Akt pathway is hypoactivated by synergistic actions of diabetes mellitus and hypercholesterolemia resulting in advanced coronary artery disease.

Atherosclerosis is an inflammatory process leading to enhanced cellular proliferation, apoptosis, and vasa vasorum (VV) neovascularization. While both diabetes mellitus (DM) and hypercholesterolemia (HC) predispose to atherosclerosis, the precise interaction of these risk factors is unclear. Akt is a central node in signaling pathways important for inflammation, and we hypothesized that DM/HC would lead to aberrant Akt signaling and advanced, complex atherosclerosis. DM was induced in pigs by streptozotocin and HC by a high-fat diet. Animals were randomized to control (non-DM, non-HC), DM only, HC only, and DM/HC groups. Coronary artery homogenates were analyzed by immunoblotting for proteins involved in the Akt pathway, including phosphorylated (p)-Akt (Ser473), p-GSK-3beta (Ser9), activated NF-kappaB p65, and VEGF. Immunohistochemical staining for Ki67 (cell proliferation), terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) (apoptosis), and von Willebrand factor (vWF) (neovascularization) was performed. Neovascularization was visualized with micro-computerized tomography (CT). Only DM/HC animals developed advanced atherosclerosis and showed decreased p-Akt (Ser473) and p-GSK-3beta (Ser9) levels (P < 0.01 and P < 0.05, respectively). DM/HC arteries demonstrated increased cellular proliferation (P < 0.001), apoptosis (P < 0.01), and activation of NF-kappaB p65 (P < 0.05). Induction of DM/HC also resulted in significant VV neovascularization by enhanced VEGF expression (P < 0.05), increased vWF staining (P < 0.01), and increased density by micro-CT. In conclusion, DM and HC synergistically resulted in complex atherosclerosis associated with attenuated p-Akt (Ser473) levels. Aberrant Akt signaling correlated with increased inflammation, cellular proliferation, apoptosis, and VV neovascularization. Our results revealed a synergistic effect of DM and HC in triggering abnormal Akt signaling, resulting in advanced atherosclerosis.

[1]  C. Fernández-Hernando,et al.  Absence of Akt1 Reduces Vascular Smooth Muscle Cell Migration and Survival and Induces Features of Plaque Vulnerability and Cardiac Dysfunction During Atherosclerosis , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[2]  J. Zweier,et al.  Ox-LDL modifies the behaviour of bone marrow stem cells and impairs their endothelial differentiation via inhibition of Akt phosphorylation , 2009, Journal of cellular and molecular medicine.

[3]  L. Lerman,et al.  Prevention of vasa vasorum neovascularization attenuates early neointima formation in experimental hypercholesterolemia , 2009, Basic Research in Cardiology.

[4]  T. Franke,et al.  PI3K/Akt: getting it right matters , 2008, Oncogene.

[5]  A. Baldwin,et al.  Maintenance of constitutive IkappaB kinase activity by glycogen synthase kinase-3alpha/beta in pancreatic cancer. , 2008, Cancer research.

[6]  Ping Zhang,et al.  Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development , 2008, Nature Medicine.

[7]  J. Arnal,et al.  Chronic Estradiol Administration In Vivo Promotes the Proinflammatory Response of Macrophages to TLR4 Activation: Involvement of the Phosphatidylinositol 3-Kinase Pathway1 , 2008, The Journal of Immunology.

[8]  R. Wilensky,et al.  Site-Specific Atherogenic Gene Expression Correlates With Subsequent Variable Lesion Development in Coronary and Peripheral Vasculature , 2008, Arteriosclerosis, thrombosis, and vascular biology.

[9]  J. Luyendyk,et al.  Genetic Analysis of the Role of the PI3K-Akt Pathway in Lipopolysaccharide-Induced Cytokine and Tissue Factor Gene Expression in Monocytes/Macrophages1 , 2008, The Journal of Immunology.

[10]  Pascale G. Charest,et al.  The regulation of cell motility and chemotaxis by phospholipid signaling , 2008, Journal of Cell Science.

[11]  M. Birnbaum,et al.  Loss of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. , 2007, Cell metabolism.

