Effect of Natriuretic Peptide Family on the Oxidized LDL–Induced Migration of Human Coronary Artery Smooth Muscle Cells
Abstract The migration of medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in atherosclerotic lesions. The present study examined the possible effect of a novel endothelium-derived relaxing peptide, C-type natriuretic peptide (CNP), on oxidized low-density lipoprotein (LDL)–induced migration of cultured human coronary artery SMCs by the Boyden’s chamber method. The effect of CNP was compared with that of atrial and brain natriuretic peptides (ANP and BNP, respectively). Oxidized LDL stimulates SMC migration in a concentration-dependent manner between 20 and 200 μg/mL. This stimulation was chemotactic in nature but was not chemokinetic. By contrast, native LDL was without significant activity. CNP-22 clearly inhibited SMC migration stimulated with 200 μg/mL oxidized LDL in a concentration-dependent manner between 10−9 and 10−6 mol/L. ANP-(1-28) and BNP-32 also inhibited oxidized LDL–induced SMC migration at concentrations of 10−7 and 10−6 mol/L, but these effects were weaker than the effect of CNP-22. Such inhibition by these natriuretic peptides was paralleled by an increase in the cellular level of cGMP. Oxidized LDL–induced migration was significantly inhibited by a stable analogue of cGMP, 8-bromo-cGMP, or an activator of the cytosolic guanylate cyclase, sodium nitroprusside. These natriuretic peptides did not suppress the cell adhesion either in the absence or presence of oxidized LDL. These data indicate that oxidized LDL stimulates migration of human coronary artery SMCs and that natriuretic peptides, especially CNP, inhibit this stimulated SMC migration, at least in part, through a cGMP-dependent process. Taken together with the finding that oxidized LDL is present in the intima, CNP may play a role as a local antimigration factor during the process of intimal thickening in hypercholesterolemia-induced coronary atherosclerosis.
Elevated levels of LDL cholesterol are an important risk factor for coronary atherosclerosis and cardiovascular morbidity.1 2 3 4 LDL is considered to be the main atherogenic class of lipoproteins. However, recent evidence strongly suggests that oxidatively modified LDL possesses more atherogenic properties than does native LDL.5 Actually, oxidized LDL is shown to contribute to the migration of bovine aortic medial SMCs into the intima,6 which is an important process of the initiation and/or the progression of atherosclerosis.7 8
Natriuretic peptides are a family of hormones involved in the control of fluid balance. ANP and BNP are two members of this family9 10 11 that are both secreted through the coronary sinus from the heart.12 13 14 15 CNP is the most recently identified member of this family, which was first identified in the porcine brain.16 Subsequently, CNP is shown to be present in cultured human endothelial cells and plasma.17 Furthermore, Suga et al18 have demonstrated that CNP is produced by cultured bovine vascular endothelial cells, and this production is markedly augmented by transforming growth factor-β. In addition to its vasorelaxant and natriuretic effects, CNP is shown to be able to inhibit the serum- and PDGF-induced mitogenesis.19 However, the effect of CNP on the oxidized LDL–induced human coronary artery SMC migration and the mechanisms of this effect remain to be clarified.
The objectives of the present study were (1) to determine whether oxidized LDL stimulates migration of cultured SMCs derived from human coronary artery and, if so, (2) to examine the possible effect of human CNP-22 on oxidized LDL–induced migration and (3) to investigate the mechanism of this effect in these cells. In addition, we examined in these cells the effects of human ANP-(1-28) and human BNP-32, the major circulating forms of ANP and BNP in humans, on oxidized LDL–induced migration.
Materials and Methods
SMC basal medium (SmBM) and human coronary artery SMCs were purchased from Clonetics Corp. Trypsin and Versine were purchased from GIBCO Laboratories. Synthetic human ANP-(1-28), human BNP-32, and human CNP-22 were purchased from the Peptide Institute. 8-Bromo-cGMP, sodium nitroprusside, IBMX, human LDL, and BSA were purchased from Sigma Chemical Co. Flasks and multiwell plates were purchased from Becton Dickinson & Co.. The cGMP assay kit was purchased from Yamasa Shoyu Co, Ltd. Diff-Quick staining solution was purchased from Green-cross Corp.
Oxidation of LDL
Before oxidation, LDL was dialyzed against three changes of PBS to remove EDTA. Then LDL was oxidized at a concentration of 500 μg/mL by exposure to 10 μmol/L CuSO4 for 24 hours at room temperature, followed by dialysis at 4°C for 24 hours against three changes of PBS.20 21 The extent of lipid peroxidation was estimated as for thiobarbituric acid–reactive substances. Tetramethoxypropane was used as a standard, and results are expressed as nanomoles of malondialdehyde equivalents per 100 μg protein. The average degree of oxidation for native LDL and oxidized LDL were 0.3±0.1 and 4.1±0.4 nmol malondialdehyde equivalents per 100 μg protein, respectively.
