Aya Shiraki

The glucagon-like peptide 1 analog liraglutide reduces TNF-α-induced oxidative stress and inflammation in endothelial cells

OBJECTIVE Glucagon-like peptide 1 (GLP-1), one of the incretin hormones, has been reported to increase positive inotropic activity in cardiac myocytes and protect against myocardial injury. However, the effects upon endothelial cells and the mechanisms involved are not fully understood. We assessed the hypothesis that GLP-1 has protective effects against inflammation and oxidative stress on human endothelial cells. METHODS AND RESULTS The effects of the GLP-1 analog liraglutide upon TNF-α-induced injury of the human umbilical vein endothelial cells (HUVECs) were evaluated. First, ROS induced by TNF-α was measured by staining with CM-H(2)DCFDA. Intracellular ROS production of HUVECs was significantly decreased in a dose-dependent manner until 30 nM while liraglutide inhibited the induction of gp91(phox) and p22(phox), subunit of NADPH oxidase, by TNF-α. In addition, protein levels of SOD-2, catalase and GPx were significantly increased by liraglutide. Second, rapid translocation of PKC-α into the membrane following TNF-α was evident. Liraglutide significantly inhibited this very rapid TNF-α-induced translocation of PKC-α into membrane at 2.5 min. Third, liraglutide significantly inhibited NF-κB activation and upregulated I-κB family while phosphorylation of IKK-α/β, which is upstream of NF-κB signaling, was also downregulated after 15 min of TNF-α treatment. Finally, liraglutide inhibited apoptosis of HUVEC and expression of Pentraxin-3 induced by TNF-α. CONCLUSION Liraglutide exerts marked anti-oxidative and anti-inflammatory effects on endothelial cells with inhibition of PKC-α, NADPH oxidase, NF-κB signaling and upregulation of protective anti-oxidative enzymes.

Possible effects of glimepiride beyond glycemic control in patients with type 2 diabetes: a preliminary report

BackgroundThe purpose of this study was to elucidate the effects of glimepiride on the levels of biomarkers related to cardiovascular regulation in patients with type 2 diabetes mellitus.Methods and resultsThirty-four patients with type 2 diabetes received glimepiride for 24 weeks. Significant decreases in the levels of glyceraldehyde-derived advanced glycation end products, (glycer-AGE: toxic AGE), eotaxin and fibroblast growth factor (FGF)-2 were recognized after the administration of glimepiride. Moreover, there were trends for there to be increases in the levels of granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF), and decreases in the levels of fractalkine, soluble CD40 ligand (sCD40L), macrophage inflammatory protein (MIP)-β, vascular endothelial growth factor (VEGF) and soluble receptor for AGE (sRAGE).ConclusionsGlimepiride may have potent anti-oxidative, anti-inflammatory and angiogenic properties and it may potentially repair tissue damage by decreasing the levels of toxic AGE and increasing colony-stimulating factors.

EGCG, a green tea catechin, attenuates the progression of heart failure induced by the heart/muscle-specific deletion of MnSOD in mice.

BACKGROUND Manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme affected in heart/muscle-specific MnSOD-deficient mice (H/M-SOD2-/-), which develop progressive congestive heart failure and exhibit pathology typical of dilated cardiomyopathy. METHODS In this study we investigated the beneficial effects of epigallocatechin gallate (EGCG) on the cardiac remodeling and telomere biology in H/M-SOD2-/- mice. H/M-SOD2-/- mice were divided into three groups: those receiving normal drinking water (KO), a low dose of EGCG (L: 10mg/L), and a high dose of EGCG (H: 100mg/L) beginning at eight weeks of age and lasting for eight weeks. RESULTS The mice in the KO group exhibited significantly dilated cardiac remodeling with reduced contractility, which was prevented by the administration of EGCG. Although the mortality of KO mice was about 50% at 16 weeks of age, the mice that received EGCG had a high survival rate. The cardiac dilatation with reduced cardiac contraction in KO mice was prevented by EGCG treatment. The levels of myocardial oxidative stress and free fatty acids were lower in the group treated with EGCG compared with the KO group. The increased expression of nitric oxide synthase 2, nitrotyrosine, fatty acid synthase, Toll-like receptor 4, and Sirt1 in the KO mice were prevented by EGCG treatment. The shortening of the telomere length, decreased telomerase activity in KO mice were also prevented by EGCG. CONCLUSIONS H/M-SOD2-/- mice receiving EGCG have a lower mortality rate and exhibit less inflammation and a better preserved cardiac function and telomere biology.

