Peripheral Arterial Diseases
Conditions
Keywords
cilostazol, endothelial progenitor cells, vasculogenesis, angiogenesis, peripheral arterial disease, biomarkers
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
Interventions
DRUGCilostazol
One tablet (100 mg) twice per day for 12 weeks
DRUGDummy Placebo
One tablet twice per day for 12 weeks
Sponsors
Department of Health, Executive Yuan, R.O.C. (Taiwan)
National Cheng-Kung University Hospital
Study design
Allocation
RANDOMIZED
Intervention model
PARALLEL
Primary purpose
TREATMENT
Masking
QUADRUPLE (Subject, Caregiver, Investigator, Outcomes Assessor)
Eligibility
Sex/Gender
ALL
Age
20 Years to No maximum
Healthy volunteers
No
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
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| 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
| Measure | Time frame | Description |
|---|---|---|
| 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
| Measure | Time frame | Description |
|---|---|---|
| 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. |
Countries
Taiwan
Outcome results
None listed