Pressure Promotes Angiotensin II–Mediated Migration of Human Coronary Smooth Muscle Cells Through Increase in Oxidative Stress
Angiotensin II–mediated oxidative stress may play a role in the pathogenesis of coronary atherosclerosis. We examined the effects of pressure on the angiotensin II–mediated increase in oxidative stress and migration of cultured human coronary smooth muscle cells (SMCs). Increased pressure (100 mm Hg) by helium gas for 48 hours increased angiotensin II–mediated oxidative stress as evaluated by flow cytometry and SMC migration (from 15.9±2.2 to 32.0±2.4 cells per 4 high-power fields, P<0.05; n=8). The pressure-induced increases in oxidative stress observed appear to involve phospholipase D (PLD) and protein kinase C (PKC), inasmuch as the indirect PLD inhibitor suramin, at 100 μmol/L, and the PKC inhibitor chelerythrine, at 1 μmol/L, completely blocked the increase in angiotensin II–mediated oxidative stress induced by pressure. Pressure-induced increase in angiotensin II–mediated oxidative stress was inhibited by diphenylene iodonium chloride, an NADPH oxidase inhibitor, by 79% (P<0.05, n=8). Losartan (1 μmol/L), its active metabolite E3174 (1 μmol/L), and the antioxidant N-acetylcysteine (100 mmol/L) but not PD123319 (1 μmol/L) also blocked pressure-induced increases in angiotensin II–mediated oxidative stress and SMC migration (P<0.05, n=8). These findings suggest a novel cellular mechanism whereby pressure regulates the angiotensin II–mediated migration of SMCs, possibly via angiotensin II type 1 receptors, and which involves PLD-mediated, PKC-mediated, and NADPH oxidase–mediated increases in oxidative stress.
Recent studies have demonstrated that physical or mechanical factors such as shear stress and stretch (or tension) contribute to vascular remodeling.1–4⇓⇓⇓ The physical forces exerted on the blood vessel wall by the passage of intravascular blood are flow and pressure. Pressure is exerted radially at right angles to the axis of flow and leads to tangential strain on the wall, causing it to stretch (tension). However, in studies using vessels or vascular rings, it is impossible to separate the effects that are possibly due to pure pressure from those that are due to stretch (tension) induced by pressure. Notably, though, a method in which pure pressure can be applied to cultured cells without shear stress and stretch has been reported.5 Pressure might result in increased oxidative stress in smooth muscle cells (SMCs), which may contribute to pressure-mediated changes in SMC function.6 The molecular pathogenesis of essential hypertension is still not known in detail, although increased production of oxygen reactive species has been reported in experimental models of hypertension7 and in patients with essential hypertension.8
Many clinicians have suggested that essential hypertension may be related to the renin-angiotensin system. Angiotensin II is a multifunctional hormone that affects both the contraction and growth of vascular SMCs through a complex series of intracellular signaling events initiated by the interaction of angiotensin II with angiotensin II type 1 (AT1) receptors. The cellular response to angiotensin II is multiphasic, involving (1) stimulation within seconds of phospholipase C (PLC) and Ca2+ mobilization, (2) activation within minutes of phospholipase D (PLD) and protein kinase C (PKC) mobilization, and (3) stimulation after a period of hours of NADH/NADPH oxidase activity.9 Recent studies have shown that angiotensin II can stimulate the oxidative stress9 that may potentiate SMC migration.10 Enhanced migration of vascular SMCs plays an important role in the atherogenesis.11
Accordingly, the objectives of the present study were to determine whether elevated pressure enhances the angiotensin II–mediated increase in oxidative stress and the migration of cultured SMCs derived from human coronary artery and, if it does, to examine the effects of losartan and its active metabolite (E3174)12 on the pressure-enhanced angiotensin II–mediated increase in oxidative stress and SMC migration. In addition, we examined the mechanism by which pressure enhances angiotensin II–mediated oxidative stress and SMC migration.
Human coronary artery SMCs (Clonetics Corp) were cultured in SMC basal medium containing human epidermal growth factor (0.5 ng/mL), human fibroblast growth factor (2 ng/mL), 5% FCS, 50 μg/mL gentamicin sulfate, and 50 μg/mL amphotericin B.13 Cells were identified as SMCs according to their morphological and immunohistochemical characteristics as previously reported.14
A pressure-loading apparatus was assembled as reported by Hishikawa et al,5 with some modifications. SMCs were seeded in 25-cm2 flasks. The flasks were clamped tightly between 2 iron plates, and the top of each flask was sealed with a rubber cap. The rubber cap was pierced with a needle connected to tubing attached to a 3-way rotary valve, a sphygmomanometer, and a pressure valve. Compressed helium gas was pumped in to raise the internal pressure. While the helium gas was being pumped in, no prepacked room air was released, so that the partial pressures of the gases originally contained in the flasks, such as oxygen, nitrogen, and carbon dioxide, remained constant.
