Wall shear stress activation of EGR1 and KLF2 transcription in valvular cells is mediated by the ERK1/2 mitogen-activated protein kinase pathway

Abstract

The aortic valve leaflet is composed of 2 cell types, an interstitial layer of fibroblast-like cells named valve interstitial cells (VICs), and an outer-layer of valve endothelial cells (VECs). The VECs are exposed to different stresses, in particular wall shear stress (WSS). Alteration of hemodynamics can lead to remodeling of the valve leaflet, which eventually result in calcified or degenerative valve. EGR1 and KLF2 are known as flow responsive transcription factors. However, the pathway activating these factors in response to WSS in the valve is not well understood. Our study aims to decipher how EGR1 and KLF2 are activated in response to the WSS in valvular cells. We used a unique fluid activation device that applies physiologically relevant pulsatile WSS to identify the signal required to activate EGR1 and KLF2. We used in vitro and ex vivo approaches to assess the downstream effect of EGR1 and KLF2. VECs exposed to the WSS were treated with U0126 (ERK1/2 inhibitor). Interestingly, treatment of the VECs with U0126 shows a downregulation of EGR1 and KLF2 transcription factors suggesting that the MAPK pathway and more precisely ERK1/2 is required to the activation of these factors. We found that addition of EGR1 adenovirus in the VECs results in the transcriptional activation of NOS3. Using luciferase assay we identified that both EGR1 and KLF2 activate NOS3 promoter. Our ex vivo model using explanted valves confirmed these data. In addition, we investigate which receptor could activate the MAPK pathway in response to the WSS. VECs exposed to the WSS and treated with different recombinant molecules to identify receptor involved in the upregulation of EGR1. Altogether, our data suggest a link between the MAPK pathway and the remodeling and/or proliferation of valve cells. In conclusion, EGR1 and KLF2 are upregulated in VECs exposed to WSS and this activation dependents on the MAP kinase pathway. EGR1 activates the transcription of NOS3 an essential regulator of valve homeostasis.