Decoding the Sex-Specific Architecture of Carotid Plaque Instability

Sex has a major influence on stroke incidence, clinical presentation, and outcomes¹. In general, men have a higher overall incidence of stroke at younger ages, whereas women experience more strokes over their lifetime, potentially due to greater longevity and increased risk associated with aging. Compared with men, women are also more likely to present with atypical stroke symptoms, experience delayed diagnosis, and have poorer functional outcomes after stroke². Many biological factors, including sex hormones, genetics, vascular physiology, and immune responses, may potentially contribute to these established sex differences in stroke.

Sex differences have also been noted in atherosclerotic plaque morphology. Specifically, plaques from male patients are more likely to exhibit rupture-prone features than plaques from female patients, including a thinner fibrous cap and a larger lipid core³. Because plaque rupture and surface erosion are major causes of ischemic stroke, the greater tendency of men to develop rupture-prone plaques provides one plausible explanation for the higher stroke incidence observed in men at younger ages. However, the mechanisms that drive the sexual dimorphism in plaque vulnerability remain incompletely understood.

Technological advances in single-cell RNA sequencing, multi-omics analysis, lineage tracing in preclinical models, and human organoid systems have reshaped our understanding of atherosclerosis. These approaches have revealed key cellular events that contribute to plaque progression and instability, including inflammation and phenotypic transitions of vascular smooth muscle cells (VSMCs) into inflammatory and macrophage-like states⁴. To dissect sex-specific molecular and cellular mechanisms underlying plaque vulnerability, Jae Hyun Byun and colleagues, in their article published in this issue of ATVB, utilize modern transcriptomic technologies to analyze carotid atherosclerotic plaques from both sexes at single-cell resolution while preserving spatial context⁵. The resulting high-resolution atlas provides novel insight into sex-based mechanisms of carotid plaque vulnerability and may help guide the future development of sex-informed therapies for stroke patients.

A major strength of this study is its direct analysis of human carotid atherosclerotic plaques, which enhances the translational relevance of the findings to plaque instability and ischemic stroke. Because the interplay among endothelial cells, VSMCs, inflammatory cells, and extracellular matrix is a major determinant of plaque instability, high-resolution cellular mapping within the structural context of carotid plaques is essential. An additional strength is the multimodal approach, which integrates single-cell RNA sequencing of more than 64,000 cells with subcellular-resolution spatial transcriptomics. This strategy allows the researchers to identify not only which cells are present, but also where each cell is located within critical plaque regions, including the fibrotic cap and the lipid core. By comparing molecular signatures and spatial patterns between stable and unstable plaques from symptomatic male and female patients (3 patients per group for single-cell analysis, 1 patient per group for spatial transcriptomics), the authors uncover cellular and spatial mechanisms that drive plaque instability, some shared by both sexes and others deployed in a sex-specific manner (Figure 1).

Figure 1. Proposed sex-specific cellular mechanisms driving instability of human carotid plaques.

A, Schematic of study design. B, Major cell populations identified in carotid plaques of both sexes by single-cell RNA sequencing. C, Sex-specific remodeling of vascular smooth muscle cells, macrophages, and T-lymphocytes during the transition from stable to unstable plaques. D, Prominent sex-specific intercellular communication networks in unstable plaques. E, Sex-specific cellular remodeling within the spatial context of unstable plaques revealed by spatial transcriptomics. This schematic is generated with assistance of ChatGPT.

Their findings confirm that inflammation is a central feature of unstable plaques in both sexes that have been previous reported⁶. However, they also reveal that the three dominant plaque-resident cell types, VSMCs, T-lymphocytes, and macrophages, undergo fundamentally different transformations depending on sex. Although inflammatory VSMC transition is prominent in both male and female unstable plaques, its spatial distribution differs markedly between sexes, suggesting distinct contributions of VSMC plasticity to plaque destabilization (Figure 1). Sex-specific spatial patterns are also observed for other VSMC subtypes as well as T-lymphocytes and macrophages, underscoring the presence of compartmentalized, sex-driven microenvironments within carotid plaques.

The significance of VSMC phenotypic switching in atherosclerosis is supported by extensive experimental and human data, including comprehensive VSMC fate-mapping and single-cell genomics studies in both mouse and human atherosclerotic plaques⁷. In addition to confirming the presence of multiple VSMC subtypes in carotid plaques, Byun and colleagues show that VSMC phenotypic transition and spatial distribution are shaped by both progression and sex. They show that in males, the transition from a stable to an unstable plaque is marked by a pronounced phenotypic shift in VSMCs. Stable male plaques are characterized by a dominant population of contractile VSMCs, which help maintain the structural integrity of the fibrotic cap. However, in unstable male lesions, these stabilizing cells are largely replaced by inflammatory synthetic and macrophage-like VSMCs. Because a thick, collagen-rich fibrous cap provides structural integrity and separates the thrombogenic lipid core from the circulation, the replacement of stabilizing VSMC phenotypes by inflammatory and macrophage-like VSMCs provides a plausible cellular basis for fibrous cap weakening in male plaques. Strikingly, the study shows that these inflammatory VSMCs are not confined to the lipid core. They also occupy the fibrotic cap, where they form “immune-rich niches” together with activated T-lymphocytes and macrophage-like VSMCs. The expansion of a pro-inflammatory microenvironment, coupled with widespread loss of stabilizing VSMC phenotypes, may help explain the higher rupture risk historically observed in men.

