Abstract Body (Do not enter title and authors here): Introduction
It is now recognized that the quality of lipoprotein particles may impact atherogenesis more than their plasma concentration. HDL and LDL can be separated into five subfractions increasingly electronegative subfractions (H1 to H5 and L1 to L5), with H1 and L1 being beneficial and H5 and L5 being dysfunctional. This study examines the effects of H5 and L5 on the viability of endothelial cells (ECs) and monocytes, key components of vascular immunity.

Hypothesis
We hypothesize that L5 induces cytotoxic responses in monocytes (THP-1 cells) and bovine aortic ECs (BAECs), with H5 exacerbating these effects, thereby promoting inflammatory and atherogenic processes in atherosclerosis.

Methods
H5 and L5 were fractionated by anion-exchange chromatography. Cells were treated with 50 µg/mL of L1, L5, H1, and H5 for 24 hours. Cell viability was assessed using a fluorescence-based viability detection method after trypan blue exclusion staining. Image-based cytometric quantification was performed using the Nexcelom Cellometer. Lipidomics was used to characterize L5 and H5, and transcriptomic analysis was performed on BAECs treated with H1 and H5 to identify differentially expressed genes.

Results
H5 slightly and L5 moderately reduced cell viability, while their combination significantly decreased both cell numbers and viability (Figures A and B). In contrast, H1 and L1 increased cell numbers compared to PBS control (Figure C). Lipidomic analysis revealed that H5 and L5 have higher triglyceride and lower phosphatidylcholine content than H1 and L1. Transcriptomic analysis indicated that H5 is associated with changes in cell cycle regulation and cellular senescence, affecting DNA damage response and chromosome organization.

Conclusion
L5 and H5, particularly in a synergistic manner, significantly impact EC and monocyte viability, impairing vascular innate immunity and contributing to atherosclerosis pathogenesis. Conversely, H1 and L1 increase cell proliferation, indicating their non-cytotoxic role and potential nutrient provision. Lipidomic and transcriptomic analyses further support the distinct roles of these lipoprotein subclasses in impaired vascular innate immunity and their contribution to atherogenic processes.