Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells.

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-ß Peptide and APP-CTFß Levels in Neuronal Cells.

Autophagy is a vital pathway for the removal of ß-amyloid peptide (Aß) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aß and C-terminal APP fragment ß subunit (APP-CTFß) by up-regulating autophagy.When N2a cells were exposed to soluble Aß1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 µM) or resveratrol (20 µM) prevented these Aß1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aß overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aß and APP-CTFß expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aß and APP-CTFß were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aß was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aß induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aß clearance and increases cell viability.

7α-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells.

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7α-Hydroxycholesterol (7αOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7αOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit α (p19) and the IL-12 subunit ß (p40). However, treatment with 7-ketocholesterol (7K) and 7ß-hydroxycholesterol (7ßOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7ßOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7αOHChol as well as secretion of IL-23 enhanced by 7αOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7αOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.

Inhibition of HMGB1-induced angiogenesis by cilostazol via SIRT1 activation in synovial fibroblasts from rheumatoid arthritis.

High mobility group box chromosomal protein 1 (HMGB-1) released from injured cells plays an important role in the development of arthritis. This study investigated the anti-angiogenic effects of cilostazol in collagen-induced arthritis (CIA) of mice, and the underlying mechanisms involved. The expressions of HIF-1α, VEGF, NF-κB p65 and SIRT1 in synovial fibroblasts obtained from rheumatoid arthritis (RA) patients were assessed by Western blotting, and in vitro and in vivo angiogenesis were analyzed. Tube formations by human microvascular endothelial cells (HMVECs) were significantly increased by direct exposure to HMGB1 or to conditioned medium derived from HMGB1-stimulated RA fibroblasts, and these increases were attenuated by cilostazol, the latter of which was blocked by sirtinol. HMGB1 increased the expression of HIF-1α and VEGF and concomitantly increased nuclear NF-κB p65 and DNA binding activity, but these effects of HMGB1 were inhibited by cilostazol. SIRT1 protein expression was time-dependently decreased (3-24 hr) by HMGB1, which was recovered by pretreatment with cilostazol (1-30 µM) or resveratrol, accompanying with increased SIRT1 deacetylase activity. In the tibiotarsal joint tissues of CIA mice treated with vehicle, HIF-1α- and VEGF-positive spots and CD31 staining were markedly exaggerated, whereas SIRT1 immunofluorescence was diminished. These variables were wholly reversed in cilostazol (30 mg/kg/day)-treated mice. Furthermore, number of blood vessels stained by von Willebrand factor antibody was significantly lower in cilostazol-treated CIA mice. Summarizing, cilostazol activated SIRT1 and inhibited NF-κB-mediated transcription, thereby suppressing the expression of HIF-1α and VEGF. In addition, cilostazol caused HIF-1α deacetylation by enhancing SIRT1 activity and reduced VEGF production, thereby had an anti-angiogenic effect in vitro studies and in CIA murine model.

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

The accumulation of plaques of ß-amyloid (Aß) peptides, a hallmark of Alzheimer's disease, results from the sequential cleavage of amyloid precursor protein (APP) by activation of ß- and γ-secretases. However, the production of Aß can be avoided by alternate cleavage of APP by α-and γ-secretases. We hypothesized that cilostazol attenuates Aß production by increasing a disintegrin and metalloproteinase 10 (ADAM10)/α-secretase activity via SIRT1-coupled retinoic acid receptor-ß (RARß) activation in N2a cells expressing human APP Swedish mutation (N2aSwe). To evoke endogenous Aß 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 Aß 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 Aß 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 RARß and ADAM10, and the cilostazol-stimulated ADAM10 elevation was significantly attenuated by LE135 (a RARß inhibitor), sirtinol, and RARß-gene silencing. In conclusion, cilostazol suppresses the accumulations of FL-APP and Aß by activating ADAM10 via the upregulation of SIRT1-coupled RARß.

Attenuation of ß-amyloid-induced tauopathy via activation of CK2α/SIRT1: targeting for cilostazol.

ß-Amyloid (Aß) 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 GSK3ß. Aß 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 Aß accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3ß 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 Aß 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.