In contrast, female plaques appear to follow a more localized and perhaps more insidious route to destabilization. Unlike male plaques, unstable female plaques retain contractile VSMCs within the fibrotic cap, which may help preserve cap integrity and strength. However, the study identifies a “profound expansion” of inflammatory synthetic VSMCs within the lipid core. This spatially restricted remodeling suggests that female plaque instability may not depend primarily on global cap replacement, but rather on inflammatory remodeling within vulnerable plaque compartments. Such a pattern may help explain why traditional histological classifications do not fully capture risk in women.

The findings made on immune cells by Byun and colleagues add another important layer to this sex-specific framework. The authors demonstrated that T-lymphocytes are not merely increased as a generic marker of inflammation. Instead, distinct T-cell phenotypes emerge in male and female plaques. In unstable male plaques, activated naive T-lymphocytes are enriched and spatially associated with inflammatory VSMC niches, suggesting that stromal inflammation and T-cell activation may reinforce one another. In female plaques, unstable lesions exhibit enrichment of cytotoxic T-lymphocytes and B-lymphocyte-mixed T-lymphocytes. These findings point to a more adaptive immune-oriented program in female plaque instability, potentially involving antigen-driven T-cell and B-cell interactions. Such chronic immune activation may be relevant to the poorer long-term recovery and greater post-stroke disability frequently observed in female stroke survivors.

Expanding the accumulated knowledge on macrophages in atherogenesis, Byun and colleagues reveal sex-specific remodeling of macrophages in carotid plaques. In both sexes, unstable plaques show increases in monocyte-derived inflammatory and T-lymphocyte-mixed macrophage populations, supporting the idea that macrophage activation is a shared feature of plaque vulnerability. However, the dominant macrophage programs differ by sex. In males, inflammatory macrophages are more prominent in unstable plaques, consistent with an acute innate inflammatory environment that may cooperate with inflammatory VSMCs to promote matrix remodeling and plaque rupture. In females, unstable plaques show greater enrichment of foam-cell macrophages, while T-lymphocyte-mixed macrophages are also more abundant in female plaques. These observations suggest that macrophages in female plaques may participate not only in lipid handling and foam-cell biology, but also in adaptive immune crosstalk. Thus, macrophages appear to contribute to instability in both sexes, but through different inflammatory architectures.

Although the influence of sex and disease stage on VSMC phenotypic switching is convincingly demonstrated by Byun and colleagues, the mechanisms underlying these novel and interesting phenomena remain unclear. Is the expansion of inflammatory synthetic VSMCs observed in male unstable plaques driven by genetic and epigenetic factors intrinsic to male cells, by male sex hormone, or by signals from neighboring immune cells? Why contractile VSMCs retained in the fibrous cap of female plaques? What role do T-lymphocytes and macrophages play in shaping sex-specific VSMC phenotypic switching? Are immune cells the initiators, amplifiers, or consequences of VSMC remodeling? These fundamental questions warrant futures investigations, especially in atherosclerosis models in which genetic, epigenetic, hormonal, and signaling mechanisms can be experimentally manipulated to establish causal relationships.

Because the transcriptomic data produced by single-cell RNA sequencing contains information on the expression of ligands, receptors, and cofactors, investigators can infer intercellular communication networks using analytic tools such as CellChat⁸. Such analyses provide valuable insight into how immune cells and residential vascular cells may influence one another, but they do not provide spatial information because of the inherent limitation of dissociated single-cell data. This obstacle can be addressed by applying the proximity and neighborhood analyses to spatial transcriptomic data⁹. Using CellChat and spatial neighborhood analyses, Byun and colleagues uncover distinct intercellular communication networks in plaques from each sex. They find that although VSMCs are dominant signaling hubs in both sexes, the pathways they activate are sex specific. In males, unstable plaques are characterized by enhanced VSMC-to-stromal and VSMC-to-immune crosstalk, including IFNγ and VCAM-mediated signaling, pathways known to promote vascular inflammation and plaque rupture. In females, unstable lesions are characterized by enhanced innate immune signaling and macrophage-to-macrophage communication, involving pathways such as ICAM and IL16. These findings reinforce the concept that plaque instability in men and women may arise through distinct cellular conversations.

In the era of precision medicine, this work has important clinical implications. By demonstrating that plaque instability is not a uniform process, Byun and colleagues challenge the adequacy of current sex-agnostic approach to diagnosis and treatment. The observation that patients of different sexes may have fundamentally different biological drivers of plaque vulnerability supports the need for sex-informed assessment of stroke risk and sex-informed therapeutic development. For instance, stabilizing a male plaque may require interventions that preserve contractile VSMCs, prevent inflammatory VSMC transition, or interrupt maladaptive stromal-immune crosstalk. In females, therapeutic emphasis may shift toward modulating macrophage-centered signaling, adaptive immune crosstalk, lipid core inflammation, or T-cell and B-cell interactions. More broadly, the molecular and cellular mechanisms uncovered by this study may guide future discovery of targeted therapies that benefit all patients by accounting for the biological diversity of plaque instability.

In conclusion, this paper serves as an important bridge between bench and bedside. By leveraging the CosMx Spatial Molecular Imaging together with single-cell RNA sequencing, the authors provide a comprehensive atlas of sex-specific vascular biology in human carotid atherosclerosis. Although the sample size is modest, the high resolution of the data provides a strong foundation for larger-scale validation studies. As cardiovascular medicine moves toward precision prevention and treatment, this study reminds us that sex is not merely a demographic variable. It is a fundamental biological lens through which atherosclerotic plaque biology should be studied, interpreted, and ultimately treated.