Integrated human carotid plaque transcriptomics and macrophage perturbation link BCAP31 to endoplasmic reticulum stress, inflammation, and apoptosis

Background Atherosclerotic plaque progression couples chronic inflammation to dysregulated cell survival, but the cellular context and regulatory role of endoplasmic reticulum (ER)-associated death responses in human carotid disease remain incompletely resolved. Methods We integrated paired bulk transcriptomes from 32 carotid plaque and macroscopically intact tissue pairs, laser-captured macrophages from five ruptured and six stable plaques, whole-plaque transcriptomes from 27 intraplaque-hemorrhage and 16 non-hemorrhage lesions, and single-cell RNA sequencing from three patients with matched atherosclerotic core and proximal adjacent tissue. BCAP31 was evaluated by siRNA-mediated depletion in oxidized low-density lipoprotein (oxLDL)-exposed THP-1-derived macrophages, with PCR, immunofluorescence, ELISA, apoptosis assays, and 4-phenylbutyrate treatment. Results Paired bulk analysis showed coordinated increases in apoptosis, ferroptosis, pyroptosis, necroptosis, inflammasome, inflammatory-response, and TNF/NF-κB programs. Ruptured plaque macrophages showed increases in ferroptosis, pyroptosis, and necroptosis scores, whereas intraplaque-hemorrhage plaques showed broader activation across all prespecified programs. Patient-level single-cell pseudobulk analysis localized a directionally consistent increase in BCAP31 to macrophages, although the estimate did not remain significant after transcriptome-wide correction in the three-patient dataset. Cross-sectional state ordering placed higher BCAP31 expression toward plaque-enriched HLA-C1QC, APOC1-SPP1-remodeling, and SPP1-lipid-loaded macrophage states, while predicted macrophage-to-vascular-wall SPP1 signaling increased in all three patient pairs. BCAP31 was also increased in paired plaque, rupture-macrophage, and intraplaque-hemorrhage cohorts and belonged to a plaque-associated coexpression module. In macrophages derived from THP-1 cells and treated with oxLDL, BCAP31 knockdown increased ER-stress transcripts, nuclear DDIT3, inflammatory cytokine expression and release, and Annexin V-positive apoptosis. The chemical chaperone 4-phenylbutyrate attenuated these effects. Conclusion Human plaque transcriptomics and macrophage perturbation support a stress-adaptive role for BCAP31 in ER-stress-associated inflammation and apoptosis. Increased tissue expression and the adverse effects of depletion indicate that plaque-associated upregulation may reflect a compensatory response.