Abstract Tu0066: The Role of Type VIII Collagen in Modulating Endothelial Cell Stiffness in Inflammation

Background: Vascular stiffness is a key biomechanical property involved in various vascular pathologies such as hypertension, and while tissue-level properties play a large role, the mechanobiology of vascular cells, such as endothelial cells, are also involved. Type VIII collagen (COL8) is a non-fibrillar, network-forming collagen highly expressed in endothelial cells (ECs). In contrast to fibrillar collagen, the impact of non-fibrillar collagens, such as COL8, on vascular cell stiffness remains poorly understood. Characterizing the relationship between COL8 expression and endothelial stiffness could provide valuable insights into the mechanisms regulating cell mechanotype in vascular inflammation. Methods/Results: The single-cell stiffness of ECs was characterized using atomic force microscopy (AFM), parallel microfiltration (PMF), and deformability cytometry (DC). Results revealed that treatment with the inflammatory cytokine, TNF-α, induces a biphasic, time-dependent effect on EC stiffness: short-term (24 hours) treatment decreased cellular stiffness, while long-term (96 hours) treatment significantly increased stiffness. Interestingly, TNF-α treatment also led to similar biphasic changes in the expression of COL8A1, which encodes one of the two alpha chains of COL8, paralleling the observed changes in EC stiffness. Inhibition of COL8A1 expression via siRNA transfection reduced EC stiffness, as measured by AFM, PMF, and DC assays, suggesting that COL8A1 plays a role in mediating the TNF-α-induced changes in endothelial mechanotype. Bulk RNA sequencing of COL8A1 knockdown ECs indicated that downregulated genes were enriched in pathways related to cytoskeleton modulation, including microtubule stability and microfilament binding, supporting a novel role for the non-fibrillar COL8A1 in impacting cellular stiffness. Conclusion: Our findings suggest that ECs undergo dynamic, biphasic changes in mechanotype in response to inflammatory stimulation by TNF-α, and that these changes are at least partially mediated by COL8A1 through cytoskeletal modulation. Key Words: Endothelial cell, Inflammation, Cell stiffness, COL8, Cytoskeleton, mechanobiology.