INTRODUCTION

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MANY CLINICAL INVESTIGATIONS have established that an elevation of either systolic or diastolic blood pressure is a powerful independent risk factor for coronary artery disease (15, 18). Furthermore, experimental studies demonstrated that hypertension enhances the development of atherosclerosis in hypercholesterolemic animals (6, 21). In Watanabe heritable hyperlipidemic rabbits, renovascular hypertension induced by the one-kidney, one-clip Goldblatt model significantly increased the area and extent of atherosclerosis (6). However, the precise mechanisms whereby hypertension facilitates the development of atherosclerosis are poorly understood, although several mechanisms have been proposed, such as involvement of the renin-angiotensin system and the sympathetic nerve system.

Hemodynamic forces such as shear stress and stretch force on the vessel wall have profound influences on the function and structure of vascular cells via modulation of expression of various genes (13, 25). It has been reported that stretch force on smooth muscle cells exerts a variety of biological responses, including increases in intracellular calcium concentration, enhancement of cell proliferation, modulation of cell phenotype, and regulation of the expression of contractile proteins (3, 4, 28). Very recently, Meyer and colleagues (22) demonstrated that stretch force on vessel walls augmented the uptake of low-density lipoprotein (LDL) into aortic walls. Because the degree of stretch force on vessel walls depends on blood pressure, the alteration of hemodynamic forces in the hypertensive state might be one of the potential mechanisms whereby hypertension facilitates atherosclerosis.

Recently, accumulating evidence suggests that increased oxidative stress in vasculature plays a pivotal role in atherogenesis (1). Oxidative modification of LDL has been considered a key step of the generation of macrophage-derived foam cells and the initiation of atherosclerosis. Oxidized LDL has profound biological activities on vascular cells, including inhibition of endothelium-dependent vasodilation and production of cytokines (7, 31). Furthermore, the oxidative stress on vascular cells might contribute to atherosclerosis through redox-sensitive gene expression such as vascular cell adhesion molecule, monocyte chemotactic protein-1, and others (19, 27, 29).

To assess the influence of stretch force on the modification of LDL by smooth muscle cells, we have utilized a culture model in which cultured cells can be subjected to persistent stretch force. In the present study, the effects of stretch force on smooth muscle cell-mediated oxidative modification of LDL and production of superoxide anion were examined.

	METHODS

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Cell culture and exposure of cultured cells to stretch force. As previously described, rat aortic smooth muscle cells (RASM) were isolated from a rat thoracic aorta by enzymatic digestion (16). Bovine aortic endothelial cells (BAEC) were obtained by scraping with a knife the internal surface of the aorta excised from a freshly slaughtered cow (12). Cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. RASM and BAEC used in the present study were from passages 10-16 and 5-8, respectively.

LDL preparation and modification. Human LDL (density 1.019-1.063 g/ml) was isolated from the plasma of healthy volunteers by sequential ultracentrifugation as previously described (10). The isolated LDL was dialyzed against 150 mM NaCl and 1 mM EDTA, stored at 4°C, and used within 1 mo. Before being used in experiments, LDL was freshly dialyzed with PBS and was sterilized by passage through a 0.22-µm filter (Millipore, Bedford, MA). Protein concentrations were determined by the method of Bradford with bovine serum albumin as a standard protein.

Assessment of extent of oxidation of LDL by cultured cells. The degree of LDL oxidation by cultured cells was assessed by measurement of thiobarbituric acid-reacting substances (TBARS). At the end of incubation of LDL with cultured cells, the culture medium was collected and centrifuged (1,500 g for 10 min) to remove detached cells. After 0.2 mM butylated hydroxytoluene was added to the medium to stop the reaction, TBARS of LDL in the medium was assayed. Formation of TBARS of LDL samples was measured with a Wako test kit (Osaka, Japan). Briefly, LDL was mixed with 1.0 ml of thiobarbituric acid solvents. After being heated at 100°C for 60 min, fluorescent reaction products were assayed on a spectrofluorometer (F-2000, Hitachi, Japan) with excitation at 515 nm and emission at 553 nm. Malondialdehyde (MDA) was used as a standard. Values are expressed as nanomoles of MDA equivalents per milligram of LDL protein.

Measurement of superoxide anion released from vascular smooth muscle cells. As previously described, the production of superoxide anion was measured as the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (11). RASM cultured on stretch dishes were washed twice with phenol red-free MEM and incubated with 1 ml of phenol red-free MEM containing 1 mg/ml cytochrome c with or without 75 U/ml SOD. After incubation for 4 h, the medium was collected and the absorbance was immediately read at 550 nm. In stretch experiments, stretch force was imposed on cells by stretching dishes during the incubation period. Superoxide-specific reduction of cytochrome c was expressed as the difference in absorbance between cells incubated with SOD and those without SOD, using an extinction coefficient of 21 mM¹ · cm¹.

Materials. All reagents were purchased from Sigma Chemical, except where specified.

Statistical analysis. Comparison of data between control and stretch experiments was made by unpaired t-test. P values < 0.05 were considered significant.

