Ki Whan Hong

Remnant lipoprotein particles induce apoptosis in endothelial cells by NAD(P)H oxidase-mediated production of superoxide and cytokines via lectin-like oxidized low-density lipoprotein receptor-1 activation: prevention by cilostazol.

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 &mgr;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)-&agr; and interleukin-1&bgr;, DNA fragmentation, and cell death. Cilostazol (1 to 100 &mgr;mol/L) significantly suppressed not only NAD(P)H oxidase–dependent superoxide production but also TNF-&agr; and interleukin-1&bgr; 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-&agr;, and interleukin-1&bgr; and DNA fragmentation. Conclusions—RLPs stimulated NAD(P)H oxidase–dependent superoxide formation and induction of cytokines in HUVECs 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.

17 β-Estradiol prevents focal cerebral ischemic damages via activation of Akt and CREB in association with reduced PTEN phosphorylation in rats

This study aimed to assess the signaling pathway of the neuroprotective action of estrogen in the cerebral ischemic injury evoked by subjecting rats to 2‐h occlusion of the middle cerebral artery (MCA) followed by 24‐h reperfusion. Rats received 17 β‐estradiol (1, 4 and 10 mg/kg, i.p.) 24 h before and 5 min after the completion of 2‐h MCA occlusion. The cerebral infarct area was consistently observed in the cortex and striatum of the left hemisphere. Increased terminal deoxynucleotidyl transferase‐mediated deoxyuridine–biotin nick‐end labeling (TUNEL)‐positive cells and DNA fragmentation in the penumbral zone were significantly reduced by 17 β‐estradiol. In line with these results, 17 β‐estradiol significantly increased Akt and cyclic AMP response element binding protein (CREB) with increased Bcl‐2 protein in the ischemic area, whereas the elevated the phosphatase and tensin homolog deleted from chromosome10 (PTEN) phosphorylation was significantly reduced with decreased Bax protein and cytochrome c release. Inhibition of DNA fragmentation, PTEN phosphorylation, and Akt activation by 17 β‐estradiol were antagonized by iberiotoxin, a maxi‐K channel blocker. Taken together, it is suggested that suppression of cerebral ischemic injury by 17 β‐estradiol may be ascribed to the maxi‐K channel opening‐coupled downregulation of PTEN phosphorylation and upregulation of Akt and CREB phosphorylation with resultant increase in Bcl‐2 protein and decrease in Bax protein and cytochrome c release.

4-Hydroxynonenal enhances MMP-2 production in vascular smooth muscle cells via mitochondrial ROS-mediated activation of the Akt/NF-κB signaling pathways

4-Hydroxynonenal (HNE) accumulates at atherosclerotic lesions, but its role in the progression of atherosclerosis is not clear. Considering the role of matrix metalloproteinases (MMP) in plaque destabilization, we investigated the mechanism by which HNE induces MMP production in vascular smooth muscle cells (VSMC). VSMC stimulated by HNE (1.0 microM) produced enzymatically active MMP-2 with an increased promoter activity, which was abolished by mutation of the NF-kappaB binding site in the promoter region. The increased NF-kappaB activity with subsequent MMP-2 production by HNE was significantly attenuated by transfection with Akt siRNA as well as by pretreatment with the PI3K/Akt inhibitors LY294002 (10 microM) and SH-5 (1.0 microM). The phosphorylation of Akt occurred as early as 5 min in VSMC exposed to HNE and was markedly attenuated by inhibition of mitochondrial reactive oxygen species (ROS). Furthermore, the impact of mitochondrial ROS on HNE-induced Akt phosphorylation with subsequent MMP-2 production was also demonstrated in mitochondrial function-deficient VSMC, as well as in cells transfected with manganese superoxide dismutase. Taken together, these results suggest that HNE enhances MMP-2 production in VSMC via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways.

