Letter to the Editor Cilostazol enhances mobilization and proliferation of endothelial progenitor cells and collateral formation by modifying vasculo-angiogenic biomarkers in peripheral arterial disease ☆ Ting-Hsing Chao a, ⁎ ,1,2 , Shih-Ya Tseng b,1,2 , I-Chih Chen c,1,2 , Yi-Shan Tsai d,2 , Yao-Yi Huang e,2 , Ping-Yen Liu a,2 , Horng-Yih Ou a,2 , Yi-Heng Li a,2 , Hua-Lin Wu f,2 , Chung-Lung Cho b,2 , Liang-Miin Tsai a,2 , Jyh-Hong Chen a,2 a Department of Internal Medicine, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan b Department of Biological Science, National Sun Yat-Sen University, Kaohsiung, Taiwan c Department of Internal Medicine, Tainan Municipal Hospital, Tainan, Taiwan d Department of Radiology, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan e Department of Emergency Medicine, National Cheng Kung University Hospital, Tainan, Taiwan f Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan article info Article history: Received 18 December 2013 Accepted 30 December 2013 Available online 11 January 2014 Keywords: Cilostazol Progenitor cells Angiogenesis Peripheral arterial disease Biological markers Substantial evidence has elucidated the significance of postnatal vasculogenesis for neovascularization and vascular remodeling by peripheral endothelial progenitor cells (EPCs) [1]. Some exogenous factors [1,2] have been reported to be beneficial in the EPC mechanisms of action, including vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF)-1α, adiponectin and thrombomodulin (TM) [2]. The number and function of circulating EPCs have been reported to be inversely associated with the presence of coronary risk factors as well as the existence of atherosclerotic diseases [1]. Cilostazol is a commercially available drug that has antiplatelet and vasodilatory activity and is indicated for peripheral artery disease (PAD)-related intermittent claudication [3]. We and other investigators have found that cilostazol may have beneficial effects on EPCs in vitro [3,4] and can provide vasculo-angiogenic effects in murine hindlimb ischemia [3,5]. Therefore, we hypothesized that cilostazol can en- hance mobilization and proliferation of EPCs and collateral forma- tion by modifying vasculo-angiogenic biomarkers in PAD. This prospective, double-blind, randomized placebo-controlled trial consecutively enrolled 44 patients with mild-to-moderate PAD who had ankle-brachial index less than 0.9 in one or both legs without obvious intermittent claudication. Exclusion criteria were listed in the registered study protocol (Clinicaltrials.org registration number NCT01952756). All study participants provided signed informed consent, and this study followed the regulation of the ethics committee of the National Cheng Kung University Hospital, where all data were collected. Eligible subjects were randomly assigned to cilostazol 200 mg (n = 24) or dummy placebo (n = 20) daily for 12 weeks using unrestricted randomization and sealed envelopes for allocation concealment. Serum concentrations of biomarkers were measured by enzyme-linked immunosorbent assay [6]. The isolation of human early EPCs was performed according to standard protocols [3]. The quanti fication of colony formation by EPCs was performed and the chemotactic motility, proliferation/viability (XTT) and apoptosis of EPCs were measured as previously described [3]. Peripheral mononuclear cells were washed and analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) with 10 5 events in the lymphocyte gate. EPCs, which were defined as negative for CD45 and positive for CD34 and kinase insert domain receptor (KDR), and apoptotic ECs, which were defined as negative for CD45 and positive for CD146 and annexin V, were selected using a gating strategy [7]. Dual-energy multi-sliced computed tomographic angiography (CTA) was performed on a 128-row dual source CT in dual-energy scan protocol (80 kVp and 140 kVp) (Siemens Somatom Definition; Siemans AG, Medical Solutions, Forchheim, Germany) [8]. CTA data were transferred to an external multimodality workplace (Syngo MMWP VA 21A, Siemans AG, Medical Solutions, Forchheim, Germany) for post-processing. Follow- up CTA was performed after 3 months of treatment in 36 participants who had severe stenosis in at least one major artery demonstrated on International Journal of Cardiology 172 (2014) e371–e374 ☆ Clinical Trial Registration: Clinicaltrials.org registration number NCT01952756. ⁎ Corresponding author at: Department of Internal Medicine, Division of Cardiology, National Cheng Kung University College of Medicine and Hospital, No. 138, Sheng-Li Road, North District, Tainan 704, Taiwan. Tel.: +886 6 2353535x2383; fax: +886 6 2753834. E-mail address: chaotinghsing@yahoo.com.tw (T.-H. Chao). 1 T.–H. C., S.-Y. T. and I.–C. C. contributed equally to this work. 2 These authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. 0167-5273/$ – see front matter © 2014 Elsevier Ireland Ltd. 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