	RESULTS

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Oxidative modification of LDL by cultured vascular smooth muscle cells. TBARS of LDL was 0.7 ± 0.5 nmol MDA/mg before experiments were started. As shown in Table 1, incubation of LDL with RASM for 24 h in Ham's F-10 medium resulted in the oxidative modification of LDL as indicated by increases in TBARS to 24.5 ± 2.3 nmol MDA/mg. Incubation of LDL in a cell-free system in Ham's F-10 medium caused oxidative modification to a much smaller degree than that in the presence of cells. TBARS of LDL in MEM, which contains no ferrous ion, was not increased by incubation with RASM as well as in the cell-free system (Table 1). The extent of LDL oxidation in MEM supplemented with 5 µM ferrous ion was similar to that in Ham's F-10 medium. Diethylenetriaminepentaacetic acid (DTPA), a divalent metal ion chelator, significantly attenuated the oxidation of LDL. These results suggest that the presence of micromolar concentrations of metal ions is necessary for LDL oxidation in vitro.

Stretch force augments smooth muscle cell-mediated oxidative modification of LDL. To determine whether mechanical stretch force on smooth muscle cells might influence the oxidative modification of LDL, stretch force was imposed on RASM cultured on laminin-precoated silicone dishes. Imposition of stretch force on RASM by stretching dishes by 10 and 20% enhanced LDL oxidation to 131.3 ± 17.2 and 149.3 ± 15.1% of control, respectively (n = 5 experiments, P < 0.05). The enhancement of LDL oxidation by stretch force was inhibited by coincubation with SOD (Fig. 1). DTPA also suppressed stretch-induced LDL oxidation by 52.2 ± 4.5% (n = 3). Because the extracellular matrix has profound influences on cellular function, we performed the same experiments using fibronectin-coated silicone dishes. In fibronectin-coated dishes, stretching RASM by 20% similarly enhanced LDL oxidation to 150.3 ± 14.5% of control static cells (n = 3 experiments). The conditioned medium from vascular smooth muscle cells on which stretch force had been imposed for 24 h did not affect the oxidation of LDL, indicating that diffusible substances were not involved. In contrast with vascular smooth muscle cells, stretch force had no effect on endothelial cell-mediated LDL oxidation (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   A: effect of stretch force on vascular smooth muscle cell-mediated low-density lipoprotein (LDL) oxidation. Native LDL (50 µg/ml) isolated from healthy human plasma was incubated with rat aortic smooth muscle cells (RASM) in F-10 medium for 24 h, and degree of oxidation of LDL was assessed by measurement of thiobarbituric acid-reacting substances (TBARS) of LDL. RASM were cultured on deformable silicone dishes, and stretch force was imposed by stretching them 10 or 20%. In some experiments, 75 U/ml of superoxide dismutase (SOD) were coincubated. Data are means ± SD of triplicates from 5 separate experiments. * P < 0.05 vs. with SOD; # P < 0.05 vs. control cells. B: effect of stretch force on endothelial cell-mediated LDL oxidation. Native LDL (50 µg/ml) was incubated with bovine aortic endothelial cells (BAEC) in F-10 medium for 24 h, and degree of oxidation of LDL was assessed by measurement of TBARS of LDL. In stretched cells, stretch force was imposed on BAEC by stretching dishes by 20%. Data are means ± SD of triplicates from 3 separate experiments.

Stretch force increases superoxide anion production from vascular smooth muscle cells. We next examined the effect of stretch force on superoxide anion production from cultured vascular smooth muscle cells. Control static RASM produced superoxide anion at the rate of 4.50 ± 0.10 nmol · mg¹ · h¹. Stretch force increased superoxide anion production to 7.22 ± 0.80 nmol · mg¹ · h¹ (n = 5, P < 0.05; Fig. 2). In fibronectin-coated stretch dishes, the amounts of superoxide anion released from control cells and stretched cells were 4.30 and 7.35 nmol · mg¹ · h¹, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of stretch force on superoxide anion production from cultured vascular smooth muscle cells. RASM were cultured on deformable silicone dishes, and stretch force was imposed on RASM by stretching dishes by 20%. Superoxide anion released from RASM was detected by SOD-inhibitable reduction of cytochrome c. Data are means ± SD of triplicates from 5 separate experiments. * P < 0.05 vs. control.

Inhibitor of NADH/NADPH oxidase abolishes LDL oxidation by cultured smooth muscle cells. Various origins of superoxide anion in vessel walls have been proposed, including lipoxygenase, cyclooxygenase, and NADH/NADPH oxidase. To determine the source of superoxide in RASM, various enzyme inhibitors were tested next. We used indomethacin, 5,8,11,14-eicosatetraynic acid (ETYA), and diphenyliodonium (DPI) as inhibitors of cyclooxygenase, lipoxygenase, and NADH/NADPH oxidase, respectively. As shown in Table 3, DPI (100 µM), an inhibitor of NADH/NADPH oxidase, significantly prevented smooth muscle cell-mediated oxidative modification, whereas ETYA (50 µM) and indomethacin (10 µM) were without effect. These concentrations of inhibitors were reported to be enough to inhibit each enzyme system in smooth muscle cells without cytotoxicity.

