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Background: Calcification of the aortic valve leads to increased leaflet stiffness and consequently to the development of calcific aortic valve disease. However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified that dipeptidyl peptidase-4 (DPP-4, also known as CD26) increases valvular calcification and promotes calcific aortic valve disease progression., Methods: We obtained the aortic valve tissues from humans and murine models (wild-type and endothelial nitric oxide synthase-deficient-mice) and cultured the valvular interstitial cells (VICs) and valvular endothelial cells from the cusps. We induced osteogenic differentiation in the primary cultured VICs and examined the effects of the DPP-4 inhibitor on the osteogenic changes in vitro and aortic valve calcification in endothelial nitric oxide synthase-deficient-mice. We also induced calcific aortic stenosis in male New Zealand rabbits (weight, 2.5-3.0 kg) by a cholesterol-enriched diet+vitamin D2 (25 000 IU, daily). Echocardiography was performed to assess the aortic valve area and the maximal and mean transaortic pressure gradients at baseline and 3-week intervals thereafter. After 12 weeks, we harvested the heart and evaluated the aortic valve tissue using immunohistochemistry., Results: We found that nitric oxide depletion in human valvular endothelial cells activates NF-κB in human VICs. Consequently, the NF-κB promotes DPP-4 expression, which then induces the osteogenic differentiation of VICs by limiting autocrine insulin-like growth factor-1 signaling. The inhibition of DPP-4 enzymatic activity blocked the osteogenic changes in VICs in vitro and reduced the aortic valve calcification in vivo in a mouse model. Sitagliptin administration in a rabbit calcific aortic valve disease model led to significant improvements in the rate of change in aortic valve area, transaortic peak velocity, and maximal and mean pressure gradients over 12 weeks. Immunohistochemistry staining confirmed the therapeutic effect of Sitagliptin in terms of reducing the calcium deposits in the rabbit aortic valve cusps. In rabbits receiving Sitagliptin, the plasma insulin-like growth factor-1 levels were significantly increased, in line with DPP-4 inhibition., Conclusions: DPP-4-dependent insulin-like growth factor-1 inhibition in VICs contributes to aortic valve calcification, suggesting that DPP-4 could serve as a potential therapeutic target to inhibit calcific aortic valve disease progression., (© 2017 American Heart Association, Inc.)

This study investigated trends in the prescription of antidiabetic medications for patients with type 2 diabetes, focusing on changing patterns of prescriptions and the cost of drugs during the last 10 years. Retrospective data on patients with type 2 diabetes aged 30 years or older were analyzed using information from the National Health Information Database collected by the National Health Insurance Service in Korea from January 2002 to December 2013. We identified patients with type 2 diabetes who had at least one service claim in each year during the study period. The prescribing information was collected and fixed-dose combination tablets were counted as each of their constituent classes. The total number of adults with type 2 diabetes who were treated using antidiabetic agents increased from 0.87 million in 2002 to 2.72 million in 2013 in Korea. Among antidiabetic medications in 2002, sulfonylurea (SU) was the most commonly used agent (87.2%), and metformin was the second (52.9%). However, in 2013, the use of metformin increased to 80.4% of the total antidiabetic prescriptions. The use of dipeptidyl peptidase-4 (DPP-4) inhibitor increased remarkably after release in late 2008 and composed one-third of the market share with 1 million prescriptions (38.4%) in 2013. Among the prescriptions for monotherapy, only 13.0% were metformin in 2002, but the amount increased to 53.2% by 2013. In contrast, the use of SU declined dramatically from 75.2% in 2002 to 30.6% in 2013. Dual and triple combinations steadily increased from 35.0% and 6.6% in 2002 to 44.9% and 15.5% in 2013, respectively. In 2013, SU with metformin (41.7%) and metformin with DPP-4 inhibitor (32.5%) combination were most frequently prescribed. The total antidiabetic medication cost increased explosively from U.S. $70 million (82.5 billion won) in 2002 to U.S. $4 billion (480 billion won) in 2013.The use of antidiabetic agents and their costs have been increasing steadily. Metformin is the most commonly used drug recently. The use of DPP-4 inhibitor increased significantly over the past decade, whereas the use of SU decreased. However, SUs still remain the most commonly prescribed second-line agents with metformin in 2013., Competing Interests: The authors declare no conflict of interest.

