We previously proposed that the production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetes complications. The association between the pathogenesis of diabetes and its complications and mitochondrial biogenesis has been recently reported. Because metformin has been reported to exert a possible additional benefit in preventing diabetes complications, we investigated the effect of metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on mtROS production and mitochondrial biogenesis in cultured human umbilical vein endothelial cells. Treatment with metformin and AICAR inhibited hyperglycemia-induced intracellular and mtROS production, stimulated AMP-activated protein kinase (AMPK) activity, and increased the expression of peroxisome proliferator-activated response-␥ coactivator-1␣ (PGC-1␣) and manganese superoxide dismutase (MnSOD) mRNAs. The dominant negative form of AMPK␣1 diminished the effects of metformin and AICAR on these events, and an overexpression of PGC-1␣ completely blocked the hyperglycemiainduced mtROS production. In addition, metformin and AICAR increased the mRNA expression of nuclear respiratory factor-1 and mitochondrial DNA transcription factor A (mtTFA) and stimulated the mitochondrial proliferation. Dominant negative-AMPK also reduced the effects of metformin and AICAR on these observations. These results suggest that metformin normalizes hyperglycemia-induced mtROS production by induction of MnSOD and promotion of mitochondrial biogenesis through the activation of AMPK-PGC-1␣ pathway. Diabetes 55:120 -127, 2006

Abstract-Vascular smooth muscle cell (VSMC) proliferation is a critical event in the development and progression of vascular diseases, including atherosclerosis. We investigated whether the activation of adenosine monophosphate-activated protein kinase (AMPK) could suppress VSMC proliferation and inhibit cell cycle progression. Treatment of human aortic smooth muscle cells (HASMCs) or isolated rabbit aortas with the AMPK activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) induced phosphorylation of AMPK and acetyl Co-A carboxylase. AICAR significantly inhibited HASMC proliferation induced by both platelet-derived growth factor-BB (PDGF-BB) and fetal calf serum (FCS). Treatment with AICAR inhibited the phosphorylation of retinoblastoma gene product (Rb) induced by PDGF-BB or FCS, and increased the expression of cyclin-dependent kinase inhibitor p21 CIP but not that of p27 KIP . Pharmacological inhibition of AMPK or overexpression of dominant negative-AMPK inhibited both the suppressive effect of AICAR on cell proliferation and the phosphorylation of Rb, suggesting that the effect of AICAR is mediated through the activation of AMPK. Cell cycle analysis in HASMCs showed that AICAR significantly increased cell population in G0/G1-phase and reduced that in S-and G2/M-phase, suggesting AICAR induced cell cycle arrest. AICAR increased both p53 protein and Ser-15 phosphorylated p53 in HASMCs, which were blocked by inhibition of AMPK. In isolated rabbit aortas, AICAR also increased Ser-15 phosphorylation and protein expression of p53 and inhibited Rb phosphorylation induced by FCS. These data suggest for the first time that AMPK suppresses VSMC proliferation via cell cycle regulation by p53 upregulation. Therefore, AMPK activation in VSMCs may be a therapoietic target for the prevention of vascular diseases.

