Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway  Shih-Ya Tseng, MS, Ting-Hsing Chao, MD, Yi-Heng Li, PhD, Ping-Yen Liu, PhD, Cheng-Han Lee, PhD, Chung-Lung Cho, PhD, Hua-Lin Wu, PhD, Jyh-Hong Chen, PhD  Journal of Vascular Surgery  Volume 63, Issue 4, Pages 1051-1062.e3 (April 2016) DOI: 10.1016/j.jvs.2014.10.103 Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 1 Cilostazol increases colony formation and proliferation by human early endothelial progenitor cells (EPCs) preincubated with high glucose (HG; 25 mmol/L). A, The representative figure illustrates human early EPCs (BF denotes bright field). B-D, Most cells are shown to simultaneously take up DiI-acetylated low-density lipoprotein (DiI-acLDL; red) and bind fluorescein isothiocyanate (FITC)-labeled lectin (green). E-H, Immunofluorescence detection (green) of expression of CD34, vascular endothelial growth factor-receptor 2 (VEGF-R2), von Willebrand factor (VWF), and CD31 in human early EPCs are shown in order. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Original magnification ×200. I, Representative flow cytometry assay shows that most early EPCs were positive for CD45, a leukocytes surface marker. The percentages of cells positive for KDR (VEGF-R2), CD34, and CD146 were identified while the CD45+ subpopulation was gated. The addition of cilostazol to cultures significantly stimulated (J) colony formation and (K) proliferation by human early EPCs, which was attenuated with protein kinase A inhibitor (PKAI) and compound C, an adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. Forskolin (a cyclic AMP activator) and AICAR (an AMPK activator) serve as positive controls. The results are presented as mean ± standard error of the mean (n = 3). *P < .05, **P < .01, and ***P < .001, significantly different from the vehicle + HG-treated cells. #P < .05, ##P < .01, and ###P < .001, significantly different from the cilostazol (100 μM) + HG-treated cells. @@@P < .001, significantly different compared with normal control. AICAR, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide; BrdU, 5-bromo-2'-deoxyuridine; CFU, colony-forming unit. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 2 Cilostazol promotes adhesion of endothelial progenitor cells (EPCs) to human umbilical vein endothelial cells (HUVECs). A-C, DiI-labeled EPCs (red) were allowed to adhere to a monolayer of HUVECs (blue; 4′,6-diamidino-2-phenylindole). Representative photographs illustrate (A) adhesion of normal control EPCs, (B) EPCs with high glucose (HG), (C) and cilostazol (100 μM) + HG. D, Cilostazol restored HG-inhibited adhesion function of EPCs to HUVECs, and inhibitors significantly blocked the cilostazol effect. Values are presented as mean ± standard error of the mean (n = 3). *P < .05 and **P < .01, significantly different from the vehicle + HG-treated cells. #P < .05, significantly different from the cilostazol (100 μM) + HG-treated cells. @@@P < .001, significantly different compared with normal control. AICAR, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide; PKAI, protein kinase A inhibitor. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 3 Cilostazol enhances endothelial functions in human umbilical vein endothelial cells (HUVECs) pretreated with high glucose (HG). Cilostazol stimulated messenger RNA expression of (A) adenosine monophosphate-activated protein kinase-α1 (AMPK-α1) and (B) phosphorylation of AMPK α1 (p-AMPK-α1) in a dose-dependent manner with peak improvement at 100 μM cilostazol. Cilostazol enhanced (C) proliferation, (D) migration, (E) antiapoptosis, and (F) nitrite production in HUVECs, which were reversed with inhibitors. Values are presented as mean ± standard error of the mean (n = 3). **P < .01 and ***P < .001, significantly different from vehicle + HG-treated cells. #P < .05, ##P < .01, and ###P < .001, significantly different from cilostazol (100 μM) + HG-treated cells. @@P < .01 and @@@P < .001, significantly different compared with normal control. AICAR, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide; BrdU, 5-bromo-2'-deoxyuridine; PKAI, protein kinase A inhibitor. