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Abstract Background Patients with cancer treated with immune checkpoint inhibitors are at increased risk of myocardial infarction and ischemic stroke. The mechanism is incompletely understood but may involve accelerated atherosclerosis due to plaque inflammation. Pre-clinical studies may provide insight in these mechanisms. Aim To assess the effects of modulating co-inhibitory immune checkpoint proteins on atherosclerosis progression in animal models. Methods A systematic review was performed in MEDLINE, Embase, Web of Science, and Scopus up to September 2023. Animal studies were included if the effect of modulation of programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and/or lymphocyte-activation gene 3 (LAG-3) on atherosclerotic plaque size was evaluated. Secondary outcomes were plaque composition and systemic inflammation. The ratios of means (RoM) across the studies were pooled in a random effects meta-analysis. Results 1,655 studies were identified of which twelve were included. All studies used an atherosclerotic mouse model (Apoe-/-, Ldlr-/-, or ApoE3*Leiden) and either evaluated pharmacological or genetic modulation of co-inhibitory immune checkpoint proteins. Upon inhibition, atherosclerotic plaque size in the aorta was 49% higher in exposed mice compared to control mice (RoM, 1.49; 95% CI, 1.12-1.82; I2=86%). Plaque composition was predominantly characterized by a greater abundance of CD4+ T cells, CD8+ T cells, and macrophages. Studies stimulating co-inhibitory immune checkpoint proteins corroborated these findings and demonstrated that atherosclerotic plaque size was reduced by 28% in treated mice compared to controls (RoM, 0.72; 95% CI, 0.65-0.80; I2=85%). This reduction was paralleled by a decrease in the number of macrophages and T cells in plaques. Conclusion Immune checkpoint inhibition leads to plaque inflammation and a significant increase in murine atherosclerotic plaque size. These changes may reflect the cause of the increased risk of myocardial infarction and ischemic stroke in patients treated with immune checkpoint inhibitors.

CD40 signalling is involved in chronic inflammation, which is increasingly recognized as an important factor in initiation and progression of various cardiovascular diseases. Inhibition of the CD40-TRAF6 pathway was previously shown to be effective in preventing atherosclerosis and obesity in mice (Seijkens, JACC 2018, Chatzigeorgiou, PNAS 2014). In the present study we investigated the effect of CD40-TRAF6 inhibitor, compound 6877002 on left ventricular (LV) and coronary microvascular function in an adult swine model of familial hypercholesterolemia (FH). For this purpose, 10 adult male FH swine (3 years old, homozygous LDLR R84C mutation) received an atherogenic diet (10% lard and 0.75% cholesterol) for 9 months. During the last 3 months, 5 swine were subjected to treatment with the 6877002 compound (0.16 mg/kg i.v), while 5 animals received the vehicle i.v. At the end of treatment, swine were anesthetized and LV performance was studied under closed-chest conditions by pressure-volume loops constructed at baseline and during cardiac preload reduction. Subsequently, small coronary arteries (±250 μm diameter) were harvested from the LV and endothelium-dependent response to bradykinin in the presence and absence of endothelial nitric oxide (eNOS) blockade with LNAME, as well as endothelium-independent responses to the nitric oxide donor SNAP were studied in vitro. 6877002-treated animals showed a lower LV end-diastolic pressure (0±2 mmHg) as compared to vehicle-treated animals (6±1 mmHg, p≤0.05), and trends towards a reduced LV stiffness, reflected in a lower slope of the end diastolic pressure-volume relationship (sEDPVR: 0.16±0.02 mmHg/ml vs 0.20±0.01 mmHg/ml, p=0.07), and a higher ejection fraction (EF 46±2% vs 40±2%, p=0.08). Data from isolated small coronary arteries indicated that endothelium-independent vasorelaxation in response to SNAP was similar in both 6877002- and vehicle-treated animals, indicating unaltered vascular smooth smooth mucle cell responsiveness to NO. However, 6877002 treatment improved endothelium-dependent vasorelaxation to bradykinin (p≤0.05), as