Cilostazol As a Reagent involved in Rho Study 2013

IL-23 production was elevated by lipoteichoic acid (LTA), 
which increased the activation of RhoA in association with increased the nuclear translocation of NF-kB
and its DNA-binding activity.

Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA),
Y27632 (Rho-kinase) or BAY11-7082 (NF-kB)
inhibited IL-23 production by LTA.

Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-kB activation.


Cilostazol suppressed the TLR2-mediated activation of RhoA,
decreased NF-kB activity with down-regulated IL-23 production, and these effects were reversed by RpcAMPS,
as an inhibitor of cAMP-dependent protein kinase.

 Abbreviations

• CIA, collagen induced arthritis;
• IL-23, interleukin-23;
• LTA, lipoteichoic acid;
• RA, rheumatoid arthritis;
• TLR2, toll like receptor 2

 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.

Graphical abstract

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Abbreviations

• CIA, collagen induced arthritis;
• IL-23, interleukin-23;
• LTA, lipoteichoic acid;
• RA, rheumatoid arthritis;
• TLR2, toll like receptor 2

 

 

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.

http://www.sciencedirect.com.scopeesprx.elsevier.com/science/article/pii/S0167527314001703

2.1. Reagents and antibodies

Lipoteichoic acid (LTA),
BAY11-7082 and
Y27632, were obtained from Sigma (St. Louis, MO).


Rp-cAMPS was purchased
from Alexis (San Diego, CA).
Clostridium botulinum exoenzyme C3 transferase (exoenzyme C3) was from Upstate Biotechnology (Lake Placid, NY. Anti-TLR2 antibody was from Abcam (Cambridge, MA).


NF-kB p65, IkBa, Histone H1and
RhoA antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).


TLR2 neutralizing antibody was from eBioscience (San Diego, CA). IgG isotype control antibody (R&D systems, Minneapolis, MN).


Cilostazol (OPC-13013),
[6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)-quinolinone, >98.5% purity by HPLC,
mean particle size, 14–28 (mean, 20) mm] was donated by Otsuka
Pharmaceutical Co. Ltd. (Tokushima, Japan),

and was dissolved in dimethyl sulfoxide to produce
a 10 mM stock solution.


https://www.blogger.com/blogger.g?blogID=4733967785175105509#editor/target=post;postID=2313110350264441265



Furthermore,

Cilostazol is a commercially available drug that has antiplatelet and vasodilatory activity and is indicated for peripheral artery disease (PAD)-related intermittent claudication [3].

 

 

We and other investigators have found that cilostazol may have beneficial effects on EPCs in vitro [3] and [4] and can provide vasculo-angiogenic effects in murine hindlimb ischemia [3] and [5].

 

 

Therefore, we hypothesized that cilostazol can enhance mobilization and proliferation of EPCs and collateral formation by modifying vasculo-angiogenic biomarkers in PAD.

 

 

 

This prospective, double-blind, randomized placebo-controlled trial consecutively enrolled 44 patients with mild-to-moderate PAD who had ankle-brachial index less than 0.9 in one or both legs without obvious intermittent claudication. Exclusion criteria were listed in the registered study protocol (Clinicaltrials.org registration number NCT01952756).

 

 

All study participants provided signed informed consent, and this study followed the regulation of the ethics committee of the National Cheng Kung University Hospital, where all data were collected.

Eligible subjects were randomly assigned to cilostazol 200 mg (n = 24) or dummy placebo (n = 20) daily for 12 weeks using unrestricted randomization and sealed envelopes for allocation concealment. Serum concentrations of biomarkers were measured by enzyme-linked immunosorbent assay [6].

 

 

The isolation of human early EPCs was performed according to standard protocols [3].

 

 

The quantification of colony formation by EPCs was performed and the chemotactic motility, proliferation/viability (XTT) and apoptosis of EPCs were measured as previously described [3].


http://www.sciencedirect.com.scopeesprx.elsevier.com/science/article/pii/S0167527314001703

シロスタゾール（２） ２０１３

Probucol and cilostazol exert a combinatorial anti-atherogenic effect in cholesterol-fed rabbits

 

 

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Abstract

Introduction

Probucol (PB) and cilostazol (CZ) both exhibit anti-atherogenic effects. However, their combinatorial effects are unclear. This study was designed to investigate their combinatorial anti-atherogenic effect in cholesterol-fed rabbits.