[12]  R. Wilensky,et al.  The molecular basis of vulnerable plaque: potential therapeutic role for immunomodulation , 2007, Current opinion in cardiology.

[13]  P. Shah.  Molecular mechanisms of plaque instability , 2007, Current opinion in lipidology.

[14]  Lewis C. Cantley,et al.  AKT/PKB Signaling: Navigating Downstream , 2007, Cell.

[15]  E. Ritman,et al.  Increased spatial vasa vasorum density in the proximal LAD in hypercholesterolemia--implications for vulnerable plaque-development. , 2007, Atherosclerosis.

[16]  S. Pennathur,et al.  Mechanisms for oxidative stress in diabetic cardiovascular disease. , 2007, Antioxidants & redox signaling.

[17]  A. Lipovsky,et al.  S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt. , 2006, Molecular cell.

[18]  F. Gruber,et al.  Oxidized Phospholipids Stimulate Angiogenesis Via Autocrine Mechanisms, Implicating a Novel Role for Lipid Oxidation in the Evolution of Atherosclerotic Lesions , 2006, Circulation research.

[19]  Brian A. Hemmings,et al.  Only Akt1 Is Required for Proliferation, while Akt2 Promotes Cell Cycle Exit through p21 Binding , 2006, Molecular and Cellular Biology.

[20]  Ji Luo,et al.  The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism , 2006, Nature Reviews Genetics.

[21]  Peter Libby,et al.  The immune response in atherosclerosis: a double-edged sword , 2006, Nature Reviews Immunology.

[22]  T. Sawamura,et al.  Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase Published, JLR Papers in Press, March 7, 2006. , 2006, Journal of Lipid Research.

[23]  D. Green,et al.  Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. , 2006, Molecular cell.

[24]  Erik L Ritman,et al.  Correlation of Vasa Vasorum Neovascularization and Plaque Progression in Aortas of Apolipoprotein E−/−/Low-Density Lipoprotein−/− Double Knockout Mice , 2005, Arteriosclerosis, thrombosis, and vascular biology.

[25]  E. Edelman,et al.  Vascular Neointimal Formation and Signaling Pathway Activation in Response to Stent Injury in Insulin-Resistant and Diabetic Animals , 2005, Circulation research.

[26]  M. Hofker,et al.  Nuclear Factor &kgr;B Signaling in Atherogenesis , 2005 .

[27]  R. Urrutia,et al.  Glycogen Synthase Kinase-3β Participates in Nuclear Factor κB–Mediated Gene Transcription and Cell Survival in Pancreatic Cancer Cells , 2005 .

[28]  Bing-Hua Jiang,et al.  Apigenin inhibits VEGF and HIF‐1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[29]  D. Guertin,et al.  Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex , 2005, Science.

[30]  B. Manning.  Balancing Akt with S6K , 2004, The Journal of cell biology.

[31]  Gieri Cathomas,et al.  Arterial Neovascularization and Inflammation in Vulnerable Patients: Early and Late Signs of Symptomatic Atherosclerosis , 2004, Circulation.

[32]  F. Agani,et al.  Vascular Endothelial Growth Factor Transcriptional Activation Is Mediated by Hypoxia-inducible Factor 1α, HDM2, and p70S6K1 in Response to Phosphatidylinositol 3-Kinase/AKT Signaling* , 2004, Journal of Biological Chemistry.

[33]  Juan J. Badimon,et al.  Plaque Neovascularization Is Increased in Ruptured Atherosclerotic Lesions of Human Aorta: Implications for Plaque Vulnerability , 2004, Circulation.

[34]  Xianglin Shi,et al.  trans-3,4,5′-Trihydroxystibene Inhibits Hypoxia-Inducible Factor 1α and Vascular Endothelial Growth Factor Expression in Human Ovarian Cancer Cells , 2004, Clinical Cancer Research.

[35]  R. Virmani,et al.  Morphologic Findings of Coronary Atherosclerotic Plaques in Diabetics: A Postmortem Study , 2004, Arteriosclerosis, thrombosis, and vascular biology.

[36]  Jongkyeong Chung,et al.  Akt: versatile mediator of cell survival and beyond. , 2002, Journal of biochemistry and molecular biology.

[37]  Yong Liao,et al.  HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation , 2001, Nature Cell Biology.

