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

Sponsor

National Cheng-Kung University Hospital (Other)

Overall Status

Completed

CT.gov ID

NCT01952756

Collaborator

Department of Health, Executive Yuan, R.O.C. (Taiwan) (Other)

Enrollment

Location

Arms

Duration (Months)

2.2

Patients Per Site Per Month

Study Details

Study Description

Brief Summary

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.

Condition or Disease	Intervention/Treatment	Phase
Peripheral Arterial Diseases	Drug: Cilostazol Drug: Dummy Placebo	Phase 4

Detailed Description

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

Study Design

Study Type:

Interventional

Actual Enrollment :

44 participants

Allocation:

Randomized

Intervention Model:

Parallel Assignment

Masking:

Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)

Primary Purpose:

Treatment

Official Title:

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

Study Start Date :

Jan 1, 2012

Actual Primary Completion Date :

Sep 1, 2013

Actual Study Completion Date :

Sep 1, 2013

Arms and Interventions

Arm	Intervention/Treatment
Active Comparator: Cilostazol One tablet (100 mg) twice per day for 12 weeks	Drug: Cilostazol One tablet (100 mg) twice per day for 12 weeks Other Names: Pletaal (brand name)
Placebo Comparator: Dummy Placebo One tablet twice per day for 12 weeks	Drug: Dummy Placebo One tablet twice per day for 12 weeks Other Names: Placebo Control

Arm

Intervention/Treatment

Active Comparator: Cilostazol

One tablet (100 mg) twice per day for 12 weeks

Drug: Cilostazol

One tablet (100 mg) twice per day for 12 weeks

Other Names:

Pletaal (brand name)

Placebo Comparator: Dummy Placebo

One tablet twice per day for 12 weeks

Drug: Dummy Placebo

One tablet twice per day for 12 weeks

Other Names:

Placebo

Control

Outcome Measures

Primary Outcome Measures

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

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.

Other Outcome Measures

Viability (Proliferation) of EPCs [3 months]
250,000 cells are seeded in each well of a 96-well plate and the cells are added with 200 μl of the culture medium and incubated at 37°C. Medium change is performed 3 days later. On 7th day, the plate is then re-incubated with 100 μl fresh medium and additional 50 μl of 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2 Hydrogen-Tetrazolium-5-Carboxanilide reagent, and further incubated in dark at 37°C for 4 h. After incubation, the orange colored complex formed is read at 450 nm using a microplate reader with a 450 nm reference filter.

Eligibility Criteria

Criteria

Ages Eligible for Study:

20 Years and Older

Sexes Eligible for Study:

All

Accepts Healthy Volunteers:

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