Materials and Methods

Rabbits were fed a cholesterol diet with PB or CZ alone or both PB and CZ for 16 weeks. The plasma levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, C-reactive protein, superoxide dismutase, malondialdehyde, and nitric oxide (NO) were measured. The aortic atherosclerotic lesions were grossly and microscopically evaluated. Additionally, in vitro experiments were conducted using human umbilical vein endothelial cells.

Results and Conclusion

We found that the PB group and the PB + CZ group exhibited a reduction in the lesion areas (70% in the PB + CZ group, 56% in the PB group) compared with the vehicle group. However, although PB alone and PB + CZ led to a reduction in the lesion size, the histological analysis revealed that only PB + CZ significantly decreased the macrophage accumulation and smooth muscle cell proliferation in the lesions compared with the vehicle group. The plasma levels of total cholesterol in the PB + CZ group were decreased compared with the vehicle group, Moreover, PB + CZ exerted obvious anti-oxidant and anti-inflammatory effects. Interestingly, the PB + CZ treatment led to a marked increase in the levels of plasma NO. The in vitro experiments showed that the combinatorial treatment up-regulated the levels of NO and protein S-nitrosylation in endothelial cells treated with oxidized LDL. In summary, these results suggest that PB and CZ exert combinatorial anti-atherogenic effects.

Abbreviations

• CRP, C-reactive protein;
• CZ, cilostazol;
• eNOS, endothelial nitric oxide synthase;
• HCD, high cholesterol diet;
• HDL, high-density lipoproteins;
• HDL-C, high-density lipoprotein cholesterol;
• HUVECs, human umbilical vein endothelial cells;
• LDL-C, low-density lipoprotein cholesterol;
• MDA, malondialdehyde;
• MФ, macrophages;
• NO, nitric oxide;
• PB, probucol;
• ROS, reactive oxygen species;
• SMC, smooth muscle cell;
• SOD, superoxide dismutase;
• TC, total cholesterol

Keywords

• Probucol;
• Cilostazol;
• Atherosclerosis;
• S-nitrosylation;
• Rabbits

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Introduction

Atherosclerosis is a major cause of mortality in both developed and developing countries [1], and hypercholesterolemia is an important risk factor for the development of atherosclerosis. The extensive use of lipid-lowering agents, such as statins, has led to a marked reduction in the number of cardiovascular events in recent years. Despite this achievement, many patients with cardiovascular disease cannot be effectively treated with the use of statins alone [2]. Therefore, it is necessary to develop new therapeutics for these patients.

 

 

Probucol (PB) not only is a lipid-lowering agent but also possesses strong antioxidant properties against low-density lipoproteins (LDL). To date, many studies have shown that PB exhibits anti-atherogenic effects [3]. It has been shown that PB exerts its anti-atherogenic effects through diverse molecular mechanisms, including anti-inflammatory effects, the inhibition of smooth muscle cell (SMC) proliferation, the enhancement of the expression of scavenger receptor class B type I, and the improvement of the functions of high-density lipoproteins (HDL) to enhance reverse cholesterol transport [4], [5], [6] and [7].

 

 

Cilostazol (CZ) is a specific phosphodiesterase type III inhibitor that is currently used for the treatment of thrombotic vascular disease due to its anti-platelet aggregation functions [8]. As an anti-thrombotic drug, CZ is a preferred alternative to aspirin because CZ has fewer side effects, such as increased bleeding time [9]. Previous studies have shown that CZ also exhibits anti-atherogenic effects [10]. In vitro studies have revealed that CZ suppresses the production of intracellular reactive oxygen species (ROS) [11], and increases the production of nitric oxide (NO) [12]. Furthermore, CZ promotes reverse cholesterol transport and inhibits the inflammation and proliferation of SMCs [13], [14], [15] and [16].

 

 

Because both PB and CZ have anti-atherogenic effects, we hypothesized that the combination of minimal doses of PB and CZ may exert additional beneficial effects and may result in a greater athero-protective response. If so, this combinatorial therapy may provide a novel strategy for the treatment and prevention of atherosclerosis for those patients who cannot be effectively treated by statins.

 

 

In the current study, we compared the anti-atherogenic effects of the combination of PB and CZ in cholesterol-fed rabbits with those obtained with either PB or CZ alone. Our results showed that the combination of PB and CZ clearly reduced the aortic atherosclerotic lesion area, and markedly inhibited macrophage (MФ) accumulation and SMC proliferation in the lesions compared with the vehicle group.

 

 

The anti-atherogenic effects of PB and CZ may be collectively mediated by multiple mechanisms, including anti-oxidant effects, anti-inflammatory effects, a decrease in the plasma lipid levels, an increase in the levels of NO, and the maintenance of the endothelial protein S-nitrosylation.

