Monocytes defined by platelet interactions and oxidative stress signaling underlie HIV-associated atherosclerosis

Background: Monocytes contribute to atherosclerosis (AS) by migrating into inflamed endothelium and differentiating into foam cells. In people living with HIV (PLWH), chronic inflammation increases AS risk, yet the role of specific monocyte clusters remains unclear. We investigated how distinct monocyte populations contribute to vascular pathology in early HIV-associated AS. Methods: We profiled 123,965 circulating monocytes using single-cell RNA-seq and integrated plasma microparticle proteomics in 32 individuals stratified by HIV and AS status. Supervised learning identified cluster- and disease-specific signatures, validated by platelet-monocyte co-cultures, RT-qPCR, bulk RNA-seq, flow cytometry and ELISA. Results: Seven monocyte clusters were identified, including a subset characterized by platelet–monocyte complexes (PMCs). Bulk RNA-seq of platelet–monocyte co-cultures revealed platelet-driven upregulation of genes involved in inflammation, lipid metabolism, oxidative stress, and endothelial adhesion. PMC-derived macrophages secreted higher levels of TGF-ß and IL-10, displayed decreased CD14 and increased CD80/CD86 while retaining CD36, and promoted endothelial-to-mesenchymal transition (decreased CDH5, PECAM1; increased S100A4) and vascular inflammation (increased ICAM1, VCAM and IL-6, CCL2 secretion). Additionally, CD14+ monocytes from HIV+AS- and HIV+AS+ groups showed enhanced ROS-NRF2 pathway activities, supported by increased basal and H2O2-induced p90RSK phosphorylation, indicating oxidative stress priming. Conclusions: Two monocyte clusters contribute independently to vascular immune dysregulation in PLWH. PMC-derived macrophages promote endothelial dysfunction while adopting a profibrotic cytokine profile. CD14+ monocytes show heightened oxidative signaling and stress responses, consistent with vascular activation. Together, these mechanisms may accelerate AS development in HIV, even in the absence of traditional cardiovascular risk factors. Overall design: Fresh blood from an ongoing URMC cohort was used to isolate monocytes and platelets for in vitro coculture experiments. Monocytes were cultured alone or cocultured with collagen-activated platelets at monocyte-to-platelet ratios of 1:100 and harvested at 24 hours (n=7) and 6 days (n=8). After coculture, monocytes were re-isolated and processed for RNA extraction to evaluate platelet-induced transcriptional changes.