Takashi Nomiyama

Inhibition of Monocyte Adhesion to Endothelial Cells and Attenuation of Atherosclerotic Lesion by a Glucagon-like Peptide-1 Receptor Agonist, Exendin-4

OBJECTIVE Exogenous administration of glucagon-like peptide-1 (GLP-1) or GLP-1 receptor agonists such as an exendin-4 has direct beneficial effects on the cardiovascular system. However, their effects on atherosclerogenesis have not been elucidated. The aim of this study was to investigate the effects of GLP-1 on accumulation of monocytes/macrophages on the vascular wall, one of the earliest steps in atherosclerogenesis. RESEARCH DESIGN AND METHODS After continuous infusion of low (300 pmol · kg−1 · day−1) or high (24 nmol · kg−1 · day−1) dose of exendin-4 in C57BL/6 or apolipoprotein E–deficient mice (apoE−/−), we evaluated monocyte adhesion to the endothelia of thoracic aorta and arteriosclerotic lesions around the aortic valve. The effects of exendin-4 were investigated in mouse macrophages and human monocytes. RESULTS Treatment with exendin-4 significantly inhibited monocytic adhesion in the aortas of C57BL/6 mice without affecting metabolic parameters. In apoE−/− mice, the same treatment reduced monocyte adhesion to the endothelium and suppressed atherosclerogenesis. In vitro treatment of mouse macrophages with exendin-4 suppressed lipopolysaccharide-induced mRNA expression of tumor necrosis factor-α and monocyte chemoattractant protein-1, and suppressed nuclear translocation of p65, a component of nuclear factor-κB. This effect was reversed by either MDL-12330A, a cAMP inhibitor or PKI14-22, a protein kinase A–specific inhibitor. In human monocytes, exendin-4 reduced the expression of CD11b. CONCLUSIONS Our data suggested that GLP-1 receptor agonists reduced monocyte/macrophage accumulation in the arterial wall by inhibiting the inflammatory response in macrophages, and that this effect may contribute to the attenuation of atherosclerotic lesion by exendin-4.

Osteopontin mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance in mice

Obesity is associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and macrophage infiltration into adipose tissue, which may contribute to the development of insulin resistance. During immune responses, tissue infiltration by macrophages is dependent on the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine that promotes monocyte chemotaxis and cell motility. In the present study, we used a murine model of diet-induced obesity to examine the role of osteopontin in the accumulation of adipose tissue macrophages and the development of insulin resistance during obesity. Mice exposed to a high-fat diet exhibited increased plasma osteopontin levels, with elevated expression in macrophages recruited into adipose tissue. Obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on diet-induced obesity, body composition, or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of inflammation, both in adipose tissue and systemically. Taken together, these results suggest that osteopontin may play a key role in linking obesity to the development of insulin resistance by promoting inflammation and the accumulation of macrophages in adipose tissue.

Anagliptin, a DPP-4 Inhibitor, Suppresses Proliferation of Vascular Smooth Muscles and Monocyte Inflammatory Reaction and Attenuates Atherosclerosis in Male apo E-Deficient Mice

Dipeptyl peptidase-4 (DPP-4) inhibitors modulate the progression of atherosclerosis. To gain insights into their mechanism of action, 9-wk-old male apolipoprotein E (apoE)-deficient mice were fed a DPP-4 inhibitor, anagliptin-containing diet. The effects of anagliptin were investigated in, a monocyte cell line, human THP-1 cells, and rat smooth muscle cells (SMCs). Treatment with anagliptin for 16 wk significantly reduced accumulation of monocytes and macrophages in the vascular wall, SMC content in plaque areas, and oil red O-stained area around the aortic valve without affecting glucose tolerance or body weight. Serum DPP-4 concentrations were significantly higher in apoE-deficient mice than control mice, and the levels increased with aging, suggesting the involvement of DPP-4 in the progression of atherosclerosis. Indeed, soluble DPP-4 augmented cultured SMC proliferation, and anagliptin suppressed the proliferation by inhibiting ERK phosphorylation. In THP-1 cells, anagliptin reduced lipopolysaccharide-induced TNF-α production with inhibiting ERK phosphorylation and nuclear translocation of nuclear factor-κB. Quantitative analysis also showed that anagliptin reduced the area of atherosclerotic lesion in apoE-deficient mice. These results indicated that the anti-atherosclerotic effect of anagliptin is mediated, at least in part, through its direct inhibition of SMC proliferation and inflammatory reaction of monocytes.

