S. Y. Shalaby1, G. Chitragari1, B. J. Sumpio1, J. Kurita1, B. Sumpio1 1Yale University School Of Medicine,Vascular Surgery,New Haven, CT, USA
Introduction: Extracellular signal regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction under laminar flow. Atherosclerotic lesions predominate in areas of disturbed flow but the mechanism of this phenomenon is not well defined. Previously reported research indicate ERK5 activation under laminar flow induces ERK5 activation and production of atheroprotective molecules. However, the characterization of ERK5 activation under different flow patterns has not been investigated.
Methods: Confluent HUVECs were serum starved for 2 hours with 1% Fetal Bovine Serum (FBS) then seeded on fibronectin coated glass slides. HUVECs were exposed to CLF, TFF, or PFF in a parallel plate flow chamber controlled by a computerized pump for up to 2 or 4 hours at 37° C in a humidified CO2 environment. HUVECs incubated in 1% FBS, 20% FBS, or hydrogen peroxide culture medium under static conditions served as control and positive stimulant, respectively. At the end of experimentation, cell lysates were prepared and immunoblotted with antibodies to phospho-ERK5 and total ERK5. ERK5 activity was assessed by the levels of phosphorylated ERK5. The densitometric mean ± SEM is calculated and analyzed by student t-test. p < 0.05 is considered significant.
Results: Levels of ERK5 decreased with all flow conditions as previously reported. TFF and CLF exhibited sustained ERK5 phosphorylation in HUVECs stimulated for up to 4 hours (Figure. 1). PFF had transient phosphorylation of ERK5 at 2 hours, which returned to baseline levels at 4 hours of exposure to flow.
Conclusion: Exposure of HUVEC to different types of shear stress results in varying patterns of activation of ERK5. Activation of ERK5 with TFF suggests a role in the pathogenesis of atherosclerosis and vascular remodeling under disturbed flow conditions.