The migration of SMCs was assayed by using a modified version of the Boyden chamber method with microchemotaxis chambers (Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp), as previously reported.15 In this experiment, polycarbonate filters with pores 12 μm in diameter were used. SMCs were allowed to grow for 48 hours in high-pressure (100 mm Hg) or normal-pressure (0 mm Hg) medium with 0.5% FCS in the presence or absence of suramin or chelerythrine. Migration activity was calculated as the mean number of migrated cells observed in 4 high-power fields (HPFs) and is given as the mean value of 4 measurements.
PLD Activity Measured by Ethanolamine Release
SMCs were allowed to grow for 48 hours in high or normal pressure with 0.5% FCS. SMCs in 25-cm2 flasks were cultured in medium containing [3H]ethanolamine (5 μCi/mL per flask) for 24 hours (the latter half of the 48-hour period with pressure) to label cellular phosphatidylethanolamine. After stimulation with angiotensin II (10 nmol/L) for 10 minutes and removal of the labeling medium, the cells were washed twice with buffer A (20 mmol/L HEPES [pH 7.4], 120 mmol/L NaCl, and 5.6 mmol/L glucose). The reaction was terminated by gentle scraping. Fractionation of ethanolamine metabolites from the aqueous phase was performed on Dowex 50 w H+-packed columns as previously described.16
Cell Fractionation and PKC Assay
SMCs were allowed to grow for 48 hours in high- or normal-pressure medium with 0.5% FCS in the presence or absence of suramin or chelerythrine. After incubation with 10 nmol/L angiotensin II for 10 minutes, PKC activity was measured by a modified version of a method previously reported with use of the Amersham PKC assay system.16
Assay of Intracellular Oxidative Stress
Intracellular oxidative stress levels were measured by using a fluorescent dye, carboxydichlorofluorescein (CDCFH) diacetate bis-acetoxymethyl (AM) ester (Molecular Probe Corp). CDCFH diacetate bis-AM ester is a nonpolar compound that is converted into a nonfluorescent polar derivative (CDCFH) by cellular esterases after incorporation into cells. CDCFH is membrane impermeable and rapidly oxidized to the highly fluorescent carboxydichlorofluorescein (CDCF) in the presence of intracellular hydrogen peroxide and peroxidases. For assays, the medium was replaced with Hanks’ solution containing 5 μmol/L CDCFH diacetate bis-AM ester at appropriate times after stimulation. After 5 minutes of incubation at room temperature, the fluorescence intensity was measured by flow cytometry as previously reported.17 The excitation wavelength was 510 to 530 nm. Relative fluorescence intensities were calculated by using untreated control cells as a standard.
SMCs were visualized through a fluorescence microscope (Olympus IX70, ×400 water immersion objective lens) via a camera (Olympus PM-C 35DX). To elicit fluorescent images, the preparation was illuminated with a 200-W mercury lamp. The light was passed through a quartz collector, heat filter, and an excitation filter to epi-illuminate the preparation. Fluorescence emission from the sample was passed through a bandpass filter (515 nm) and onto the camera.
Statistical analysis was performed by ANOVA and the Scheffé modified t test.18 Values of P<0.05 were considered significant.
