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

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

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

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.

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

cilostazol, endothelial progenitor cells, vasculogenesis, angiogenesis, peripheral arterial disease, biomarkers

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.

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.

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.

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

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Biscetti F, Pecorini G, Straface G, Arena V, Stigliano E, Rutella S, Locatelli F, Angelini F, Ghirlanda G, Flex A. Cilostazol promotes angiogenesis after peripheral ischemia through a VEGF-dependent mechanism. Int J Cardiol. 2013 Aug 10;167(3):910-6. doi: 10.1016/j.ijcard.2012.03.103. Epub 2012 Apr 2.

Chao TH, Chen IC, Li YH, Lee PT, Tseng SY. Plasma Levels of Proprotein Convertase Subtilisin/Kexin Type 9 Are Elevated in Patients With Peripheral Artery Disease and Associated With Metabolic Disorders and Dysfunction in Circulating Progenitor Cells. J Am Heart Assoc. 2016 May 20;5(5):e003497. doi: 10.1161/JAHA.116.003497. Erratum In: J Am Heart Assoc. 2016;5(7). pii: e002090. doi: 10.1161/JAHA.116.002090.