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CLINICAL STUDIES |
From the Department of Cardiovascular Diseases, the First Affiliated Hospital, Medical School of Zhejiang University, People's Republic of China.
Address for reprints: XingXiang Wang, Department of Cardiovascular Diseases, the First Affiliated Hospital, Medical School of Zhejiang University, 79 Qingchun Road, Hangzhou 310003, Zhejiang Province, P.R. China.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Endothelial progenitor cell • nicotine • vasculogenesis • coronary artery disease • laser scanning confocal microscope
The effects of nicotine on EPCs have not been reported so far. Therefore, in the present study, we investigated whether nicotine had influences on the EPC number and activity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cellular Staining
Fluorescent chemical detection of EPCs was performed on attached MNCs after 7 days in culture. Direct fluorescent staining was used to detect dual binding of FITC-labeled Ulex europaeus agglutinin (UEA-1; Sigma) and 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine (DiI)-labeled acetylated low-density lipoprotein (acLDL; Molecular Probe). Cells were first incubated with acLDL at 37°C and later fixed with 2% paraformaldehyde for 10 min. After being washed, the cells were reacted with UEA-1 (10 µg/mL) for 1 h. After the staining, samples were viewed with an inverted fluorescent microscope (Leica) and further demonstrated by a laser-scanning confocal microscope (LSCM, Leica). Cells demonstrating double-positive fluorescence were identified as differentiating EPCs.20,21 Two or 3 independent investigators evaluated the number of EPCs per well by counting 15 randomly selected high-power fields (x200) with an inverted fluorescent microscope.
Flow Cytometry Analysis
Fluorescence-activated cell sorting (FACS) detection of EPCs was performed on attached MNCs after 7 days in culture. Mononuclear cells were detached with 0.25% trypsin followed by repeated gentle flushing through a pipette tip. Cells (2 x 105) were incubated for 30 min at 4°C with anti-vascular endothelium (VE)-cadherin (Chemicon), phycoerythrin-conjugated monoclonal antibodies against a kinase insert domain-containing receptor (KDR, R&D), CD34, and AC133 (Miltenyi Biotec). An FITC-conjugated anti-mouse antibody (Vector) was added for staining with VE-cadherin. Isotype-identical antibodies served as controls. After treatment, the cells were fixed in 1% paraformaldehyde. Quantitative FACS was performed on a FACStar flow cytometer (COULTER).20,21
Protocols
Cells were serum depleted for 32 h before experiments. To demonstrate a concentration-dependent effect of nicotine (free base, purchased from Sigma Chemical) on EPCs, cells were incubated with 10-12 mol/L, 10-10 mol/L, 10-8 mol/L, 10-6 mol/L, and 10-4 mol/L nicotine for 32 h. To determine the reaction time course, cells were treated with 10-8 mol/L nicotine for 12, 18, 24, 32, and 48 h.
Migration Assay
The EPC migration was evaluated by using a modified Boyden chamber assay. In brief, isolated EPCs were detached using 0.25% trypsin, harvested by centrifugation, resuspended in 500 µL M199, and counted, and then 2 x 104 EPCs were placed in the upper chamber of a modified Boyden chamber (Qiling Medical Equipment Factory, Jiangsu, China). VEGF in serum-free M199 media was placed in the lower compartment of the chamber. After a 24-h incubation at 37°C, the lower side of the filter was washed with PBS and fixed with 2% paraformaldehyde. For quantification, cells were stained with a Giemsa solution. Cells migrating into the lower chamber were counted manually in 3 random microscopic fields.21
Cell Adhesion Assay
After 32 h of incubation with nicotine, human EPCs were washed with PBS and gently detached with 0.25% trypsin. After centrifugation and resuspension in M199 and 5% FBS, identical cell numbers were replated onto fibronectin-coated culture dishes and incubated for 30 min at 37°C. Adherent cells were counted by independent blinded investigators.16
EPC Proliferation Assay
The effect of nicotine on EPC proliferation was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. After being cultured for 7 days, EPCs were digested with 0.25% trypsin and then cultured in a serum-free medium in a 96-well culture plate (200 µL per well), to which was added nicotine (to make a series of final concentrations: 10-12 mol/L, 10-10 mol/L, 10-8 mol/L, 10-6 mol/L, and 10-4 mol/L). Each concentration included 6 wells, and the serum-free medium served as a control. After being cultured for 24 h, EPCs were supplemented with 10 µL MTT (5 g/L, Fluka Co. Product) and incubated for another 4 h. Then the supernatant was discarded by aspiration, and the EPC preparation was shaken with 200 µL dimethyl sulphoxide (DMSO) for 10 min, before the OD value was measured at 490 nm.
