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Superoxide counteracts low-density lipoprotein-induced human aortic smooth muscle cell proliferation
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 3, 157–162. 2007 DOI: 10.1263/jbb.104.157
© 2007, The Society for Biotechnology, Japan
Superoxide Counteracts Low-Density Lipoprotein-Induced Human Aortic Smooth Muscle Cell Proliferation Chi-Chao Yin,1 Tin-Kwang Lin,2 and Kuang-Tse Huang1* Department of Chemical Engineering, National Chung Cheng University, Chia-Yi 621, Taiwan1 and Department of Cardiology, Buddhist Tzuchi Dalin General Hospital, Chia-Yi 622, Taiwan2 Received 30 March 2007/Accepted 26 May 2007
Previously, we demonstrated that the level of intracellular O2– is increased by low-density lipoprotein (LDL) in human aortic smooth muscle cells (HASMCs). The exact role of O2– in the LDLinduced proliferation of HASMCs, however, has not been determined. In this study, we found that the increase in the concentrations of intracellular O2– induced by native and oxidized LDL increased SMC-nitric oxide (NO) uptake rate. Moreover, the treatment of HASMCs with diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, significantly increased NO uptake rate owing to the increase in intracellular O2– concentrations. Although native and oxidized LDL decreased soluble guanylyl cyclase (sGC) protein content, they still caused a net increase in cyclic GMP production in HASMCs. In addition, when cyclic GMP production was normalized by sGC protein content and NO uptake rate, it was found to be positively dependent on the level of intracellular H2O2. Finally, we simulated cell proliferation stimulated by native and oxidized LDL as a linear function of intracellular O2– and H2O2 concentrations, demonstrating that O2– negatively modulated the native and oxidized LDL-stimulated HASMC proliferation through the increase in NO uptake rate. [Key words: low-density lipoprotein, nitric oxide, cyclic GMP, superoxide, hydrogen peroxide, smooth muscle cells]
the increase in the level of intracellular O2– induced by LDL may counteract the effect of LDL on VSMC proliferation via an increase in O2–-mediated NO uptake rate. To determine effect of O2– on VSMC proliferation, we first measured the rate of NO uptake by VSMCs (VSMCNO uptake rate) at various nLDL and oxLDL concentrations. The involvement of O2– in VSMC-NO uptake rate increase was further confirmed by measuring intracellular cGMP production and changing intracellular O2– content using diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor. Finally, we measured the change of the cell cycle profile to serve as an indicator of cell proliferation and determined the correlation between O2– concentrations and cell proliferation using a mathematical model. Our results showed that the increase in intracellular O2– concentrations induced by nLDL and oxLDL increased VSMC-NO uptake rate, and which in turn stimulated cGMP production. The increased intracellular O2– content counteracted the effect of H2O2 on VSMC proliferation in the presence of exogenous NO and oxyhemoglobin (oxyHb).
Elevated levels of plasma low-density lipoprotein (LDL) cholesterols are associated with an increased risk of cardiovascular disease. LDL is usually oxidized by macrophages or endothelial cells in the subendothelial space (1, 2). The resulting oxidized LDL (oxLDL) has been shown to enhance vascular smooth muscle cell (VSMC) proliferation, apoptosis, impairment of endothelium-dependent relaxation, and generation of proinflammatory cytokines (3–6). Both the levels of H2O2 and O2– are elevated by native LDL (nLDL) and oxLDL. Previously, we demonstrated that in the absence of exogenous NO, human aortic VSMC proliferation stimulated by nLDL and oxLDL correlates well with the level of intracellular H2O2 but not with that of O2– (7). On the contrary, exogenous O2– has been reported to increase cell proliferation in rat and human aortic VSMCs (8–10). It remains unclear regarding the role of O2– in the LDL-stimulated cell proliferation in human aortic VSMCs in the presence of nitric oxide (NO). O2– reacts with NO at an extremely high rate (k = 1.6 ×1010 –1 –1 M s ) (11). Previous evidence demonstrated that O2– may serve as a mediator for the bioactivity of NO in VSMCs (12). In addition, NO has been shown to inhibit VSMC proliferation through cyclic GMP (cGMP)-dependent and -independent pathways (13, 14). Therefore, it is possible that
MATERIALS AND METHODS Cell culture Human aortic smooth muscle cells (Cascade Biologics, Portland, OR, USA) were maintained in Dulbecco’s modified Eagle’s medium/F-12 1: 1 mixture supplemented with smooth muscle growth supplement (Cascade Biologics), 10% cosmic calf serum (HyClone, Logan, UT, USA), 2 mM glutamine, 2
* Corresponding author. e-mail: [email protected] phone: +886-5-272-0411 ext. 33487 fax: +886-5-272-1206 157
