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.

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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.


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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