Varma, Shubha, Brajesh K. Lal, Ruifang Zheng, Jerome W. Breslin, Satoshi Saito, Peter J. Pappas, Robert W. Hobson II, and Walter N. Durán. Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction. Am J Physiol Heart Circ Physiol 289: H1744– H1751, 2005. First published June 17, 2005; doi:10.1152/ajpheart.01088. 2004—Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM D-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM D-glucose (control) exhibited log-phase growth on days 7–10. D-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM D-glucose. D-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM Dglucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM D-glucose. We conclude that D-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.
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