Culture of SMCs
Human coronary artery SMCs were cultured in SmBM containing human epidermal growth factor (0.5 ng/mL), human fibroblast growth factor (2 ng/mL), insulin (5 μg/mL), 5% FBS, 50 μg/mL gentamicin sulfate, and 50 μg/mL amphotericin B. Cells were identified as SMCs according to their morphological and growth characteristics.22 Cultures were maintained at 37°C with atmospheric air and 5% CO2. Cells were subcultured after treatment with 0.25% trypsin and 0.02% EDTA. Subconfluent SMCs between the 4th and 8th passages were used for the experiments.
Migration of SMCs was assayed by a modification of the Boyden’s chamber method using microchemotaxis chambers (Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp), as previously reported.23
In this experiment, polycarbonate filters with pores of 12 μm in diameter were used. In all experiments, collagen-coated filters were used. Briefly, the membranes were treated with 0.5N acetic acid and then incubated for 48 to 72 hours at 25°C in a collagen solution (100 μg/mL type I collagen in 0.5N acetic acid).24 They were then air-dried. Cultured SMCs were trypsinized and suspended at a concentration of ≈5.0×105 cells/mL in SmBM supplemented with 0.4% BSA. The cell number was counted with an electronic cell counter (model ZB1, Coulter Electronics). A volume of 200 μL of SMC suspension was placed in the upper chamber, and 40 μL of medium/0.4% BSA containing oxidized LDL or native LDL was placed in the lower chamber. The chamber was incubated at 37°C under 5% CO2 in air for 3, 6, and 9 hours. After incubation, SMCs on the upper side of the filter were scraped off, and the filter was removed. The SMCs that had migrated to the lower side of the filter were fixed in ethanol, stained with Diff-Quick staining solution, and counted under a microscope (magnification ×400) for quantification of SMC migration. Migration activity is calculated as the mean number of migrated cells observed in 4HPF and is given as the mean value of four measurements. In experiments to determine the effect of native LDL or oxidized LDL on SMC migration, 20, 50, 100, and 200 μg/mL of these lipoproteins were added to the lower chamber.
Oxidized LDL–induced migration may be separated into chemotactic and chemokinetic components. The chemotactic component was determined by the addition of 200 μg/mL oxidized LDL to the lower chamber only, whereas the chemokinetic component was determined with 200 μg/mL oxidized LDL added to either the upper chamber only or to the upper and lower chamber.6
In experiments to determine the effects of human ANP-(1-28), human BNP-32, and human CNP-22 on oxidized LDL–induced SMC migration, various concentrations (10−9, 10−8, 10−7, and 10−6 mol/L) of these natriuretic peptides were added to the lower chamber in addition to 200 μg/mL oxidized LDL.
In separate experiments, to determine the effects of 8-bromo-cGMP or sodium nitroprusside on oxidized LDL–stimulated SMC migration, these agents were added to the lower chamber in addition to 200 μg/mL oxidized LDL.
SMC adhesion to the filter was assayed under conditions identical to the SMC migration assay by using microchemotaxis chamber and polycarbonate filters with pores of 12-μm diameter that had been precoated with type I collagen as previously described.24 Cultured SMCs were trypsinized and suspended at a concentration of ≈5.0×105 cells per milliliter in SmBM supplemented with 0.4% BSA. A 200-μL volume of SMC suspension was placed in the upper chamber, and 40 μL of medium/0.4% BSA containing oxidized LDL, ANP, BNP, CNP, or a combination of these factors was placed in the lower chamber. The chamber was incubated at 37°C under 5% CO2 in air for 6 hours. After incubation, the filter was removed and gently washed to remove nonattached cells. The adherent SMCs on both upper and lower sides of the filter were fixed in ethanol, stained with Diff-Quick staining solution, and counted under a microscope (×400) for quantification of SMC adhesion.
