Hyperglycemia Induced Modulation of eNOS and ICAM-1 In Endothelial Cells by MEK5/ERK5/KLF2 Pathway

Author(s): Rohit Patel, Johnna F. Varghese and Umesh C. S. Yadav*

Abstract: Aim: To investigate the role of Erk5 in hyperglycemia (HG)-induced Endothelial dysfunction (ED) via modulation of eNOS and ICAM-1 in primary Human umbilical vein endothelial cells (pHUVECs). Methods: pHUVECs were stimulated with different doses of glucose for different time intervals. MTT and Acridine Orange/ Ethidium Bromide (AO/EB) assays were done to examine cell viability and cytotoxicity. Western blotting to assess the protein expression and monocyte adhesion assay to determine monocyte adherence to pHUVECs and tube formation assay to determine endothelial characteristics were performed. Results: The results indicated that HG marginally decreased the viability of pHUVECs by 48 h of incubation and also decreased the expression of eNOS while increased that of ICAM-1. Incubation of pHUVECs with HG down regulated Erk-5 level, an indicator of compromised pHUVECs homeostasis. SiRNA-mediated silencing of Erk5 appeared to add to HG-mediated effects on eNOS and ICAM-1 as well as on monocyte adhesion. Further, HG decreased the tube formation ability of pHUVECs, which was again augmented when Erk5 was inhibited using synthetic inhibitor BIX02189. Furthermore, HG down regulated Mek5 and Klf2, the upstream and downstream molecules in Erk5 signalling. Inhibition of Erk5 further decreased Klf2 in HG-treated pHUVECs. Conclusion: In conclusion, HG down regulated Erk5 in pHUVECs causing ED by significantly decreasing eNOS and increasing ICAM-1 levels, which were phenotypically observed as increased monocytes adhesions and decreased tube formation ability of pHUVECs. These effects were likely regulated by the Mek5/Erk5/Klf2 pathway.

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