compared to vessels isolated from vehicle-treated animals. Moreover, eNOS blockade with LNAME blunted the endothelium-dependent vasorelaxation to bradykinin only in vessels from 6877002-treated animals, but not in those from the vehicle-treated swine, suggesting that NO bioavailability was completely lost in the vehicle-treated FH-animals, which was at least partially restored by treatment with 6877002 Conclusion: – Initation of CD40-TRAF6 pathway inhibition in adult FH swine receiving a high fat diet reduced LV end-diastolic pressure and enhanced LV passive compliance. The improved LV performance was accompanied by an amelioration of coronary microvascular endothelial dysfunction, characterized by the partial restoration of NO bioavailability. Funding: Grant 2020B008 RECONNEXT This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Introduction: Atherosclerosis, the underlying cause of cardiovascular disease (CVD), is a lipid-driven chronic inflammatory disease. We have identified the co-stimulatory CD40-CD40L immune checkpoint dyad as a key driver of inflammation during atherosclerosis. CD40 and CD40L exert cell-type divergent functions via different signaling pathways. Hypothesis: We hypothesize that CD40 on endothelial cell (ECs) induces endothelial-to-mesenchymal transition (EndMT)-mediated vascular inflammation in atherosclerosis. Methods & Results: Using single cell RNAseq, we found that CD40 (TNFRSF5) is expressed on PDE3A + GJA5 + and KDR + COL15A1 + ECs of advanced human carotid atherosclerotic plaques. CD40 is highly expressed in the endothelial lining of human and mouse atherosclerotic plaques, and colocalizes with the EndMT markers fibronectin and collagen and adhesion molecule ICAM-1. CD40L activation of human aortic endothelial cells (HAECs) results in expression of the EndMT markers fibronectin, vimentin, sm22α, FSP1 and snail. Activation of CD40 in EC induces filamin A dependent translocation of CD40 into lipid rafts, increases TNF-receptor associated factor (TRAFs) -2, -3 and 6 levels, induces Akt signaling and increases expression of VCAM1 and CCL2. Conclusions: The endothelial cell has been more and more regarded as a cell type that plays a major role in driving inflammation. Our data suggest that the CD40-CD40L axis plays a significant role in EC-mediated inflammation in atherosclerosis by driving EndMT.

Cardiovascular disease is the global #1 cause of mortality and morbidity. The majority of cardiovascular diseases is caused by atherosclerosis, a lipid-driven, inflammatory disease of the middle- and large-sized arteries. The disease is characterized by the formation of atherosclerotic plaques throughout the arterial tree. Over the years, insights into the pathogenesis of atherosclerosis have shifted from a ‘lipid-driven’ model to a ‘response-to-injury’ perspective, and more recently to a ‘lipid-driven inflammatory disease’ viewpoint. We are now aware that a network of multiple immune cell-types and -subsets of the innate and adaptive immune system inhabit our arteries. Intricate interactions between these immune cellsubsets, non-immune cells, and local environmental substances such as lipids, cell debris and calcium cause a fluidic balance of pro-inflammatory and regulatory responses. A dysregulation of this balance towards a pro-inflammatory milieu drives atherosclerotic disease progression. Although we have acknowledged that atherosclerosis is an inflammatory disease, state-of-theart treatments are still based on lipid lowering, anti-hypertensive and lifestyle-changing strategies. In the past decade, clinical phase I, II and III trials targeting the immune system revealed that patients tolerate immunotherapy, show decreased inflammation and/or had a reduction in cardiovascular endpoints. However, the search for novel immunotherapeutic targets and treatment regimens as well as stratification of patients who would benefit from such treatments to combat atherosclerotic cardiovascular disease is only just beginning. In this review article, we will highlight the newest insights on the different cell subsets and components of the immune system in atherosclerosis and elaborate on current and future immunotherapeutics to treat atherosclerotic cardiovascular disease.