The cell monolayers were washed twice with PBS and then stimulated for 2, 5, 10, 30, 60, 180, and 360 minutes with different concentrations of human ANP-(1-28), human BNP-32, or human CNP-22 dissolved in SmBM that contained 5×10−4 mol/L IBMX. The reaction was stopped by rapid aspiration and the addition of 2 mL of ice-cold 65% ethanol, as previously described.25 After evaporation by a centrifugal evaporator, the dry residue was dissolved in an assay buffer. cGMP levels were determined by radioimmunoassay performed with the cGMP assay kit, as previously described.26
We examined whether oxidized LDL increases the production of NO in cultured human coronary artery SMCs and, if so, whether natriuretic peptides augment this production. Cultured SMCs were assayed for NO production by measuring the stable end product of NO, NO2, and NO3, as previously reported.27 Cultured SMCs were incubated for 6 hours in the absence or presence of 200 μg/mL oxidized LDL or 10−6 mol/L CNP-22. NO2/NO3 concentrations were expressed as nanomoles per 107 cells per 6 hours.
Calculations and Analysis
The statistical significance of differences in the results was evaluated using an unpaired ANOVA, and probability values were calculated by Scheffé’s method.28 All values were expressed as the mean±SD.
Effect of Oxidized LDL on SMC Migration
Fig 1A⇓ shows time-dependent effects of various concentrations (20, 50, 100, and 200 μg/mL) of oxidized LDL on human coronary artery SMC migration. Oxidized LDL stimulated cell migration in a concentration-dependent manner. SMC migration increased during the initial 6 hours of incubation, after which the rate of increase slightly declined. Therefore, subsequent studies on SMC migration were done with cells incubated for 6 hours. By contrast, native LDL was without significant activity even after 9 hours.
Fig 1B⇑ shows the checkerboard analysis of migration activity of oxidized LDL. The chemotactic component was determined by the addition of oxidized LDL to the lower chamber only, whereas the chemokinetic effect was determined with oxidized LDL added to either the upper chamber only or to both the upper and lower chamber. The presence of positive concentration gradients of the oxidized LDL across the filter rather than equal concentration indicates that the migration-stimulatory effect of this lipoprotein is chemotactic in nature for human coronary artery SMCs (Fig 1B⇑).
Effects of Natriuretic Peptides on Oxidized LDL– Induced SMC Migration and cGMP Level
Fig 2⇓ shows effects of various concentrations (10−9, 10−8, 10−7, and 10−6 mol/L) of human ANP-(1-28), human BNP-32, and human CNP-22 on SMC migration after stimulation with 200 μg/mL oxidized LDL. ANP-(1-28) and BNP-32 significantly inhibited oxidized LDL–induced migration at concentrations of 10−7 and 10−6 mol/L. CNP-22 significantly inhibited the oxidized LDL effect between 10−9 and 10−6 mol/L. The potency of CNP-22 was significantly greater than that of ANP-(1-28) or BNP-32 (P<.05).
Effects of natriuretic peptides on cellular cGMP level in cells treated with 200 μg/mL oxidized LDL are shown in Fig 3⇓. Cellular cGMP levels rapidly increased after treatment of the cells with natriuretic peptides, and the increased cGMP levels were sustained over 6 hours (Fig 3A⇓). The increase in cellular cGMP levels induced by natriuretic peptides for 30 minutes was concentration dependent (Fig 3B⇓). In parallel with the inhibition by ANP-(1-28), BNP-32, and CNP-22 on oxidized LDL–induced SMC migration, cellular cGMP increased after treatment with these natriuretic peptides (Figs 2⇑ and 3B⇓). Actually, CNP-22 was stronger than ANP-(1-28) or BNP-32 in inhibiting SMC migration and increasing cGMP levels.
The effects of ANP, BNP, and CNP on the adhesion of SMCs to the filter in the absence or presence of oxidized LDL are also examined. ANP, BNP, and CNP (10−6 mol/L) had no significant effect on cell adhesion, either in the absence or presence of 200 μg/mL oxidized LDL (data not shown).
Effects of 8-Bromo-cGMP and Sodium Nitroprusside on SMC Migration
To determine whether the inhibitory effects of natriuretic peptides on SMC migration after stimulation with oxidized LDL are causally linked to the increase in cellular cGMP, we examined the effect of a cGMP analogue, 8-bromo-cGMP, on 200 μg/mL oxidized LDL–stimulated SMC migration. 8-Bromo-cGMP inhibited oxidized LDL–stimulated migration in a concentration-dependent manner between 10−6 and 10−4 mol/L (Fig 4A⇓).
Approaching the issue from another point of view, the effect of an activator of the cytosolic guanylate cyclase, sodium nitroprusside, on 200 μg/mL oxidized LDL–induced migration was examined. Sodium nitroprusside inhibited oxidized LDL–induced migration in a concentration-dependent manner between 10−7 and 10−5 mol/L (Fig 4B⇑).