Objective: Nucleotide-binding oligomerization domain protein 2 (NOD2) stimulates diverse inflammatory responses resulting in differential cellular phenotypes. To identify the role of NOD2 in vascular arterial obstructive diseases, we investigated the expression and pathophysiological role of NOD2 in a vascular injury model of neointimal hyperplasia., Methods and Results: We first analyzed for neointimal hyperplasia following femoral artery injury in NOD2(+/+) and NOD2(-/-) mice. NOD2(-/-) mice showed a 2.86-fold increase in neointimal formation that was mainly composed of smooth muscle (SM) α-actin positive cells. NOD2 was expressed in vascular smooth muscle cells (VSMCs) and NOD2(-/-) VSMCs showed increased cell proliferation in response to mitogenic stimuli, platelet-derived growth factor-BB (PDGF-BB), or fetal bovine serum, compared with NOD2(+/+) VSMCs. Furthermore, NOD2 deficiency markedly promoted VSMCs migration in response to PDGF-BB, and this increased cell migration was attenuated by a phosphatidylinositol 3-kinase inhibitor. However, protein kinase C and c-Jun N-terminal kinase inhibitors exerted negligible effects. Moreover, muramyl dipeptide-stimulated NOD2 prevented PDGF-BB-induced VSMCs migration., Conclusion: Functional NOD2 was found to be expressed in VSMCs, and NOD2 deficiency promoted VSMCs proliferation, migration, and neointimal formation after vascular injury. These results provide evidence for the involvement of NOD2 in vascular homeostasis and tissue injury, serving as a potential molecular target in the modulation of arteriosclerotic vascular disease.

Objective: To explore whether α-lipoic acid (ALA), a naturally occurring antioxidant, inhibits neointimal hyperplasia by inducing apoptosis of vascular smooth muscle cells and to examine its potential effects on reendothelialization and platelet aggregation., Methods and Results: Restenosis and late stent thrombosis, caused by neointimal hyperplasia and delayed reendothelialization, are significant clinical problems of balloon angioplasty and drug-eluting stents. ALA treatment strongly induced apoptosis of vascular smooth muscle cells and enhanced the expression and cytoplasmic localization of Nur77, which triggers intrinsic apoptotic events. Small interfering RNA-mediated downregulation of Nur77 diminished this proapoptotic effect of ALA. Moreover, ALA increased p38 mitogen-activated protein kinase phosphorylation, and inhibition of p38 mitogen-activated protein kinase completely blocked ALA-induced vascular smooth muscle cell apoptosis and Nur77 induction and cytoplasmic localization. In balloon-injured rat carotid arteries, ALA enhanced Nur77 expression and increased TUNEL-positive apoptotic cells in the neointima, leading to inhibition of neointimal hyperplasia. This preventive effect of ALA was significantly reduced by infection of an adenovirus encoding Nur77 small hairpin (sh)RNA. Furthermore, ALA reduced basal apoptosis of human aortic endothelial cells and accelerated reendothelialization after balloon injury. ALA also suppressed arachidonic acid-induced platelet aggregation., Conclusions: ALA could be a promising therapeutic agent to prevent restenosis and late stent thrombosis after angioplasty and drug-eluting stent implantation.

Objective: Fatty acids increase reactive oxygen species generation and cell apoptosis in endothelial cells. The peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1alpha) is a transcriptional coactivator that increases mitochondrial biogenesis and fatty acid oxidation in various cells. This study was undertaken to investigate the possible preventive effect of PGC-1alpha on endothelial apoptosis and its molecular mechanism., Methods and Results: Treatment with linoleic acid in cultured human aortic endothelial cells increased reactive oxygen species generation and cell apoptosis. These effects appeared to be mediated by increases in cytosolic fat metabolites, ie, fatty acyl CoA, diacylglycerol, and ceramide, and consequent decreases in ATP/ADP translocase activity of adenine nucleotide translocator. Adenoviral overexpression of PGC-1alpha prevented linoleic acid-induced increases in reactive oxygen species generation and cell apoptosis in human aortic endothelial cells by increasing fatty acid oxidation, decreasing diacylglycerol and ceramide, and increasing ATP/ADP translocase activity. In isolated aorta, PGC-1alpha overexpression prevented linoleic acid-induced decrease in endothelium-dependent vasorelaxation, and this effect was abolished by adenine nucleotide translocator1 shRNA., Conclusions: PGC-1alpha regulates reactive oxygen species generation and apoptosis in endothelial cells by increasing fatty acid oxidation and enhancing ATP/ADP translocase activity. Measures to increase PGC-1alpha expression or ATP/ADP translocase activity in vascular cells may aid in the prevention or treatment of atherosclerosis.