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 4  Cilostazol promotes in vitro capillary-like tube formation of high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). (A) The representative photomicrographs illustrate HUVECs cultured on Matrigel (BD Biosciences, San Jose, Calif) to form a capillary-like tube network, (B) but the HG-treated cells did not mature and failed to form networks. These effects were restored with (C) cilostazol, (D) forskolin, or (E) 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). The beneficial effect of cilostazol was significantly suppressed by coincubation with (F) protein kinase A inhibitor (PKAI) and (G) compound C. Original magnification ×100. H, Capillary-like structures were scored. Values are presented as mean ± standard error of the mean (n = 3). *P < .05 and **P < .01, significantly different from vehicle + HG-treated cells. #P < .05, significantly different from cilostazol + HG-treated cells. @@P < .01, significantly different compared with normal control. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 5 Cilostazol (Cilo) activates phosphorylation of signaling molecules in human umbilical vein endothelial cells (HUVECs) pre-treated with high glucose (HG). A, HG-mediated diminished phosphorylation (p) of protein kinase A (PKA), adenosine monophosphate-activated protein kinase (AMPK), acetyl-coenzyme A carboxylase (ACC), Akt, and endothelial nitric oxide synthase (eNOS) were restored with cilostazol. The stimulatory effect of cilostazol on the phosphorylation of signaling molecules was abolished with inhibitors. B, Transfection complexes containing short hairpin (sh)-AMPKα1 (target-pLk0.1 plasmid) or sh-pLK0.1 (nontarget pLk0.1 plasmid) negative control constructs were used for knockdown experiment. Efficient AMPK knockdown was performed. C, The stimulatory effect of cilostazol on phosphorylation of signaling molecules was attenuated in sh-AMPK-transfected HUVECs (n = 3 for each protein). AICAR, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide; M, mannitol; N, normal control; PKAI, protein kinase A inhibitor. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 6 Cilostazol shows better improvement on animal-based assays in hind limb ischemia. A, Cilostazol accelerated flow recovery. B, Capillaries in the leg muscles were visualized by anti-CD31 immunostaining (red), and nuclei were counterstained with hematoxylin (blue). C, Capillary density was significantly higher in cilostazol-treated mice. D, Cilostazol increased the circulating number of CD34+ cells. E, Western blotting in ischemic hind limbs showed increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)/acetyl-coenzyme A carboxylase (ACC) and Akt/endothelial nitric oxide synthase (eNOS) in cilostazol-treated mice. Values are presented as mean ± standard error of the mean (n = 12 in each animal group). *P < .05 and **P < .01 denote significant difference from the vehicle-treated control mice. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 7 The beneficial effects of cilostazol on animal-based assays in hind limb ischemia were attenuated with adenosine monophosphate-activated protein kinase (AMPK) knockdown. A, Blood flow recovery was markedly retarded in mice with AMPK knockdown (short hairpin [sh]-AMPK). B, Capillaries (red) and nuclei (blue) in the leg muscles are shown. C, Capillary density was significantly lower in AMPK-knockdown mice. D, The circulating number of CD34+ cells was significantly lower in mice with sh-AMPK after day 14. E, Efficient AMPK knockdown is demonstrated. F, Downregulated phosphorylation of acetyl-coenzyme A carboxylase (p-ACC) and Akt/endothelial nitric oxide synthase (eNOS) were observed in AMPK-knockdown mice. Values are presented as mean ± standard error of the mean (n = 12 in each animal group).*P < .05 and **P < .01 denote significant difference from vector-treated mice. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 8 Schematic representation shows the vasculoangiogenic effects of cilostazol in hyperglycemia mediated by the interaction of a broad signaling network. ACC, Acetyl-coenzyme A carboxylase; AMPK, adenosine monophosphate-activated protein kinase; cAMP, cyclic AMP; eNOS, endothelial nitric oxide synthase; PKA, protein kinase A. Journal of Vascular Surgery 2016 63, 1051-1062.e3DOI: (10.1016/j.jvs.2014.10.103) Copyright © 2016 Society for Vascular Surgery Terms and Conditions