BACKGROUND: The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme EZH2 (enhancer of zeste homolog 2) mediates the H3K27me3 (trimethylation of histone H3 lysine 27) and is pivotal in controlling T cell responses. METHODS: To detail the role of T cell EZH2 in atherosclerosis, we used human carotid endarterectomy specimens to reveal plaque expression and geography of EZH2. Atherosclerosis-prone Apoe (apolipoprotein E)–deficient mice with CD (cluster of differentiation) 4 + or CD8 + T cell–specific Ezh2 deletion (Ezh2 cd4 -knockout [KO], Ezh2 cd8 -KO) were analyzed to unravel the role of T cell Ezh2 in atherosclerosis and T cell–associated immune status. RESULTS: EZH2 expression is elevated in advanced human atherosclerotic plaques and primarily expressed in the T cell nucleus, suggesting the importance of canonical EZH2 function in atherosclerosis. Ezh2 cd4 -KO, but not Ezh2 cd8 -KO, mice showed reduced atherosclerosis with fewer advanced plaques, which contained less collagen and macrophages, indicating that Ezh2 in CD4 + T cells drives atherosclerosis. In-depth analysis of CD4 + T cells of Ezh2 cd4 -KO mice revealed that absence of Ezh2 results in a type 2 immune response with increased Il-4 (interleukin 4) gene and protein expression in the aorta and lymphoid organs. In vitro, Ezh2 -deficient T cells polarized macrophages toward an anti-inflammatory phenotype. Single-cell RNA-sequencing of splenic T cells revealed that Ezh2 deficiency reduced naive, Ccl5 + (C-C motif chemokine ligand 5) and regulatory T cell populations and increased the frequencies of memory T cells and invariant natural killer T (iNKT) cells. Flow cytometric analysis identified a shift toward Th2 (type 2 T helper) effector CD4 + T cells in Ezh2 cd4 -KO mice and confirmed a profound increase in splenic iNKT cells with increased expression of Plzf (promyelocytic leukemia zinc finger), which is the characteristic marker of the iNKT2 subset. Likewise, Zbtb16 ([zinc finger and BTB domain containing 16], the Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2 cd4 -KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3–chromatin immunoprecipitation followed by quantitative polymerase chain reaction showed that T cell–Ezh2 regulates the transcription of the Il-4 and Zbtb16 genes. CONCLUSIONS: Our study uncovers the importance of T cell EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4 + T cells drives type 2 immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis.

Abstract Studies suggest varying atherosclerotic cardiovascular disease (ASCVD) prevalence across arterial beds. Factors such as smoking expedite ASCVD progression in the abdominal aorta, while diabetes accelerates plaque development in lower limb arteries, and hypertension plays a significant role in ASCVD development in the coronary and carotid arteries. Moreover, superficial femoral atherosclerosis advances slower compared with atherosclerosis in coronary and carotid arteries. Furthermore, femoral atherosclerosis exhibits higher levels of ossification and calcification, but lower cholesterol concentrations compared with atherosclerotic lesions of other vascular beds. Such disparities exemplify the diverse progression of ASCVD across arterial beds, pointing towards differential mechanistic pathways in each vascular bed. Hence, this review summarizes current literature on immune-inflammatory mechanisms in various arterial beds in ASCVD to advance our understanding of this disease in an aging society with increased need of vascular bed and patient-specific treatment options.

Introduction: Atrial fibrillation (AF) is prevalent in patients with atherosclerotic coronary and carotid artery disease and is associated with elevated plasma inflammatory markers, such as Interleukin (IL)-6. In carotid artery atherosclerosis, macrophages are considered a local marker of inflammation. Hypothesis: We hypothesize that AF is independently associated with carotid plaque inflammation. Aims: In patients undergoing carotid endarterectomy (CEA), we aimed to determine whether carotid plaques inflammation is associated with AF. Approach: In this prospective cohort study, consecutive patients with carotid artery disease undergoing CEA were enrolled. From the plaques removed during surgery, fixed in formalin and embedded in paraffin blocks, sections were stained with antibodies against CD68 to detect macrophages (Fig.1A) . Digital copies of the slides were created by Motic Easy Scan and histologically annotated with QuPath. The percentage of CD68+ cells was defined as (CD68+ positive cells/total nuclei) x 100. Flash-frozen plaque segments were analyzed by ELISA for IL-6 concentration. Baseline demographics and clinical data, including AF diagnosis, were extracted from the electronic health records. Results: A total of 106 patients undergoing CEA, with a median age of 73 [67-78] years and comprising 83 (78.3%) males, were included in the study. AF was diagnosed prior to CEA in 13 (12.3%) patients. Univariate logistic regression showed that plaque CD68+ cell content was associated with AF (OR 1.048 [95% CI 1.010 – 1.088]; p = 0.013). Additionally, in Mann-Whitney test IL-6 levels were significantly over twofold higher in plaques of patients with AF compared to those without AF (82.4 [64.9-111.7] pg/mg vs 37.0 [18.4-93.3] pg/mg; p=0.020) (Fig. 1B) . Conclusions: The current study demonstrates an association between carotid atherosclerotic plaque macrophage content, IL6 levels and AF, supporting the role of inflammation in the pathogenesis of AF in these patients. Further studies are needed to determine the potential of plaque inflammation as a therapeutic target in AF.