Objective: Clusterin is induced in vascular smooth muscle cells (VSMCs) during atherosclerosis and injury-induced neointimal hyperplasia. However, its functional roles in VSMCs and endothelial cells remain controversial and elusive. This study was undertaken to clarify the role of clusterin in neointimal hyperplasia and elucidate its mechanism of action., Methods and Results: Adenovirus-mediated overexpression of clusterin (Ad-Clu) repressed TNF-alpha-stimulated expression of MCP-1, fractalkine, ICAM-1, VCAM-1, and MMP-9, leading to inhibition of VSMC migration. Both Ad-Clu and secreted clusterin suppressed VSMC proliferation by inhibiting DNA synthesis, but not by inducing apoptosis. Ad-Clu upregulated p53 and p21(cip1/waf1) but downregulated cyclins D and E, leading to suppression of pRb phosphorylation and subsequent induction of G1 arrest in VSMCs. Clusterin deficiency augmented VSMC proliferation in vitro and accelerated neointimal hyperplasia in vivo, but concomitantly impaired reendothelialization in wire-injured murine femoral arteries. Moreover, Ad-Clu significantly reduced neointimal thickening in balloon-injured rat carotid arteries. Clusterin also diminished TNF-alpha-induced apoptosis of human umbilical vein endothelial cells and restored endothelial nitric oxide synthase expression suppressed by TNF-alpha., Conclusions: These results suggest that upregulation of clusterin during vascular injury may be a protective response against, rather than a causative response to, the development of neointimal hyperplasia.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.

Objective: Lipid accumulation in vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis in obese subjects. We showed previously that alpha-lipoic acid (ALA) activates AMP-activated protein kinase (AMPK) and reduces lipid accumulation in skeletal muscle of obese rats. Here, we investigated whether ALA improves endothelial dysfunction in obese rats by activating AMPK in endothelial cells., Methods and Results: Endothelium-dependent vascular relaxation was impaired, and the number of apoptotic endothelial cells was higher in the aorta of obese rats compared with control rats. In addition, triglyceride and lipid peroxide levels were higher, and NO synthesis was lower. Administration of ALA improved all of these abnormalities. AMPK activity was lower in aortic endothelium of obese rats, and ALA normalized it. Incubation of human aortic endothelial cells with ALA activated AMPK and protected cells from linoleic acid-induced apoptosis. Dominant-negative AMPK inhibited the antiapoptotic effects of ALA., Conclusions: Reduced AMPK activation may play an important role in the genesis of endothelial dysfunction in obese rats. ALA improves vascular dysfunction by normalizing lipid metabolism and activating AMPK in endothelial cells.

Objective: Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and regulates production of reactive oxygen species in macrophages. Previous studies have shown that selective genetic disruption of UCP2 in bone marrow cells results in excess accumulation of monocytes/macrophages in the vascular wall of hypercholesterolemic low-density lipoprotein receptor-deficient (LDLR-/-) mice. Here we investigated whether UCP2 regulates expression of genes involved in monocyte recruitment., Methods and Results: UCP2 overexpression in THP1 monocytes, which induced a 10-fold increase in mitochondrial UCP2 protein levels, reduced steady-state level of intracellular reactive oxygen species (ROS) and H2O2-induced ROS production. THP1 monocytes with UCP2 overexpression showed lower intracellular calcium levels and less H2O2-triggered intracellular calcium mobilization, and less protein and mRNA levels of beta2 integrins, most notably CD11b. UCP2 overexpression reduced beta2 integrin-mediated firm adhesion of monocytes to either tumor necrosis factor-alpha (TNF-alpha)-stimulated human aortic endothelial cell (HAEC) monolayers or to plates coated with intercellular adhesion molecule-1, not vascular cell adhesion molecule-1. UCP2 overexpression also inhibited cell spreading and actin polymerization in monocytes treated with TNF-alpha and monocyte chemoattractant protein-1 (MCP-1), and reduced MCP-1-induced transmigration of monocytes through HAEC monolayers., Conclusions: Mitochondrial UCP2 in circulating monocytes may prevent excessive accumulation of monocytes/macrophages in the arterial wall, thereby reducing atherosclerotic plaque formation.