Ernest Diez Benavente

Background: Coronary microvascular dysfunction (CMD) is a common cause of ischemic symptoms in patients with angina and non-obstructive coronary arteries (ANOCA), predominantly women. Neutrophils may contribute to CMD via vascular inflammation and endothelial injury. We therefore investigated neutrophil and coronary endothelial cells in women with ANOCA. Methods: In this cross-sectional study, we collected blood samples from ANOCA patients (n=123, 84% women) undergoing coronary function testing. Complete blood counts (CELL-DYN, n=123) and neutrophil activation (AQUIOS, n=76) were assessed. Isolated neutrophils were profiled using single-cell RNA-seq (n=11). Guidewire-derived coronary endothelial cells (n=7) isolated during coronary function testing were cultured to passage 2 for bulk RNA-seq. Results: Of 103 women with ANOCA, 23 (22%) were diagnosed with CMD (CFRdim ) was inversely associated with CFR at baseline. Single-cell RNA-seq identified four neutrophil clusters, including interferon (IFN)-activated and immature neutrophils. The IFN-activated cluster was expanded in patients with lower CFR and its proportion was inversely associated with surface CD62L expression. Immature neutrophils showed reduced translational and increased inflammatory gene expression in patients with low CFR. Coronary endothelial cells from patients with increased CD62L ^dim neutrophils showed higher proliferative/metabolic and lower IFN, complement and coagulation pathway activity. Conclusion: Reduced CFR in women with ANOCA is associated with neutrophil activation, expansion of an IFN-activated neutrophil population, and altered immature neutrophil transcriptional programs. Neutrophil activation is accompanied by distinct endothelial signatures. These findings implicate neutrophil–endothelial interactions in CMD and identify IFN-associated neutrophil states as targets for mechanistic investigation. Clinical Perspective: What is new?: This study provides the first characterization of circulating neutrophils in coronary microvascular dysfunction (CMD), identifying an increased proportion of interferon (IFN)-activated neutrophils and an IFN-enriched immature neutrophil phenotype Reduced neutrophil surface CD62L expression was associated with altered endothelial gene expression and increased endothelial proliferative and metabolic activity, suggesting a potential neutrophil-endothelial interaction in CMD What are the clinical implications?: Neutrophil activation and IFN signaling emerge as candidate pathways for further investigation into the mechanisms underlying CMD and as potential targets for future therapeutic research The association between lower CFR in women and an increased IFN-activated neutrophil population supports further investigation of sex-specific immune mechanisms in CMD.

Background: Heart failure (HF) is a systemic syndrome affecting multiple organs. Organ age gaps (OAGs), quantifying organ-level ageing from plasma proteomics, are linked to organ-specific disease including HF, but have been studied exclusively cross-sectionally. We examined how organ-specific ageing evolves within individuals over the course of HF and whether it carries prognostic information beyond established HF markers. Methods: We studied two complementary cohorts with repeated plasma proteomic profiling: 557 individuals including those at risk for HF, pre-HF, and newly diagnosed HF (HELPFul, mainly HFpEF) and 382 patients with established HF (Bio-SHiFT, all LVEF < 50%). OAGs for five organs plus a conventional clock were z-scored. Within each cohort, we modelled longitudinal OAG trajectories using linear mixed-effects models, and, in Bio-SHiFT, related OAGs to the primary endpoint using Cox models with progressive adjustment up to NT-proBNP and troponin T. Results: Baseline OAGs differed across HF severity in both cohorts. Longitudinally, 50 HELPFul individuals with LVDD showed increasing artery and immune OAGs over 4.6 years. In Bio-SHiFT, event-free patients had stable trajectories, whereas those with adverse events showed sharply accelerating multi-organ ageing over 2.1 years (immune β = 0.36 z-score per year, 95% CI 0.26–0.47; heart β = 0.29, 95% CI 0.17–0.40). Higher OAGs predicted the primary endpoint, and the kidney, lung, and immune OAGs remained prognostic after full adjustment including NT-proBNP and troponin T. Conclusion: In this exploratory study, multi-organ ageing accelerated before adverse events, and the kidney, lung, and immune OAGs predicted outcome independently of conventional severity markers, supporting a dynamic, organ-level view of HF risk that warrants confirmation in larger cohorts.