FSL-1, a Toll-like Receptor 2/6 Agonist, Induces Expression of Interleukin-1α in the Presence of 27-hydroxycholesterol.

We investigated the question of whether cholesterol catabolite can influence expression of inflammatory cytokines via Toll-like receptors (TLR) in monocytic cells. Treatment of THP-1 monocytic cells with 27-hydroxycholesterol (27OHChol) resulted in induction of gene transcription of TLR6 and elevated level of cell surface TLR6. Addition of FSL-1, a TLR6 agonist, to 27OHChol-treated cells resulted in transcription of the IL-1α gene and enhanced secretion of the corresponding gene product. However, cholesterol did not affect TLR6 expression, and addition of FSL-1 to cholesterol-treated cells did not induce expression of IL-1α. Using pharmacological inhibitors, we investigated molecular mechanisms underlying the expression of TLR6 and IL-1α. Treatment with Akt inhibitor IV or U0126 resulted in significantly attenuated expression of TLR6 and IL-1α induced by 27OHChol and 27OHChol plus FSL-1, respectively. In addition, treatment with LY294002, SB202190, or SP600125 resulted in significantly attenuated secretion of IL-1α. These results indicate that 27OHChol can induce inflammation by augmentation of TLR6-mediated production of IL-1α in monocytic cells via multiple signaling pathways.

The cholesterol-binding antibiotic nystatin induces expression of macrophage inflammatory protein-1 in macrophages.

Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1α and MIP-1ß, however, upon exposure to nystatin, significantly elevated expression of MIP-1α and MIP-1ß was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1α and MIP-1ß was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.

RhoA/ROCK-dependent pathway is required for TLR2-mediated IL-23 production in human synovial macrophages: suppression by cilostazol.

IL-23 is produced by antigen presenting cells and plays critical roles in immune response in rheumatoid arthritis. In this study, we investigated whether the RhoA/Rho-kinase pathway is required to elevate TLR2-mediated IL-23 production in synovial macrophages from patients with rheumatoid arthritis (RA), and then examined the suppressive effect of cilostazol on these pathways. IL-23 production was elevated by lipoteichoic acid (LTA), a TLR2 ligand, and this elevation was more prominent in RA macrophages than in those from peripheral blood of normal control. LTA increased the activation of RhoA in association with increased the nuclear translocation of NF-κB and its DNA-binding activity. Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA), Y27632 (Rho-kinase) or BAY11-7082 (NF-κB) inhibited IL-23 production by LTA. Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-κB activation. Cilostazol suppressed the TLR2-mediated activation of RhoA, decreased NF-κB activity with down-regulated IL-23 production, and these effects were reversed by Rp-cAMPS, as an inhibitor of cAMP-dependent protein kinase. The expression of IL-23, which colocalized with CD68⁺ cells in knee joint of CIA mice, was significantly attenuated by cilostazol along with the decreased severity of arthritis. Taken together, the RhoA/Rho-kinase pathway signals TLR2-stimulated IL-23 production in synovial fluid macrophages via activation of NF-κB. Thus it is summarized that cilostazol suppresses TLR2-mediated IL-23 production by suppressing RhoA pathway via cAMP-dependent protein kinase activation.

Differential regulation of CC chemokine ligand 2 and CXCL8 by antifungal agent nystatin in macrophages.

The polyene antifungal antibiotic nystatin can interact with cholesterol, thereby altering the composition of the plasma membrane in eukaryotic cells. We investigated whether nystatin influences responses to the infection by inducing expression of chemokines. THP-1 macrophages rarely expressed CC chemokine ligand 2 (CCL2) and CXCL8. However, nystatin dose-dependently increased CCL2 and CXCL8 expression at the mRNA and protein levels. To understand the molecular mechanisms of the antifungal agent, we identified cellular factors activated by nystatin and those involved in nystatin-induced upregulation of CCL2 and CXCL8. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK1/2, p38 MAPK, and JNK. Treatment with cholesterol, LY294002, Akt inhibitor IV, U0126, and SP6001250 resulted in abrogation or significant attenuation of nystatin-induced CCL2 expression. Nystatin-mediated CXCL8 expression was attenuated in the presence of Akt inhibitor IV and SP6001250. These results indicate that exposure of human macrophages to nystatin can lead to differential regulation of CCL2 and CXCL8 via the activation of multiple cellular kinases. We propose that upregulation of CCL2 and CXCL8 contributes to pharmacological effects of nystatin.

