Anthony Wayne Orr

Background: Premature coronary artery disease (pCAD) is a distinct presentation of CAD. Using the All of Us (AoU) Research Program data, we compared pCAD with later-onset CAD across cardiometabolic, polygenic, and genome-wide domains. Methods: We conducted a retrospective, cross-sectional case-case analysis of All of Us Research Program (AoU) participants with established CAD. pCAD was defined as first CAD event before age 55 years in males and before age 65 in females; later-onset CAD was defined as the first CAD event at or after age threshold. Pre-index cardiometabolic measures were extracted from electronic health records and modelled with logistic regression. Using 607,719 variants, a CAD polygenic risk score (PRS) was constructed using published summary statistics and short-read whole-genome sequencing data (srWGS). Results were standardized within self-reported race groups, and evaluated overall, by race, and with a PRS x race interaction. A case-case genome-wide association study (GWAS) compared pCAD with later-onset CAD. Results: The CAD cohort included 49,919 participants, including 18,455 with pCAD and 31,464 with later-onset CAD. Participants with pCAD had higher BMI, LDL cholesterol and diastolic blood pressure, and lower HDL cholesterol, serum calcium, and systolic blood pressure. Higher BMI and LDL cholesterol were positively associated with higher odds of pCAD, whereas higher HDL cholesterol, systolic blood pressure, and serum calcium were inversely associated. PRS was associated with greater odds of pCAD in the genomic models (OR per 1-SD higher PRS, 1.14; 95% CI, 1.12–1.17; P P =5.8x10 ^-9 ). A total of 137 variants met an exploratory threshold of P -5 . Conclusions: Premature onset of CAD is characterized by a distinct cardiometabolic profile driven by modifiable risk factors, greater polygenic burden, and genomic signals associated with earlier disease timing. A European-derived PRS did not transfer to Black participants, who were overrepresented in pCAD. Together, these findings support pCAD as a clinically and genetically distinct presentation of CAD and highlight the need for ancestry-specific validation of genomic risk tools in diverse cohorts.

BACKGROUND: Apoptotic cell clearance by macrophages, termed efferocytosis , resolves inflammation and promotes tissue repair, whereas defective efferocytosis contributes to chronic inflammatory diseases such as atherosclerosis. Putrescine biosynthesis, driven by the rate-limiting enzyme ODC1 (ornithine decarboxylase 1), has emerged as a key efferocytosis-induced metabolic pathway that promotes inflammation resolution. However, the mechanisms regulating ODC1 during atherosclerosis remain undefined. Here, we investigated the mechanisms controlling ODC1 and putrescine biosynthesis and identified a novel integrin αv–FAK (focal adhesion kinase)–Rac1 (Rac family small GTPase 1)–STAT3 (signal transducer and activator of transcription 3)–MYC (MYC proto-oncogene) signaling axis that becomes progressively impaired during atherosclerosis. METHODS: We performed metabolomic and cytokine profiling of human atherosclerotic plaques and assessed ODC1 expression in lesional macrophages from humans and mice. Mechanistic studies used apoptotic cell– or polystyrene bead–exposed macrophages, Ldlr ^{–/ –} mice with hematopoietic integrin αv deficiency fed a Western-type diet, single-nuclei RNA sequencing, and bulk RNA sequencing. RESULTS: Putrescine levels negatively correlated with inflammatory mediators, and ODC1 expression in lesional macrophages declined with disease progression in humans and mice. Engulfment of apoptotic cells or polystyrene beads increased ODC1 expression through an integrin αv– and actin polymerization–dependent mechanism. Hematopoietic integrin αv deficiency exacerbated lesion size and necrosis, reduced efferocytosis, and decreased fibrous cap thickness. Single-nuclei RNA sequencing showed no changes in lesional cell composition but revealed activation of inflammatory pathways specifically in integrin αv–deficient macrophages. Mechanistically, phagocytosis-induced ODC1 expression relied on integrin αv–mediated STAT3 activation and MYC-dependent Odc1 transcription. Bulk RNA sequencing showed that apoptotic cell–exposed human macrophages lacking integrin αv upregulated atherogenic mediators and inflammatory pathways, an effect reversed by restoring putrescine levels. CONCLUSIONS: Together, these findings uncover a previously unrecognized mechanistic link between phagocytosis and macrophage metabolism, which we identify as a case of mechanometabolism, and demonstrate how its dysregulation compromises inflammation resolution and promotes atherosclerosis.