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mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml gentamicin at 37°C under 5% CO2 humidified atmosphere. To minimize the effect of serum on cell proliferation and production of intracellular O2– and to enable the cells to adapt to their altered growth environment more successfully, the serum concentration was reduced from 10% to 0.3% sequentially before experiments. First, the serum concentration was reduced to 1% for 1 d, and then the cells were trypsinized and subcultured at 40% of cell confluence. One day after the passaging, the serum concentration was further reduced to 0.3% by exchanging the medium. The investigations were carried out 18 h after the last adjustment of serum concentration. The VSMCs used in the experiments underwent 5–7 passages. Preparation of oxyHb solution The blood from human median cubital vein was collected into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). OxyHb was purified as described previously (12, 15). Isolation and oxidative modification of LDL LDL was prepared by ultracentrifugation of plasma from healthy donors as described previously (16). Briefly, the density of plasma was first adjusted to 1.09 g/ml by adding KBr granules. The adjusted serum was layered between various densities of KBr solutions that were prepared by mixing different amounts of KBr (1.346 g/ml) and NaCl (1.005 g/ml) solutions. The ultracentrifugation was performed at 29,000 ×g, 12°C for 23 h and LDL subfractions were collected in the density range, ρ=1.019–1.063 g/ml. Partial oxidization (75% of complete oxidation) of LDL was carried out by dialyzing LDL against Dulbecco’s phosphate-buffered saline (DPBS) (pH 7.4, 34°C) containing 5 µM CuCl2 for about 7.5 h. The oxidation of LDL was monitored on the basis of absorbance at 234 nm in a UVVisible spectrophotometer (Varian, Palo Alto, CA, USA) and was terminated by adding 10 mM EDTA. The resulting oxidized LDL was further dialyzed three times against EDTA-free DPBS to remove EDTA. Competition assay for measuring rate of NO uptake by VSMCs This method was described previously (12). Briefly, after the incubation of VSMCs with nLDL and oxLDL for 18 h, each set of the experiments consists of four samples, each of which contained oxyHb; oxyHb with spermine NONOate (SpNO); VSMCs and oxyHb; VSMCs and oxyHb with SpNO in a 24-well cell culture dish. MetHb production in the solution was measured spectrophotometrically using a plate reader (µQuant Universal Microplate Spectrophotometer; Bio-Tek, Winooski, VT, USA). VSMC-NO uptake rate was defined in the following equation: rsmc = kSMC[O2–][NO]
ments, the VSMCs were washed three times with DPBS, incubated with 5 µM HE or DHR123 in a serum-free medium at 37°C for 30 min, and then mobilized with DPBS containing 0.02% EDTA and 0.25% trypsin. FACS was run using a FACScalibur (Becton, Dickinson and Company). The mean fluorescence intensity of the stained VSMCs was obtained after adjusting the mean fluorescence intensity of the untreated/unstained control to 5. Determination of cell cycle profiles of VSMCs The DNA content of VSMCs was detected by FACS using fluorescent dye propidium iodide (Ex: 287 and 488 nm; Em: 602 nm) (17). After treatment with nLDL and oxLDL in the presence and absence of oxyHb and diethylenetriamine NONOate (DETA-NO) for 18 h, cells were washed three times with DPBS, mobilized with trypsin, and centrifuged at 150 ×g for 5 min. Cells were then fixed with absolute ethanol for at least 1 h at 4°C. After incubation, cells were washed twice with DPBS, resuspended in DPBS/DNA extraction buffer (0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8) 1:1 mixture for 5 min, and then incubated for 30 min at room temperature with 3.8 mM sodium citrate buffer containing 50 µg/ml propidium iodide and 0.5 µg/ml RNase A. FACS was run using a FACScalibur
(1) –
where kSMC is the rate constant for SMC-NO reaction, [O2 ] is the intracellular O2– concentration, and [NO] is the NO concentration in bulk medium. kSMC[O2–]/kHb as an indicator of VSMC-NO uptake rate was calculated using the following equation: [ oxyHb] kSMC[O2–]/kHb = ([metHb]cf − [metHb])/(rv ln(------------------------0- )) (2) [ oxyHb] where kHb is the rate constant for oxyHb-NO reaction. [metHb] and [metHb]cf are the metHb concentrations in the samples with and without VSMCs, respectively. rv is the volumetric ratio of VSMCs to solution and [oxyHb]0 is the initial concentration of oxyHb in the medium. To investigate the effect of intracellular O2– concentrations on NO uptake rate, VSMCs were incubated with 7.5 mM DETC at the last 30 min of various treatments. Estimation of intracellular O2– and H2O2 concentrations Hydroethidine (HE) (Ex: 535 nm; Em: 610 nm) and dihydrorhodamine 123 (DHR123) (Ex: 488 nm; Em: 530 nm) were used for fluorescence-activated cell sorting (FACS) to evaluate intracellular contents of O2– and H2O2, respectively (7). After various treat-
FIG. 1. Effect of nLDL and oxLDL on rate of NO uptake by VSMCs in the presence and absence of DETC (A). VSMC-NO uptake rate was plotted against the mean HE-emitted fluorescence intensity (B). All data represent means ± S.E. for n = 3.