The NR4A orphan nuclear receptor NOR1 is induced by platelet-derived growth factor and mediates vascular smooth muscle cell proliferation

Members of the nuclear hormone receptor superfamily function as key transcriptional regulators of inflammation and proliferation in cardiovascular diseases. In addition to the ligand-dependent peroxisome proliferator-activated receptors and liver X receptors, this family of transcription factors includes a large number of orphan receptors, and their role in vascular diseases remains to be investigated. The neuron-derived orphan receptor-1 (NOR1) belongs to the ligand-independent NR4A subfamily, which has been implicated in cell proliferation, differentiation, and apoptosis. In this study, we demonstrate NOR1 expression in vascular smooth muscle cells (SMC) of human atherosclerotic lesions. In response to mitogenic stimulation with platelet-derived growth factor (PDGF), SMC rapidly express NOR1 through an ERK-MAPK-dependent signaling pathway. 5′-Deletion analysis, site-directed mutagenesis, and transactivation experiments demonstrate that PDGF-induced NOR1 expression is mediated through a cAMP-response element-binding protein (CREB)-dependent transactivation of the NOR1 promoter. Consequently, short interfering RNA-mediated depletion of CREB abolished PDGF-induced NOR1 expression in SMC. Furthermore, PDGF induced Ser-133 phosphorylation of CREB and subsequent binding to the CRE sites of the endogenous NOR1 promoter. Functional analysis demonstrated that PDGF induces NOR1 transactivation of its consensus NGFI-B-response elements (NBRE) in SMC. We finally demonstrate that SMC isolated from NOR1-deficient mice exhibit decreased cell proliferation and characterize cyclin D1 and D2 as NOR1 target genes in SMC. These experiments indicate that PDGF-induced NOR1 transcription in SMC is mediated through CREB-dependent transactivation of the NOR1 promoter and further demonstrate that NOR1 functions as a key transcriptional regulator of SMC proliferation.

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury

Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.

The polymorphism of manganese superoxide dismutase is associated with diabetic nephropathy in Japanese type 2 diabetic patients

AbstractWe evaluated the relationship of an alanine or valine polymorphism at amino acid sequence 16 [Val(16)Ala] of manganese superoxide dismutase (Mn-SOD) with diabetes and diabetic nephropathy in Japanese type 2 diabetic patients. Val(16)Ala genotyping of Mn-SOD was done by polymerase chain reaction-restriction fragment length polymorphism with a restriction enzyme (Bsaw I) in 478 Japanese type 2 diabetic patients and 261 nondiabetic Japanese healthy subjects. The genotype distribution of diabetic and nondiabetic subjects was then compared, and the association of genotype with diabetic nephropathy was evaluated in the diabetic patients. The allele frequency and genotype of the diabetic patients were not different from those of the healthy nondiabetic subjects. The VV type showed a significantly higher frequency in the diabetic patients with nephropathy than did the AA or VA type [VV type: normoalbuminuria 70.8%, microalbuminuria 84.8% (P = 0.0057), macroalbuminuria 84.1% (P = 0.0128)]. Furthermore, logistic regression analysis showed that this polymorphism is associated with diabetic nephropathy independently (odds ratio = 0.461925, P = 0.03). Accordingly, the Val(16)Ala polymorphism of Mn-SOD may be unrelated to the etiology of type 2 diabetes, but it seems to be associated with diabetic nephropathy in Japanese type 2 diabetic patients.

Activation of Peroxisome Proliferator-Activated Receptor γ Suppresses Telomerase Activity in Vascular Smooth Muscle Cells

Activation of the peroxisome proliferator-activated receptor (PPAR) &ggr;, the molecular target for insulin sensitizing thiazolidinediones used in patients with type 2 diabetes, inhibits vascular smooth muscle cell (VSMC) proliferation and prevents atherosclerosis and neointima formation. Emerging evidence indicates that telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation. In the present study, we demonstrate that ligand-induced and constitutive PPAR&ggr; activation inhibits telomerase activity in VSMCs. Telomerase reverse transcriptase (TERT) confers the catalytic activity of telomerase, and PPAR&ggr; ligands inhibit TERT expression through a receptor-dependent suppression of the TERT promoter. 5′-deletion analysis, site-directed mutagenesis, and transactivation studies using overexpression of Ets-1 revealed that suppression of TERT transcription by PPAR&ggr; is mediated through negative cross-talk with Ets-1–dependent transactivation of the TERT promoter. Chromatin immunoprecipitation assays further demonstrated that PPAR&ggr; ligands inhibit Ets-1 binding to the TERT promoter, which is mediated at least in part through an inhibition of Ets-1 expression by PPAR&ggr; ligands. In VSMCs overexpressing TERT, the efficacy of PPAR&ggr; ligands to inhibit cell proliferation is lost, indicating that TERT constitutes an important molecular target for the antiproliferative effects of PPAR&ggr; ligands. Finally, we demonstrate that telomerase activation during the proliferative response after vascular injury is effectively inhibited by PPAR&ggr; ligands. These findings provide a previously unrecognized mechanism for the antiproliferative effects of PPAR&ggr; ligands and support the concept that PPAR&ggr; ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases.