Background: Contemporary cardiovascular disease (CVD) risk equations may not fully capture cumulative biological aging or long-term exposure burden. DNA methylation (DNAm) biomarkers may capture aging- and exposure-related biology, but their incremental prognostic value beyond clinical risk-factor models like PREVENT remains uncertain. To our knowledge, no prior study has benchmarked DNAm-based biomarkers with PREVENT. Methods: In a population-based cohort study, we analyzed NHANES 1999-2002 participants with DNAm biomarkers and mortality follow-up. We derived a DNAmScore from candidate DNAm biomarkers using elastic-net Cox regression with repeated nested cross-validation. A PREVENT-like clinical model was defined as a Cox model fit in NHANES using PREVENT predictors. Weighted Cox models estimated the association between DNAmScore and mortality after adjustment for PREVENT-like clinical predictors. We then compared the PREVENT-like clinical model, DNAmScore alone, and a combined model (PREVENT-like clinical predictors plus DNAmScore) using cross-fitted C-index, time-dependent AUC, calibration, and Brier score. Results: Our cohort included 2,282 participants; 597 and 937 deaths occurred by 10 and 15 years, respectively. After adjustment for PREVENT-like clinical predictors, the cross-fitted DNAmScore was strongly associated with all-cause mortality (HR per 1-SD increase, 2.43; 95% CI, 1.97–2.99). At 10 years, AUCs were 0.791 for the PREVENT-like model, 0.791 for DNAmScore, and 0.803 for the combined model. At 15 years, corresponding AUCs were 0.825, 0.822, and 0.835. Compared with the PREVENT-like model, the combined model improved AUC by 0.013 (95% CI, 0.006–0.020) at 10 years and 0.010 (95% CI, 0.004–0.015) at 15 years. The combined model had lower Brier scores at all three horizons with similar calibration. DNAmScore remained associated with CVD mortality after clinical adjustment. Conclusions: DNAmScore identified residual biological risk beyond PREVENT-like clinical predictors, with strong independent mortality associations and modest, consistent improvements in cross-fitted prediction performance. These findings support development and external validation of CVD-specific DNAm biomarkers. What Is Known: Contemporary cardiovascular risk models may not fully capture cumulative biological aging and exposure burden. DNA methylation biomarkers are associated with mortality, but their incremental predictive value beyond clinical risk predictors remains uncertain. What the Study Adds: In a nationally representative NHANES cohort, a cross-fitted composite DNAmScore remained strongly associated with mortality after adjustment for PREVENT predictors. Adding DNAmScore produced modest improvements in discrimination and prediction error at 5, 10, and 15 years, and DNAmScore was also associated with cardiovascular mortality in secondary analyses.

BACKGROUND: Macrophages are key players in the pathogenesis of atherosclerosis. They trigger immune responses through their cell-surface receptors. However, how macrophages regulate those receptors in response to proinflammatory stimuli is not completely understood. Endocytic membrane trafficking involving receptor internalization, followed by endosomal transport and recycling of the internalized receptors, plays essential roles in balancing cell-surface receptors to meet cellular needs. Here, we explored the role of the endocytic regulator EHD (c-terminal Eps15 [Epidermal growth factor receptor substrate 15] homology domain) 1 in immune responses in macrophages and determined its contribution to atherosclerosis progression. METHODS: EHD1 expression profiles in mouse and human plaques were determined by single-cell RNA sequencing and immunofluorescence staining. Bone marrow transplantation by transplanting bone marrow cells from Ehd1 ^−/− or littermate wild-type mice to irradiated Ldlr ^−/− mice was performed to determine the effect of EHD1 deletion on atherosclerosis progression. In vitro mechanistic studies, including inflammation signaling and endocytosis assays, were performed in bone marrow–derived macrophages. RESULTS: EHD1 expression in macrophages is enhanced as atherosclerosis progresses in both mice and humans. Histological analysis of aortic root sections from bone marrow transplantation mice showed that EHD1 deletion reduces lesion size. Single-cell RNA sequencing of aortic CD (cluster of differentiation) 45 ⁺ cells demonstrated that EHD1 deletion attenuates proinflammatory responses and cell–cell interactions. Mechanistic studies revealed that EHD1 accelerates the endocytic recycling of TNFR2 (tumor necrosis factor receptor 2) and activates NF-κB (nuclear factor kappa B), leading to increased expression of inflammatory cytokines. Moreover, EHD1 interacts with retromer and stabilizes sortilin, a retrograde cargo of retromer and a risk factor for atherosclerosis. CONCLUSIONS: EHD1 promotes inflammation by enhancing TNFR2−NF-κB signaling and stabilizing sortilin, leading to accelerated atherosclerosis. Our study reveals novel roles for EHD1-mediated membrane trafficking in macrophage function and paves the way to innovative therapeutic strategies that aim to address dysregulated membrane trafficking in atherosclerosis.

Neutrophil-mediated persistent inflammation and neutrophil extracellular trap formation (NETosis) are critical in the pathogenesis of deep vein thrombosis (DVT). Identifying the mechanisms controlling these proinflammatory and prothrombotic functions is critical for designing and developing new therapeutics for DVT. However, how neutrophils acquire these phenotypes during the early stages of DVT remains poorly understood. Here, multiomic analyses (RNA sequencing, proteomics, and flow cytometry) of bone-marrow neutrophils 3 hours after DVT, revealed consistent overexpression of CD14. Concurrently, mice with DVT exhibited significantly elevated plasma granulocyte colony-stimulating factor (G-CSF) and neutrophil hyperactivation. Mechanistic studies using a geometric deep learning model (DeepPBS) and chromatin immunoprecipitation revealed that elevated G-CSF drives CD14 overexpression by upregulating the transcription factor C/EBPα (leucine zipper CCAAT-enhancer binding protein α). Importantly, neutrophil-specific CD14 knockdown using a novel lentiviral short hairpin RNA approach significantly improved DVT outcomes by lowering thrombus burden, thrombosis incidence, and intrathrombus neutrophil and citrullinated histone H3 accumulation. Studies in G-CSF stimulated primary human neutrophils revealed that CD14 inhibition reduces markers of inflammation and NETosis (activated gasdermin D, citrullinated histone H3, and S100A8/A9), while increasing apoptosis, thus demonstrating translational relevance. Collectively, our study uncovers an essential role of neutrophil CD14 in DVT pathogenesis and establishes it as a promising therapeutic target for DVT.

