Background-Remnant lipoprotein particles (RLPs), products of lipolytic degradation of triglyceride-rich lipoprotein derived from VLDL, exert atherogenesis. In this study, we observed how RLPs induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) and cilostazol prevented cell death. Methods and Results-RLPs were isolated from the plasma of hyperlipidemic patients by use of an immunoaffinity gel mixture of anti-apolipoprotein A-1 and anti-apolipoprotein B-100 monoclonal antibodies. RLPs (50 g/mL) significantly increased superoxide formation in HUVECs associated with elevated gp91phox mRNA and protein expression and Rac1 translocation, accompanied by increased production of tumor necrosis factor (TNF)-␣ and interleukin-1␤, DNA fragmentation, and cell death. Cilostazol (1 to 100 mol/L) significantly suppressed not only NAD(P)H oxidase-dependent superoxide production but also TNF-␣ and interleukin-1␤ release and restored viability. RLPs activated a lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which was not inhibited by cilostazol. Treatment of HUVECs with monoclonal antibody for LOX-1 attenuated RLP-mediated production of superoxide, TNF-␣, and interleukin-1␤ and DNA fragmentation. Conclusions-RLPs stimulated NAD(P)H oxidase-dependent superoxide formation and induction of cytokines inHUVECs via activation of LOX-1, consequently leading to reduction in cell viability with DNA fragmentation, and cilostazol exerts a cell-protective effect by suppressing these variables.

This study examined the protective effects of cilostazol on cerebral infarcts produced by subjecting rats to 2-h occlusion of the left middle cerebral artery followed by 24-h reperfusion. The ischemic cerebral infarct consistently involved the cortex and striatum. The infarct size was significantly reduced, when rats received 10 mg/kg cilostazol intravenously 5 min or 1 h after the completion of 2-h ischemia. Cyclic AMP level was significantly elevated in the cortex of 4-and 12-h reperfusion (P Ͻ 0.01) following treatment with cilostazol (10 mg/kg, 5 min after 2-h ischemia) accompanied by decreased tumor necrosis factor-␣ level. Samples from the regions corresponding to the penumbra showed markedly reduced Bcl-2 protein level and, in contrast, high levels of Bax protein and cytochrome c release. Cilostazol decreased Bax protein and cytochrome c release and increased the levels of Bcl-2 protein. Cilostazol (10

This work describes the pharmacological inhibition by cilostazol and its metabolites, OPC-13015 and OPC-13213, of the apoptosis in the human umbilical vein endothelial cells (HUVECs) damaged by lipopolysaccharide (LPS) in comparison with its analog, cilostamide. Cilostazol and OPC-31213 caused a significant suppression of cell death induced by LPS (1 g/ml) in a concentration-dependent manner but a modest suppression by cilostamide and OPC-13015. These compounds potently inhibited the 5,5-dimethyl-1-pyrroline-1-oxide (DMPO)/ ⅐ OH adduct formation and significantly reduced the increased intracellular reactive oxygen species (ROS) and tumor necrosis factor-␣ (TNF-␣) production induced by LPS (1 g/ml). An apoptotic death of HUVECs by 1 g/ml LPS (DNA ladders on electrophoresis) was strongly suppressed by all these compounds. Incubation with LPS caused a marked decrease in Bcl-2 protein, which was significantly reversed by cilostazol and its analogs. The greatly increased Bax protein expression and cytochrome c release by LPS were, in contrast, suppressed by cilostazol and, to a lesser degree, by others. In conclusion, cilostazol and its analogs exert a strong protection against apoptotic cell death by scavenging hydroxyl radicals and intracellular ROS with reduction in TNF-␣ formation and by increasing Bcl-2 protein expression and decreasing Bax protein and cytochrome c release.