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(Becton, Dickinson and Company). Western blot analysis of soluble guanulyl cyclase (sGC) Human aortic VSMCs were lysed using 20 mM Tris buffer (pH 7.4) containing 0.1% sodium dodecylsulfate (SDS) and 0.1 mM phenylmethylsulfonyl fluoride. Proteins of equal amounts (40 µg/lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with PBS containing 5% nonfat milk and 0.05% Tween-20 for 1 h at room temperature. The membranes were then incubated for 1.5 h with a rabbit polyclonal antibody against sGC (1:500; Calbiochem, San Diego, CA, USA) and a mouse monoclonal antibody against β-actin (1 : 10000; Chemicon, Temecula, CA, USA). For immunodetection, the membranes were incubated 1 h with a goat anti-rabbit IgG (1 : 10000; Sigma, St. Louis, MO, USA) and rat anti-mouse IgG (1 : 10000; BD Biosciences, San Jose, CA, USA) conjugated with horseradish peroxidase. The signals were detected using the ECL detection system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and were analyzed by densitometry. Total protein concentration was determined by bicinchoninic acid protein assays (Pierce, Rockford, IL, USA). Measurement of intracellular cGMP The VSMCs were treated with phosphodiesterase inhibitor 0.2-mM 3-isobutyl-1-methylxanthine (Sigma) 30 min before the competition assay. At the end of competition experiments, the cells were lysed to measure intracellular cGMP concentration using a direct cGMP kit (Assay Designs, Ann Arbor, MI, USA). Modeling of cell proliferation We hypothesized that cell proliferation induced by nLDL and oxLDL is a linear function of intracellular levels of H2O2 and O2–: cell proliferation = k1[O2–] + k2[H2O2] + constant
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eration. The estimates of the coefficients in Eq. 3 for the presence and absence of oxyHb and DETA-NO were obtained separately from linear least square regression using Excel 2003 (Microsoft, Seattle, WA, USA).
RESULTS AND DISCUSSION Effect of O2– on VSMC-NO uptake rate in the challenge of nLDL and oxLDL To determine whether the increase in the levels of intracellular O2– induced by nLDL and oxLDL correlates with VSMC-NO uptake rate, we measured NO
(3)
– 2
[O ] and [H2O2] are the normalized mean fluorescence intensity of the VSMCs loaded respectively with HE and DHR123. The ratio of cells in S, G2, and M phases indicated the degree of cell prolif-
FIG. 2. Changes in sGC protein expression level after treatment of VSMCs with nLDL and oxLDL. After treatment of nLDL and oxLDL (20 and 100 µg/ml) for 12 h, the sGC protein expression in VSMCs was analyzed by western blotting. All data represent means ± S.E. for n=2.
FIG. 3. Effect of nLDL and oxLDL on NO-cGMP signaling. Treatment of nLDL and oxLDL (20 and 100 µg/ml) dose-dependently increased intracellular cGMP production in the presence of spNO and oxyHb. Pretreatment of VSMCs with 7 mM DETC for 30 min further increased intracellular cGMP production in various samples (A). cGMP production normalized by sGC protein content and VSMC-NO uptake rate from control (square), the cells treated with 20 µg/ml nLDL (circle), 100 µg/ml nLDL (triangle), 20 µg/ml oxLDL (inverted triangle), or 100 µg/ml nLDL (diamond) was dependent on the intracellular level of H2O2 (B). All data represent means ± S.E. for n = 3.
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uptake rate in NO competition experiments and compared it with the corresponding intracellular O2– concentration. Figure 1 shows that VSMC-NO uptake rate was increased in the cells treated with nLDL and oxLDL (20 µg/ml and 100 µg/ml, respectively) for 18 h (Fig. 1A) and positively depended on the level of intracellular O2– (Fig. 1B). These results are consistent with our previous finding that VSMC(A7r5)-NO uptake rate is positively proportional to the intracellular O2– concentration, which was altered by varying cell confluence and serum concentration (12). The treatment of VSMCs with 7.5 mM DETC (an SOD inhibitor) further increased the level of intracellular O2– and VSMC-NO uptake rate. Notably, the increase in intracellular O2– level alone induced by DETC through the inhibition of SOD cannot completely explain the significant effect of DETC on NO uptake rate (Fig. 1B). DETC is able to remove Cu2+ from Cu/Zn-SOD forming the DETC-Cu2+ complex (18). However, the DETC-Cu2+ complex is not able to bind to NO molecules; therefore, it is unlikely to contribute to the increase in NO uptake rate induced by DETC (19). On the other hand, DETC-Fe2+ has been used as a spin trap for detecting NO (20–22). Moreover, LDL has been demonstrated to increase the level of intracellular free iron in hu-
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man aortic endothelial cells and VSMCs (23). Therefore, it is plausible that the binding of NO by the intracellular DETC-Fe2+ complex may account for the further increase in VSMC-NO uptake rate. Enhancement of NO-cGMP signaling by nLDL and oxLDL To investigate whether the increase in NO uptake rate induced by nLDL and oxLDL in VSMCs increases intracellular cGMP production, we measured both the level of sGC protein and intracellular cGMP production after the competition experiment. Treatments of VSMCs with nLDL and oxLDL caused a dose-dependent decrease in sGC protein level (Fig. 2). In particular, oxLDL had a greater effect of reducing the content of sGC protein. The production of cGMP was increased by treatments of VSMCs with nLDL and oxLDL, even though the sGC protein content in these treated cells was low (Fig. 3A). In addition, the administration of DETC further enhanced cGMP production through the increase in the rate of O2–-mediated NO uptake, implying that the Fe2+(DETC)2NO complex may preserve, at least in part, the bioactivity of NO. If we normalized cGMP production by the content of sGC and NO uptake rate, we found that the normalized cGMP production was dependent on intracellular H2O2 concentration. OxLDL has been shown to
FIG. 4. Effect of DETA-NO and oxyHb on nLDL- and oxLDL-stimulated VSMC proliferation. After incubation of nLDL and oxLDL (20 and 100 µg/ml) for 18 h, the DNA profiles of human aortic VSMCs were analyzed by FACS using propidium iodide (50 µg/ml) (A, B). All data represent means ± S.E. for n = 3.
