Improving risk prediction in patients with carotid atherosclerosis : risk scoring, diagnostic imaging and molecular biomarkers

<p dir="ltr">Stroke is the third leading cause of death in Sweden, with approximately 20 000 strokes occurring annually. Of these, 10% result in death within the first month. The majority of strokes in Sweden (86%) are ischemic rather than hemorrhagic. Carotid stenosis is estimated to contribute to 8-18% of ischemic strokes. However, this may be underestimated, as emerging evidence suggests that embolic strokes of undetermined source can also originate from non-stenotic carotid plaques.</p><p dir="ltr">Currently, the decision to perform carotid revascularization in addition to optimized medical therapy is primarily based on the degree of luminal narrowing and the presence of neurological symptoms. Ideally, individuals at risk - those with vulnerable plaque features - would be identified before a stroke occurs. This PhD project aimed to improve the risk prediction in carotid patients by integrating clinical profiles with plaque biological phenotypes, imaging-derived morphological characteristics, and long-term cardiovascular outcomes.</p><p dir="ltr">Study I was a translational analysis of 101 symptomatic patients with available plaque microarray data. Patients were stratified into high or low stroke risk groups based on CAR and ABCD2 scores. Differential gene expression and gene set enrichment analyses were conducted between high- and low-risk plaques for each risk score. Both CAR and ABCD high-risk plaques exhibited upregulation of genes and biological processes associated with plaque vulnerability, including extracellular matrix degradation, iron ion metabolism, angiogenesis and foam cell differentiation. Notably, the ATP-binding cassette transporter gene ABCB5, previously unassociated with atherosclerosis, was significantly upregulated in high-risk plaques for both scoring systems. Immunohistochemical analysis localized ABCB5 expression to macrophages and neovascularized regions within plaques, suggesting a potential role in intraplaque hemorrhage.</p><p dir="ltr">Study II, an observational cohort study of 1033 patients, investigated long-term all-cause mortality following carotid endarterectomy and their mortality rate in comparison to an age- and sex matched population. Follow-up data were obtained from national inpatient and death registries. No significant difference in all-cause mortality was observed between asymptomatic and symptomatic patients. However, men exhibited higher mortality, which persisted after adjustments. Increasing age and diabetes were also predictors of mortality. Among women, smoking and cardiac disease were independent risk factors, whereas in men, lipid-lowering and antiplatelet therapies were associated with improved survival, an effect not observed in women. Compared to the matched population, all patient groups experienced excess mortality during the five first years post carotid endarterectomies, with women exhibiting a 2.4-fold higher mortality rate.</p><p dir="ltr">Study III was a translational study including 238 symptomatic patients with completed ultrasound examination and available RNA sequencing (RNAseq) data. Patients were stratified by stenosis severity into low-grade (<50%) and high- grade (70-99%) groups. In a subset of patients with available computed tomography angiography, plaque morphology was also assessed. Despite differences in stenosis grade, the groups were largely similar in clinical and imaging characteristics. Dimensionality reduction analyses and differential gene expression analyses revealed no major transcriptomic differences between groups. Nevertheless, HIF3A emerged as the most upregulated gene in low-grade plaques. Immunohistochemistry showed HIF3A to be co-localized with macrophages in neovessel-rich regions, like HIF1A. In smooth muscle cells, HIF3A was localized near the nucleus, whereas HIF1A was found in the surrounding extracellular matrix.</p><p dir="ltr">Study IV was an observational cohort study of 961 patients undergoing carotid endarterectomy, evaluating long-term risk of major adverse cardiovascular events (MACE) and its components in relation to sex, preoperative symptom status, and predicted risk using CAR and SCORE2/SCORE2-OP models. Follow-up data on stroke and acute coronary syndromes obtained from national registries were validated through manual review of medical records. CEA-treated patients exhibited a persistently high long-term risk of MACE, with similar rates observed in both asymptomatic and symptomatic patients. Men had a higher risk of MACE compared to women, which persisted through the adjustments. Patients classified as high-risk by CAR and SCORE2 had higher crude MACE rates, although these associations were attenuated after adjustments.</p><p dir="ltr">In summary, carotid plaques from patients with high CAR and ABCD2 scores exhibit biological processes for plaque vulnerability. Low-grade stenosis plaques were similar to high-grade plaques. Although long-term stroke risk after CEA was low, patients remain at high risk for MACEs. A history of stroke is a strong predictor of long-term MACE. Men experience higher rates of all-cause mortality and MACE, while women show excess mortality during the first five years post-CEA.</p><h3>List of scientific papers</h3><p dir="ltr">I. Clinical Risk Scores for Stroke Correlate with Molecular Signatures of Vulnerability in Symptomatic Carotid Patients. <b>Katarina Wadén</b>, Eva Karlöf, Sampath Narayanan, Mariette Lengquist, Göran K. Hansson, Ulf Hedin, Joy Roy, Ljubica Matic. iScience. 2022 Apr 8;25(5):104219. <a href="https://doi.org/10.1016/j.isci.2022.104219" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.isci.2022.104219</a></p><p dir="ltr">II. Long Term Mortality Rate in Patients Treated with Carotid Endarterectomy. <b>Katarina Wadén</b>, Rebecka Hultgren, Maria loanna Kotopouli, Peter Gillgren, Joy Roy, Ulf Hedin, Ljubica Matic. Eur J Vasc Endovasc Surg. 2023 Jun;65(6):778-786. <a href="https://doi.org/10.1016/j.ejvs.2023.02.079" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.ejvs.2023.02.079</a></p><p dir="ltr">III. Atherosclerotic Plaque Instability in Symptomatic Non-significant Carotid Stenosis. Paul Cyréus, <b>Katarina Wadén</b>, Sofie Hellberg, Otto Bergman, Mariette Lengquist, Eva Karlöf, Andrew Buckler, Ljubica Matic, Joy Roy, David Marlevi, Melody Chemaly, Ulf Hedin. JVS Vasc Sci. 2025 Jan 17:6:100280. <a href="https://doi.org/10.1016/j.jvssci.2025.100280" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.jvssci.2025.100280</a></p><p dir="ltr">IV. Sex-specific Differences in Long-Term Risk of Stroke and Major Adverse Cardiovascular Events after Carotid Endarterectomy. <b>Katarina Wadén</b>, Paul Cyreus, Antti Siika, Joy Roy, Ulf Hedin, Ljubica Matic, Rebecka Hultgren [Manuscript]</p>