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

This study shows that 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2(1H)-quinolinone (cilostazol) suppresses the atherosclerotic lesion formation in the low-density lipoprotein receptor (Ldlr)-null mice. Ldlr-null mice fed a high cholesterol diet showed multiple plaque lesions in the proximal ascending aorta including aortic sinus, accompanied by increased macrophage accumulation with increased expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). Supplementation of cilostazol (0.2% w/w) in diet significantly decreased the plaque lesions with reduced macrophage accumulation and suppression of VCAM-1 and MCP-1 in situ. Increased superoxide and tumor necrosis factor-␣ (TNF-␣) production were significantly lowered by cilostazol in situ as well as in cultured human umbilical vein endothelial cells (HUVECs). TNF-␣-induced increased inhibitory B␣ degradation in the cytoplasm and nuclear factor-B (NF-B) p65 activation in the nuclei of HUVECs were reversed by cilostazol (1 ϳ 100 M) as well as by (E)-3[(4-t-butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085) (10 M), suggesting that cilostazol strongly inhibits NF-B activation and p65 translocation into the nuclei. Furthermore, in gel shift and DNA-binding assay, cilostazol inhibited NF-B/DNA complex and nuclear DNA-binding activity of the NF-B in the nuclear extracts of the RAW 264.7 cells. Taken together, it is suggested that the antiatherogenic effect of cilostazol in cholesterol-fed Ldlr-null mice is ascribed to its property to suppress superoxide and TNF-␣ formation, and thereby reducing NF-B activation/transcription, VCAM-1/MCP-1 expressions, and monocyte recruitments.Evidence accumulates that atherogenesis is closely related to the inflammatory and proliferative responses of the endothelium after injury (Ross, 1993). During early stages of the atherosclerosis, adhesion and chemoattractant molecules, including vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), are secreted by the activated endothelial cells in the atherosclerotic lesions, by which the immune cells and monocytes are recruited and migrated into the intimal area of the vascular wall (Reape and Groot, 1999). Reactive oxygen species and TNF-␣ are critically implicated not only in the induction of endothelial apoptosis (Dimmeler et al., 1998) but also in the development and progression of atherosclerotic lesions in humans (Meyer et al., 1999).Inactive NF-B resides in the cytoplasm bound by its inhibitory subunit, IB␣ (Pahl, 1999). Inflammatory stimuli including TNF-␣ and endotoxin lead to degradation of IB␣ by its phosphorylation pathway (Chen et al., 1995), which allows translocation of active NF-B into the nucleus, where it regulates gene expression and binds to the promoter of the target genes such as VCAM-1 and MCP-1.The low-density lipoprotein receptor (Ldlr)-null mouse is an animal model of homozygous familial hypercholesterolemia characterized by an absence of functional LDL receptors. Th...

Toll-like receptor (TLR)-4 signaling promotes cytokine synthesis in vascular smooth muscle cells (VSMC). However, it is unknown how TLR-4 regulates interleukin-6 (IL-6) in VSMC. Therefore, the present study investigated cellular factors involved in TLR-4-mediated IL-6 in VSMC in terms of MAPK and transcription elements. Exposure of aortic smooth muscle cells to TLR4-specific lipopolysaccharide (LPS) not only enhanced IL-6 release but also induced IL-6 transcript via promoter activation. The promoter activation was attenuated by dominant-negative MKK1 and to a lesser extent by dominant-negative MKK3, but not by dominant-negative MKK4. IL-6 promoter activity was diminished by U0126 or SB202190, but not by SP600125. Co-transfection with dominant negative CCAAT/enhancer binding protein or with IkappaB suppressed LPS-induced promoter activation, whereas the promoter activity was not influenced by dominant negative c-Jun. Mutation in the IL-6 promoter region at the binding site of NF-kappaB or C/EBP impaired promoter activation in response to LPS. Further impairment occurred when both NF-kappaB- and C/EBP-binding sites were mutated. LPS-induced IL-6 promoter activation was also prevented by pretreatment with epigallocatechin 3-gallate, curcumin, and resveratrol. The present study reports that TLR4-agonistic LPS induces IL-6 through transcriptional activation in VSMC and ERK1/2, p38 MAPK, NF-kappaB, and C/EBP play active roles in that process.