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TABLE 1. Estimation of parameters of linear regression for cell proliferation as a function of intracellular O2– and H2O2 concentrationsa Cell proliferationb DETA-NO+ Hb
Normalizedc
[O2–] + − Control 0.20 0.20 0.99 0.22 0.21 1.00 0.21 0.21 1.00 LDL-100 µg/ml 0.20 0.24 1.89 0.23 0.25 1.87 0.21 0.25 1.90 oxLDL-20 µg/ml 0.41 0.40 1.31 0.42 0.43 1.38 0.42 0.44 1.33 oxLDL-100 µg/ml 0.56 0.68 2.39 0.53 0.63 2.37 0.57 0.68 2.42 −0.059 −0.001 k1 0.166 0.181 k2 constant 0.199 0.029 0.988 0.997 R2 a Cell proliferation = k1[O2–]k2[H2O2] + constant b The ratio of cells in S, G2, and M phases was used to indicate the degree of cell proliferation. c The concentrations of intracellular O2– and H2O2 were normalized to the average of control.
significantly increase the protein expression of CuZnSOD in human aortic VSMCs (7). Furthermore, CuZnSOD/H2O2 has been reported to catalyze the degradation of s-nitrosoglutathione (GSNO) (24–26). Therefore, the higher normalized cGMP production in the oxLDL-treated VSMCs might be due to the higher levels of intracellular H2O2 and SOD for releasing NO from intracellular GSNO. Superoxide counteracted nLDL- and oxLDL-stimulated cell proliferation through increase in NO uptake rate Previous studies demonstrated that NO can inhibit VSMC proliferation via a sGC/cGMP dependent pathway by changing the expression level and activity of cell cycle regulatory proteins (27, 28). To test whether O2–-mediated NO uptake is involved in VSMC proliferation stimulated by nLDL and oxLDL, we simulated VSMC proliferation using a model on the basis of the data of intracellular DNA status, H2O2, and O2–. Figures 4A and 4B show that the presence of oxyHb and the NO donor DETA-NO decreased the ratio of cells in S, G2, and M phases. In particular, the addition of the NO donor showed the most significant effect on the 100 µg/ml oxLDL-treated cells that had the highest level of intracellular O2–. When we normalized the fluorescence intensities of O2– and H2O2 against the controls with and without DETANO, we found that cell proliferation was well expressed as a linear function of normalized O2– and H2O2 concentrations (Table 1). The coefficients of normalized O2– concentration in the equation in the presence and absence of oxyHb and DETA-NO were respectively −0.059 and −0.001, indicating that intracellular O2– had an inhibitory effect on nLDL- and oxLDL-stimulated VSMC proliferation and was strongly dependent on the presence of NO. On the other hand, the coefficients of normalized H2O2 concentration with and without oxyHb and DETA-NO were similar, indicating that the effect of H2O2 on cell proliferation was nearly independent of NO. Notably, our model generated using limited data did not exclude the effect of other factors on cell prolif-
[H2O2] 0.99 1.01 0.99 1.17 1.24 1.20 2.12 2.14 2.19 3.62 3.27 3.66
eration. In summary, the levels of intracellular O2– altered by nLDL and oxLDL correlated well with VSMC-NO uptake rate. Treatment of VSMCs with DETC increased NO uptake rate through the increase in intracellular O2– concentration. Although nLDL and oxLDL decreased sGC protein content, they still caused a net increase in cGMP production in VSMCs. Furthermore, O2– was able to counteract nLDLand oxLDL-stimulated VSMC proliferation through the increase in NO uptake rate. These findings may provide new insights into the therapeutic strategies of atherosclerosis caused by oxLDL. ACKNOWLEDGMENTS This work was supported by grants NSC 94-2214-E-194-007 and NSC 95-2221-E-194-006 from the National Science Council, Taiwan and a grant from Buddist Tzuchi Dalin General Hospital, Taiwan.