In Vitro Vasculogenesis Assay
The in vitro vasculogenesis assay was performed with the In Vitro Angiogenesis Assay Kit (Chemicon). The protocol was performed according to the manufacturer's instructions. Briefly, ECMatrixTM solution was thawed on ice overnight, then mixed with 10 x ECMatrixTM Diluent and placed in a 96-well tissue culture plate at 37°C for 1 h to allow the matrix solution to solidify. The EPCs were harvested as described above and replated (10,000 cells per well) on top of the solidified matrix solution. Cells were grown with nicotine or vehicle control and incubated at 37°C for 24 h. Tubule formation was inspected under an inverted light microscope at x200 magnification. Tubule formation was defined as a structure exhibiting a length 4 times its width.22,23 Five independent fields were assessed for each well, and the average number of tubules/x200 field was determined.
Statistical Analysis
Results were expressed as means ± standard deviation (SD) of data from experiments repeated 6 times (n = 6). Differences between group means were assessed by an unpaired Student t test for single comparisons and by analysis of variance for multiple comparisons. Values of P < .05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Nicotine on EPCs Number
Incubation of isolated human MNCs with nicotine increased the number of differentiated, adherent EPCs in a concentration-dependent manner, at nicotine concentrations of 10-12 to 10-8 mol/L. The peak increase in the EPC number was observed at concentrations of nicotine 10-8 mol/L, similar to those in the blood of smokers (Figures 2 and 3A). In contrast, compared with control, a marked decrease in cell number was observed when EPCs were incubated with the highest concentration of nicotine tested, 10-4 mol/L. In time course experiments performed with a nicotine concentration of 10-8 mol/L, an increase of the EPC number became apparent at 18 h and reached the maximum at 32 h (Figure 3B).
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Effect of Nicotine on EPC Proliferation
The effect of nicotine on EPC proliferation was assayed using an MTT assay (Figure 4). Nicotine dose-dependently increased EPC proliferative activity at nicotine concentrations of 10-12 to 10-8 mol/L, maximal at 10-8 mol/L nicotine (P < .01). However, EPC proliferative activity was significantly lower than control at a high nicotine concentration (10-4 mol/L). In time course experiments performed with a nicotine concentration of 10-8 mol/L, an increase of EPC proliferative activity became apparent at 18 h (P < .05) and reached the maximum at 32 h (P < .01).
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Effect of Nicotine on EPC Migration
The effects of nicotine on EPC migration were analyzed in a modified Boyden chamber assay (Figure 5). Nicotine profoundly improved cell migration at nicotine concentrations of 10-12 to 10-8 mol/L, maximal at 10-8 mol/L nicotine (P < .01). In contrast, compared with control, a marked decrease in cell migration was observed when EPCs were incubated with the highest concentration of nicotine tested, 10-4 mol/L. Nicotine (10-8 mol/L) also time dependently improved EPC migratory activity, which became apparent at 18 h (P < .05) and reached the maximum at 32 h (P < .01).
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Effect of Nicotine on EPC Adhesiveness
To study the possibility that nicotine alters the adhesiveness of cultured human EPCs, EPCs were incubated with nicotine for 32 h. After being replated on fibronectin-coated dishes, EPCs preexposed to nicotine exhibited a significant increase in the number of adhesiveness cells at 30 min at nicotine concentrations of 10-12 to 10-8 mol/L (Figure 6). The increase in the number of adhesiveness cells occurred dose dependently, with a maximal effect achieved at 10-8 mol/L. In contrast, the inhibitory effects on EPC adhesive activity occurred at a nicotine concentration of 10-4 mol/L. In addition, EPCs were incubated with 10-8 mol/L nicotine for the respective time points (12, 18, 24, 32, and 48 h); as a result, nicotine time dependently improved EPC adhesive activity, which became apparent at 18 h (P < .05) and reached the maximum at 32 h (P < .01).