Superoxide anion can modulate vascular smooth muscle tone and is potentially involved in diabetic vascular complications. The present study was undertaken to characterize both vascular production and the enzymatic source of superoxide anion in type 2 diabetic rats. In the thoracic aorta of OLETF rats, endotheliumdependent relaxation was markedly attenuated compared with that of control (LETO) rats in association with a significant increase in superoxide production (2,421.39 ؎ 407.01 nmol ⅐ min -1 ⅐ mg -1 ). The increased production of superoxide anion was significantly attenuated by diphenyleneiodonium (DPI; 10 mol/l), an inhibitor of NAD(P)H oxidase. The production of superoxide anion in response to NADH as a substrate was markedly increased in the vascular homogenates, but NADPH, arachidonic acid, xanthine, and succinate produced only small increases in chemiluminescence. In line with these results, studies using various enzyme inhibitors, such as DPI, allopurinol, rotenone, N G -monomethyl-L-arginine, and indomethacin, suggest that the predominant source of superoxide anion in vascular particulate fraction is NADH-dependent membranebound oxidase. Furthermore, the expression of p22phox, a major component of vascular NAD(P)H oxidase, was markedly increased in the aorta from OLETF rats compared with that of LETO rats. These findings suggest that upregulated expression of p22phox mRNA and enhanced NADH oxidase activity contribute to the impaired endothelium-dependent vasodilation in OLETF rats. Diabetes 51:522-527, 2002

High-mobility group box protein 1 (HMGB1), a nonhistone nuclear protein and a cytokine mediator, is implicated in the pathogenesis of rheumatoid arthritis (RA). Extracellular HMGB1 binds to its receptors and triggers downstream signal cascade leading to the perpetuation of synovitis and local tissue invasion. Here, we investigated a novel role of HMGB1 in regulating hypoxia-inducible factor (HIF)-1α to mediate angiogenesis in RA synovium. HIF-1α mRNA levels and activities in synovial fibroblasts from RA patients were enhanced by HMGB1. Pharmacological inhibition of TLR4 and NF-kappaB activation blocked the HMGB1-dependent upregulation of HIF-1α mRNA expression and its activity, suggesting the involvement of transcriptional regulation. HMGB1 stimulated expression of vascular endothelial growth factor (VEGF), and inhibition of HIF-1α attenuated HMGB1-induced VEGF. Conditioned media derived from HMGB1-stimulated synovial fibroblasts enhanced tube formation in human microvascular endothelial cells by upregulating HIF-1α. In the joint tissues of mice with collagen-induced arthritis, treatment with anti-HMGB1 neutralizing antibody prevented blood vessel formation in association with decreased expression of HIF-1α. These observations support the idea that increased HMGB1 induces an extension of inflamed synovium by accelerating angiogenesis in RA through enhancement of HIF-1α activation. Therefore, inhibition of HMGB1 could prove beneficial for the treatment of angiogenesis in RA.Keywords: Fibroblast r HIF-1α r angiogenesis r HMGB1 r Rheumatoid arthritis Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by hyperplastic synovium in association with immunemediated inflammatory synovitis involving the ingress of abundant Correspondence: Prof. Chi Dae Kim e-mail: chidkim@pusan.ac.kr leucocytes and enhanced angiogenesis. Furthermore, blood vessel growth supports the proliferation of inflammatory synovial pannus and ingress of inflammatory leukocytes into synovial tissues during the development and progression of arthritis [1].High-mobility group box protein 1 (HMGB1), a nuclear DNAbinding protein, is released by passive diffusion from necrotic cells and activated macrophages, and is found extracellularly in abundance in synovitis. Furthermore, HMGB1 levels are reportedwww.eji-journal.eu Eur. J. Immunol. 2015. 45: 1216-1227 Molecular immunology 1217 to be higher in RA synovial fluid than in the synovial fluid of osteoarthritis (OA) [2][3][4]. In addition, it has been demonstrated that released HMGB1 binds to its receptors, such as, TLR2, TLR4, and RAGE [5,6], and that these mediate inflammatory responses by inducing the productions of proinflammatory cytokines, such as, 8]. Furthermore, Lin et al. [9] have demonstrated HMGB1 initiates TLR4-dependent neovascularization in cornea. It has also been reported that hypoxia increases extracellular HMGB-1, which colocalizes with tissue h...