Endothelial Progenitor Cells Participate in Nicotine-Mediated Angiogenesis
Christopher Heeschen, MD*, ,
Edwin Chang, PhD*,
Alexandra Aicher, PhD and
John P. Cooke, MD, PhD*,*
* Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California
Molecular Cardiology, J.W. Goethe University, Frankfurt, Germany
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Figure 1 Nicotine increases capillary density in the ischemic hind limb. Murine model of hind limb ischemia: nicotine stimulates angiogenesis as assessed by an increase in capillary density. Systemic treatment with nicotine resulted in a significantly higher angiogenic response as compared with local administration of nicotine into the ischemic hind limb (n = 7 each group). PBS = phosphate-buffered saline. *p < 0.01 versus control; **p < 0.05 versus local nicotine.
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Figure 2 Quantitation of endothelial progenitor cells by flow cytometry. Frequency of CD34+ and Flk-1+ cells in control animals without ischemia, with ischemia, and with ischemia and nicotine treatment (A). Histogram showing number of endothelial progenitor cells in the bone marrow at increasing intervals after induction of ischemia and initiation of oral nicotine (B). For each group, n = 5. *p < 0.01 versus control for each respective time point.
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Figure 3 Incorporation of mobilized endothelial progenitor cells (EPCs). Representative cross sections of ischemic hind limbs of female animals treated with vehicle (A) or nicotine (B), respectively. All endothelial cells stain for CD31 (fluorescein-isothiocyanate-labeled; green). Those endothelial cells that are derived from the male transgenic animal also express ß-galactosidase (red fluorescent phycoerythrin [PE]-labeled secondary antibody) resulting in a double stain that is yellow. Histogram showing mean values for capillary density and percentage of EPC-containing vessels (from an analysis of 10 randomly selected high-power fields in each group) (C). Nicotine administration increased capillary density as well as the number of vessels containing endothelial cells that had been derived from EPC. For each group, n = 5. Open bars = capillary density (CD31+); solid bars = EPC-containing vessels. *p < 0.01 versus phosphate-buffered saline (PBS).
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Figure 4 Nicotine pre-treatment increases endothelial progenitor cell (EPC) number in vitro. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DilacLDL) uptake (A and B) of isolated mononuclear cells was determined by fluorescence microscopy. Treatment of the cells with nicotine for 48 h increased the number of adherent DilacLDL+ cells per high-power (HP) field in a dose-dependent fashion (C). For each group, n = 5. *p < 0.01 versus control.
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Figure 5 Nicotine stimulates endothelial progenitor cell (EPC) transmigration. Nicotine stimulates EPC transmigration through human umbilical vein endothelial cell (HUVEC) monolayer: transmigrated cells at the bottom of the porous membrane in the vehicle (A) and nicotine (B) group. Endothelial progenitor cells were labeled with CellTracker before co-incubation. Significantly more transmigrated EPCs were observed when EPCs were pre-treated with nicotine (C). Pre-treatment of the endothelial cell monolayer with nicotine also increased EPC transmigration, and the effects of nicotine treatment on EPCs or HUVECs appeared to be additive with each other, or with stem-cell-derived factor (SDF)-1 (C). For each group, n = 5. *p < 0.01 versus control.
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