Effect of Cilostazol Endothelial Progenitor Cells and Collateral Formation in Peripheral Occlusive Artery Disease (PAOD)

Study Overview
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Brief Summary
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The number and function of circulating endothelial progenitor cells (EPCs) are inversely associated with coronary risk factors and atherosclerotic diseases such as PAOD. This double-blind, randomized, placebo-controlled trial to evaluate the effects of cilostazol on human early EPCs and angiogenesis as well as the potential mechanisms of action in patients with mild-to-moderate PAOD.

Detailed Description
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titration of drugs run-in period: eligible subjects are screened and baseline blood samples are obtained study period: 12 weeks 24 subjects with cilostazol and 20 subjects with dummy placebo On the first day after the end of the study period, the follow-up data are obtained by the same procedure blood sampling and measurement of serum biomarkers obtained from peripheral veins in all study subjects at the run-in period and the end of the treatment period of the study sent for isolation, cell culture, and assays of human EPCs also stored for enzyme-linked immunosorbent assay (Stromal cell derived factor-alfa1, adiponectin, soluble thrombomodulin, vascular endothelial growth factor) assays of human EPCs colony formation by EPCs quantification of EPCs and apoptotic endothelial cells chemotactic motility, proliferation/viability and apoptosis assays collateral vessels formation and distal run-off assessed by dual-energy multi-slice computed tomography angiography echocardiographic examinations to evaluate left ventricular functions

Official Title
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Cilostazol Enhances the Number and Functions of Circulating Endothelial Progenitor Cells and Collateral Formation Assessed by Dual-energy 128-row CT Angiography Mediated Through Multiple Mechanisms in Patients With Mild-to-moderate PAOD

Conditions
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Peripheral Arterial Diseases

Intervention / Treatment
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* Drug: Cilostazol
* Drug: Dummy Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: ankle-brachial index (ABI) less than 0.9 in one or both legs but no obvious symptoms of intermittent claudication Exclusion Criteria: obvious symptoms of intermittent claudication severe PAD (Fontaine grading > 3) or critical limb ischemia in at least one leg severe liver dysfunction (transaminases >10 times of upper normal limit, history of liver cirrhosis, or hepatoma) > stage 4 chronic kidney disease (end-stage renal disease with chronic dialysis not excluded) left ventricular ejection fraction <50% by echocardiography documented active malignancy chronic inflammatory disease planned coronary intervention or endovascular therapy or bypass surgery within 3 months known drug allergy history for cilostazol current use of cilostazol or any other cAMP-elevator premenopausal women

Ages Eligible for Study
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Minimum Age: 20 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Cilostazol<br>One tablet (100 mg) twice per day for 12 weeks | Drug: Cilostazol<br>* One tablet (100 mg) twice per day for 12 weeks<br>* Other names: Pletaal (brand name);|
| Placebo Comparator: Dummy Placebo<br>One tablet twice per day for 12 weeks | Drug: Dummy Placebo<br>* One tablet twice per day for 12 weeks<br>* Other names: Control;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Circulating EPCs Number | Peripheral blood mononuclear cells (one million cells in each) are suspended in 100 µL phosphate-buffered saline and incubated for 30 min with monoclonal antibodies against human peridinin chlorophyll protein-conjugated cluster of differentiation antigen-45, phycoerythrin-conjugated anti-human cluster of differentiation antigen-34 antibody and anti-human kinase insert domain receptor (KDR) antibody conjugated with Alexa Flour 647. Cells are washed and analyzed on a FACSCalibur flow cytometer with 100,000 events in the lymphocyte gate. EPCs, which are defined as negative for cluster of differentiation antigen-45 and positive for cluster of differentiation antigen-34 and KDR. Based on the peripheral blood mononuclear cell counts, the absolute number of circulating EPCs/µL is calculated. | 3 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Colony Formation by EPCs | Peripheral blood mononuclear cells are isolated by density gradient centrifugation according to standard protocols. After centrifugation, cells are washed, resuspended in M199 medium supplemented with 20% (vol/vol) fetal bovine serum, 10 ng/ml vascular endothelial growth factor, 2 ng/ml basic-fibroblast growth factor, 10 ng/ml epidermal growth factor and 2 ng/ml insulin growth factor, and cultured in 24-well plates coated with human fibronectin for 7 days. EPCs cells are confirmed by uptake of acetyl-low density lipoprotein and lectin and by the expression of EPC markers. Cells are harvested after 7 days, fixed and stained with crystal violet reagent. The colony densities are quantified with an Olympus microscope at 100-fold magnification using an imaging measurement software. | 3 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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cilostazol, endothelial progenitor cells, vasculogenesis, angiogenesis, peripheral arterial disease, biomarkers