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

Phase 4

Completed

• Conditions: Peripheral Arterial Diseases
• Interventions: Drug: Dummy Placebo; Drug: Cilostazol

• Registration Number: NCT01952756
• Lead Sponsor: National Cheng-Kung University Hospital

Brief Summary

1. The number and function of circulating endothelial progenitor cells (EPCs) are inversely associated with coronary risk factors and atherosclerotic diseases such as PAOD.

2. 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

1. titration of drugs

1. run-in period: eligible subjects are screened and baseline blood samples are obtained

2. 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

3. 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)

2. assays of human EPCs

1. colony formation by EPCs

2. quantification of EPCs and apoptotic endothelial cells

3. chemotactic motility, proliferation/viability and apoptosis assays

3. collateral vessels formation and distal run-off assessed by dual-energy multi-slice computed tomography angiography

4. echocardiographic examinations to evaluate left ventricular functions

Study Timeline

• Study Start: 2012-01
• Primary Completion: 2013-09
• Study Completion: 2013-09
• Study First Submitted: 2013-09-21
• Study First Submitted QC: 2013-09-25
• Study First Posted: 2013-09-30
• Status Verified: 2014-07
• Last Update Submitted: 2014-07-16
• Last Update Posted: 2014-07-18

Recruitment & Eligibility

• Status: COMPLETED
• Sex: All
• Target Recruitment: 44

Inclusion Criteria

• ankle-brachial index (ABI) less than 0.9 in one or both legs but no obvious symptoms of intermittent claudication

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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

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Study & Design

• Study Type: INTERVENTIONAL
• Study Design: PARALLEL

Arm && Interventions

Group	Intervention	Description
Dummy Placebo	Dummy Placebo	One tablet twice per day for 12 weeks
Cilostazol	Cilostazol	One tablet (100 mg) twice per day for 12 weeks

Primary Outcome Measures

Name	Time	Method
Circulating EPCs Number	3 months	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.

Secondary Outcome Measures

Name	Time	Method
Colony Formation by EPCs	3 months	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.

Trial Locations

Locations (1)

National Cheng Kung University Hospital; 🇨🇳 Tainan, Taiwan