Vascular NAD(P)H Oxidase Triggers Delayed Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats

Background and Purpose– To clarify the role of vascular NAD(P)H oxidase in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), both the activity and/or activation mechanisms of NAD(P)H oxidase in the cerebral vasculature and the effect of oxidase inhibition on SAH-induced cerebral vasospasm were assessed. Methods— The changes in the luminal perimeter of the middle cerebral artery were measured histologically after SAH was induced according to a 2-hemorrhage model in rats. The NAD(P)H oxidase activity in the cerebral vasculature was measured with a lucigenin assay at different time intervals from 12 hours to 14 days after injection of autologous blood into cisterna magna. The membrane translocation of p47phox and the protein expression of membrane subunits (gp91phox and p22phox) of NAD(P)H oxidase were analyzed using Western blot analysis. Results— The luminal perimeter of the middle cerebral artery started to decrease on day 1 and peaked on day 5 after a second injection of blood, and these changes were significantly ameliorated by treatment with an NAD(P)H oxidase inhibitor, diphenyleneiodonium. At 24 hours after the second injection of blood, both vascular production of superoxide anion and NAD(P)H oxidase activity were markedly increased with enhanced membrane translocation of p47phox, but by 48 hours the enzyme activity had regained normal values. However, no significant changes in the expression of gp91phox and p22phox were observed throughout the experiments. Conclusions— These findings suggest that the activation of NAD(P)H oxidase through enhanced assembly of the oxidase components in the early stages of SAH might contribute to the delayed cerebral vasospasm in SAH rats.

Cilostazol reduces brain lesion induced by focal cerebral ischemia in rats--an MRI study.

To investigate the effects of cilostazol on the hemispheric ischemic lesion, we monitored the apparent diffusion coefficient (ADC) and T2 images by MRI techniques in comparison with histology at the terminal of and after 24-h reperfusion following 2-h occlusion of middle cerebral artery (MCA). The ADC values of tissue water and T2-weighted images were quantified by high field magnetic resonance. No significant difference was observed by ADC image among vehicle and cilostazol treatment groups when measured during MCA occlusion. Oral treatment with cilostazol 30 mg/kg two times at 5 min and 4 h significantly suppressed the hemispheric lesion area and volumes when detected by ADC, T2 images and histology, but 3 and 10 mg/kg cilostazol were without effect. Cilostazol (30 mg/kg) significantly reduced the increased cerebral water content at the ischemic hemisphere compared with vehicle group. In line with these results, the neurological deteriorations were much improved in the cilostazol-treated group. Taken together, it is concluded that post-treatment with cilostazol exerts a potent protective effect against cerebral infarct size by reducing the cytotoxic edema.

Neuroprotection by cilostazol, a phosphodiesterase type 3 inhibitor, against apoptotic white matter changes in rat after chronic cerebral hypoperfusion.

In the present study, we elucidated effect of cilostazol to prevent the occurrence of vacuolation and rarefaction of the white matter in association with apoptosis induced by bilateral occlusion of common carotid arteries in the male Wistar rats. Rats orally received vehicle (DMSO) or 60 mg kg(-1) day(-1) (orally) cilostazol for 3, 7, 14 or 30 days. In the vehicle group, increased vacuolation and rarefactions in the white matter were accompanied by extensive activation of both microglial and astroglial cells with suppression of oligodendrocytes in association with increased TNF-alpha production, caspase-3 immunoreactivity and TUNEL-positive cells in the white matter including optic tract. Post-treatment with cilostazol (60 mg kg(-1) day(-1)) strongly suppressed not only elevated activation of astroglia and microglia but also diminished oligodendrocytes following chronic cerebral hypoperfusion. In conclusion, cilostazol (60 mg kg(-1) day(-1), orally) significantly reduced the apoptotic cell death in association with decreased TNF-alpha production and caspase-3-positive cells in the white matter of rat brains subjected to bilateral occlusion of common carotid arteries, consequently ameliorating vacuoles and rarefaction changes in the white matter.

Cilostazol Suppresses Superoxide Production and Expression of Adhesion Molecules in Human Endothelial Cells via Mediation of cAMP-Dependent Protein Kinase-Mediated Maxi-K Channel Activation