This study shows cilostazol effect to prevent remnant lipoprotein particle (RLP)-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). Upon incubation of HUVECs with RLP (50 g/ml), adherent monocytes significantly increased by 3.3-fold with increased cell surface expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, E-selectin, and monocyte chemoattractant protein-1 (MCP-1). Cilostazol (ϳ1-100 M) concentration dependently repressed these variables as did (E)3-[(4-t-butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085) (10 M), a specific nuclear factor-B (NF-B) inhibitor. Cilostazol effects were significantly antagonized by iberiotoxin (1 M), a maxi-K channel blocker. RLP significantly increased expression of lectin-like receptor for oxidized low-density lipoprotein (LDL) (LOX-1) receptor protein. Upon transfection with antisense LOX-1 oligodeoxynucleotide (As-LOX-1), LOX-1 receptor expression was reduced, whereas HUVECs with sense LOX-1 oligodeoxynucleotide did express high LOX-1 receptor. RLP-stimulated superoxide and tumor necrosis factor-␣ levels were significantly lowered with decreased expression of VCAM-1 and MCP-1 by transfection with As-LOX-1 as did polyinosinic acid (10 g/ml, a LOX-1 receptor inhibitor). RLP significantly degraded inhibitory B␣ in the cytoplasm and activated nuclear factor-B (NF-B) p65 in the nucleus of HUVECs with increased luciferase activity of NF-B, all of which were reversed by cilostazol (10 M), BAY 11-7085, and polyinosinic acid. Together, cilostazol suppresses RLP-stimulated increased monocyte adhesion to HUVECs by suppression of LOX-1 receptorcoupled NF-B-dependent nuclear transcription via mediation of the maxi-K channel opening.Atherosclerosis is known as chronic inflammatory processes resulting from interaction between oxidized low-density lipoprotein (Ox-LDL), macrophages, lymphocytes, and other cellular elements of the arterial wall (Ross, 1999). Recent clinical evidence has suggested that endothelial dysfunction elicited by Ox-LDL is critically important in the pathogenesis of atherosclerosis, in that inflammation plays a central role in its development (Ross, 1999;Blake and Ridker, 2001). Since Nakajima et al. (1993) have developed a simple and rapid assay method for determination of remnant lipoprotein particles (RLP)-cholesterol, i.e., chylomicron and VLDL remnants, a number of reports have focused on the role of RLP, derived from VLDL and chylomicrons, as an atherogenic factor (Hodis, 1999). It has been shown that RLP elicit endothelial vasomotor dysfunction in human coronary arteries and in isolated rabbit aorta . Endothelium-derived reactive oxygen species initiate and propagate free radical chain reactions in polyunsaturated fatty acid in RLP (Doi et al., 2000). Ox-LDL elicits endothelial dysfunction by enhancing expresArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.077826. ABBREVIATIONS:Ox-LDL, oxidized low-density lipoprot...

The accumulation of plaques of β-amyloid (Aβ) peptides, a hallmark of Alzheimer's disease, results from the sequential cleavage of amyloid precursor protein (APP) by activation of β- and γ-secretases. However, the production of Aβ can be avoided by alternate cleavage of APP by α-and γ-secretases. We hypothesized that cilostazol attenuates Aβ production by increasing a disintegrin and metalloproteinase 10 (ADAM10)/α-secretase activity via SIRT1-coupled retinoic acid receptor-β (RARβ) activation in N2a cells expressing human APP Swedish mutation (N2aSwe). To evoke endogenous Aβ overproduction, the culture medium was switched from medium containing 10% fetal bovine serum (FBS) to medium containing 1% FBS, and cells were cultured for 3∼24 hr. After depletion of FBS in media, N2aSwe cells showed increased accumulations of full-length APP (FL-APP) and Aβ in a time-dependent manner (3-24 hr) in association with decreased ADAM10 protein expression. When pretreated with cilostazol (10-30 μM), FL-APP and Aβ levels were significantly reduced, and ADAM10 and α-secretase activities were restored. Furthermore, the effect of cilostazol on ADAM10 expression was antagonized by pretreating Rp-cAMPS and sirtinol and by SIRT1-gene silencing. In the N2aSwe cells overexpressing the SIRT1 gene, ADAM10, and sAPPα levels were significantly elevated. In addition, like all-trans retinoic acid, cilostazol enhanced the protein expressions of RARβ and ADAM10, and the cilostazol-stimulated ADAM10 elevation was significantly attenuated by LE135 (a RARβ inhibitor), sirtinol, and RARβ-gene silencing. In conclusion, cilostazol suppresses the accumulations of FL-APP and Aβ by activating ADAM10 via the upregulation of SIRT1-coupled RARβ.