Abstract
Endothelial-to-mesenchymal transition (EndMT) is the process of endothelial cells undergoing molecular changes that shift their phenotype from that of endothelial cells to that of mesenchymal-like cells. It is a crucial developmental process that has been implicated in various physiological and pathological conditions. EndMT has gained attention as a potential therapeutic target for cardiovascular disease processes, including atherosclerosis, myocardial fibrosis, and vascular calcification. In addition to the assessment of endothelial and mesenchymal markers, the behavioral mechanics of endothelial cells, such as migration and invasion, are often used to identify endothelial cells that have undergone EndMT. However, whether cell chirality may be another mechanobiological marker of EndMT remains unclear. In this study, we aimed to develop an accessible micropatterning platform and created a stereolithography (SLA) 3D printing-based polydimethylsiloxane (PDMS) protein-stamp fabrication platform to create customized patterns of ECM proteins to study endothelial cell chirality during EndMT. Human aortic endothelial cells (HAECs) were treated with the inflammatory cytokine tumor necrosis factor-α (TNF-α), which resulted in the downregulation of the endothelial marker ENOS3 and the upregulation of the mesenchymal markers N-cadherin and transgelin, supporting the induction of EndMT. HAECs were seeded onto fibronectin stripe micropatterns, and cell chirality was measured using custom cell-profiling software. HAECs treated with TNF-α exhibited a shift in cell orientation by approximately 18°, supporting altered cell chirality during TNF-α-induced EndMT. Our work innovates novel methods of studying EndMT by developing a flexible and cost-effective protein-stamp fabrication and image analysis pipeline. This pipeline can be used by researchers to study the endothelial cell chirality in response to EndMT induction.