Effect of Pressure on Angiotensin II–Mediated Migration of Human Coronary SMCs
Human coronary SMCs were pressurized for 24 hours or 48 hours. There were no significant changes in cell migration at 24 hours. However, pressurization for 48 hours at 50, 100, and 150 mm Hg significantly increased SMC migration (at 0 mm Hg, 16.5±2.6 cells/4 HPFs; at 50 mm Hg, 22.2±2.2 cells/4 HPFs [P<0.05 versus 0 mm Hg group]; at 100 mm Hg, 31.0±2.2 cells/4 HPFs [P<0.05 versus 0 mm Hg group]; and at 150 mm Hg, 32.4±3.8 cells/4 HPFs [P<0.05 versus 0 mm Hg group]; n=8). The increment in cell migration at 100 mm Hg was 85% (Figure 1). Suramin (an indirect PLD inhibitor) at 100 μmol/L, chelerythrine (a specific PKC inhibitor) at 1 μmol/L, and N-acetyl-l-cysteine (NAC, Sigma Chemical Co; an antioxidant) as well as losartan and its active metabolite E3174 (AT1 receptor antagonists) at 0.1 to 1 μmol/L but not PD123319 (an angiotensin II type 2 receptor antagonist) at 0.1 to 1 μmol/L (Figure 1) significantly reduced the magnitude of increase in SMC migration by pressure, followed by the addition of 0.1 μmol/L angiotensin II with or without other agents as follows: for angiotensin II alone, 31.0±2.2 cells/4 HPFs; for angiotensin II+0.1 μmol/L losartan, 24.0±2.2 cells/4 HPFs (P<0.05 versus angiotensin II group); for angiotensin II+1 μmol/L losartan, 18.2±1.2 cells/4 HPFs (P<0.05 versus angiotensin II group); for angiotensin II+10 μmol/L losartan, 6.4±1.2 cells/4 HPFs (P<0.05 versus angiotensin II group); for angiotensin II+0.1 μmol/L E3174, 19.0±2.2 cells/4 HPFs (P<0.05 versus angiotensin II group); for angiotensin II+1 μmol/L E3174, 15.8±2.0 cells/4 HPFs (P<0.05 versus angiotensin II group); for angiotensin II+0.1 μmol/L PD123319, 30±2.4 cells/4 HPFs; and for angiotensin II+1 μmol/L PD123319, 28.4±2.6 cells/4 HPFs; n=8). However, these compounds at these concentrations had no effect on SMC migration in normal-pressure (0 mm Hg) medium without angiotensin II (data not shown).
We also performed checkerboard analysis. The presence of positive concentration gradients of angiotensin II across the filter rather than equal concentrations indicates that the migration-stimulatory effect of angiotensin II is chemotactic in nature for human coronary SMCs in normal-pressure– and high-pressure–treated SMCs. Figure 2 shows the checkerboard analysis of migration activity of angiotensin II. In pressure (100 mm Hg)–treated and angiotensin II–treated SMCs, a chemokinetic effect cannot be completely ruled out.
Involvement of PLD and PKC in Angiotensin II–Mediated SMC Migration
Increases and decreases in PLD and PKC were examined by measurement of PLD and membrane-bound (particulate) PKC activities in the presence of angiotensin II. PLD and membrane-bound PKC activities were increased by pressure (Table). These increases were significantly reduced by NAC (100 mmol/L), suramin (100 μmol/L), and losartan (1 μmol/L) but not by PD123319 (1 μmol/L), although these compounds at these concentrations had no effect on either PLD or PKC activity in normal-pressure medium (data not shown). Suramin (100 μmol/L) and chelerythrine (1 μmol/L) each completely inhibited the pressure (100 mm Hg)–mediated and angiotensin II (0.1 μmol/L)–mediated increase in PLD and PKC activity, respectively (Table). These compounds at the concentrations used did not cause loss of cells in the confluent state. After incubation, <5% of cells were found in the supernatant media, as confirmed by comparison of percent LDH activity present with total LDH activity (3% to 5%). Cell viability was also checked by trypan blue staining, which confirmed that >95% of the cells were alive.
Effects of Pressure on SMC Intracellular Oxidative Stress
Pressurization for 48 hours at 50, 100, and 150 mm Hg significantly increased angiotensin II–mediated SMC oxidative stress (for 0 mm Hg, 78.2±7.2 arbitrary units [AU]; for 50 mm Hg, 100.2±9.6 AU [P<0.05 versus 0 mm Hg group]; for 100 mm Hg, 140.0±6.2 AU [P<0.05 versus 0 mm Hg group]; and for 150 mm Hg, 146.8±11.6 AU [P<0.05 versus 0 mm Hg group]; n=8). Chronic high-pressure treatment for 48 hours increased oxidative stress by 71% (Figure 3). Suramin (100 μmol/L), chelerythrine (1 μmol/L), NAC (100 mmol/L), and losartan and its active metabolite E3174 (0.1 to 1 μmol/L) but not PD123319 (0.1 to 1 μmol/L) (Figure 3) also decreased intracellular oxidative stress (for angiotensin II alone, 140±6.2 AU; for angiotensin II+0.1 μmol/L losartan, 118±8.2 AU [P<0.05 versus angiotensin II group]; for angiotensin II+1 μmol/L losartan, 84.2±7.4 AU [P<0.05 versus angiotensin II group]; for angiotensin II+10 μmol/L losartan, 30.4±3.6 AU [P<0.05 versus angiotensin II group]; for angiotensin II+0.1 μmol/L E3174, 98±8.6 AU [P<0.05 versus angiotensin II group]; for angiotensin II+1 μmol/L E3174, 118.4±6.2 AU [P<0.05 versus angiotensin II group]; for angiotensin II+0.1 μmol/L PD123319, 136.4±9.4 AU; and for angiotensin II+1 μmol/L PD123319, 132.4±8.4 AU; n=8). Figure 4 shows representative findings for the effects of chronic high-pressure treatment on angiotensin II–mediated oxidative stress in coronary SMCs as visualized by fluorescence microscopy and measured by flow cytometry.