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Effects of Nicotine on EPC Vasculogenesis
Recent studies have demonstrated that circulating EPCs home to sites of neovascularization and differentiate into endothelial cells in situ11,13 in a manner consistent with a process termed vasculogenesis. The invitro vasculogenesis assay was to simulate this process and was used in this study to investigate the ability of EPCs to participate in neovascularization, which is the most important activity of EPCs. The response of the EPCs to nicotine is depicted in Figure 7. Tubule number increased in a dose-response manner to nicotine concentrations (10-12 to 10-8 mol/L) at 24 h of incubation, with peak production at 10-8 mol/L nicotine. Moreover, tubules in the nicotine wells were qualitatively different and less complex than those in the control wells. In contrast, the inhibitory effects on EPC in vitro vasculogenesis activity occurred at high nicotine concentrations (10-6 to 10-4 mol/L).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nicotine has a very complex pharmacology. Most previous studies on the cellular effects of the products of cigarette smoke, including nicotine, have shown them to be deleterious, and investigators have therefore assumed constituents of cigarette smoke to be associated with cellular injury. For example, investigations of the effect of nicotine on ECs have indicated that nicotine may be associated with cell loss and desquamation.24 These features suggest cellular toxicity in response to nicotine. Our studies are consistent with cytotoxic effects of nicotine. However, cytotoxicity was only seen at high nicotine concentrations (> 10-6 mol/L).
Recently, nicotine has been reported to increase proliferation and tube formation of ECs in an in vitro assay. Furthermore, in a murine model of hind-limb ischemia, intramuscular injections of nicotine increased capillary density, enhanced collateral size and number, and augmented blood flow. Thus, nicotine has been regarded as being able to enhance neovascularization via stimulating angiogenesis.5-7 However, the formation of new blood vessels (neovascularization) includes 2 different processes: vasculogenesis and angiogenesis. Studies have demonstrated that circulating EPCs home to sites of neovascularization and differentiate into endothelial cells in situ11,13 in a manner consistent with a process termed vasculogenesis. The present study suggested that nicotine increased the EPC number and promoted EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity, maximal at a concentration of nicotine (10-8 mol/L) similar to that in the blood of smokers. Given the well-established role of EPCs participating in neovascularization, our findings may indicate that nicotine at physiologically relevant concentrations increases neovascularization, at least in part via stimulating vasculogenesis.
However, these findings seem counterintuitive. Tobacco use accelerates coronary and peripheral arterial diseases. Tobacco cessation is a mainstay of therapy for these patients. Moreover, with logistic analysis, smoking has recently been revealed as the major independent predictor for the reduction of EPC levels of patients with CAD. Accordingly, it is surprising that nicotine would increase the EPC number with enhanced functional activity. However, tobacco smoke is a complex mixture of more than 4000 chemical constituents, and the effect of nicotine delivered via the use of tobacco may be quite different. Indeed, there are several molecules in cigarette smoke that are toxic to ECs (eg, cadmium, reactive oxygen species)4 and may impair EPCs, too. Thus, the net effect of cigarette smoke on EPCs might be quite different from that of nicotine alone, which at least in part has been demonstrated by some clinical trials of nicotine therapy to aid smoking cessation in smokers with cardiovascular disease. In these clinical trials, nicotine therapy has shown no evidence of increased risk.25 In fact, smokers who continue to smoke but smoke fewer cigarettes while using nicotine patches, despite higher levels of nicotine in the blood, demonstrate less ischemia during exercise compared to before nicotine medication.26
The mechanisms by which nicotine increases EPC numbers and activity remain to be determined. There are several possible scenarios by which nicotine could increase the number of circulating EPCs. One explanation might be decreased apoptosis of premature progenitor cells. Indeed, CD34-positive EPCs were shown to be very sensitive to apoptosis induction. Moreover, nicotine has been reported to be able to protect apoptotic endothelial cells. Another explanation is that nicotine may interfere with the signaling pathways regulating EPC differentiation or mobilization.
Although the pathophysiological effect of nicotine increasing the EPC number and activity is not well understood, the following scheme is postulated to explain the potential significance of our results: nicotine is believed to function as a tumor promoter.27,28 Neovascularization, known to take place in tumors, correlates not only with tumor growth but also with a tumor's metastatic potential. Furthermore, EPCs have been demonstrated to significantly incorporate into tumor neovasculature13 and are regarded as an important in vitro target of angiostatin,29 which is known to possess potent antitumor and antiangiogenic properties in vivo.30,31 If nicotine promotes EPC proliferation and tumor growth, then nicotine could be of significance in the vascular support of malignant tumors, specifically in nicotine-mediated tumor neovascularization.
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FOOTNOTES |
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Submitted for publication November 9, 2003; Revised version accepted May 9, 2004.
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