Cardiovascular disease remains the leading cause of death in women and a major contributor to disability, yet the evidence guiding diagnosis and treatment is still largely derived from studies in which men and male-pattern disease phenotypes have predominated. Women exhibit distinct cardiovascular physiology that contributes to sex differences in cardiovascular health and disease across the life course. However, these biological differences rarely inform clinical decision-making and remain incompletely understood, owing to a research ecosystem that inadequately addresses sex-specific biology. Conditions such as spontaneous coronary artery dissection, Takotsubo syndrome and coronary vasomotor disorders disproportionately affect women but were historically under-recognized as distinct clinical entities. Consequently, they remain understudied, with limited mechanistic insight and few evidence-based therapeutic strategies. In this Perspective, we advocate strengthening the translational understanding of sex-biased mechanisms and integrating these insights into diagnostics and therapeutics, with a focus on female-predominant conditions. We outline a roadmap for translational research that embeds sex as a biological variable and illustrates how advances in emerging technologies can accelerate the development of an evidence base that improves cardiovascular care for women.

BACKGROUND: Atherosclerosis, an inflammatory driver of coronary artery disease, manifests as unstable atheromatous plaques and stable fibrous plaques. Although atheromatous plaques have been extensively studied, fibrous plaques, particularly in women aged floralwhite and GRN _yellowgreen , were enriched for inflammatory and actin remodeling pathways, respectively. Single-cell RNA-sequencing confirmed sex-specific network activity in plaque vascular smooth muscle cells. Subcellular phenotyping identified a sex-specific gene expression program within GRN _yellowgreen enriched for contractile and vascular development pathways. Bayesian network modeling identified MYH9 (myosin heavy chain 9) as a key driver gene. Elevated MYH9 abundance was associated with increased smooth muscle cell content and reduced lipid content in female carotid plaques compared with males, consistent with fibrous plaque features. Proteomic analyses confirmed MYH9 upregulation in female fibrous plaques and association with stable plaque characteristics. CONCLUSIONS: These findings identify MYH9 as a regulator of female-biased fibrous plaque biology and highlight the importance of sex-specific network regulation in atherosclerosis.

Background: Angina with nonobstructive coronary arteries (ANOCA) is a heterogeneous condition encompassing distinct endotypes representing different underlying pathophysiological mechanisms. Endothelial dysfunction is considered a central hallmark of ANOCA. However, studying patient-derived endothelial cells (ECs) remains challenging due to the limited availability of disease-specific endothelial samples. We therefore aimed to assess the feasibility of isolating and culturing ECs from catheterization material obtained during routine coronary function testing in ANOCA patients. Methods: Catheterization material was collected from 79 ANOCA patients (84% female, age 58±10 years) undergoing coronary function testing. ECs were isolated, expanded and characterized using immunostaining, flow cytometry, gene expression profiling and functional assays. Results: EC isolation was successful in 43% of cases and resulted in 34 primary EC cultures that were expanded up to passage 10. Isolation success was independent of clinical or procedural characteristics. Isolated cells exhibited typical EC morphology and expressed EC markers confirmed by immunostaining, flow cytometry and gene expression analyses. EC marker gene expression remained largely stable over passages. However, stress- and defense-related gene expression programs increased over time, while proliferation-related processes decreased. Functional assays demonstrated that the coronary catheterization-derived ECs showed typical properties of wound healing, angiogenesis, activation responses upon stimuli and monocyte adhesion. Conclusions: This study demonstrates the feasibility of isolating and expanding ECs directly from catheterization material collected during routine coronary function testing in ANOCA patients. These patient-derived ECs retain characteristic endothelial features and functionality. This approach offers primary EC cultures to study the mechanisms underlying endothelial dysfunction in ANOCA. Graphic Perspectives: Clinical perspective: What is new?: We established a method to isolate and culture endothelial cells from routine coronary catheterization material in patients with ANOCA, enabling direct study of patient-specific endothelial dysfunction. The patient-derived endothelial cells exhibit characteristic morphology, express canonical endothelial markers and retain functional properties consistent with endothelial physiology. What are the clinical implications?: This approach provides a clinically relevant platform to investigate mechanisms underlying ANOCA and may support the development of personalized diagnostic and therapeutic strategies. Broader application of this method could facilitate translational research in other vascular pathologies where access to endothelial tissue is limited.

