Faculty Bibliography

Coronary artery disease (CAD) is a major cause of global morbidity and mortality. Macrophages play a central role in orchestrating this disease process. In 2016, we initiated the STARNET primary blood macrophage study, followed by the multi-ethnic NGS-PREDICT primary blood macrophage study in 2018. We applied integrative systems genetics analysis to explore and validate the role of macrophage gene regulatory co-expression networks (GRNs) in clinically significant CAD. This study included 318 CAD cases and 134 CAD-free controls in STARNET, and 95 CAD cases and 35 CAD-free controls in NGS-PREDICT. Primary leukocytes were isolated from blood and differentiated into macrophages in vitro, followed by RNA extraction and deep sequencing (RNAseq). In STARNET, we analyzed differentially expressed genes, inferred macrophage GRNs, assessed the phenotypic associations and functions of these GRNs, and determined their key driver genes. Integrative analysis of STARNET expression quantitative traits (eQTLs) with genotype data from genome-wide association studies was performed to determine the content of CAD candidate genes in these GRNs, and their contributions to CAD heritability. Five independent RNAseq datasets were used to retrospectively validate CAD-associated macrophage GRNs, followed by prospective validation in the NGS-PREDICT study. Using the STARNET datasets, we identified 23 macrophage GRNs. Of these, GRN_GREEN stood out as being causally associated with CAD severity (SYNTAX score) and comprised 729 genes and 90 key drivers, with the top key driver being NEIL1. GRN_GREEN accounted for 3.73% of CAD heritability and contained 34 candidate genes previously identified by GWAS of CAD. Functional analysis of GRN_GREEN revealed a large portion of genes involved in the biological process of SRP-dependent co-translational protein targeting to the membrane. GRN_GREEN replicated retrospectively in five independent human arterial wall RNAseq datasets, and prospectively in the NGS-PREDICT study. To prevent clinically significant CAD, GRN_GREEN and its top key driver NEIL1 may be suitable therapeutic targets to modify SRP-dependent co-translational targeting of proteins to the endoplasmic reticulum in macrophages.

Cardiovascular diseases including atherosclerosis and heart failure, arise from the intricate interplay of metabolic, immune, and neural dysregulation within vascular and cardiac tissues: This review focuses on integrating recent advances in metabolic and immune crosstalk of the cardiac vasculature that affects cardiometabolic health and disease progression. Coronary and lymphatic endothelial cells regulate cardiac metabolism, and their dysfunction is linked to cardiovascular diseases. Lymphatics maintain tissue homeostasis, including clearing metabolic waste, lipids, and immune cells, and their maladaptation in metabolic diseases worsens outcomes. Altered vascular endothelial metabolism in heart failure drives immune-mediated inflammation, fibrosis, and adverse cardiac remodeling. Concurrently, artery tertiary lymphoid organs formed in the adventitia of advanced atherosclerotic arteries, serve as pivotal neuroimmune hubs, coordinating local immunity through T and B cell activation and neurovascular signaling via artery-brain circuits. T cells within plaques and artery tertiary lymphoid organs undergo clonal expansion as a result of peripheral tolerance breakdown, with proinflammatory CD4⁺ and CD8⁺ subsets amplifying atherosclerosis, effects further shaped by systemic immune activation. Therapeutic strategies targeting endothelial cell metabolism, lymphatic dysfunction, neuroimmune crosstalk, and T cell plasticity hold promise for integrated cardiovascular disease management.

BACKGROUND:New evidence has emerged that plastic polymers and their chemical additives, particularly di-2-ethylhexylphthalate (DEHP), contribute to cardiovascular disease (CVD). Phthalates are commonly used in the production of plastic materials and have been linked to increased oxidative stress, metabolic dysfunction, and cardiovascular disease. Estimates of phthalate-attributable cardiovascular mortality have been made for the US, but global estimates are needed to inform ongoing negotiations of a Global Plastics Treaty. METHODS:Cardiovascular mortality data from the Institute for Health Metrics and Evaluation (IHME) and regional DEHP exposure estimates from several sources were used to estimate burden. Hazard ratios of CV mortality were calculated using published exposure estimates, and country-level cardiovascular mortality rates were used to calculate excess deaths and years of life lost (YLL) due to DEHP exposure. FINDINGS/RESULTS:In 2018, an estimated 356,238 deaths globally were attributed to DEHP exposure, representing 13.497% of all cardiovascular deaths among individuals aged 55-64. Of these, 349,113 were attributed to the use of plastics. Geographic disparities were evident, with South Asia and the Middle East suffering the greatest percentage of cardiovascular deaths attributable to DEHP exposure (16.807%). The Middle East, South Asia, East Asia, and the Pacific accounted for the largest shares of DEHP-attributable CVD deaths (73.163%). Globally, DEHP resulted in 10.473 million YLL. INTERPRETATION/CONCLUSIONS:Plastics pose a significant risk to increased cardiovascular mortality, disproportionately impacting regions which have developing plastic production sectors. The findings underscore the need for urgent global and local regulatory interventions to kerb mortality from DEHP exposure. FUNDING/BACKGROUND:Bloomberg Philanthropies and the National Institutes of Health.

Immune checkpoint inhibitor (ICI) therapies can increase the risk of cardiovascular events in survivors of cancer by worsening atherosclerosis. Here we map the expression of immune checkpoints (ICs) within human carotid and coronary atherosclerotic plaques, revealing a network of immune cell interactions that ICI treatments can unintentionally target in arteries. We identify a population of mature, regulatory CCR7⁺FSCN1⁺ dendritic cells, similar to those described in tumors, as a hub of IC-mediated signaling within plaques. Additionally, we show that type 2 diabetes and lipid-lowering therapies alter immune cell interactions through PD-1, CTLA4, LAG3 and other IC targets in clinical development, impacting plaque inflammation. This comprehensive map of the IC interactome in healthy and cardiometabolic disease states provides a framework for understanding the potential adverse and beneficial impacts of approved and investigational ICIs on atherosclerosis, setting the stage for designing ICI strategies that minimize cardiovascular disease risk in cancer survivors.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1β. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1β release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1β levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

Genetic and experimental evidence suggests that Alzheimer's disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages, including microglia, to damage of lipid-rich tissues like the brain. Recently, sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues, we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors, BHLHE40/41, in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia, resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing, increased cholesterol efflux and storage, and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues.

Patients with coronavirus disease 2019 (COVID-19) present increased risk for ischemic cardiovascular complications up to 1 year after infection. Although the systemic inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques remains unknown. Here we report that SARS-CoV-2 viral RNA is detectable and replicates in coronary lesions taken at autopsy from severe COVID-19 cases. SARS-CoV-2 targeted plaque macrophages and exhibited a stronger tropism for arterial lesions than adjacent perivascular fat, correlating with macrophage infiltration levels. SARS-CoV-2 entry was increased in cholesterol-loaded primary macrophages and dependent, in part, on neuropilin-1. SARS-CoV-2 induced a robust inflammatory response in cultured macrophages and human atherosclerotic vascular explants with secretion of cytokines known to trigger cardiovascular events. Our data establish that SARS-CoV-2 infects coronary vessels, inducing plaque inflammation that could trigger acute cardiovascular complications and increase the long-term cardiovascular risk.