Sarah Gorey

Background: Inflammation is an emerging target for stroke prevention, but additional therapeutic candidates are needed. Experimental and observational studies implicate tumor necrosis factor (TNF) in atherosclerotic plaque progression and cardiovascular events. We integrated plasma proteomics from population-based studies and prospective stroke cohorts with single-cell and spatial transcriptomic profiling of human atherosclerotic plaque to investigate TNF signaling in stroke pathogenesis. Methods: We assessed associations between 34 TNF-superfamily proteins (Olink Explore) and incident ischemic stroke among 47,529 UK Biobank participants without cardiovascular disease. We performed an individual-participant data (IPD) meta-analysis of four prospective cohorts with ischemic stroke (n=2,180) to examine associations between TNF-α and recurrent vascular events. We characterised plaque-level TNF biology using single-cell RNA sequencing of 259,116 cells (62 donors) and Xenium spatial transcriptomics (12 donors) with human carotid plaques. Results: In UK Biobank, higher circulating TNF pathway proteins were independently associated with incident stroke after multivariable adjustment, including TNF, TNFR1, and TNFR2 (Hazard Ratio [HR] per SD increase, 1.14, [95% CI 1.07–1.20], 1.22, [1.14–1.31], and 1.15 [1.09–1.21], respectively. An additional 15 TNF superfamily members were also associated with incident stroke. In the IPD analysis of stroke cohorts, TNF-α was associated with recurrent stroke (risk ratio [RR] 1.50, 95% CI 1.14-1.98, top vs. bottom third of TNF-α) and MACE (RR 1.54, 1.18-2.02) after adjustment for cardiovascular risk factors and secondary prevention medications (537 MACE events, 6793 person-years follow up). In single-cell RNA plaque sequencing, TNF and TNF pathway genes were broadly expressed across immune cell populations. In spatial transcriptomics, TNF detection increased progressively from media to fibrous cap (Odds Ratio 2.32 vs media, 95% CI 1.94–2.78, p<0.001). At the fibrous cap, CD8+ effector T cells demonstrated 4.1-fold enrichment for TNF expression despite comprising only 3% of fibrous cap cells. Conclusions: TNF signaling is independently associated with incident ischemic stroke and recurrent MACE after stroke. TNF is enriched in human carotid plaque at the fibrous cap, in macrophages and CD8+ effector T cells. These results support evaluation of TNF-targeted therapies for stroke prevention.

The residual recurrence risk after atrial fibrillation (AF)-related stroke is high despite anticoagulation, thereby necessitating new therapies. The importance of inflammation in AF is increasingly recognized. However, patients with AF-related stroke were excluded from recent trials of anti-inflammatory therapies. It is uncertain whether associations between IL-6/high-sensitivity C-reactive protein (hsCRP) and poststroke recurrence are modified by AF status. In this study, we aimed to analyze the association between IL-6/hsCRP and recurrence according to AF history. We leveraged individual participant data from studies identified by systematic review. We analyzed associations between IL-6/hsCRP and recurrent stroke/major adverse cardiovascular events (MACEs) (defined as fatal or nonfatal recurrent stroke or major coronary events) using multivariable Cox regression analyses (conditional logistic regression for 1 study) adjusted for age, sex, index event, cardiovascular risk factors, and medication use, stratified by AF status. Data from 11 prospective studies with 10,080 patients (2,134 with AF, mean age 70 years, 59.3% male) were included. During 21,080 person-years of follow-up, 1,677 patients had MACEs and 1,342 had recurrent stroke. Inflammatory markers were higher in patients with AF, irrespective of stroke severity and timing of measurement, with elevated levels persisting well beyond the acute phase. hsCRP was associated with recurrent MACEs in patients with AF (adjusted risk ratio [aRR] 1.14, 95% CI 1.04-1.25, per log_e-unit increase) and without AF (aRR 1.08, 1.03-1.13) (P_interaction 0.30). There were similar associations on per-quarter analysis in patients with AF (aRR 1.51, 1.04-2.18) and without AF (aRR 1.32, 1.10-1.59) (Q4 vs Q1). No association was observed between hsCRP and recurrent stroke in either group. For IL-6, there was no evidence of interaction according to AF status for MACEs (P_interaction 0.57) or recurrent stroke (P_interaction 0.82). The aRR per log_e unit for MACE was 1.17 (1.07-1.27) in patients without AF and 1.10 (0.91-1.34) in those with AF. The corresponding aRR for recurrent stroke was 1.15 (1.05-1.25) and 1.12 (0.91-1.37) in patients without and with AF, respectively. These data highlight the importance of inflammatory mechanisms in vascular recurrence irrespective of AF, provide rationale for the inclusion of patients with AF in trials of anti-inflammatory therapy, and support a selection approach in future trials based on elevated inflammatory marker levels, rather than stroke etiology alone.