Multiple Signaling Molecules are Involved in Expression of CCL2 and IL-1ß in Response to FSL-1, a Toll-Like Receptor 6 Agonist, in Macrophages.

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1ß and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1ß in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1ß, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1ß. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1ß. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1ß via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.

Roles of 7-ketocholesterol on the homeostasis of intracellular cholesterol level.

Atherosclerotic plaque contains materials, such as cholesterol, oxysterols, cell debris, modified fatty acids, and infiltrated cells. Among them, cholesterol is the major component in plaque. Cholesterol is known to originate from the influx of extracellular materials, but this explanation is not enough for the cholesterol accumulation observed in atherosclerotic plaque. This study examined the origins of cholesterols in plaques. The main focus was to determine if the intracellular cholesterol levels are affected by oxysterols in human vascular smooth muscle cells. The results showed that the cholesterol levels increased in response to a 7-ketocholesterol (7K)-treatment in a dose-dependent manner. Eight enzymes involved in cholesterol biosynthesis were examined. Among them, squalene epoxidase (SQLE) was increased by 7K but not by 7α-hydroxycholesterol, 27-hydroxycholesterol (27OH-chol), or cholesterol. The 7K-induced SQLE expression was suppressed in the presence of the enzyme inhibitor SB203580 but not by UO126 and SP600125. The SQLE immunoreactivity was detected in the atherosclerotic plaque of the aortic roots from apoE mice. In addition, 7K increased the cholesterol level and SQLE expression in murine bone marrow-derived macrophages. This suggests that 7K increases the intracellular cholesterol level through an elevation of SQLE expression, which might affect the progress of cholesterol accumulation in the atherosclerotic lipid core.

Protection by cilostazol against amyloid-ß(1-40)-induced suppression of viability and neurite elongation through activation of CK2α in HT22 mouse hippocampal cells.

Amyloid-ß peptide (Aß) deposits in the brain are critical in the neurotoxicity induced by Aß. This study elucidates the underlying signaling pathway by which cilostazol protects HT22 neuronal cells from Aß(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 Aß 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, Aß(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-3ß phosphorylation at Ser9 and ß-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, Aß-stimulated GSK-3ß phosphorylation at Tyr 216 was decreased by cilostazol in the control but not in the CK2α siRNA-transfected cells. Furthermore, the Aß (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-3ß phosphorylation at Ser9/ß-catenin phosphorylation at Ser675, thereby suppressing Aß(1-40)-induced neurotoxicity and improving neurite elongation.

Extracellular Nucleotides Can Induce Chemokine (C-C motif) Ligand 2 Expression in Human Vascular Smooth Muscle Cells.

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y(1), P2Y(6), and P2Y(11) receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD(+), an agonist of the human P2Y(11) receptor, and NADP(+) as well as ATP, an agonist for P2Y(1) and P2Y(11) receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD(+) and NADP(+) was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD(+) and NADP(+) activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD(+)- and NADP(+)-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.

Peptidoglycan enhances secretion of monocyte chemoattractants via multiple signaling pathways.

Peptidoglycan (PG) is detected in a high proportion in inflammatory cell-rich regions of human atheromatous plaques. In the present study, we determined the cellular factors involved in PG-mediated chemokine expression in mononuclear cells in order to understand the molecular mechanisms of inflammatory responses to bacterial pathogen-associated molecular patterns in the diseased artery. Exposure of human monocytic leukemia THP-1 cells to PG resulted in not only enhanced secretion of CCL2 and CCL4 but also profound induction of their gene transcripts, which were abrogated by oxidized 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine, an inhibitor of Toll-like receptors (TLRs)-2/4, but not by polymyxin B. PG enhanced phosphorylation of Akt and mitogen-activated protein kinases and activated protein kinase C. Pharmacological inhibitors such as SB202190, SP6001250, U0126, Akt inhibitor IV, rapamycin, and RO318220 significantly attenuated PG-mediated up-regulation of CCL2 and CCL4. We propose that PG contributes to vascular inflammation in atherosclerotic plaques by upregulating expression of mononuclear cell chemoattractants via TLR-2, protein kinase C, Akt, mTOR, and mitogen-activated protein kinases.