Deletion of Apoptosis Inhibitor of Macrophage (AIM)/CD5L Attenuates the Inflammatory Response and Infarct Size in Acute Myocardial Infarction

Background An excessive inflammatory response after myocardial infarction (MI) increases myocardial injury. The toll‐like receptor (TLR)‐4 is activated by the recognition of endogenous ligands and is proinflammatory when there is myocardial tissue injury. The apoptosis inhibitor of the macrophage (AIM) is known to provoke an efflux of saturated free fatty acids (FFA) due to lipolysis, which causes inflammation via the TLR‐4 pathway. Therefore, this study investigated the hypothesis that AIM causes a proinflammatory response after MI. Methods and Results The left anterior descending coronary artery was ligated to induce MI in both AIM‐knockout (AIM−/−) and wild‐type (WT) mice. After 3 days, the inflammatory response from activation of the TLR‐4/NFκB pathway was assessed, and infarct size was measured by staining with triphenyltetrazolium chloride. In addition, left ventricular remodeling was examined after 28 days. Although the area at risk was similar between AIM−/− and WT mice, the infarct size was significantly smaller in AIM−/− mice (P=0.02). The heart weight–to–body weight ratio and myocardial fibrosis were also decreased in the AIM−/− mice, and the 28‐day survival rate was improved (P<0.01). With the reduction of plasma FFA in AIM−/− mice, myocardial IRAK4 and NFκB activity were decreased (all P<0.05). Moreover, there was a reduction in myeloperoxidase activity and inducible nitric oxide synthase as part of the inflammatory response (P<0.01, P=0.03, respectively). Furthermore, NFκB DNA‐binding activation via TLR‐4, neutrophil infiltration, and inflammatory mediators were decreased in AIM−/− mice. Conclusions The deletion of AIM reduced the inflammatory response and infarct size and improved survival after myocardial infarction.

Pentraxin-3 regulates the inflammatory activity of macrophages

Background and aims Pentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aimed to investigate the function of PTX3 in macrophages. Methods PMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis. Results In the presence of PTX3, macrophage interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and MCP-1 levels were reduced significantly (−39%, P=0.007; −21%, P=0.008; and −67%, P=0.0003, respectively), whilst activated transforming growth factor-β (TGF−β) was detected in the THP-1 macrophages (P=0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced nuclear factor-kappa B (NF-κB) activation by 35% (P=0.002), which was induced by TNF-α in THP-1 macrophages. Furthermore, silencing of PTX3 in apoptotic cells resulted in increased macrophage binding, elevated expression rate of HLA-DR (+30%, P=0.015) and CD86 (+204%, P=0.004) positive cells, and induction of IL-1β (+36%, P=0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner. Conclusions We identified PTX3 as a novel regulator of macrophage activity, and this function suggests that PTX3 acts to resolve inflammation.

Efficacy and safety of sitagliptin in elderly patients with type 2 diabetes mellitus: DPP-4 inhibitor in elderly T2DM patients

The aim of the present study was to assess the efficacy and safety of sitagliptin in elderly patients with type 2 diabetes mellitus.

Azelnidipine Inhibits the Differentiation and Activation of THP-1 Macrophages through the L-Type Calcium Channel

Aim: Recently, calcium channel blockers (CCBs) have been reported to reduce atherosclerosis with anti-inflammatory or antiatherosclerotic effects in vivo. It is well established that monocytes and macrophages play important roles in promoting atherosclerosis. However, the effects of CCBs on macrophage activation remain unclear. The aim of this study was to evaluate the effects of azelnidipine, a dihydropyridine L-type CCB, on the activation of macrophages and to clarify the mechanisms of the effects of CCBs on atherosclerosis. Methods: THP-1 monocytes, a human leukemic cell line, were stimulated with 50 ng/mL of phorbol-12-myristate-13-acetate (PMA) 1 h after pretreatment with 10 µM azelnidipine or dimethyl sulfoxide (DMSO), and harvested. Results: Azelnidipine blocked the expression of intercellular adhesion molecule-1 quantified by FACS analysis. The expression levels of Apo E and MMP9, which are markers of macrophage differentiation, were inhibited by azelnidipine as evaluated by quantitative RT-PCR. The level of LOX-1 mRNA, a scavenger receptor, was also reduced significantly by pretreatment with 10 µM azelnidipine. Azelnidipine also lowered the uptake of acetylated LDL. The expression of the L-type calcium channel Cav1.2 was 10-fold higher after 24 h of PMA stimulation. A knockdown of the CACNA1C gene, which encodes Cav1.2 protein in humans, with siRNA blocked the effect of reducing adhesion by azelnidipine, indicating that the effects of azelnidipine on macrophage differentiation were expressed through the CACNA1C gene. Conclusion: Our results suggest that azelnidipine has potent antiatherosclerotic properties by inhibition of macrophage activation through Cav1.2.