BACKGROUND: Sex and atherosclerotic plaque histology are intertwined, with fibrous plaques being more prevalent in women. Plaque erosion, a significant contributor to acute coronary syndromes, is linked to fibrous plaques and is more prevalent in women than men. We hypothesize that the molecular drivers of histologically determined fibrous plaques differ between men and women. METHODS: Human end-stage atherosclerotic plaques were isolated from carotid endarterectomy patients included in the Athero-Express Biobank. Fibrous plaques were histologically assessed, linked to clinical characteristics, and processed for protein, bulk RNA, single-cell RNA, and DNA methylation data. We leveraged sex-differential gene expression and deconvolution analyses to uncover sex-biased molecular and cellular mechanisms. Spatial transcriptomics localized gene expression patterns in plaques. Furthermore, we studied the female-biased processes in human plaque endothelial cells and vascular smooth muscle cells stimulated with TGF-β (transforming growth factor-β), with or without SMAD3 (SMAD family member 3) inhibition. RESULTS: Of 1889 atherosclerotic plaques (1309 male and 580 female), fibrous lesions were observed in 50% of female and 31% of male patients. Compared with patients with atheromatous plaques (n=494), women with fibrous plaques exhibited a high prevalence of smoking, while men with fibrous plaques presented more often with diabetes. Female fibrous plaques were characterized by smooth muscle cell–driven ECM (extracellular matrix) remodeling, TGF-β response, and endothelial-to-mesenchymal transition, localized to the fibrous cap. Conversely, male plaques were linked to macrophage-mediated inflammation proximal to the core, dependent on diabetes. Finally, we experimentally confirmed these female-biased mechanisms, showing that TGF-β induced endothelial-to-mesenchymal transition in endothelial cells and ECM remodeling in vascular smooth muscle cells, both partly reversed by SMAD3 inhibition. CONCLUSIONS: Women and men with end-stage fibrous atherosclerotic plaques exhibit distinct clinical and molecular profiles. These mechanisms might be candidate pathways to understand plaque erosion from a molecular point of view and may provide promising targets for atherosclerosis therapies, as they account for both sex and plaque phenotype.

Plasmodium vivax elimination is challenged by dormant liver stages (hypnozoites) that can reactivate months after initial infection resulting in relapses. Relapsing infections confound antimalarial clinical efficacy trials due to the inability to distinguish between recurrences arising from blood-stage treatment failure (recrudescence), reinfection or relapse. Genetic relatedness of paired parasite isolates, measured by identity-by-descent (IBD), can provide important information on whether individuals have had single or multiple mosquito inoculations, thus informing on recurrence origin. We developed a high-throughput amplicon sequencing assay comprising 93 multi-SNP (microhaplotype) markers to determine IBD between P. vivax clinical isolates. The assay was evaluated in 745 global infections, including 128 infection pairs from a randomized controlled trial (RCT) (ClinicalTrials.gov NCT01680406). Simulations demonstrate low error in pairwise IBD estimation at the panel (RMSE < 0.12) and IBD-based networks illustrate strong clustering by geography. IBD analysis in the RCT demonstrates a lower frequency of suspected relapses or recrudescence in patients treated with primaquine compared to those without primaquine; the impact is greater when paired with chloroquine than with artemether-lumefantrine. Our results demonstrate the potential to derive new information on P. vivax treatment and transmission using IBD generated by amplicon sequencing data that can be further improved with time-to-event models.