Abstract Background Transforming growth factor beta (TGFβ) and its receptors (TGFβRs) are widely expressed by most human and murine cells and are implicated in several physiological and pathological conditions. In the context of atherosclerosis, TGFβ has been identified as a pleiotropic molecule involved in both pro- and anti-atherogenic processes, with its effects varying depending on the cell type it signals through. Purpose Given that myeloid cells play a major role in atherogenesis, we sought out to unravel the involvement of myeloid TGFβ signaling in atherosclerosis. Methods Aortic leukocytes from ApoE-/- mice fed a Western diet (WD) for 20 weeks were subjected to CITE-Seq analysis to assess the expression profile of Tgfβ and Tgfβrs. To investigate the effect of myeloid Tgfβ signaling on atherosclerosis, LysMCre+Tgfβr1flox/floxTgfβr2flox/floxmT/mGflox/floxApoE-/- (M-Tgfβr1/2dkoApoE-/-) mice and their LysMCre- littermates (ApoE-/-) were fed a WD for 6 weeks. Leukocyte phenotyping was assessed by flow cytometry (FC) and qPCR. Atherosclerotic plaque size in the aortic root was assessed by immunohistochemistry. Results CITE-Seq revealed that aortic ApoE-/- leukocytes highly expressed Tgfb1, whereas Tgfb2 and Tgfb3 were very lowly expressed. Tgfbr1 was mainly expressed in myeloid cells with macrophages and neutrophils showing the highest expression. Tgfbr2 was primarily expressed in T and B cells, while still showing a high level of expression in macrophages. Importantly, Tgfbr3 was not expressed in myeloid cells. Thus, to study the effect of myeloid Tgfβ signaling on atherosclerosis and to mitigate the potential for aberrant signaling that might arise from deleting only one receptor, we generated ApoE-/- mice with myeloid lineage tracing mT/mG alleles and combined deletion of Tgfbr1 and Tgfbr2 in myeloid cells using the LysMCre model. FC analysis of eGFP+ cells, i.e., cells where LysMCre is active, revealed a ~60-80% Tgfbr1/2 deletion efficiency in bone marrow-derived macrophages (BMDM) and in blood, splenic and BM CD11b+ cells, which was accompanied by decreased Smad2/3 signaling. M-Tgfβr1/2dkoApoE-/- mice showed a ~35% increase in blood and splenic CD11b+ cells and a ~25% increase in splenic macrophages compared to ApoE-/- controls, while BM CD11b+ cells were not affected. Tgfbr1/2-deficient BMDMs had increased Il-6, Il-1b and CD38 expression compared to controls, suggesting that Tgfbr1/2-deficient macrophages have acquired a pro-inflammatory phenotype. Interestingly, M-Tgfβr1/2dkoApoE-/- mice had ~30% fewer blood and splenic T cells compared to controls, which was explained by decreased naïve CD4+ and CD8+ T cells. M-Tgfβr1/2dkoApoE-/- mice had a ~50% increase in atherosclerotic plaque burden compared to ApoE-/- controls. Conclusion Our data suggest that impaired myeloid Tgfβ signaling leads to a pro-atherogenic environment in ApoE-/- mice. These effects may be mediated by an altered crosstalk between T cells and Tgfβr1/2-deficient macrophages.