Cilostazol enhances integrin-dependent homing of progenitor cells by activation of cAMP-dependent protein kinase in synergy with Epac1.

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 ß1, 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, N(6)-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 ß1 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.

Induction of heme oxygenase-1 expression by cilostazol contributes to its anti-inflammatory effects in J774 murine macrophages.

The effects of cilostazol on stimulating heme oxygenase (HO)-1 expression including signal pathways and suppression of inflammatory cytokines and molecules were studied. Cilostazol stimulation time (1-8 h)- and concentration (1-30 µM)-dependently increased the HO-1 mRNA and protein expression associated with increased HO-1 activity, as did cobalt protoporphyrin IX (1-3 µM) in J774 macrophages. In addition, cilostazol (1-30 µM) concentration-dependently reduced lipopolysaccharide (LPS)-mediated nitrite and TNF-α production, in accordance with the inhibition of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in the J774 macrophages, as did CoPP (1 µM). In parallel with these results, LPS-induced IκBα degradation and NF-κB nuclear translocation were significantly decreased after treatment with cilostazol as well as with CoPP. These effects of cilostazol and CoPP were significantly reversed by Zn protoporphyrin IX (ZnPP). The effects of cilostazol on IκBα expression and nitrite production were not manifested in the cells transfected with HO-1 small interfering RNA. In the J774 macrophages, cilostazol time (0-180min)- and concentration (1-100µM)-dependently increased the nuclear expression of NF-E2 related factor (Nrf2) and antioxidant response element (ARE) activity (3.70±0.45 fold, P<0.01). PI3-kinase and Akt play a role in the major signal pathways with cilostazol-induced HO-1 expression. In summary, cilostazol suppressed production of anti-inflammatory cytokines and molecules via inhibition of NF-κB activation, through a mechanism involving up-regulation of cyclic AMP-dependent protein kinase activation-coupled Nrf2-linked HO-1 expression in J774A.1 macrophages.

Cilostazol enhances apoptosis of synovial cells from rheumatoid arthritis patients with inhibition of cytokine formation via Nrf2-linked heme oxygenase 1 induction.

OBJECTIVE: To assess the effects of cilostazol in inhibiting proliferation and enhancing apoptosis in synovial cells from patients with rheumatoid arthritis (RA). METHODS: Synovial cell proliferation was measured by MTT assay. The expression of NF-kappaB, IkappaBalpha, Bcl-2, Bax, heme oxygenase 1 (HO-1), and Nrf2 was determined by Western blotting. RESULTS: Cilostazol suppressed synovial cell proliferation by arresting the G(2)/M phases of the cell cycle, and this was reversed by KT5720, an inhibitor of protein kinase A. Cilostazol increased the number of TUNEL-positive cells, with increased cytochrome c release and apoptosis-inducing factor translocation as well as increased caspase 3 activation. Cilostazol (10 microM) and cobalt protoporphyrin IX (CoPP) increased HO-1 messenger RNA and protein expression. These effects were suppressed by zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor. Cilostazol and CoPP significantly increased IkappaBalpha in the cytosol and decreased NF-kappaB p65 expression in the nucleus. Increased expression of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 induced by lipopolysaccharide was attenuated by cilostazol and CoPP, and this was reversed by ZnPP. In mice with collagen-induced arthritis treated with cilostazol (10 and 30 mg/kg/day), paw thickness was decreased with increased apoptotic cells in the joints. In synovial cells transfected with small interfering RNA (siRNA) targeting HO-1, cilostazol did not suppress expression of TNFalpha, IL-1beta, and IL-6, in contrast to findings with negative control cells. Cilostazol- and CoPP-induced HO-1 expression was diminished in cells transfected with Nrf2 siRNA. CONCLUSION: Cilostazol suppressed proliferation of synovial cells from RA patients by enhancing apoptosis, and also inhibited cytokine production via mediation of cAMP-dependent protein kinase activation-coupled Nrf2-linked HO-1 expression.

Cilostazol enhances neovascularization in the mouse hippocampus after transient forebrain ischemia.

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.