 

 

シロスタゾール & ROCK （１）

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

• So Youn Park^a,
• Sung Won Lee^c,
• Won Suk Lee^{a, b},
• Byung Yong Rhim^b,
• Seung Jin Lee^a,
• Sang Mo Kwon^d,
• Ki Whan Hong^a,
• Chi Dae Kim^{a, b, ,}

• ^a Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Gyeongnam, South Korea
• ^b Department of Pharmacology, School of Medicine, Pusan National University, Gyeongnam, South Korea
• ^c Department of Internal Medicine, College of Medicine, Dong-A University, Busan, South Korea
• ^d Department of Physiology, School of Medicine, Pusan National University, Gyeongnam, South Korea

Received 20 June 2013, Accepted 13 August 2013, Available online 22 August 2013

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DOI: 10.1016/j.bcp.2013.08.017

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  Open Access

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Abstract

IL-23 is produced by antigen presenting cells and plays critical roles in immune response in rheumatoid arthritis (LA).

 

 

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.

 

 

 

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Graphical abstract

Figure options

• Download full-size image
• Download as PowerPoint slide

Abbreviations

• CIA, collagen induced arthritis;
• IL-23, interleukin-23;
• LTA, lipoteichoic acid;
• RA, rheumatoid arthritis;
• TLR2, toll like receptor 2

Keywords

• RA synovial macrophages;
• RhoA;
• TLR2;
• IL-23;
• Cilostazol

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1. Introduction

Rheumatoid arthritis (RA) is a common autoimmune and chronic inflammatory joint disease characterized by increased infiltration of macrophages, proliferation of synovial fibroblast with joint destruction [1]. When abundant monocytes/macrophages in the synovial fluid of RA patients are activated, they produce high levels of cytokines and chemokines, such as interleukin-1β (IL-β), tumor necrosis factor α (TNFα), IL-6, and MCP-1, which contribute to chronic inflammation and joint destruction [2] and [3].

Toll-like receptors (TLRs) are conserved receptors that recognize pathogen-associated molecular patterns, and play important roles in innate and adaptive immunity [4]. TLR2 are mainly expressed on cells, such as macrophages and dendritic cells; and act as primary sensors by recognizing diverse ranges of stimuli [5]. The lipoteichoic acid (LTA) and peptidoglycan are recognized mainly by TLR-2 [6]. It has been reported that TLR2 stimulation causes the preferential induction of IL-8 and IL-23 p19 [7]. IL-23 is involved in autoimmune diseases like RA and psoriasis, in which the cellular function of IL-23 is associated with the self-reactive productions of IL-17, IL-6, and TNF-α, and thus IL-23 plays a critical role in development of autoimmune inflammation [8]. Furthermore, they reported that mice deficient in IL-23 (p19^−/−) were relatively resistant to the development of joint and bone inflammation in a collagen-induced arthritis (CIA) model.

The Rho-GTPase family of monomeric RhoA, Rac1 and Cdc42 is known to cycle between the inactivated GDP-bound state and the activated GTP-bound state [9]. In the active state, Rho is implicated in various cellular processes, such as the cell cycle, cytoskeletal regulation, cellular growth and apoptosis [10] and [11]. Furthermore, it has been reported that RhoA is a key regulator of transcription factors, NF-κB [12], and that inhibition of Rho-kinase reduces the severity of synovial inflammation in rats with CIA [13].

Although it has been demonstrated that IL-23 production plays a crucial role in inflammatory reactions associated with rheumatoid arthritis [14], the signal pathway by which TLR2 induces IL-23 production in RA synovial macrophages has not been defined. On the other hand, cilostazol, a type-III phosphodiesterase inhibitor, has been reported to have anti-inflammatory effects due to the cAMP-dependent protein kinase activation-coupled suppression of NF-κB gene transcription [15].

Therefore, in the present study, we undertook to investigate the signal transduction pathways responsible for TLR2-mediated IL-23 production in synovial fluid macrophages from RA patients: in particular, the present study highlighted implication of the RhoA/ROCK signal pathway in the regulation of TLR2-mediated IL-23 production in RA macrophages. We found that increased IL-23 production by TLR2 involves the activation of NF-κB via a RhoA/ROCK pathway. Further, cilostazol was found to inhibit TLR2-mediated IL-23 production by suppressing RhoA activity via the activation of cAMP-dependent protein kinase, and to suppress the expression of IL-23 in the knee joints of CIA mice.