Investigation of oxidized low-density lipoprotein (oxLDL)-induced inflammation in atherosclerosis using a paper-based...

Atherosclerosis is driven by complex interactions between endothelial cells, smooth muscle cells, and immune cells, yet conventional in vitro models often fail to capture these multicellular dynamics. Here, we present a biomimetic paper-based immuno-vascular model that vertically integrates endothelial cells, smooth muscle cells, and THP-1-derived macrophages to simulate the vascular intima environment. Using this model, CD36-mediated inflammatory responses were investigated under oxLDL stimulation. The results revealed that CD36 expression was the highest in multicellular co-cultures, indicating synergistic effects of oxLDL and macrophage-derived paracrine signaling. The model also maintained homogeneous protein distribution and high cell viability over extended culture periods, demonstrating spatially precise assessment of immune-vascular crosstalk, including layer-to-layer propagation of inflammatory signaling consistent with translaminar paracrine communication. These immunoassay-based findings were independently corroborated within the same experimental batch by layer-separated RT-qPCR of CD36, IL-6, IL-1β, and TNF-α performed on RNA recovered directly from the destacked paper layers, which reproduced the same rank order of conditions at the transcript level. These findings highlight the critical role of intercellular signaling in potentiating inflammatory responses and validate the model as a versatile tool for studying early atherogenic processes. Beyond physiological relevance, the model offers reproducible fabrication, experimental accessibility, and compatibility with in situ molecular analyses, supporting mechanistic studies, hypothesis-driven experimentation, and early-stage therapeutic screening in cardiovascular research.