title: Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction.
abstract: 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 d-glucose. 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.
authors.affiliation: Department of Surgery, UMDNJ-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07101, USA.
authors.forename: Shubha
authors.initials: S
authors.lastname: Varma
authors.forename: Brajesh K
authors.initials: BK
authors.lastname: Lal
authors.forename: Ruifang
authors.initials: R
authors.lastname: Zheng
authors.forename: Jerome W
authors.initials: JW
authors.lastname: Breslin
authors.forename: Satoshi
authors.initials: S
authors.lastname: Saito
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Pappas
authors.forename: Robert W
authors.initials: RW
authors.lastname: Hobson
authors.forename: Walter N
authors.initials: WN
authors.lastname: Durán
mesh_terms: D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism* / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D005947:Glucose; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; C494918:AKT1 protein, human