PPARα Agonists Suppress Osteopontin Expression in Macrophages and Decrease Plasma Levels in Patients With Type 2 Diabetes

Osteopontin (OPN) is a proinflammatory cytokine implicated in the chemoattraction of monocytes and the development of atherosclerosis. Peroxisome proliferator–activated receptor (PPAR)α, a ligand-activated transcription factor with pleiotropic anti-inflammatory effects in macrophages, is the molecular target for fibrates, which are frequently used to treat dyslipidemia in patients with type 2 diabetes at high risk for cardiovascular disease. In the present study, we examined the regulation of OPN by PPARα agonists in macrophages and determined the effect of fibrate treatment on OPN plasma levels in patients with type 2 diabetes. Treatment of human macrophages with the PPARα ligands bezafibrate or WY14643 inhibited OPN expression. PPARα ligands suppressed OPN promoter activity, and an activator protein (AP)-1 consensus site conferred this repression. Overexpression of c-Fos and c-Jun reversed the inhibitory effect of PPARα ligands on OPN transcription, and, in chromatin immunoprecipitation assays, PPARα ligands inhibited c-Fos and phospho–c-Jun binding to the OPN promoter. Moreover, c-Fos and phospho–c-Jun protein expression was inhibited by PPARα agonists, indicating that PPARα ligands suppress OPN expression through negative cross talk with AP-1–dependent transactivation of the OPN promoter. This inhibitory effect of PPARα ligands on OPN expression was absent in PPARα-deficient macrophages, suggesting a receptor-mediated mechanism of OPN suppression. Finally, treatment of type 2 diabetic patients with bezafibrate significantly decreased OPN plasma levels. These results demonstrate a novel mechanism whereby PPARα ligands may impact macrophage inflammatory responses and decrease early proinflammatory markers for cardiovascular disease.

Deficiency of the NR4A Orphan Nuclear Receptor NOR1 Decreases Monocyte Adhesion and Atherosclerosis

Rationale The orphan nuclear receptor NOR1 is a member of the evolutionary highly conserved and ligand-independent NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily have been characterized as early response genes regulating essential biological processes including inflammation and proliferation; however, the role of NOR1 in atherosclerosis remains unknown. Objective The goal of the present study was to determine the causal contribution of NOR1 to atherosclerosis development and to identify the mechanism by which this nuclear receptor participates in the disease process. Methods and Results In the present study, we demonstrate expression of NOR1 in endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through a nuclear factor &kgr;B–dependent transactivation of the NOR1 promoter. Overexpression of NOR1 in human endothelial cells increased the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule-1, whereas NOR1 deficiency altered adhesion molecule expression in response to inflammatory stimuli. Transient transfection experiments and chromatin immunoprecipitation assays revealed that NOR1 induces VCAM-1 promoter activity by binding to a canonical response element for NR4A receptors in the VCAM-1 promoter. Further functional studies confirmed that NOR1 mediates monocyte adhesion by inducing VCAM-1 and intercellular adhesion molecule-1 expression in endothelial cells. Finally, we demonstrate that NOR1 deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE−/− mice by decreasing the macrophage content of the lesion. Conclusions In concert, these studies identify a novel pathway underlying monocyte adhesion and establish that NOR1 serves a previously unrecognized atherogenic role in mice by positively regulating monocyte recruitment to the vascular wall.

Deficiency of the NR4A Neuron-Derived Orphan Receptor-1 Attenuates Neointima Formation After Vascular Injury

Background— The neuron-derived orphan receptor-1 (NOR1) belongs to the evolutionary highly conserved and most ancient NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily function as early-response genes regulating key cellular processes, including proliferation, differentiation, and survival. Although NOR1 has previously been demonstrated to be required for smooth muscle cell proliferation in vitro, the role of this nuclear receptor for the proliferative response underlying neointima formation and target genes trans-activated by NOR1 remain to be defined. Methods and Results— Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in NOR1−/− mice compared with wild-type mice. In vitro, NOR1-deficient smooth muscle cells exhibit decreased proliferation as a result of a G1→S phase arrest of the cell cycle and increased apoptosis in response to serum deprivation. NOR1 deficiency alters phosphorylation of the retinoblastoma protein by preventing mitogen-induced cyclin D1 and D2 expression. Conversely, overexpression of NOR1 induces cyclin D1 expression and the transcriptional activity of the cyclin D1 promoter in transient reporter assays. Gel shift and chromatin immunoprecipitation assays identified a putative response element for NR4A receptors in the cyclin D1 promoter, to which NOR1 is recruited in response to mitogenic stimulation. Finally, we provide evidence that these observations are applicable in vivo by demonstrating decreased cyclin D1 expression during neointima formation in NOR1-deficient mice. Conclusions— These experiments characterize cyclin D1 as an NOR1-regulated target gene in smooth muscle cells and demonstrate that NOR1 deficiency decreases neointima formation in response to vascular injury.