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© 2007, The Society for Biotechnology, Japan
Superoxide Counteracts Low-Density Lipoprotein-Induced Human Aortic Smooth Muscle Cell Proliferation Chi-Chao Yin,1 Tin-Kwang Lin,2 and Kuang-Tse Huang1* Department of Chemical Engineering, National Chung Cheng University, Chia-Yi 621, Taiwan1 and Department of Cardiology, Buddhist Tzuchi Dalin General Hospital, Chia-Yi 622, Taiwan2 Received 30 March 2007/Accepted 26 May 2007
Previously, we demonstrated that the level of intracellular O2– is increased by low-density lipoprotein (LDL) in human aortic smooth muscle cells (HASMCs). The exact role of O2– in the LDLinduced proliferation of HASMCs, however, has not been determined. In this study, we found that the increase in the concentrations of intracellular O2– induced by native and oxidized LDL increased SMC-nitric oxide (NO) uptake rate. Moreover, the treatment of HASMCs with diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, significantly increased NO uptake rate owing to the increase in intracellular O2– concentrations. Although native and oxidized LDL decreased soluble guanylyl cyclase (sGC) protein content, they still caused a net increase in cyclic GMP production in HASMCs. In addition, when cyclic GMP production was normalized by sGC protein content and NO uptake rate, it was found to be positively dependent on the level of intracellular H2O2. Finally, we simulated cell proliferation stimulated by native and oxidized LDL as a linear function of intracellular O2– and H2O2 concentrations, demonstrating that O2– negatively modulated the native and oxidized LDL-stimulated HASMC proliferation through the increase in NO uptake rate. [Key words: low-density lipoprotein, nitric oxide, cyclic GMP, superoxide, hydrogen peroxide, smooth muscle cells]
the increase in the level of intracellular O2– induced by LDL may counteract the effect of LDL on VSMC proliferation via an increase in O2–-mediated NO uptake rate. To determine effect of O2– on VSMC proliferation, we first measured the rate of NO uptake by VSMCs (VSMCNO uptake rate) at various nLDL and oxLDL concentrations. The involvement of O2– in VSMC-NO uptake rate increase was further confirmed by measuring intracellular cGMP production and changing intracellular O2– content using diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor. Finally, we measured the change of the cell cycle profile to serve as an indicator of cell proliferation and determined the correlation between O2– concentrations and cell proliferation using a mathematical model. Our results showed that the increase in intracellular O2– concentrations induced by nLDL and oxLDL increased VSMC-NO uptake rate, and which in turn stimulated cGMP production. The increased intracellular O2– content counteracted the effect of H2O2 on VSMC proliferation in the presence of exogenous NO and oxyhemoglobin (oxyHb).
Elevated levels of plasma low-density lipoprotein (LDL) cholesterols are associated with an increased risk of cardiovascular disease. LDL is usually oxidized by macrophages or endothelial cells in the subendothelial space (1, 2). The resulting oxidized LDL (oxLDL) has been shown to enhance vascular smooth muscle cell (VSMC) proliferation, apoptosis, impairment of endothelium-dependent relaxation, and generation of proinflammatory cytokines (3–6). Both the levels of H2O2 and O2– are elevated by native LDL (nLDL) and oxLDL. Previously, we demonstrated that in the absence of exogenous NO, human aortic VSMC proliferation stimulated by nLDL and oxLDL correlates well with the level of intracellular H2O2 but not with that of O2– (7). On the contrary, exogenous O2– has been reported to increase cell proliferation in rat and human aortic VSMCs (8–10). It remains unclear regarding the role of O2– in the LDL-stimulated cell proliferation in human aortic VSMCs in the presence of nitric oxide (NO). O2– reacts with NO at an extremely high rate (k = 1.6 ×1010 –1 –1 M s ) (11). Previous evidence demonstrated that O2– may serve as a mediator for the bioactivity of NO in VSMCs (12). In addition, NO has been shown to inhibit VSMC proliferation through cyclic GMP (cGMP)-dependent and -independent pathways (13, 14). Therefore, it is possible that
MATERIALS AND METHODS Cell culture Human aortic smooth muscle cells (Cascade Biologics, Portland, OR, USA) were maintained in Dulbecco’s modified Eagle’s medium/F-12 1: 1 mixture supplemented with smooth muscle growth supplement (Cascade Biologics), 10% cosmic calf serum (HyClone, Logan, UT, USA), 2 mM glutamine, 2
* Corresponding author. e-mail: [email protected] phone: +886-5-272-0411 ext. 33487 fax: +886-5-272-1206 157