BACKGROUND: Uncertainty remains whether inflammation is implicated in poststroke recurrence in patients without atherosclerosis. We evaluated the contribution of atherosclerosis status to the association between inflammatory markers and major adverse cardiovascular events (MACE) poststroke. METHODS: We performed an individual-participant data meta-analysis of 11 prospective cohorts (12 countries, 1995–2017). Studies included patients with ischemic stroke/transient ischemic attack and measured IL (interleukin)-6/hsCRP (high-sensitivity C-reactive protein) postevent. We analyzed the association between IL-6/hsCRP and recurrent stroke/MACE using multivariable Cox regression analyses (conditional logistic regression for 1 study). Analyses were stratified by the presence/absence of atherosclerosis (definition: prior history of coronary disease, peripheral artery disease, or large artery atherosclerotic stroke) and adjusted for cardiovascular risk factors/preventative medication. RESULTS: Overall 10 148 patients (3448 [34.0%] had atherosclerosis) with 21 177 years of follow-up were included (1707 MACE outcomes/1353 recurrent strokes). In patients with atherosclerosis, IL-6 was independently associated with MACE (risk ratio [RR], 1.22 [95% CI, 1.08–1.37]; per log _e unit increase) and recurrent stroke (RR, 1.23 [95% CI, 1.08–1.41]). Compared with patients in the bottom quarter, those in the top quarter of IL-6 levels had double the risk of MACE (RR, 2.05 [95% CI, 1.37–3.08]) and stroke (RR, 1.97 [95% CI, 1.28–3.05]). IL-6 was also associated with MACE (RR, 1.11 [95% CI, 1.01–1.23]) but not stroke (RR, 1.08 [95% CI, 0.98–1.20]; per log _e unit) in patients without atherosclerosis. However, there was no evidence of statistical interaction between IL-6 levels and atherosclerosis status for either outcome ( P _interaction =0.25 and 0.13 for MACE/recurrent stroke, respectively). hsCRP was associated with MACE in patients with (RR, 1.12 [95% CI, 1.05–1.21]; per log _e unit) and without atherosclerosis (RR, 1.07 [95% CI, 1.01–1.14]; P _interaction =0.28). No association with recurrent stroke was observed for hsCRP with (RR, 1.06 [95% CI, 0.98–1.14]) or without atherosclerosis (RR, 0.97 [95% CI, 0.91–1.04]; P _interaction =0.18). CONCLUSIONS: IL-6/hsCRP were associated with poststroke recurrence irrespective of atherosclerosis. These data support the inclusion of patients in trials of anti-inflammatory therapies after stroke with elevated IL-6 or hsCRP, including those without prior atherosclerotic events.

Despite effective secondary prevention, including oral anticoagulant (OAC) therapy, the risk of recurrent stroke (RS) in patients with atrial fibrillation (AF) remains substantial with an annualized risk of 3.2%-6.5% per year. The reasons for this high residual risk are unclear. There is growing need for improved risk prediction tools to identify patients at greatest risk of RS in AF and to find new therapeutic targets for secondary prevention. Our objective was to perform a systematic review to investigate the association of clinical factors and echocardiographic, blood, and neuroimaging biomarkers, with stroke recurrence after AF-related stroke. We searched Embase/Ovid Medline until August 2023. Studies were included irrespective of OAC use. Risk ratios (RRs) were pooled using random effects. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. Of 5,427 records searched, 42 reports from 28 studies including 52,798 patients (5,046 RS events during follow-up) were identified. In addition to the CHA₂DS₂-Vasc score (RR 1.22, 95% CI 1.15-1.30, per point) and its individual components, the clinical factors associated with recurrence were a qualifying stroke despite OAC (RR 1.55, 1.23-1.94 [vs OAC-naïve]), sustained AF (RR 1.44, 1.19-1.75 [vs paroxysmal]), hyperlipidemia (RR 1.27, 1.05-1.53), chronic kidney disease (RR 1.86, 1.19-2.92), and malignancy (RR 4.36, 1.85-10.78). NIH Stroke Scale scores (RR 0.97, 0.95-0.99) and Asian ethnicity (RR 0.59, 0.36-0.97) were associated with a reduced risk of recurrence. Known AF was not associated with a higher risk of stroke compared with AF detected after stroke (RR 1.20, 0.93-1.55). Neuroimaging markers associated with RS included chronic lacunar (RR 1.91, 1.29-2.84) or embolic-appearing (RR 2.76, 1.32-5.77) infarcts and cerebral microbleeds (RR 1.29, 1.04-1.59). Echocardiographic markers included atrial size (RR 1.40, 1.12-1.75 per cm/m²), intracardiac thrombus (RR 1.99, 1.38-2.87), spontaneous left atrial appendage (LAA) echocardiographic contrast (RR 2.91, 1.21-6.17), or low LAA intensity variation (RR 2.81, 1.48-5.32). Data were limited for blood biomarkers. We identified several clinical factors associated with recurrence after AF-related stroke, with AF burden and stroke despite OAC of particular clinical relevance. Biomarkers of atrial cardiopathy, small vessel disease, or previous infarction were also associated with RS. Collaborative efforts are needed to identify and validate new risk factors and biomarkers of RS in AF.

