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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.

β-Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2-coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac-tau) and tau phosphorylation (P-tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3β P-Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3-30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP-dependent protein kinase-linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau-related neurodegeneration in the AD brain.

Amyloid-β peptide (Aβ) deposits in the brain are critical in the neurotoxicity induced by Aβ. This study elucidates the underlying signaling pathway by which cilostazol protects HT22 neuronal cells from Aβ(1-40) (3-30 μM)-induced deterioration of cell proliferation, viability, and neurite elongation. Cilostazol rescued HT22 cells from the apoptotic cell death induced by Aβ toxicity through the downregulation of phosphorylated p53 (Ser15), Bax, and caspase-3 and the upregulation of Bcl-2 expression, which improved neuronal cell proliferation and viability. Furthermore, Aβ(1-40) suppressed both phosphorylated CK2α protein expression and CK2 activity in the cytosol; these were concentration dependently recovered by cilostazol (3-30 μM). Cilostazol significantly increased the levels of GSK-3β phosphorylation at Ser9 and β-catenin phosphorylation at Ser675 in the cytosol and nucleus. Cilostazol effects were reversed by KT5720 (1 μM, PKA inhibitor) and TBCA (40 μM, inhibitor of CK2) and CK2α knockdown by siRNA transfection. Likewise, Aβ-stimulated GSK-3β phosphorylation at Tyr 216 was decreased by cilostazol in the control but not in the CK2α siRNA-transfected cells. Furthermore, the Aβ (10 μM)-induced suppression of neurite elongation was recovered by cilostazol; this recovery was attenuated by inhibitors such as KT5720 and TBCA and blocked by CK2α knockdown. In conclusion, increased cAMP-dependent protein kinase-linked CK2α activation underlies the pharmacological effects of cilostazol in downregulating p53 phosphorylation at Ser15 and upregulating GSK-3β phosphorylation at Ser9/β-catenin phosphorylation at Ser675, thereby suppressing Aβ(1-40)-induced neurotoxicity and improving neurite elongation.

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.

Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 μM) stimulated up-regulation of integrins β1, α1, and αv as well as 8-pCPT-2′-O-Me-cAMP (100 μM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 μM) and GGTI-298 (Rap1 inhibitor, 20 μM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N6-benzoyl-cAMP (100 μM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin β1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease. © 2011 Wiley-Liss, Inc.

Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3-28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain-derived neurotrophic factor, was up-regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca-1/vascular endothelial growth factor receptor-2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell-derived factor-1alpha-positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up-regulated by cilostazol in mouse bone marrow-derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow-derived stem cells to areas of injured tissues. CD31-positive cells were colocalized with almost all bromodeoxyuridine-positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow-derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. (c) 2010 Wiley-Liss, Inc.