PS 13-17 APOPTOSIS INHIBITOR OF MACROPHAGE ACTIVATES INFLAMMATORY RESPONSE AFTER ACUTE MYOCARDIAL INFARCTION

Objective: The persistent inflammation response after myocardial infarction increases myocardial injury, and causes heart failure due to cardiac dysfunction and remodeling. Toll-like receptors (TLR)-4 was activated by the recognition of not only pathogen-associated molecular patterns but also endogenous ligands and it serves as proinflammatory function in circumstances involving myocardial tissue injury. Whereas, it was reported the apoptosis inhibitor of macrophage (AIM) provoke an efflux of saturated free fatty acid (FFA), one of the TLR4 ligands, due to lipolysis, which causes the chronic inflammation via TLR-4 pathway in adipose tissue. This study investigated the hypothesis that AIM activates a proinflammatory response after myocardial infarction. Design and Method: The ligation of the left anterior descending coronary artery to induced myocardial infarction was performed on both AIM-deficient(AIM−/−) mice and wild type (WT) mice. After 3 days, the inflammation responses were assessed in TLR-4/NFkB activation pathway. And infarct size/area-at-risk was measured by staining with Evans blue and triphenyltetrazolium chloride. In addition, the left ventricular remodeling was examined after 28 days. Results: AIM−/− mice showed significantly smaller infarct size compared with WT mice given similar area at risk (20.8 ± 0.6% vs. 27.8 ± 1.9%, p = 0.02). In AIM−/− mice with reduction of plasma FFA, IRAK4 and NFkB activity were decreased on myocardium (p = 0.02, 0.03 vs. WT, respectively). Moreover, there was the reduction of myeloperoxidase activity and iNOS in the inflammation response (p < 0.01, p = 0.03, respectively). We found that the NFkB DNA-binding activation via TLR-4, neutrophil infiltration, and inflammatory mediator were decreased in AIM−/− mice compared with WT mice after 3days. And, the heart weight to body weight ratio, myocardial fibrosis of AIM−/− mice were decreased, and a survival rate improved after 28 days (p < 0.01). Conclusions: AIM−/− mice demonstrated less inflammation response and smaller infarction size after myocardial infarction, suggesting AIM may activate a proinflammatory of TLR-4 after myocardial infarction.

PS 13-05 PENTRAXIN-3 REGULATES THE INFLAMMATORY ACTIVITY OF MACROPHAGES

Objective: Pentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aims to investigate the function of PTX3 in macrophages. Design and Method: PMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis. Results: In the presence of PTX3, macrophage interleukin-1b (IL-1b), tumor necrosis factor-alpha (TNF-a) and MCP-1 levels were reduced significantly (−39%, P = 0.007; -21%, P = 0.008; and-67%, P = 0.0003, respectively), whilst the activated transforming growth factor-b (TGF-b) was detected in the THP-1 macrophages (P = 0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced the nuclear factor-kappa B (NF-&kgr;B) activation by 35% (P = 0.002), which was induced by TNF-a in THP-1 macrophages. Furthermore, the silencing of PTX3 in apoptotic cells resulted in the increased macrophage binding, elevated expression rate of HLA-DR (+30%, P = 0.015) and CD86 (+204%, P = 0.004) positive cells, and induction of IL-1b (+36%, P = 0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner. Conclusions: We identified PTX3 as a novel regulator of macrophage activity, which suggests that PTX3 acts to resolve inflammation.

PS 13-13 EGCG, A GREEN TEA CATECHIN, ATTENUATES THE PROGRESSION OF HEART FAILURE INDUCED BY THE HEART/MUSCLE-SPECIFIC DELETION OF MnSOD IN MICE

Objective: Manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme affected in heart/muscle-specific MnSOD-deficient mice (H/M-SOD2−/−), which develop progressive congestive heart failure and exhibit pathology typical of dilated cardiomyopathy. Design and Method: In this study we investigated the beneficial effects of epigallocatechin gallate (EGCG) on the cardiac remodeling and telomere biology in H/M-SOD2 −/−mice. Methods: H/M-SOD2−/− mice were divided into three groups: those receiving normal drinking water (KO), a low dose of EGCG (L: 10 mg/L) and a high dose of EGCG (H: 100 mg/L) beginning at eight weeks of age and lasting for eight weeks. Results: The mice in the KO group exhibited significantly dilated cardiac remodeling with reduced contractility, which was prevented by the administration of EGCG. Although the mortality of KO mice was about 50% at 16 weeks of age, the mice that received EGCG had a high survival rate. The cardiac dilatation with reduced cardiac contraction in KO mice was prevented by EGCG treatment. The levels of myocardial oxidative stress and free fatty acids were lower in the group treated with EGCG compared with the KO group. The increased expression of nitric oxide synthase 2, nitrotyrosine, fatty acid synthase, Toll-like receptor 4 and Sirt1 in the KO mice were prevented by EGCG treatment. The shortening of the telomere length, decreased telomerase activity in KO mice were also prevented by EGCG. Conclusions: H/M-SOD2−/− mice receiving EGCG have a lower mortality rate and exhibit less inflammation and a better preserved cardiac function and telomere biology.