancy-related disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/26352450v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1332b09org.highwire.dtl.DTLVardef@4e7c49org.highwire.dtl.DTLVardef@c1b980org.highwire.dtl.DTLVardef@799767_HPS_FORMAT_FIGEXP M_FIG C_FIG Shared genetic variants across hypertension, blood pressure traits, and preeclampsia converge on immune regulatory genes linking gene regulation to clinical phenotypes. GWAS summary statistics for hypertension, SBP, DBP, and preeclampsia were intersected to identify 4,792 shared variants, of which 4,663 were tested in cis-eQTL analyses in whole blood from 180 African American women (left). Shared variants regulate immune-related genes through cis-eQTL effects, yielding 1,837 associations involving 78 genes (FDR [&le;] 0.05). Three convergent genes emerged: C4B (upregulated), HLA-C (downregulated), and HLA-DQB1 (upregulated), with 645 associations involving 24 genes replicated across eight tissues in GTEx (center). Expression-trait analyses confirmed that C4B expression was positively associated with hypertension and SBP, HLA-C expression was negatively associated with hypertension, SBP, and DBP, and HLA-DQB1 expression was specifically associated with DBP. These genes implicate complement activation, antigen presentation, and adaptive immunity as shared mechanisms contributing to vascular dysfunction in both hypertension and preeclampsia. eQTL indicates expression quantitative trait locus; FDR, false discovery rate; GTEx, Genotype-Tissue Expression project; SBP, systolic blood pressure; DBP, diastolic blood pressure; APC, antigen-presenting cell; TCR, T-cell receptor; MHC, major histocompatibility complex.