[38]  L. Mayo,et al.  A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  C. Van Waes,et al.  Hepatocyte growth factor/scatter factor-induced activation of MEK and PI3K signal pathways contributes to expression of proangiogenic cytokines interleukin-8 and vascular endothelial growth factor in head and neck squamous cell carcinoma. , 2001, Cancer research.

[40]  R. Natarajan,et al.  Diabetes-induced accelerated atherosclerosis in swine. , 2001, Diabetes.

[41]  E. Chavakis,et al.  Oxidized LDL Inhibits Vascular Endothelial Growth Factor-Induced Endothelial Cell Migration by an Inhibitory Effect on the Akt/Endothelial Nitric Oxide Synthase Pathway , 2001, Circulation.

[42]  V. Fuster,et al.  Coronary Composition and Macrophage Infiltration in Atherectomy Specimens From Patients With Diabetes Mellitus , 2000, Circulation.

[43]  J. Woodgett,et al.  Requirement for glycogen synthase kinase-3β in cell survival and NF-κB activation , 2000, Nature.

[44]  R. Virmani,et al.  Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[45]  S. R. Datta,et al.  Cellular survival: a play in three Akts. , 1999, Genes & development.

[46]  R. Ross.  Atherosclerosis is an inflammatory disease , 1999 .

[47]  A. Weiss,et al.  Induction of NF-κB by the Akt/PKB kinase , 1999, Current Biology.

[48]  M A Konerding,et al.  Angiogenesis inhibitors endostatin or TNP-470 reduce intimal neovascularization and plaque growth in apolipoprotein E-deficient mice. , 1999, Circulation.

[49]  M. Kuzuya,et al.  Induction of macrophage VEGF in response to oxidized LDL and VEGF accumulation in human atherosclerotic lesions. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[50]  E L Ritman,et al.  Enhanced coronary vasa vasorum neovascularization in experimental hypercholesterolemia. , 1998, The Journal of clinical investigation.

[51]  A. Giaccia,et al.  Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. , 1997, Blood.

[52]  J. Testa,et al.  Membrane translocation and activation of the Akt kinase in growth factor-stimulated hematopoietic cells. , 1997, Leukemia research.

[53]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[54]  S M Schwartz,et al.  Angiogenesis in human coronary atherosclerotic plaques. , 1994, The American journal of pathology.

[55]  E. Edelman,et al.  Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries. , 1992, The Journal of clinical investigation.

[56]  A. Barger,et al.  Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis. , 1984, The New England journal of medicine.

[57]  G. Werstuck,et al.  Vascular Biology , Atherosclerosis and Endothelium Biology Valproate Attenuates Accelerated Atherosclerosis in Hyperglycemic ApoE-Deficient Mice Evidence in Support of a Role for Endoplasmic Reticulum Stress and Glycogen Synthase Kinase-3 in Lesion Development and Hepatic Steatosis , 2010 .

[58]  E. M. Sale,et al.  Protein kinase B: signalling roles and therapeutic targeting , 2007, Cellular and Molecular Life Sciences.

[59]  R. Urrutia,et al.  Glycogen synthase kinase-3beta participates in nuclear factor kappaB-mediated gene transcription and cell survival in pancreatic cancer cells. , 2005, Cancer research.

[60]  J. Woodgett,et al.  Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. , 2000, Nature.

[61]  D. Holmes,et al.  Activated nuclear factor-kappaB is present in the coronary vasculature in experimental hypercholesterolemia. , 2000, Atherosclerosis.

[62]  T. Hunter,et al.  Phosphatidylinositol 3-Kinase Signaling Controls Levels of Hypoxia-inducible Factor 11 , 2000 .

[63]  A. Weiss,et al.  Induction of NF-kappaB by the Akt/PKB kinase. , 1999, Current biology : CB.

[64]  Glycogen Synthase Kinase 3 (cid:2) and 3 (cid:3) Mediate a Glucose-Sensitive Antiapoptotic Signaling Pathway To Stabilize Mcl-1 (cid:1) , 2022 .

[65]  Glycogen Synthase Kinase 3 (cid:2) Functions To Specify Gene-Specific, NF- (cid:3) B-Dependent Transcription , 2022 .