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mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml gentamicin at 37°C under 5% CO2 humidified atmosphere. To minimize the effect of serum on cell proliferation and production of intracellular O2– and to enable the cells to adapt to their altered growth environment more successfully, the serum concentration was reduced from 10% to 0.3% sequentially before experiments. First, the serum concentration was reduced to 1% for 1 d, and then the cells were trypsinized and subcultured at 40% of cell confluence. One day after the passaging, the serum concentration was further reduced to 0.3% by exchanging the medium. The investigations were carried out 18 h after the last adjustment of serum concentration. The VSMCs used in the experiments underwent 5–7 passages. Preparation of oxyHb solution The blood from human median cubital vein was collected into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). OxyHb was purified as described previously (12, 15). Isolation and oxidative modification of LDL LDL was prepared by ultracentrifugation of plasma from healthy donors as described previously (16). Briefly, the density of plasma was first adjusted to 1.09 g/ml by adding KBr granules. The adjusted serum was layered between various densities of KBr solutions that were prepared by mixing different amounts of KBr (1.346 g/ml) and NaCl (1.005 g/ml) solutions. The ultracentrifugation was performed at 29,000 ×g, 12°C for 23 h and LDL subfractions were collected in the density range, ρ=1.019–1.063 g/ml. Partial oxidization (75% of complete oxidation) of LDL was carried out by dialyzing LDL against Dulbecco’s phosphate-buffered saline (DPBS) (pH 7.4, 34°C) containing 5 µM CuCl2 for about 7.5 h. The oxidation of LDL was monitored on the basis of absorbance at 234 nm in a UVVisible spectrophotometer (Varian, Palo Alto, CA, USA) and was terminated by adding 10 mM EDTA. The resulting oxidized LDL was further dialyzed three times against EDTA-free DPBS to remove EDTA. Competition assay for measuring rate of NO uptake by VSMCs This method was described previously (12). Briefly, after the incubation of VSMCs with nLDL and oxLDL for 18 h, each set of the experiments consists of four samples, each of which contained oxyHb; oxyHb with spermine NONOate (SpNO); VSMCs and oxyHb; VSMCs and oxyHb with SpNO in a 24-well cell culture dish. MetHb production in the solution was measured spectrophotometrically using a plate reader (µQuant Universal Microplate Spectrophotometer; Bio-Tek, Winooski, VT, USA). VSMC-NO uptake rate was defined in the following equation: rsmc = kSMC[O2–][NO]
ments, the VSMCs were washed three times with DPBS, incubated with 5 µM HE or DHR123 in a serum-free medium at 37°C for 30 min, and then mobilized with DPBS containing 0.02% EDTA and 0.25% trypsin. FACS was run using a FACScalibur (Becton, Dickinson and Company). The mean fluorescence intensity of the stained VSMCs was obtained after adjusting the mean fluorescence intensity of the untreated/unstained control to 5. Determination of cell cycle profiles of VSMCs The DNA content of VSMCs was detected by FACS using fluorescent dye propidium iodide (Ex: 287 and 488 nm; Em: 602 nm) (17). After treatment with nLDL and oxLDL in the presence and absence of oxyHb and diethylenetriamine NONOate (DETA-NO) for 18 h, cells were washed three times with DPBS, mobilized with trypsin, and centrifuged at 150 ×g for 5 min. Cells were then fixed with absolute ethanol for at least 1 h at 4°C. After incubation, cells were washed twice with DPBS, resuspended in DPBS/DNA extraction buffer (0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8) 1:1 mixture for 5 min, and then incubated for 30 min at room temperature with 3.8 mM sodium citrate buffer containing 50 µg/ml propidium iodide and 0.5 µg/ml RNase A. FACS was run using a FACScalibur
(1) –
where kSMC is the rate constant for SMC-NO reaction, [O2 ] is the intracellular O2– concentration, and [NO] is the NO concentration in bulk medium. kSMC[O2–]/kHb as an indicator of VSMC-NO uptake rate was calculated using the following equation: [ oxyHb] kSMC[O2–]/kHb = ([metHb]cf − [metHb])/(rv ln(------------------------0- )) (2) [ oxyHb] where kHb is the rate constant for oxyHb-NO reaction. [metHb] and [metHb]cf are the metHb concentrations in the samples with and without VSMCs, respectively. rv is the volumetric ratio of VSMCs to solution and [oxyHb]0 is the initial concentration of oxyHb in the medium. To investigate the effect of intracellular O2– concentrations on NO uptake rate, VSMCs were incubated with 7.5 mM DETC at the last 30 min of various treatments. Estimation of intracellular O2– and H2O2 concentrations Hydroethidine (HE) (Ex: 535 nm; Em: 610 nm) and dihydrorhodamine 123 (DHR123) (Ex: 488 nm; Em: 530 nm) were used for fluorescence-activated cell sorting (FACS) to evaluate intracellular contents of O2– and H2O2, respectively (7). After various treat-
FIG. 1. Effect of nLDL and oxLDL on rate of NO uptake by VSMCs in the presence and absence of DETC (A). VSMC-NO uptake rate was plotted against the mean HE-emitted fluorescence intensity (B). All data represent means ± S.E. for n = 3.
