Elize A. M. de Jong

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: 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.

This study provides proof of concept that assessment of the peripheral vasculature, particularly vascular smooth muscle cells measured using the LASCA technology holds potential as a noninvasive tool for detecting coronary vasomotor dysfunction. This finding highlights the potential of the LASCA technology in, for example, medication studies for coronary vasomotor dysfunction, especially when investigating whether medication improves vascular function, as repeated peripheral measurements are less invasive than invasive coronary function testing, the current gold standard.

The following are what is known: 1) wedge-pressure correction is often considered for the derivation of indices of minimal microvascular resistance, and 2) the Yong method for calculating wedge pressure-corrected index of microvascular resistance (IMR) without balloon inflation has never been validated in a Doppler-derived population and has not been tested under different physiological conditions. This study 1) adds validation for the Yong method for calculated wedge-pressure correction in a Doppler-derived study population and 2) shows significant influence of the physiological conditions on the derivation of coronary wedge pressure.