Abstract 154: Hemodynamic Regulation of Tie1 in Aortic Valve Endothelial Cells

Abstract

During the cardiac cycle, the ventricular side of the aortic valve (ventricularis) is exposed to high shear pulsatile flow, while the aortic side (fibrosa) is exposed to low shear recirculatory flow. The two sides have different transcriptional profiles, likely due to their distinct flow patterns. Tie1 is a mechanically sensitive orphan tyrosine kinase receptor found in endothelial cells (ECs). Tie1 is often found associated with Tie2, a tyrosine kinase receptor involved in EC survival. Tie1 prevents ligand binding to Tie2 until Tie1 is cleaved, via shear stress or other signals. Although studies have shown shear stress cleaves Tie1, none have been done in the aortic valve. Mechanically induced changes in Tie1 expression in the valve may be crucial to understand the effects of the hemodynamics of each side. ECs were isolated from the fibrosa and ventricularis of excised porcine aortic valves. The Flexcell 4000 Tension System was used for strain, and an insert for the Flexcell plates ( A, B, C ) was designed to apply shear stress at the same time. Full length Tie1 protein expression in porcine aortic valvular endothelial cells (pAVECs) decreased ~40% after 15% strain for 24 hours, while cleaved Tie1 endodomain levels increased ( D ). Additionally, qPCR results showed that Tie1 mRNA levels did not decrease as dramatically, also supporting Tie1 cleavage. Full length Tie1 protein expression in pAVECs also decreased ~60% after 7 hours of 10 dynes/cm 2 pulsatile bidirectional shear stress and decreased ~90% after 24 hours of 15% strain combined with 2 dynes/cm 2 pulsatile bidirectional shear stress ( E, F ). Akt, a protein kinase phosphorylated by activated Tie2, showed increased phosphorylation after 15 minutes of strain ( G ), providing evidence that cleaved Tie1 may be activating Tie2. These results show the varying responses in Tie1 expression to different shear stresses and strains and demonstrate that mechanical regulation plays an important role in its signaling in the aortic valve.