Effects of Natriuretic Peptides and 8-Bromo-cGMP on the Basal Migration Activity of Nonstimulated SMCs
Effects of natriuretic peptides and 8-bromo-cGMP on the basal migration activity of nonstimulated SMCs were examined. Neither natriuretic peptides (10−6 mol/L) nor 8-bromo-cGMP (10−4 mol/L) inhibited this basal activity (baseline, 5.4±3.2 cells/4HPF; ANP, 5.3±2.7 cells/4HPF; BNP, 5.7±3.1 cells/4HPF; CNP, 5.3±3.0 cells/4HPF; and 8-bromo-cGMP, 5.2±2.8 cells/4HPF).
Effects of Oxidized LDL and CNP on NO Production
Fig 5⇓ shows effects of oxidized LDL and CNP on NO production in cultured human coronary artery SMCs. Oxidized LDL (200 μg/mL) slightly but significantly increased NO production by 6 hours in these cells. However, CNP did not augment NO production by 6 hours either in the absence or presence of oxidized LDL.
In the present study, we showed that native LDL elicited no effects on the migration of human coronary artery SMCs, whereas oxidized LDL stimulated this migration. Furthermore, evaluation of oxidized LDL–induced migration showed that the migration-stimulatory effect of oxidized LDL was chemotactic in nature for cultured human coronary artery SMCs but was not chemokinetic. This finding seems to be compatible with the study by Autio et al,6 who reported that oxidized LDL stimulates the migration of bovine aortic SMCs. Recent evidence29 suggests that oxidized LDL is present in the intima. Therefore, these observations may raise the hypothesis that oxidation of LDL in the intima may render it chemotactic for medial SMCs, thus contributing to plaque formation and atherogenesis in the coronary artery, although we have no direct evidence in vivo at this time.
We were concerned that LDL oxidized by Cu2+ incubation may interact differently with the arterial wall than does LDL that has been oxidized in vivo.30 However, the Cu2+/oxidized LDL used in the present study shares physical and chemical characteristics with LDL oxidized in vivo, ie, an increased electrophoretic mobility on agarose gels, a high density, and a slightly greater size than native LDL.29 31 32 These observations suggest that Cu2+/oxidized LDL in the present study to a large extent mimics the biological effect of LDL oxidized in vivo. However, the present study cannot conclusively exclude the possibility that Cu2+/oxidized LDL may behave differently in vivo compared with LDL oxidized in vivo.
We have shown for the first time that human CNP-22 strongly inhibits oxidized LDL–induced migration of human coronary artery SMCs in a concentration-dependent manner. Actually, 200 μg/mL oxidized LDL–induced SMC migration was significantly inhibited by human CNP-22 at concentrations of 10−9 to 10−6 mol/L. Percent inhibition by 10−6 mol/L CNP-22 of SMC migration stimulated with 200 μg/mL oxidized LDL was ≈60%. Human ANP-(1-28) and human BNP-32 also inhibited oxidized LDL–induced SMC migration at concentrations of 10−7 and 10−6 mol/L, but these effects were weaker than that of human CNP-22. Although ANP-(1-28), BNP-32, and CNP-22 are the respective major circulating forms of ANP, BNP, and CNP in humans,12 14 15 17 the normal plasma concentrations (≈10−10 to 10−11 mol/L) are lower than those of synthetic natriuretic peptides that inhibited SMC migration in our in vitro study. However, plasma concentrations of ANP and BNP are found to be markedly high in patients with severe hypertension,13 15 congestive heart failure,13 and acute myocardial infarction.33 In addition, vascular SMCs are shown to express a large number of biologically active receptors of ANP and BNP.34 In addition, local levels of CNP in coronary vascular tissues may be much higher than plasma concentration (10−12 mol/L) of CNP,17 because it has recently been shown that a considerable amount of CNP is synthesized in and secreted from vascular endothelial cells.18 In addition, CNP is shown to be the specific ligand for a guanylate cyclase–linked receptor, termed the ANP-B receptor, which is separated for the receptor that binds ANP and BNP and is highly expressed in vascular SMCs.34 35 Taking the matter into account, our results suggest that CNP-22, by acting locally as a paracrine, inhibits the migration of human coronary artery SMCs after stimulation with oxidized LDL. It has been reported that CNP, as well as ANP and BNP, inhibits the proliferation of SMCs stimulated with FCS and PDGF.19 Consequently, the natriuretic peptide family, especially CNP, may antagonize the development of coronary atherosclerotic vascular lesions in hypercholesterolemic patients. However, this experiment was performed on cultured vascular SMCs. It has been demonstrated34 that in intact aortic media, mRNA of the ANP-A receptor and ANP-B receptor is detected and that the potency of cGMP production by ANP is at least two orders of magnitude stronger than that of CNP. By contrast, in cultured aortic SMCs, the ANP-B receptor and the clearance receptor are abundantly expressed, whereas the ANP-A receptor is minimally expressed.36 Therefore, any extrapolation from the present experiment on cultured SMCs to in vivo conditions should be carefully performed.
