Clin Res Cardiol 104, Suppl 1, April 2015

V1191 - The APJ Receptor: Involvement in PECAM-1-induced Mechanotransduction
A. Strohbach1, M. Pennewitz1, M. Glaubitz2, R. Palankar2, M. Delcea2, S. B. Felix1, R. Busch1
1Klinik u. Poliklinik für Innere Medizin B, Universitätsklinikum Greifswald, Greifswald; 2Zentrum für Innovationskompetenz - Humorale Immunreaktionen bei kardiovaskulären Erkrankun, ZIK HIKE, Universität Greifswald, Greifswald;
Aim: Application of fluid shear stress to endothelial cells (ECs) elicits not only the formation of NO via PECAM-1 induced PI3K-signaling but also PECAM-1/integrin-mediated adaptive cellular stiffening. Previously, we did show an apelin-12/APJ-dependent induction of PI3K/Akt/eNOS signaling, which resulted in an increased NO production in human ECs. Here, we present data, which suggest a flow-induced association of APJ with PECAM-1 effecting not only NO-production but also cellular adaption of the cytoskeleton.
Methods: HUVEC were transfected with specific APJ siRNA and transduced with LifeAct adenoviral vectors for F-actin and APJ. Subsequently, we applied physiological shear stress (1.5 dyne/cm²) to WT and APJ-deficient (APJ-KD) HUVEC using ibidi®Luer0.2µ-Slides. To determine interactions of APJ and PECAM-1, HUVEC were fixed and stained for PECAM-1 or vinculin after shear stress exposure, and analyzed using confocal laser scanning microscopy (CLSM). Furthermore, WT and APJ- HUVEC were subjected to Atomic Force Microscopy (AFM). Cell-stiffness was analyzed using the Hertz-Model (HM) to calculate the Young’s Modulus (YM).
Results: To show an association of APJ and PECAM-1, we determined the Manders’ coefficient under static and flow conditions via CLSM. We found a time-dependent association of APJ and PECAM-1 under shear stress only (p=0.023). We also found a time-dependent loss of cell-cell contacts in APJ-KD HUVEC (p=0.012). Additionally, APJ-KD led to an impaired formation of vinculin particles under flow conditions (p=0.023). Furthermore, gene expression analysis of WT and APJ-KD HUVEC revealed a loss of flow-induced eNOS (2.5 fold reduction, p=0.002) and PECAM-1 (2.7 fold reduction; p<0.0001) mRNA expression compared to WT static controls. PECAM-1 is known to not only initiate PI3K-dependent eNOS phosphorylation, but to play a role in adaptive stiffening of ECs subjected to mechanical forces. Therefore, we determined the influence of APJ-KD on the cellular stiffness of ECs. In accordance with the literature our AFM data show a 2-fold increase in cellular stiffness in WT HUVEC after onset of flow (p=0.032). Interestingly, static/ APJ-KD ECs did not show any significant difference to static WT controls. However, for flow-exposed WT HUVEC we found 3-fold higher YM-values compared to flow-exposed APJ-KD cells (p=0.0008). In addition, we also observed a flow-induced reorganization of actin-cytoskeleton structures in WT ECs using live cell imaging, which was impaired by APJ-silencing.
Conclusion: In HUVEC, shear stress elicits signals through PI3K/Akt/eNOS resulting in an increase in NO-production, which might be related to a mechanically induced association of APJ and PECAM-1. Additionally, the silencing of APJ results in a loss of cell-cell contacts, changes in cell morphology, and a loss of adaptive stiffening in response to the mechanical force. These results lead us to the assumption, that the APJ receptor is sufficient for PECAM-1-dependent responses to fluid flow.
Clin Res Cardiol 104, Suppl 1, April 2015
Zitierung mit Vortrags- oder Posternummer s.o.
DOI 10.1007/s00392-015-1100-4