Introduction: Acute and late inflammatory markers including high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) are associated with future vascular events after stroke. However, few longitudinal studies exist examining the intra-individual reproducibility of inflammatory biomarker measures at different timepoints after atherosclerotic stroke. We sought to examine the reproducibility of hsCRP and IL-6 in a cohort of patients with minor stroke or transient ischaemic attack (TIA) caused by ipsilateral carotid atherosclerosis. Methods: Two observational cohort studies (DUCASS and BIOVASC) were pooled. Included patients had non-severe ischaemic stroke and ipsilateral internal carotid artery stenosis (≥50%). Patients had bloods drawn within 2 weeks of their index stroke/TIA event which was stored for later analysis. All patients included were followed up at 5 years, and repeat phlebotomy was performed. Bloods were analysed for hsCRP and IL-6 using high-throughput immunochemiluminescence. Difference between baseline and follow-up blood levels and intraclass correlation (ICC) was calculated. Results: Ninety-five participants were included, median age 69 (IQR: 63–77), and 51 (53.7%) had TIA as their presenting event. When biomarkers were dichotomised (for hsCRP <2 mg/L or ≥2 mg/L, and for IL-6 <7.5 pg/mL [median] or ≥7.5 pg/mL), 68.4% (IL-6) and 65.2% (hsCRP) of participants remained in the same risk category (high or low) over time. However, when analysed as a continuous variable, ICC coefficients were low: ICC for IL-6 0.14 (95% CI: −0.06 to 0.33), ICC for hsCRP 0.05 (95% CI: −0.14 to 0.25). ICC increased after removing outliers. Clinical characteristics and treatment were not associated with observed variability. Conclusion: Our results suggest that concordance between early- and late-phase inflammatory marker risk categories is modest, and absolute levels are not highly correlated at early and late timepoints, despite associations at both times with future vascular risk. Investigators should standardise timing of phlebotomy and analysis protocols in future studies of inflammatory biomarkers.

BACKGROUND: Inflammation promotes atherogenesis. Randomized controlled trials of anti-inflammatory therapies for prevention after stroke have not yet demonstrated clear benefit. IL-6 (interleukin-6) and hsCRP (high-sensitivity C-reactive protein) are independently associated with major adverse cardiovascular events poststroke and may guide patient selection in future randomized controlled trials. Optimal timing of hsCRP/IL-6 measurement poststroke is unknown, as early blood levels may be confounded by the inflammatory response to brain infarction. METHODS: Using individual-participant data from a systematic review, we performed a time-course analysis to investigate the association between hsCRP/IL-6 and recurrent events stratified by timing of sampling. The prespecified coprimary end points after sample measurement were: (1) recurrent major adverse cardiovascular events (first major coronary event, recurrent stroke, or vascular death) and (2) recurrent stroke (ischemic, hemorrhagic, or unspecified). The poststroke dynamics of IL-6/hsCRP were analyzed by plotting their median (interquartile interval) concentrations within each tenth of the sampling timeframe. Acute/postacute phases were defined for each biomarker according to the shape of this relationship. RESULTS: There were data for 9798 patients from 11 studies (19 891 person-years follow-up, 10 observational cohorts, and 1 randomized trial). Each marker was measured once. IL-6 was markedly elevated P e IL-6), but not in the acute phase (P _interaction =0.03). After adjustment for risk factors/medication, the association remained for postacute IL-6 when analyzed per log _e unit (risk ratio, 1.16 [CI, 1.05–1.66]) and per quarter increase (risk ratio, 1.55 [CI, 1.19–2.02]; Q4 versus Q1), but not if measured acutely. Similar findings were observed for recurrent stroke. There was no evidence of time-dependent interaction with hsCRP. CONCLUSIONS: Timing of sample measurement after stroke modifies the association with recurrent major adverse cardiovascular events for IL-6 but not hsCRP. These data inform future randomized controlled trial designs incorporating biomarker-based selection of patients for anti-inflammatory therapies.

