01.03 Shear Stresses Regulate Inflammatory Changes in Endocardial Endothelial Cells leading to Fibrosis.

P. Ji1,2, S. A. Brimer1,2, H. Li1,2, L. Yu1,2, J. Grande-Allen3, S. Balaji1,2, R. K. Birla1,2, S. Keswani1,2  1Baylor College Of Medicine, TCH Pedi Surgery, Houston, TX, USA 2Texas Children’s Hospital, TCH Pedi Surgery, Houston, TX, USA 3Rice University, Bioengineering, Houston, TX, USA

Introduction: Discrete subaortic stenosis (DSS) is characterized by the development of fibrous membrane tissue in the left ventricular outflow tract (LVOT), leading to an increase in pressure gradient and altered shear flow pattern in the narrowed passage below the aortic valve in the LVOT. Except surgical resection, there are no other treatment options, as the mechanisms of fibrous membrane formation are not known. Further, DSS recurs in some patients, necessitating repeat surgeries. As endocardial endothelial cells (EEC) of the LVOT experience changes in pressure gradient and shear flow, we hypothesized that altered shear forces induce an inflammatory response by EEC, which interact with cardiac fibroblasts to govern a fibrotic phenotype that contributes to the pathophysiology of DSS.


Methods: Cone-and-plate devices were used to generate flow-induced shear stress regimes (6, 15, 35 dynes/cm^2) noted in DSS for 1, 4, 8 and 24 hours on 2D cultures of porcine EECs. Inflammatory cytokine profiles of the shear-conditioned EEC media were analyzed (Luminex Assay) and RNA was isolated and bulk RNA sequencing was performed. Differences in CD31 receptor expression among EECs after shear stress was analyzed using immunofluorescence staining. Shear-conditioned EEC supernatant was then added to cardiac fibroblasts and changes in the fibroblast fibrotic phenotype was assessed using PCR array. Data presented as mean+/-stdev and p values by ANOVA.  


Results: High Shear stresses on EEC led to a significant increase in several inflammatory cytokines including TNF- α, GM-CSF, IL-2, IL-4 and IL-8 (p<0.01). Following bioreactor treatment with varying shear stresses, the expression of CD31 receptors on the porcine EEC membrane was significantly elevated (p<0.05). RNA-seq analysis showed that the inflammatory cell signaling pathway was activated in the EEC. The bioreactor-treated EEC conditioned medium induced fibrosis in porcine fibroblasts at 48 hours after media transfer, and the RT-qPCR data showed that the fibrotic markers (α-SMA, ACTA2, TFG-β1, Col1, SMAD2 & AGTRAP) were significantly altered in porcine fibroblasts, especially under the high shear stress bioreactor treated porcine EEC medium.


Conclusion: The study demonstrates that altered shear forces induce an inflammatory response in EEC, which may lead to their interaction with cardiac fibroblasts that promotes a fibrotic phenotype, thereby contributing to DSS pathophysiology. CD31 has been identified as a crucial player in the mechanical sensing and inflammatory response of EEC. These findings hold significant implications for understanding DSS pathophysiology and pave the way for developing strategies to enhance patient management, ultimately reducing the morbidity and associated costs linked to DSS recurrence in future.