Abstract Background Atherosclerosis is a chronic inflammatory disease and the primary underlying cause of cardiovascular disease (CVD). Recently, intracellular metabolic pathways have been identified as master switches of immune cell function and thus seem promising targets for therapeutic interventions aimed at lowering inflammation and thereby atherogenesis. Decades of research have demonstrated the importance of the amino acid (AA) L-arginine (Arg) in CVD. Although most research has focused on endothelium-dependent effects of Arg by the nitric oxide synthase (NOS), Arg metabolism - mainly through its metabolizing enzyme arginase 1 (Arg1) - has also been shown to regulate immune cell responses independent of its endothelial functions. Despite these findings, the cell-specific contribution of Arg/Arg1 metabolism on atherogenesis is still largely unknown. Purpose Profiling the Arg metabolism in endothelial and immune cells to identify cell-specific changes during athero-progression. Methods To investigate Arg metabolism in atherogenesis, we fed apolipoprotein E deficient (Apoe-­/­-) mice a Western diet (WD) for 6 and 12 weeks. Chow diet-fed aged-matched Apoe-­/-­ mice were used as littermate controls and C57BL/6 mice as steady-state controls. Systemic and local Arg concentrations were measured by mass-spectrometry. Arg metabolism in organs was studied by qPCR. Arg1 expression in atherosclerotic lesions was examined by immunohistochemistry (IHC). To assess cell-specific expression of Arg-related enzymes, aortic leukocytes from WD-fed Apoe-­/-­ mice were subjected to CITE-seq analysis. The role of T cell Arg1 was determined by nor-NOHA inhibition in Jurkat cells. Results AA measurements showed that systemic Arg was reduced in early and advanced atherogenesis (p<0.02), and splenic Arg was reduced in advanced atherogenesis (p=0.006) compared with steady state. In advanced atherosclerosis, Arg1 was decreased in the liver but increased in the spleen and aorta, whereas Nos2 was increased in the liver and aorta compared with steady state. To identify the cell types driving these local changes, we performed IHC on atherosclerotic lesions. Arg1+ lesional macrophages accumulated in advanced compared to early lesions (p=0.03), whereas Arg1+ endothelial cells tended to decrease (p=0.2). Surprisingly, we also found an accumulation of Arg1+ CD4+ T cells in lesions and a further upward trend with athero-progression. CITE-seq confirmed a constitutive expression of Arg1 in lymphoid clusters. In vitro experiments with Jurkat cells showed that arginase inhibition suppressed T cell proliferation and promoted apoptosis and migration (all p<0.0001). Conclusion Our data indicate that Arg metabolism is strongly regulated during athero-progression and suggest a so far unknown role of T cell-intrinsic Arg/Arg1 metabolism in the disease. Murine atherosclerosis studies are now being performed to decipher the cell-specific contribution of Arg/Arg1 metabolism in atherosclerosis.

Abstract Background Patients with carotid artery disease undergoing carotid endarterectomy (CEA) appear to be at high risk of all-cause mortality during their follow-up. However, the possible predictors of this adverse outcome are not elucidated yet. Purpose We aimed to test cardiovascular (CV) risk factors as possible predictors of all-cause mortality in patients undergoing CEA and to determine whether there is a difference between the mortality rate of our study cohort and the general population. We hypothesize that CEA-treated patients have a higher mortality rate during follow-up after surgery than the general population, which is not related to the presence of CV risk factors. Methods We prospectively enrolled and followed up patients undergoing CEA. Univariate and multivariable Cox regression were performed to test whether common CV risk factors (demographics, laboratory values, comorbidities, and events before CEA) were predictive of all-cause mortality. We then compared the standardized mortality ratio (SMR) of our population and the age-sex-matched Minnesota (MN) general population at 5,10, and 21 years of follow-up after CEA. Results A total of 710 patients were followed for a median of 7.1 [2.9-11.9] years after CEA. Mortality occurred in 198 (27.0%) patients during the follow-up. The median time to event was 11.0 [10.0-12.0] years. Age was the only predictor of all-cause mortality in CEA-treated patients in multivariable models adjusted for known CV risk factors (Model 1: HR 1.03 [95%CI 1.01-1.06]; p=0.004; Model 2: HR 1.02 [95%CI 1.00-1.04]; p=0.023) (Table 1). However, the CEA-treated group had a higher mortality rate than would have been expected in the MN population (log-rank p<0.001) (Fig.1A). Compared to the MN population, at 5 years of follow-up only patients <60 years old had a higher SMR (4.53 [1.95-8.92]; p=0.001), whereas, at 10 years and 21 years of follow-up, our study population had a higher mortality rate at any age (Fig.1B). Conclusions The elevated mortality risk post-CEA in our cohort cannot be solely attributed to chronological aging. The underlying process of atherosclerosis likely contributes to all-cause mortality, and plaque characteristics may offer additional insight into the risk stratification of this patient group.All cause mortality predictors after CEAStudy population vs. MN population SMR