Hemodynamic shear stress regulates the apelin/APJ system in human endothelial cells

Abstract

Introduction: The apelin/APJ system is being considered to play a crucial role in cardiovascular function. Namely, the isoforms apelin-12 and apelin-13 seem to be most active in the cardio vasculature. Application of fluid shear stress to endothelial cells elicits the formation of NO via PI3K/AKT signaling and phosphorylation of the endothelial NO synthase (eNOS). We suppose that the apelin/APJ system is involved in the transduction of mechanical forces into intracellular signaling. Methods: We applied hemodynamic shear stress (1.5 dyne/cm2 for venous, 20 dyne/cm2 for arterial conditions) on HUVEC, HCAEC, and HCASMC using a parallel plate flow chamber. Cells were stimulated with apelin-12 and apelin-13. Static controls were kept under similar conditions. After shear stress exposure cells were harvested and prepared for real-time qPCR and Western Blot analysis or fixed and stained for anti-APJ (IF). The supernatant was collected for nitrite measurements. Additionally, HUVEC were transfected with APJ siRNA. Results: After application of shear stress, apelin gene and protein expression were regulated in ECs only. In HUVEC, expression of apelin was increased by 40% under low shear stress, whereas under high shear stress conditions apelin expression decreased by 50% (p<0.01). Interestingly, HCAEC showed an opposite behaviour (p<0.001). Furthermore, in ECs APJ gene and protein expression increased after exposure to shear stress (p<0.05), which could be confirmed by IF. Incubation of HUVEC with apelin-12 and -13 revealed a dose-dependent increase of NO production only after incubation with apelin-12 (P<0.01). Western Blot Analysis underlined apelin isoform dependent cellular signaling. Incubation with apelin-12 for up to 15 min lead to a significant increase in PI3 kinase and Akt phosphorylation under static and dynamic conditions (p=0.02), whereas incubation with apelin-13 had no significant influence on PI3k/Akt signalling and lead to phosphorylation of Erk 1/2 instead. In order to demonstrate shear stress dependency of the apelin/APJ system, we transfected HUVEC with APJ siRNA and exposed them to our standard flow conditions. Transfection with APJ siRNA lead to a loss of shear stress dependent eNOS up-regulation and adhesion. Conclusion: Our results show for the first time a shear stress regulated adjustment of the Apelin/APJ system in human endothelial cells depending on arterial or venous flow conditions. This is confirmed by an enhanced eNOS expression and NO production. Interestingly, apelin-12 and apelin-13 activate different signaling pathways in HUVEC and give rise for various biological responses.