To further clarify the enzymatic pathway by which pressure increases oxidative stress, SMCs were pretreated with p-chloromercuriphenylsulfonic acid (PCMPS, Sigma), an NADH oxidase inhibitor, or diphenylene iodonium chloride (DPI, Sigma), an NADPH oxidase inhibitor. Pressure-induced increase in angiotensin II–mediated oxidative stress (control, 84±8.0 AU; 100 mm Hg, 140±8.8 AU [P<0.05 versus control group]; n=8) was significantly suppressed by 10 μmol/L PCMPS (112±6.4 AU, P<0.05 versus 100 mm Hg group; n=8) and 10 μmol/L DPI (96±7.6 AU, P<0.05 versus 100 mm Hg group; n=8).
In the present study, we found that high-pressure potentiated angiotensin II–stimulated oxidative stress and migration. Losartan (10 μmol/L) blocked oxidative stress and migration (by 78% and 79%, respectively). These results suggest that high-pressure–potentiated angiotensin II–stimulated oxidative stress and migration are mediated by AT1 receptors.
In the present study, suramin and chelerythrine inhibited PLD or PKC activation (Table). Suramin inhibits PLD activity, which may decrease diacylglycerol, resulting in the suppression of PKC activity. Chelerythrine inhibits PKC activity, which may decrease PLD activity.19 We also found that high-pressure (100 mm Hg) treatment for 48 hours increased oxidative stress 1.7-fold, as directly measured by flow cytometry, and that suramin and chelerythrine, which completely inhibited the pressure (100 mm Hg)–mediated increase in PLD and PKC activity (Table), each suppressed this increase (Figure 3). These findings suggest that pressure increased the angiotensin II–mediated oxidative stress through PLD and PKC activation. Thus, our findings suggest that high pressure increases PLD activity in coronary SMCs, which, in turn, increases PKC activity.17 Inasmuch as it has been reported that the PKC pathway plays a role in increasing oxidative stress,16,20⇓ the increase in oxidative stress may play a role in the increase in the rate of migration of SMCs.11
It has been reported that pressure may enhance PLC activity.4 Because suramin is also considered to be a G-protein antagonist21 and because PKC-α, -β, and -ζ are activated by H2O2 and angiotensin II,22 the effects of H2O2 and angiotensin II are also mediated by PLC.
To determine whether angiotensin II–mediated NADH or NADPH oxidase was involved in the pressure-mediated increase in the oxidative stress we observed, we treated SMCs with PCMPS or DPI. In SMCs, PCMPS and DPI each significantly inhibited the pressure-mediated increase in oxidative stress. This suggests that NADH/NADPH oxidase is the enzyme responsible for the increase in oxidative stress. However, the lack of specificity of DPI limits this interpretation, inasmuch as DPI inhibits not only NADPH oxidase but also other flavoprotein-dependent enzymes. Interestingly, PKC also activates NADH/NADPH oxidase. These results suggest that PLD and PKC may play roles in the activation of NADH/NADPH oxidase.
In the present study, we showed that losartan prevents pressure-mediated and angiotensin II–mediated SMC migration. We also showed that losartan directly prevents an increase in oxidative stress by increased pressure and angiotensin II, a finding not observed with PD123319, suggesting that the increase in oxidative stress that was observed is mediated through AT1 receptors. We also showed that the active metabolite of losartan, E3174, directly and more potently prevents the angiotensin II–mediated increase in oxidative stress and migration of SMCs, suggesting that E3174 plays an important role in mediating the effects of losartan.12
In summary, increased static pressure increased human coronary SMC migration, possibly through AT1 receptors. This increase involves PLD-mediated, PKC-mediated, and NADH/NADPH oxidase–mediated increases in oxidative stress. Future therapeutic strategies for vascular protection in hypertensive patients may include direct targeting of the signaling pathways to prevent oxidative stress in vascular tissue.
This study was supported by grants from the Ministry of Education, Sports, Science, and Technology, Kimura Heart Foundation, and the Research Foundation for Community Medicine. We would like to thank Keiko Hirata and Sayuri Takagi for excellent technical assistance.
- Received September 23, 2001.
- Revision received October 29, 2001.
- Accepted November 7, 2001.
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