A110-07 Single Cell Contractility as a Functional Marker of Endothelial-to-mesenchymal Transition

Rationale Endothelial cells (EC) lack inherent contractility, existing in an organized cobblestone monolayer with tight cell-cell junctions; however, cytoskeleton remodeling and junction modulation occurs during embryogenesis, inflammation, and in response to mechanical forces. Endothelial-to-mesenchymal transition (EndMT) represents a dynamic continuum wherein endothelial cells adopt mesenchymal characteristics and suppress endothelial properties. EndMT has been implicated in the pathophysiology of several cardiopulmonary diseases- pulmonary hypertension, fibrosis, atherosclerosis, and malignancy. EndMT is induced by inflammatory cytokines, turbulent flow, increased cyclic strain, and microenvironment stiffness. Current techniques for measuring cellular forces are low-throughput, highly complex and have variable precision at the single cell level. A high throughput system has been developed based on fluorescently-labeled elastomeric contractible surfaces (FLECS) to serve as a functional assay of contraction at the single cell level. To date, single cell contractility of cells undergoing EndMT has not been investigated. Methods Human aortic endothelial cells (HAEC) were treated with a combination of TGF beta 5ng/mL, TNF alpha 5ng/mL, and IL 1beta 1ng/mL for 96 hours. Serial brightfield images were obtained at hours 0, 24, 48, and 96 to capture the morphologic changes associated with EndMT. Real-time qPCR was performed to assess endothelial and mesenchymal gene expression. HAEC were seeded on fibronectin coated FLECS plates (Forcyte) with Hoechst staining of nuclei and live fluorescence microscopy. Micropattern contraction was measured using proprietary analysis software (Forcyte); only micropatterns with single nuclei were included in the analysis. Statistical analyses using non-parametric testing were completed on GraphPad Prism (v10.5.0). P-values less than 0.05 were considered statistically significant. Results On serial brightfield imaging, treated HAEC underwent morphologic changes that became more pronounced with increased treatment duration. There was a 67% decrease in ENOS expression (median fold change of 0.33 n = 10; 95% CI 0.15 to 1.22; p = 0.002), a 92% decrease in CD31 expression (median fold change 0.08; n = 10; p = 0.003), and a median fold change of 1.08 in CDH2 (n = 10; 95% CI 0.67 to 1.34; p < 0.001). Fluorescent microscopy of FLECS plates showed more contracted micropatterns in the 96H treated group vs untreated controls. Micropattern analysis showed a trend towards increase in contraction in the 96H treated HAEC with a median fold increase of 1.7 compared to the untreated controls (p < 0.001). Conclusions Single cell contractility can be used as a novel functional marker of EndMT. Additional experiments are planned to further characterize contractility in EndMT with high throughput screening of potential therapeutic agents that can modulate this process.