	DISCUSSION

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The present study demonstrated that stretch force enhances smooth muscle cell-mediated oxidative modification of LDL in a superoxide anion-dependent manner. We demonstrated that stretch force increases superoxide anion production from smooth muscle cells. Furthermore, the inhibition of NADH/NADPH oxidase significantly attenuated the oxidative modification of LDL by smooth muscle cells, indicating that this oxidase system is likely involved in superoxide anion production.

The earliest event in the development of fatty streaks is the transport of LDL into the arterial wall. The LDL trapped in subendothelial space is oxidized by reactive oxygen species in the arterial wall. Because both endothelial cells and arterial smooth muscle cells were shown to produce superoxide anion and to actively oxidize LDL in vitro (11, 20), these vascular cells could be potential sources for the oxidative modification of LDL in vivo. However, according to the findings of Rajagopalan et al. (26), smooth muscle cells likely play more important roles in vascular superoxide anion production than endothelial cells, especially in the hypertensive state. In the present study stretch force increased smooth muscle cell-mediated LDL oxidation, whereas it had no effect on LDL oxidation by endothelial cells. Although it is difficult to compare the biological responses in vitro with those in vivo, it is likely that vascular smooth muscle cells as a generator of oxidant stress play a pivotal role in atherogenesis.

As shown in Tables 1 and 2, LDL oxidation by cultured smooth muscle cells depends on the presence of metal ion and is likely mediated by superoxide anion. However, cultured vascular smooth muscle cells had the ability to produce superoxide anion in MEM, which contains no ferrous ion (Fig. 2), indicating that the production of superoxide anion by this cell type requires no metal ion. Thus LDL oxidation by cultured smooth muscle cells is a metal ion-dependent and superoxide-mediated process; however, superoxide anion itself is not enough for LDL oxidation. These observations are consistent with previously published reports regarding LDL oxidation by cultured smooth muscle cells (11, 24). Although the mechanism of superoxide-mediated and metal ion-dependent oxidation of LDL is not fully understood, several mechanisms should be considered. Because catalase and mannitol were without effect on LDL oxidation by cultured smooth muscle cells, aqueous hydroxyl radical did not play a major role; however, the role of lipid-phase hydroxyl radical is not excluded. Superoxide anion and H₂O₂ might cause metal ion-dependent production of highly reactive · OH in the lipid phase via the Fenton reaction, and this might promote lipid peroxidation. Otherwise, metal ion might decompose peroxide to chain-propagating radicals according to the following reaction Superoxide anion might promote recycling of the oxidation state of the oxidant metal ion as follows There are a variety of potential enzymatic origins of superoxide in cultured vascular smooth muscle cells, including cyclooxygenase, lipoxygenase, the mitochondrial electron-transfer system, the xanthine/xanthine oxidase system, and NADH/NADPH oxidase. Recently, it has become evident that NADH/NADPH oxidase is the most important origin of superoxide anion in endothelial cells as well as vascular smooth muscle cells (9, 23). In the present study DPI attenuated the oxidative modification of LDL by RASM, indicating that NADH/NADPH oxidase system is a most likely origin of superoxide anion in vascular smooth muscle cells. We observed that stretch force increased superoxide anion production concomitant with the enhancement of LDL oxidation. Although the neutrophil NADPH oxidase has been reported to be regulated by activation of phospholipase D, the subsequential production of phosphatidic acid and diacylglycerol, and activation of protein kinase C (2, 8, 30), how vascular NADH/NADPH oxidase activity is regulated is poorly understood. The mechanism and signal pathway whereby stretch force increases superoxide anion production from smooth muscle cells via NADH/NADPH oxidase remain to be elucidated.

To evaluate the contribution of hypertension as a risk factor for atherosclerosis, it is important to consider whether hypercholesterolemia coexists or not. Experimental studies demonstrated that the severity and extent of atherosclerosis are markedly increased in the presence of hypertension (6), whereas in the absence of hyperlipidemia the intimal thickening in hypertensive state is minimal (5). Furthermore, a previous epidemiologic investigation demonstrated that hypertension was not important in populations with mean plasma cholesterol levels <160 mg/dl (14). In the epidemiologic study of a very large population, Kannel and Gordan (14) demonstrated that hypertension has an especially strong risk impact when there is coexistent hypercholesterolemia. According to the observation of Meyer et al. (22), the imposition of stretch force on the aortic wall increases uptake of LDL into the vessel wall. Taken together with our observations, when hypertension coexists with hypercholesterolemia, the accelerative effects of stretch force on LDL oxidation might synergistically facilitate the development of atherosclerosis.

In conclusion, stretch force augments smooth muscle cell-mediated oxidative modification of LDL via superoxide anion production. NADH/NADPH oxidase is likely involved in superoxide anion production by smooth muscle cells. Given the importance of oxidant stress on vasculature in atherogenesis, our results suggest that the increased oxidant stress induced by alteration of hemodynamic forces in the hypertensive state is one of the potential mechanisms whereby hypertension facilitates the extent of atherosclerosis.