Introduction: Inflammation is an emerging target for secondary prevention after stroke and randomised trials of anti-inflammatory therapies are ongoing. Fibrinogen, a putative pro-inflammatory marker, is associated with first stroke, but its association with major adverse cardiovascular events (MACE) after stroke is unclear. Materials and Methods: We did a systematic review investigating the association between fibrinogen and post-stroke vascular recurrence. Authors were invited to provide individual-participant data (IPD) and where available we did within-study multivariable analyses with adjustment for cardiovascular risk factors and medications. Adjusted summary-level data was extracted from published reports from studies that did not provide IPD. We pooled risk ratios (RR) by random-effects meta-analysis by comparing supra-median with sub-median fibrinogen levels and performed pre-specified subgroup analysis according to timing of phlebotomy after the index event. Results: Eleven studies were included (14,002 patients, 42,800 follow-up years), of which seven provided IPD. Fibrinogen was associated with recurrent MACE on unadjusted (RR 1.35, 95% CI 1.17–1.57, supra-median vs sub-median) and adjusted models (RR 1.21, 95% CI 1.06–1.38). Fibrinogen was associated with recurrent stroke on univariate analysis (RR 1.19, 95% CI 1.03–1.39), but not after adjustment (RR 1.11, 95% CI 0.94–1.31). The association with recurrent MACE was consistently observed in patients with post-acute (⩾14 days) fibrinogen measures (RR 1.29, 95% CI 1.16–1.45), but not in those with early phlebotomy (<14 days) (RR 0.98, 95% CI 0.82–1.18) (P interaction = 0.01). Similar associations were observed for recurrent stroke. Discussion and Conclusion: Fibrinogen was independently associated with recurrence after stroke, but the association was modified by timing of phlebotomy. Fibrinogen measurements might be useful to identify patients who are more likely to derive benefit from anti-inflammatory therapies after stroke.

Anti-inflammatory therapies reduce major adverse cardiovascular events (MACE) in coronary artery disease but remain unproven after stroke. Establishing the subtype-specific association between inflammatory markers and recurrence risk is essential for optimal selection of patients in randomized trials (RCTs) of anti-inflammatory therapies for secondary stroke prevention. Using individual participant data (IPD) identified from a systematic review, we analyzed the association between high-sensitivity C-reactive protein, interleukin-6 (IL-6), and vascular recurrence after ischemic stroke or transient ischemic attack. The prespecified coprimary end points were (1) any recurrent MACE (first major coronary event, recurrent stroke, or vascular death) and (2) any recurrent stroke (ischemic, hemorrhagic, or unspecified) after sample measurement. Analyses were performed stratified by stroke mechanism, per quarter and per biomarker unit increase after log_e transformation. We then did study-level meta-analysis with comparable published studies not providing IPD. Preferred Reporting Items for Systematic Review and Meta-Analyses IPD guidelines were followed. IPD was obtained from 10 studies (8,420 patients). After adjustment for vascular risk factors and statins/antithrombotic therapy, IL-6 was associated with recurrent MACE in stroke caused by large artery atherosclerosis (LAA) (risk ratio [RR] 2.30, 95% CI 1.21-4.36, p = 0.01), stroke of undetermined cause (UND) (RR 1.78, 1.19-2.66, p = 0.005), and small vessel occlusion (SVO) (RR 1.71, 0.99-2.96, p = 0.053) (quarter 4 [Q4] vs quarter 1 [Q1]). No association was observed for stroke due to cardioembolism or other determined cause. Similar results were seen for recurrent stroke and when analyzed per log_e unit increase for MACE (LAA, RR 1.26 [1.06-1.50], p = 0.009; SVO, RR 1.22 [1.01-1.47], p = 0.04; UND, RR 1.18 [1.04-1.34], p = 0.01). High-sensitivity CRP was associated with recurrent MACE in UND stroke only (Q4 vs Q1 RR 1.45 [1.04-2.03], p = 0.03). Findings were consistent on study-level meta-analysis of the IPD results with 2 other comparable studies (20,136 patients). Our data provide new evidence for the selection of patients in future RCTs of anti-inflammatory therapy in stroke due to large artery atherosclerosis, small vessel occlusion, and undetermined etiology according to inflammatory marker profile.