Effect of Cilostazol on Endothelial Progenitor Cells and Endothelial Function in High Risk for Cardiovascular Disease

Cilostazol Enhances the Number and Functions of Circulating Endothelial Progenitor Cells Mediated Through Multiple Mechanisms in Patients With High Risk for Cardiovascular Disease

1. The number and function of circulating endothelial progenitor cells (EPCs) are inversely associated with coronary risk factors and atherosclerotic diseases.
2. This double-blind, randomized, placebo-controlled trial to evaluate the effects of cilostazol on human early EPCs and endothelial function as well as the potential mechanisms of action in patients with high risk for cardiovascular disease.

Study Overview

• Status: Completed
• Conditions: Cardiovascular Diseases
• Intervention / Treatment: Drug: Cilostazol; Drug: Dummy Placebo

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

      • subjects with cilostazol and 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. measurement of flow-mediated dilatation (FMD) of left brachial artery by sonography

• Study Type: Interventional
• Enrollment (Actual): 71
• Phase: Phase 4

Contacts and Locations

Study Locations

• Taiwan
  • Tainan, Taiwan, 704
    • National Cheng Kung University Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 20 years and older (ADULT, OLDER_ADULT)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

Description

Inclusion Criteria: high-risk patients who have at least one of the following situations without pre-existing cardiovascular disease including peripheral artery disease or coronary artery disease:

• type 2 diabetes mellitus
• metabolic syndrome
• stage 3 (or more advanced) chronic kidney disease
• 2 or more coronary risk factors (male > 45 years or female > 55 years, hypertension, tobacco smoking, hyperlipidemia, family history of cardiovascular disease)

Exclusion Criteria:

• ankle-brachial index less than 0.9 or more than 1.3 in one or both legs
• significant stenosis (more than 50% as compared to reference vessel) in peripheral artery on image study
• symptoms suggesting peripheral artery disease in at least one leg
• clinical or electrocardiographic evidence of coronary artery disease
• clinical evidence of cerebrovascular disease
• severe liver dysfunction (transaminases >10 times of upper normal limit, history of liver cirrhosis, or hepatoma)
• left ventricular ejection fraction (<50% by echocardiography)
• documented active malignancy
• chronic inflammatory disease
• known drug allergy history for cilostazol
• current use of cilostazol or any other cAMP-elevator
• premenopausal women

Study Plan

How is the study designed?

Design Details

• Primary Purpose: TREATMENT
• Allocation: RANDOMIZED
• Interventional Model: PARALLEL
• Masking: QUADRUPLE

Number of Arms

2

Arms and Interventions

Participant Group / 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

What is the study measuring?

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
Viability (Proliferation) of EPCs	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.	3 months

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
FMD of the Brachial Artery	FMD in response to reactive hyperemia is measured in the left brachial artery in a quiet, temperature-controlled room after 10 min of bed rest. A high-resolution ultrasound machine equipped with a 7.5 mega Hertz linear array probe is used for the study. Arterial diameters are measured at the baseline and during reactive hyperemia. FMD is calculated as the percentage change in diameter compared with the baseline.	3 months

Collaborators and Investigators

• Sponsor: National Cheng-Kung University Hospital
• Collaborators: Department of Health, Executive Yuan, R.O.C. (Taiwan)

Publications and helpful links

General Publications

• Chao TH, Tseng SY, Li YH, Liu PY, Cho CL, Shi GY, Wu HL, Chen JH. A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade. Clin Sci (Lond). 2012 Aug 1;123(3):147-59. doi: 10.1042/CS20110432.
• 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, Tseng SY, Chen IC, Tsai YS, Huang YY, Liu PY, Ou HY, Li YH, Wu HL, Cho CL, Tsai LM, Chen JH. Cilostazol enhances mobilization and proliferation of endothelial progenitor cells and collateral formation by modifying vasculo-angiogenic biomarkers in peripheral arterial disease. Int J Cardiol. 2014 Mar 15;172(2):e371-4. doi: 10.1016/j.ijcard.2013.12.295. Epub 2014 Jan 11. No abstract available.
• 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.

Study record dates

Study Major Dates

• Study Start: January 1, 2013
• Primary Completion (Actual): March 1, 2014
• Study Completion (Actual): August 1, 2014

Study Registration Dates

• First Submitted: July 16, 2014
• First Submitted That Met QC Criteria: July 16, 2014
• First Posted (Estimate): July 18, 2014

Study Record Updates

• Last Update Posted (Estimate): October 14, 2015
• Last Update Submitted That Met QC Criteria: October 11, 2015
• Last Verified: October 1, 2015

More Information

Terms related to this study

• Keywords: cardiovascular disease; risk factors; angiogenesis; cilostazol; progenitor cells; biological markers; endothelial function
• Additional Relevant MeSH Terms: Cardiovascular Diseases; Physiological Effects of Drugs; Molecular Mechanisms of Pharmacological Action; Vasodilator Agents; Autonomic Agents; Peripheral Nervous System Agents; Enzyme Inhibitors; Fibrinolytic Agents; Fibrin Modulating Agents; Platelet Aggregation Inhibitors; Neuroprotective Agents; Protective Agents; Bronchodilator Agents; Anti-Asthmatic Agents; Respiratory System Agents; Phosphodiesterase Inhibitors; Phosphodiesterase 3 Inhibitors; Cilostazol
• Other Study ID Numbers: B-BR-101-121; NCKUH-10203022 (Other Grant/Funding Number: NCKUH)