This study shows whether increased intracellular cAMP level by cilostazol is directly coupled to its maxi-K channel activation in human endothelial cells. Cilostazol (1 μM) increased the K+ currents in the human endothelial cells by activating maxi-K channels, which was abolished by iberiotoxin (100 nM), a maxi-K channel blocker. On incubation of human coronary artery endothelial cells with tumor necrosis factor-α (TNF-α) (50 ng/ml), monocyte adhesion significantly increased with increased superoxide generation and expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) accompanied by increased degradation of inhibitory κBα in cytoplasm and activation of nuclear factor-κB p65 in nucleus. All these variables were significantly suppressed by cilostazol (10 μM), which was antagonized by iberiotoxin (1 μM) and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) (300 nM, cAMP-dependent protein kinase inhibitor), but not by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindo-lo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823) (300 nM, cGMP-dependent protein kinase inhibitor). In the human endothelial cells transfected with siRNA-targeting maxi-K channels, cilostazol did not suppress the superoxide generation, VCAM-1 and MCP-1 expressions, and monocyte adhesion as contrasted with the wild-type cells. These findings were similarly evident with (3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one (BMS-204352), a maxi-K channel opener, and forskolin and dibutyryl cAMP. In conclusion, increased cAMP level by cilostazol is directly coupled to its maxi-K channel opening action via protein kinase activation in human endothelial cells, thereby suppressing TNF-α–stimulated superoxide production and expression of adhesion molecules.

NAD(P)H oxidase participates in the signaling events in high glucose-induced proliferation of vascular smooth muscle cells.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of vascular dysfunction in diabetes mellitus, and NAD(P)H oxidase is known as the most important source of ROS in the vasculatures. To determine whether NAD(P)H oxidase is a major participant in the critical intermediary signaling events in high glucose (HG, 25 mM)-induced proliferation of vascular smooth muscle cells (VSMC), we investigated in explanted aortic VSMC from rats the role of NAD(P)H oxidase on the HG-related cellular proliferation and superoxide production. VSMC under HG condition had increased proliferative capacity that was inhibited by tiron (1 mM), a cell membrane permeable superoxide scavenger, but not by SOD, which is not permeable to cell membrane. The nitroblue tetrazolium staining in the HG-exposed VSMC was more prominent than that of VSMC under normal glucose (5.5 mM) condition, which was significantly inhibited by DPI (10 microM), an NAD(P)H oxidase inhibitor, but not by inhibitors for other oxidases such as NADH dehydrogenase, xanthine oxidase, and nitric oxide synthase. In the VSMC under HG condition, the enhanced NAD(P)H oxidase activity with increased membrane translocation of Rac1 was observed, but the protein expression of p22phox and gp91phox was not increased. These data suggest that HG-induced changes in VSMC proliferation are related to the intracellular production of superoxide through enhanced activity of NAD(P)H oxidase.

Protective effect of the phosphodiesterase III inhibitor cilostazol on amyloid β-induced cognitive deficits associated with decreased amyloid β accumulation.

Alzheimers disease (AD), which is characterized by progressive cognitive impairment, is the most common neurodegenerative disease. Here, we investigated the preventive effect of a phosphodiesterase III inhibitor, cilostazol against cognitive decline in AD mouse model. In vitro studies using N2a cells stably expressing human amyloid precursor protein Swedish mutation (N2aSwe) showed that cilostazol decreased the amyloid β (Aβ) levels in the conditioned medium and cell lysates. Cilostazol attenuated the expression of ApoE, which is responsible for Aβ aggregation, in N2aSwe. Intracerebroventricular injection of Aβ(25-35) in C57BL/6J mice resulted in increased immunoreactivity of Aβ and p-Tau, and microglia activation in the brain. Oral administration of cilostazol for 2 weeks before Aβ administration and once a day for 4 weeks post-surgery almost completely prevented the Aβ-induced increases of Aβ and p-Tau immunoreactivity, as well as CD11b immunoreactivity. However, post-treatment with cilostazol 4 weeks after Aβ administration, when Aβ was already accumulated, did not prevent the Aβ-induced neuropathological responses. Furthermore, cilostazol did not affect the neprilysin and insulin degrading enzymes involved in the degradation of the Aβ peptide, but decreased ApoE levels in Aβ-injected brain. In addition, cilostazol significantly improved spatial learning and memory in Aβ-injected mice. The findings suggest that a phosphodiesterase III inhibitor, cilostazol significantly decreased Aβ accumulation and improved memory impairment induced by Aβ(25-35). The beneficial effects of cilostazol might be explained by the reduction of Aβ accumulation and tau phosphorylation, not through an increase in Aβ degradation but via a significant decrease in ApoE-mediated Aβ aggregation. Cilostazol may be the basis of a novel strategy for the therapy of AD.

Cilostazol suppresses β-amyloid production by activating a disintegrin and metalloproteinase 10 via the upregulation of SIRT1-coupled retinoic acid receptor-β

The accumulation of plaques of β‐amyloid (Aβ) peptides, a hallmark of Alzheimers 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.