Background: Macrophages are key players in the pathogenesis of atherosclerosis. They trigger immune responses through their cell-surface receptors. However, how macrophages regulate those receptors in response to pro-inflammatory stimuli is not completely understood. Endocytic membrane trafficking involving receptor internalization, followed by endosomal transport and recycling of the internalized receptors, plays essential roles in balancing cell-surface receptors to meet cellular needs. Here, we explored the role of the endocytic regulator EHD1 in immune responses in macrophages and determined its contribution to atherosclerosis progression. Methods: EHD1 expression profiles in mouse and human plaques were determined by single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining. Bone marrow transplantation (BMT) by transplanting bone marrow cells from Ehd1 ^−/− or littermate wild-type mice to irradiated Ldlr ^−/− mice was performed to determine the effect of EHD1 deletion on atherosclerosis progression. In vitro mechanistic studies including inflammation signaling and endocytosis assays were performed in bone marrow–derived macrophages. Results: EHD1 expression in macrophages is enhanced as atherosclerosis progresses in both mice and humans. Histological analysis of aortic root sections from BMT mice showed that EHD1 deletion reduces lesion size. ScRNA-seq of aortic CD45 ⁺ cells demonstrated that EHD1 deletion attenuates pro-inflammatory responses and cell–cell interactions. Mechanistic studies revealed that EHD1 accelerates the endocytic recycling of TNFR2 and activates NF-kB, leading to increased expression of inflammatory cytokines. Moreover, EHD1 interacts with retromer and stabilizes sortilin, a retrograde cargo of retromer and a risk factor for atherosclerosis. Conclusions: EHD1 promotes inflammation by enhancing TNFR2−NF-kB signaling and stabilizing sortilin, leading to accelerated atherosclerosis. Our study reveals novel roles for EHD1-mediated membrane trafficking in macrophage function and paves the way to innovative therapeutic strategies that aim to address dysregulated membrane trafficking in atherosclerosis.

BACKGROUND: Neutrophil infiltration exacerbates brain injury after subarachnoid hemorrhage (SAH). Integrin α9, expressed on neutrophils, facilitates their adhesion and transendothelial migration, leading to aggravated inflammatory responses and neuronal apoptosis. Insufficient clearance of apoptotic neurons by microglia and infiltrating blood-derived macrophages (defective efferocytosis) contributes to persistent inflammation and poor SAH recovery. This study investigated the role of neutrophil integrin α9 in neuronal apoptosis, microglia/macrophage efferocytosis, and SAH outcomes. METHODS: Neutrophil-specific α9 ^–/– ( α9 ^fl/fl Mrp8Cre ^–/+ ) and littermate control ( α9 ^fl/fl Mrp8Cre ^–/– ) mice were subjected to the endovascular perforation model to induce SAH. Sensorimotor and cognitive function were assessed for up to 4 weeks post-SAH using neurological severity score, corner and cylinder tests, Y-maze, and novel object recognition. In vitro and in vivo functional assays were conducted to assess the effect of integrin α9–dependent neutrophil transendothelial migration on efferocytosis of apoptotic neurons. Neutrophil infiltration, cerebral inflammation, neuronal apoptosis, and MMP (matrix metalloproteinase)-9 were quantified 24 hours post-SAH. RESULTS: Mice subjected to SAH exhibited increased integrin α9 levels on infiltrated neutrophils compared with sham surgery controls. Neutrophil-specific α9 ^–/– mice demonstrated improved long-term sensorimotor and cognitive recovery, reduced neutrophil infiltration, and decreased MMP-9 expression and neuronal apoptosis. Importantly, neutrophil-specific α9 ^–/– mice exhibited reduced brain neutrophil elastase levels and enhanced efferocytosis. Mechanistic studies have revealed that the reduced transendothelial migration of α9 ^–/– neutrophils directly contributed to the enhanced microglia/macrophage efferocytosis of apoptotic neurons. Pharmacological targeting of integrin α9 with macitentan significantly improved SAH outcomes by reducing neutrophil infiltration and enhancing efferocytosis. Comparable SAH outcomes in both macitentan-treated controls and neutrophil-specific α9 ^–/– mice suggested that the therapeutic effects of macitentan were mediated by inhibition of neutrophil integrin α9. CONCLUSIONS: Our study revealed a novel role for neutrophil integrin α9 in sensorimotor function and cognitive recovery after SAH, suggesting it as a potential therapeutic target for SAH.