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(Becton, Dickinson and Company). Western blot analysis of soluble guanulyl cyclase (sGC) Human aortic VSMCs were lysed using 20 mM Tris buffer (pH 7.4) containing 0.1% sodium dodecylsulfate (SDS) and 0.1 mM phenylmethylsulfonyl fluoride. Proteins of equal amounts (40 µg/lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with PBS containing 5% nonfat milk and 0.05% Tween-20 for 1 h at room temperature. The membranes were then incubated for 1.5 h with a rabbit polyclonal antibody against sGC (1:500; Calbiochem, San Diego, CA, USA) and a mouse monoclonal antibody against β-actin (1 : 10000; Chemicon, Temecula, CA, USA). For immunodetection, the membranes were incubated 1 h with a goat anti-rabbit IgG (1 : 10000; Sigma, St. Louis, MO, USA) and rat anti-mouse IgG (1 : 10000; BD Biosciences, San Jose, CA, USA) conjugated with horseradish peroxidase. The signals were detected using the ECL detection system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and were analyzed by densitometry. Total protein concentration was determined by bicinchoninic acid protein assays (Pierce, Rockford, IL, USA). Measurement of intracellular cGMP The VSMCs were treated with phosphodiesterase inhibitor 0.2-mM 3-isobutyl-1-methylxanthine (Sigma) 30 min before the competition assay. At the end of competition experiments, the cells were lysed to measure intracellular cGMP concentration using a direct cGMP kit (Assay Designs, Ann Arbor, MI, USA). Modeling of cell proliferation We hypothesized that cell proliferation induced by nLDL and oxLDL is a linear function of intracellular levels of H2O2 and O2–: cell proliferation = k1[O2–] + k2[H2O2] + constant
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eration. The estimates of the coefficients in Eq. 3 for the presence and absence of oxyHb and DETA-NO were obtained separately from linear least square regression using Excel 2003 (Microsoft, Seattle, WA, USA).
RESULTS AND DISCUSSION Effect of O2– on VSMC-NO uptake rate in the challenge of nLDL and oxLDL To determine whether the increase in the levels of intracellular O2– induced by nLDL and oxLDL correlates with VSMC-NO uptake rate, we measured NO
(3)
– 2
[O ] and [H2O2] are the normalized mean fluorescence intensity of the VSMCs loaded respectively with HE and DHR123. The ratio of cells in S, G2, and M phases indicated the degree of cell prolif-
FIG. 2. Changes in sGC protein expression level after treatment of VSMCs with nLDL and oxLDL. After treatment of nLDL and oxLDL (20 and 100 µg/ml) for 12 h, the sGC protein expression in VSMCs was analyzed by western blotting. All data represent means ± S.E. for n=2.
FIG. 3. Effect of nLDL and oxLDL on NO-cGMP signaling. Treatment of nLDL and oxLDL (20 and 100 µg/ml) dose-dependently increased intracellular cGMP production in the presence of spNO and oxyHb. Pretreatment of VSMCs with 7 mM DETC for 30 min further increased intracellular cGMP production in various samples (A). cGMP production normalized by sGC protein content and VSMC-NO uptake rate from control (square), the cells treated with 20 µg/ml nLDL (circle), 100 µg/ml nLDL (triangle), 20 µg/ml oxLDL (inverted triangle), or 100 µg/ml nLDL (diamond) was dependent on the intracellular level of H2O2 (B). All data represent means ± S.E. for n = 3.
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uptake rate in NO competition experiments and compared it with the corresponding intracellular O2– concentration. Figure 1 shows that VSMC-NO uptake rate was increased in the cells treated with nLDL and oxLDL (20 µg/ml and 100 µg/ml, respectively) for 18 h (Fig. 1A) and positively depended on the level of intracellular O2– (Fig. 1B). These results are consistent with our previous finding that VSMC(A7r5)-NO uptake rate is positively proportional to the intracellular O2– concentration, which was altered by varying cell confluence and serum concentration (12). The treatment of VSMCs with 7.5 mM DETC (an SOD inhibitor) further increased the level of intracellular O2– and VSMC-NO uptake rate. Notably, the increase in intracellular O2– level alone induced by DETC through the inhibition of SOD cannot completely explain the significant effect of DETC on NO uptake rate (Fig. 1B). DETC is able to remove Cu2+ from Cu/Zn-SOD forming the DETC-Cu2+ complex (18). However, the DETC-Cu2+ complex is not able to bind to NO molecules; therefore, it is unlikely to contribute to the increase in NO uptake rate induced by DETC (19). On the other hand, DETC-Fe2+ has been used as a spin trap for detecting NO (20–22). Moreover, LDL has been demonstrated to increase the level of intracellular free iron in hu-
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man aortic endothelial cells and VSMCs (23). Therefore, it is plausible that the binding of NO by the intracellular DETC-Fe2+ complex may account for the further increase in VSMC-NO uptake rate. Enhancement of NO-cGMP signaling by nLDL and oxLDL To investigate whether the increase in NO uptake rate induced by nLDL and oxLDL in VSMCs increases intracellular cGMP production, we measured both the level of sGC protein and intracellular cGMP production after the competition experiment. Treatments of VSMCs with nLDL and oxLDL caused a dose-dependent decrease in sGC protein level (Fig. 2). In particular, oxLDL had a greater effect of reducing the content of sGC protein. The production of cGMP was increased by treatments of VSMCs with nLDL and oxLDL, even though the sGC protein content in these treated cells was low (Fig. 3A). In addition, the administration of DETC further enhanced cGMP production through the increase in the rate of O2–-mediated NO uptake, implying that the Fe2+(DETC)2NO complex may preserve, at least in part, the bioactivity of NO. If we normalized cGMP production by the content of sGC and NO uptake rate, we found that the normalized cGMP production was dependent on intracellular H2O2 concentration. OxLDL has been shown to
FIG. 4. Effect of DETA-NO and oxyHb on nLDL- and oxLDL-stimulated VSMC proliferation. After incubation of nLDL and oxLDL (20 and 100 µg/ml) for 18 h, the DNA profiles of human aortic VSMCs were analyzed by FACS using propidium iodide (50 µg/ml) (A, B). All data represent means ± S.E. for n = 3.