Abstract-Both statins and peroxisome proliferator-activated receptor (PPAR)␥ ligands have been reported to protect against the progression of atherosclerosis. In the present study, we investigated the effects of statins on PPAR␥ activation in macrophages. Statins increased PPAR␥ activity, which was inhibited by mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore, a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor mimicked the effects of statins. Statins inhibited the membrane translocations of Ras, RhoA, Rac, and Cdc42, and overexpression of dominant-negative mutants of RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPAR␥ activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2-or p38 MAPK-specific inhibitors abrogated statin-induced PPAR␥ activation. Statins induced cyclooxygenase (COX)-2 expression and increased intracellular 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) levels through ERK1/2-and p38 MAPK-dependent pathways, and inhibitors or small interfering RNA of COX-2 inhibited statin-induced PPAR␥ activation. Statins also activate PPAR␣ via COX-2-dependent increases in 15d-PGJ 2 levels. We further demonstrated that statins inhibited lipopolysaccharide-induced tumor necrosis factor ␣ or monocyte chemoattractant protein-1 mRNA expression, and these effects by statins were abrogated by the PPAR␥ antagonist T0070907 or by small interfering RNA of PPAR␥ or PPAR␣. Statins also induced ATP-binding cassette protein A1 or CD36 mRNA expression, and these effects were suppressed by small interfering RNAs of PPAR␥ or PPAR␣. In conclusion, statins induce COX-2-dependent increase in 15d-PGJ 2 level through a RhoA-and Cdc42-dependent p38 MAPK pathway and a RhoA-and Cdc42-independent ERK1/2 pathway, thereby activating PPAR␥. Statins also activate PPAR␣ via COX-2-dependent pathway. These effects of statins may explain their antiatherogenic actions. (Circ Res. 2007;100:1442-1451.) Key Words: cyclooxygenase Ⅲ MAPK Ⅲ macrophages Ⅲ PPAR Ⅲ statins 3 -Hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to reduce the incidence of cardiovascular events and death, and these benefits are mainly caused by their lipid-lowering effects. 1 However, recent evidence has suggested that the beneficial effects by statins are independent of their cholesterol-lowering effects. 2 Cholesterol is synthesized via the isoprenoid biosynthetic pathway. 3 In this pathway, isopentenyl-PP is the basic isoprene unit used for synthesis of all subsequent isoprenoids. 3 Among the isoprenoids, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) serve as important lipid attachments for several proteins, including the small GTP-binding protein Ras and Ras-like proteins, such as Rho, Rac, and Cdc42, whose proper membrane localization and function are dependent on isoprenylation. 3 The pleio...

Objective-To elucidate whether and how the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway in macrophages is involved in the rupture of atherosclerotic plaques. Methods and Results-Increases in macrophage-derived foam cell death in coronary atherosclerotic plaques cause the plaque to become vulnerable, thus resulting in acute coronary syndrome. The ER stress-CHOP/growth arrest and DNA damage-inducible gene-153 (GADD153) pathway is induced in the macrophage-derived cells in atherosclerotic lesions and is involved in plaque formation. However, the role of CHOP in the final stage of atherosclerosis has not been fully elucidated. Many CHOP-expressing macrophages showed apoptosis in advanced ruptured atherosclerotic lesions in wild-type mice, whereas few apoptotic cells were observed in Chop Ϫ/Ϫ mice. The rupture of atherosclerotic plaques was significantly reduced in high cholesterol-fed Chop Ϫ/Ϫ /Apoe Ϫ/Ϫ mice compared with Chop ϩ/ϩ /Apoe Ϫ/Ϫ mice. Furthermore, using mice that underwent bone marrow transplantation, we showed that expression of CHOP in macrophages significantly contributes to the formation of ruptures. By using primary cultured macrophages, we further showed that unesterified free cholesterol derived from incorporated denatured low-density lipoprotein was accumulated in the ER and induced ER stress-mediated apoptosis in a CHOP-Bcl2-associated X protein (Bax) pathway-dependent manner. Key Words: acute coronary syndrome Ⅲ CHOP Ⅲ ER stress Ⅲ Bax Ⅲ apoptosis A cute coronary syndrome, including myocardial infarction and unstable angina, is most frequently caused by an occlusive coronary thrombosis at the site of a preexisting atherosclerotic plaque. [1][2][3][4][5] The formation of coronary thrombosis is generally the result of the rupture of an atherosclerotic plaque, followed by the aggregation of platelets and the formation of fibrin. Therefore, clarification of the mechanisms by which an atherosclerotic plaque becomes vulnerable to rupture would be useful for preventing the onset of acute coronary syndrome. Atherosclerosis is a chronic inflammatory disease of the arterial wall. [1][2][3]6 Macrophages ingest an excess amount of oxidized low-density lipoprotein (LDL) and are converted into foam cells, which then secrete various inflammatory cytokines. Metalloproteinases secreted by macrophages and apoptosis of macrophage-derived foam cells affect the stability of plaques. Thus, monocytes/macrophages play a key role in the instability of atherosclerotic plaques. Conclusion-TheRecently, Myoishi et al 5 reported that the induction of apoptosis and the activation of the endoplasmic reticulum (ER) stress pathway, including the induction of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene0153 (GADD153), a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, were detected in macrophages and smooth muscle cells within ruptured plaques, but not within stable fibrous plaques, in humans. They also reported that the levels of ...