Roles for NOX4 in macrophage (DYS)function and atherogenesis

The National Heart, Lung, and Blood Institute awarded Wake Forest University Health Sciences $100,228 in a Project Grant (F31HL186704) on December 15, 2026, under the Cardiovascular Diseases Research program (CFDA 93.837).

The award funds research investigating the role of NADPH oxidase 4 (NOX4) in macrophage dysfunction and atherogenesis. The research examines how metabolic stress—specifically high-calorie diet consumption—induces NOX4 expression in monocytes and macrophages, triggering hydrogen peroxide production and protein S-glutathionylation that drives dysfunctional immune responses and accelerates atherosclerosis. The investigator will use conditional myeloid-specific NOX4 knockdown mice with an atherosclerosis-prone genetic background fed a high-calorie diet for 20 weeks to test the hypothesis that NOX4-induced proteome reprogramming enhances monocyte chemotaxis, accelerates macrophage accumulation in the vascular wall, and promotes proinflammatory macrophage polarization.

Performance occurs in Winston-Salem, North Carolina, with a period of performance through June 30, 2028. This is a training grant supporting early-career investigator development in cardiovascular research.

Abstract

Macrophages are essential for tissue and metabolic homeostasis, but in the context of metabolic disorders, they become dysfunctional and promote chronic inflammatory diseases, including atherosclerosis. Consuming a high-calorie diet (HCD) stimulates the production of hydrogen peroxide (H2O2) in blood monocytes. The resulting thiol oxidative stress promotes the formation of mixed disulfides between protein thiols and glutathione, a reaction referred to as "protein S-glutathionylation." the S-glutathionylation of proteins is a reversible posttranslational modification. However, if sustained, in most instances, leads to conformational changes within the protein, loss of function and, in some cases, degradation of the target proteins, resulting in the reprogramming of the monocyte proteome. Blood monocytes reprogrammed by HCD exposure become dysfunctional and show enhanced chemotaxis and increased recruitment to sites of inflammation, as well as dysregulated polarization profiles. However, the source of H2O2 responsible for the reprogramming of monocytes by metabolic stress and its protein targets remain largely unexplored. We identified nadph oxidase 4 (NOX4) as a novel, inducible H2O2- generating nadph oxidase in monocytes and macrophages. The central hypothesis of my proposal is therefore that metabolic stress-induced NOX4 expression in monocytes and macrophages promotes atherogenesis by triggering H2O2-induced protein S-glutathionylation and proteome reprogramming, which in turn enhances monocyte chemotactic activity, accelerates the accumulation of monocyte- derived macrophages within the vascular wall, and skews the polarization of macrophages within the vessel toward proinflammatory phenotypes. To test this hypothesis, I will use novel conditional myeloid- specific NOX4 knockdown mice with an atherosclerosis-prone ldlr KO genetic background and feed these mice a HCD for 20 weeks to induce atherogenesis. To investigate the role of NOX4 in monocyte proteome reprogramming and the overrecruitment of dysregulated macrophages into atherosclerotic lesions, I propose to employ state-of-the art redox proteomics and bulk RNA-SEQ transcriptomics combined with custom-designed qpcr-based high-throughput gene profiling approaches. In addition, I will use immunoprecipitation and western blot approaches to elucidate the molecular mechanisms responsible for dysregulating the activation patterns of key transcription factors involved in macrophage polarization and signaling, and to identify the roles of NOX4 in this process. This fellowship provides a well-structured training plan in a supportive, well-equipped research environment. The plan focuses on macrophage biology, redox biochemistry and vascular disease, using state- of-the-art omics, profiling approaches and novel transgenic mouse models. The research plan I propose will provide me with an outstanding training opportunity in an excellent training environment and prepare me for the next phase of my career.