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TABLE 1. Estimation of parameters of linear regression for cell proliferation as a function of intracellular O2– and H2O2 concentrationsa Cell proliferationb DETA-NO+ Hb
Normalizedc
[O2–] + − Control 0.20 0.20 0.99 0.22 0.21 1.00 0.21 0.21 1.00 LDL-100 µg/ml 0.20 0.24 1.89 0.23 0.25 1.87 0.21 0.25 1.90 oxLDL-20 µg/ml 0.41 0.40 1.31 0.42 0.43 1.38 0.42 0.44 1.33 oxLDL-100 µg/ml 0.56 0.68 2.39 0.53 0.63 2.37 0.57 0.68 2.42 −0.059 −0.001 k1 0.166 0.181 k2 constant 0.199 0.029 0.988 0.997 R2 a Cell proliferation = k1[O2–]k2[H2O2] + constant b The ratio of cells in S, G2, and M phases was used to indicate the degree of cell proliferation. c The concentrations of intracellular O2– and H2O2 were normalized to the average of control.
significantly increase the protein expression of CuZnSOD in human aortic VSMCs (7). Furthermore, CuZnSOD/H2O2 has been reported to catalyze the degradation of s-nitrosoglutathione (GSNO) (24–26). Therefore, the higher normalized cGMP production in the oxLDL-treated VSMCs might be due to the higher levels of intracellular H2O2 and SOD for releasing NO from intracellular GSNO. Superoxide counteracted nLDL- and oxLDL-stimulated cell proliferation through increase in NO uptake rate Previous studies demonstrated that NO can inhibit VSMC proliferation via a sGC/cGMP dependent pathway by changing the expression level and activity of cell cycle regulatory proteins (27, 28). To test whether O2–-mediated NO uptake is involved in VSMC proliferation stimulated by nLDL and oxLDL, we simulated VSMC proliferation using a model on the basis of the data of intracellular DNA status, H2O2, and O2–. Figures 4A and 4B show that the presence of oxyHb and the NO donor DETA-NO decreased the ratio of cells in S, G2, and M phases. In particular, the addition of the NO donor showed the most significant effect on the 100 µg/ml oxLDL-treated cells that had the highest level of intracellular O2–. When we normalized the fluorescence intensities of O2– and H2O2 against the controls with and without DETANO, we found that cell proliferation was well expressed as a linear function of normalized O2– and H2O2 concentrations (Table 1). The coefficients of normalized O2– concentration in the equation in the presence and absence of oxyHb and DETA-NO were respectively −0.059 and −0.001, indicating that intracellular O2– had an inhibitory effect on nLDL- and oxLDL-stimulated VSMC proliferation and was strongly dependent on the presence of NO. On the other hand, the coefficients of normalized H2O2 concentration with and without oxyHb and DETA-NO were similar, indicating that the effect of H2O2 on cell proliferation was nearly independent of NO. Notably, our model generated using limited data did not exclude the effect of other factors on cell prolif-
[H2O2] 0.99 1.01 0.99 1.17 1.24 1.20 2.12 2.14 2.19 3.62 3.27 3.66
eration. In summary, the levels of intracellular O2– altered by nLDL and oxLDL correlated well with VSMC-NO uptake rate. Treatment of VSMCs with DETC increased NO uptake rate through the increase in intracellular O2– concentration. Although nLDL and oxLDL decreased sGC protein content, they still caused a net increase in cGMP production in VSMCs. Furthermore, O2– was able to counteract nLDLand oxLDL-stimulated VSMC proliferation through the increase in NO uptake rate. These findings may provide new insights into the therapeutic strategies of atherosclerosis caused by oxLDL. ACKNOWLEDGMENTS This work was supported by grants NSC 94-2214-E-194-007 and NSC 95-2221-E-194-006 from the National Science Council, Taiwan and a grant from Buddist Tzuchi Dalin General Hospital, Taiwan.
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