(G-247) Shear Stress Mediates Endothelial-to-Mesenchymal Transition in Left Ventricular Outflow Tract Pathologies

Discrete subaortic stenosis (DSS) is a pediatric cardiovascular disease characterized by a fibro-membranous growth in the left ventricular outflow tract, often requiring open-heart surgical removal. However, this growth recurs in about 20-30% of these patients, denoted as an aggressive phenotype. If left untreated, complications such as arrhythmias and death can occur. Although the cause of this fibrotic growth is unclear, evidence suggests that altered blood flow could be the cause. The growth obstructs blood flow in the heart, resulting in increased shear stress. Two possibilities that we are exploring as a result of this increased shear are as follows: 1) Endothelial cells are being damaged by the elevated shear, attracting circulating monocytes to invade and induce fibrosis, and 2) endothelial cells may be undergoing endMT following exposure to elevated shear, resulting in fibrosis. We hypothesize that the elevated shear endothelial cells experience directly correlates with the formation of this fibrotic growth.

Human aortic endothelial cells (HAECs) were purchased from Lonza and expanded to passage 4. HAECs were seeded at a density of 500,000 cells per 60 mm petri dish and allowed to grow to confluence. HAECs were then subjected to varying levels of shear: static, 15 dynes/cm^2 (physiological), or 35 dynes/cm^2 (pathological) for a period of 3 hours or 24 hours. Uniform shear stress was applied using a cone-and-plate viscometer system. The pathological shear rate was chosen as 35 dynes/cm^2 has been shown to fall within the range for DSS. Post-shear, RT-qPCR was performed on these cells for PECAM-1, VCAM, ICAM, E-Selectin, and SNAIL1. Conditioned media was collected and saved to be applied to macrophages.

Interestingly, SNAIL1 and ICAM were significantly upregulated in the pathological shear condition at 3 hours compared to the static control. This indicates that at the early time point, endothelial cells may be promoting leukocyte migration while also undergoing endMT. At 24 hours, VCAM expression was significantly downregulated with increasing shear at 24 hours and there was no change in SNAIL1 expression. Most changes seemed to occur at 3 hours. A time point between 3 and 24 hours will be investigated. Other markers of endMT such as SLUG, vimentin, and alpha-smooth muscle actin should be probed. It would be interesting to investigate if immune cells such as monocytes and macrophages play a role in promoting endMT in this context. Future work will consist of applying conditioned media from HAECs to macrophages and exploring the role of how endothelial cells exposed to elevated shear affects surrounding cells.