Compartmentalized Regulation of Vascular Endothelial Cell Responses to Fluid Shear Stress by Phosphodiesterase 4D7 (PDE4D7)

Abstract

While fast flowing blood in linear arterial segments exposes arterial vascular endothelial cells (VECs) to high levels of laminar fluid shear stress (FSS, 15–20 dyne/cm2), slow or oscillatory blood flow, such as is present at arterial bifurcations, exposes VECs to modest levels of non‐linear FSS (1–3 dyne/cm2). These hemodynamic features make arterial bifurcations, arches and branch points especially prone to atherosclerosis. Exposing VECs to high laminar FSS promotes establishment of an adaptive (anti‐thrombotic, anti‐atherogenic and barrier forming) phenotype, an effect that is reversed following exposure of these cells to low / non‐linear levels of FSS. Previously we reported that a cAMP‐signalosome, composed of exchange protein activated by cAMP1 (EPAC1), Rap1 and a phosphodiesterase 4D (PDE4D) allowed cAMP to promote barrier functions in static VEC cultures. This signalosome was shown to associate with components of VEC adherens junctions, including VE‐cadherin (VECAD) and β‐catenin. Recently, we reported that activation of this same AJ‐associated signalosome allowed cAMP, via EPAC1‐mediated activation of Rap1, to promote expression of an adaptive phenotype even in VECs exposed to low levels of FSS. Indeed, siRNA‐mediated knockdown of EPAC1, Rap1 or PDE4D reduced the adaptive responses of VECs exposed to FSS and increased their pro‐thrombotic and pro‐angiogenic potential. Interestingly, PDE4D knockdown reduced responses in these cells to levels equivalent to those detected in cells in which EPAC1 was knocked down. We reported that this effect was due to the fact that PDE4D acts as a tether to localize EPAC1 within this important cAMP‐signalosome. Recently we identified phosphodiesterase 4D7 (PDE4D7) as the PDE4D variant that populates this VEC cAMP‐signalosome. Notably, VEC alignment in the direction of flow was reduced by knockdown of either pan‐PDE4D, selective PDE4D7 or EPAC1 when subjected to low FSS. Both Pan‐PDE4D or selective PDE4D7 siRNA‐mediated knockdown markedly reduced the ability of VECs to respond adaptively to FSS, and promoted loss of EPAC1 from the cAMP‐signalosome. In contrast, PDE4 inhibitors only modestly altered these responses in VECs exposed to various levels of FSS. These findings, together with our earlier work, are consistent with the idea that the tethering actions of PDE4D7 are dominant in this complex in VECs. Using telomerase‐immortalized human aortic endothelial cells, we created a cell‐based system in which the separate catalytic and tethering actions of PDE4D7 in controlling VEC responses to FSS could be measured. Overall, our findings are consistent with the novel idea that the EPAC1 tethering function of PDE4D7 is dominant in regulating EPAC1/Rap1‐mediated effects in VECs and that the cAMP hydrolytic function of PDE4D7 has a hyperlocalized impact within the AJ‐delimited nanodomain. Our findings pave the way for the development of displacing peptide (DP)‐based tools through which to regulate cAMP‐mediated function in VECs exposed to mal‐adaptive levels of FSS.Support or Funding InformationFunding provided by the CIHR.