Recently, Sugiyama et al37 have demonstrated that oxidized LDL actually decreases spontaneous and transforming growth factor-β1–stimulated secretion of CNP from cultured vascular endothelial cells. This finding raises the hypothesis that in the in vivo condition, oxidized LDL in atherosclerotic arterial wall may suppress CNP synthesis and that this may weaken the net antiatherogenic action of CNP. However, our recent study38 using immunohistochemical staining has shown that CNP-positive endothelial cells, SMCs, and macrophages are present in early human coronary atherosclerotic lesions. Therefore, CNP, not only in vascular endothelial cells but also in SMCs and macrophages, may play a role as a local antiatherogenic factor through the inhibition of medial SMC migration into the intima in the initial phase of coronary atherosclerosis, although we have no direct evidence at this time.
In the present study, natriuretic peptides and 8-bromo-cGMP did not inhibit the basal migration activity of nonstimulated SMCs. In addition, natriuretic peptides did not suppress the cell adhesion either in the absence or presence of oxidized LDL. Furthermore, in a trypan blue exclusion test, dead cells stained with trypan blue were not found 6 hours after treatment with 10−6 mol/L natriuretic peptides. Therefore, it is likely that the observed migration-inhibitory effects of natriuretic peptides were not due to their cytotoxicity or their suppression on cell viability.
We have obtained some evidence for a causal link between cGMP production and inhibition of SMC migration after stimulation with oxidized LDL. First, human ANP-(1-28), human BNP-32, and human CNP-22 increased cGMP levels, and these effects paralleled the inhibition of migration. Second, a cGMP analogue, 8-bromo-cGMP, and an activator of the cytosolic guanylate cyclase, sodium nitroprusside, significantly inhibited oxidized LDL–stimulated migration. These results suggest that the natriuretic peptide family, especially CNP-22, inhibits oxidized LDL–stimulated migration, at least in part, through a cGMP-dependent process. Very recently, NO is shown to inhibit angiotensin II–induced migration of rat aortic SMCs in part via a cGMP-dependent mechanism.39 This finding may support our hypothesis. However, further studies are necessary to elucidate the involvement of cGMP and its related systems in the inhibition by natriuretic peptides of oxidized LDL–induced migration of coronary artery SMCs.
Finally, we examined whether oxidized LDL or CNP augments NO release in cultured coronary artery SMCs and whether the observed inhibition by CNP of SMC migration is mediated via released NO. Oxidized LDL slightly but significantly increased NO production in these cells. However, CNP did not augment NO production either in the absence or presence of oxidized LDL. Therefore, it seems to be unlikely, at least in our experimental conditions, that the inhibition of SMC migration and elevation of cGMP by natriuretic peptides are mediated via induction of NO.
Overall, the present work suggests that oxidized LDL stimulates human coronary artery SMCs and that the natriuretic peptide family, especially CNP, inhibits this stimulation. The increase in cellular cGMP levels is likely to be involved in this inhibition. Taken together with two findings that CNP potently suppresses SMC proliferation19 and that oxidized LDL is present in the intima,30 CNP in coronary vascular tissues may antagonize the development of coronary atherosclerosis as a paracrine in hypercholesterolemic patients. However, further in vivo studies will be necessary to elucidate the exact role of endogenous CNP on the initiation or development of hypercholesterolemia-induced coronary atherosclerosis.
Selected Abbreviations and Acronyms
|4HPF||=||four high-power fields|
|ANP||=||atrial natriuretic peptide|
|BNP||=||brain natriuretic peptide|
|CNP||=||C-type natriuretic peptide|
|PDGF||=||platelet-derived growth factor|
|SMC||=||smooth muscle cell|
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan (572-690-231-646) and a grant from Osaka Heart Club. The authors thank Atsumi Ohnishi and Yuka Inoshita (Division of Hypertension and Atherosclerosis, Osaka City University Medical School) and Hiroyuki Sano (Sumitomo Pharmaceuticals, Osaka) for their technical assistance and Dr Mayumi Furuya (Suntory Institute for Biomedical Research, Osaka) and Dr Naoki Tohdoh (Discovery Research Laboratories III, Sumitomo Pharmaceuticals Research Center, Osaka) for their helpful advice.
- Received February 14, 1997.
- Accepted June 6, 1997.
- © 1997 American Heart Association, Inc.
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