Adrenomedullin Is a Potent Inhibitor of Angiotensin II–Induced Migration of Human Coronary Artery Smooth Muscle Cells
Abstract The migration of coronary artery medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerotic lesions. In the current study, we examined the possible interaction of adrenomedullin, a novel vasorelaxant peptide, and angiotensin II (Ang II) on human coronary artery SMC migration using Boyden’s chamber method. Ang II stimulated SMC migration in a concentration-dependent manner between 10−6 and 10−8 mol/L. This stimulation was clearly blocked by the Ang II type 1 receptor antagonist losartan but not by the type 2 receptor antagonist PD 123319. The migration stimulatory effect of Ang II was chemotactic in nature for cultured human coronary artery SMCs but was not chemokinetic. Human adrenomedullin clearly inhibited Ang II–induced migration in a concentration-dependent manner. Human adrenomedullin stimulated cAMP formation in these cells. Inhibition by adrenomedullin of Ang II–induced SMC migration was paralleled by an increase in the cellular level of cAMP. 8-Bromo-cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the Ang II–induced SMC migration. These results suggest that Ang II stimulates SMC migration via type 1 receptors in human coronary artery and adrenomedullin inhibits Ang II–induced migration at least partly through a cAMP-dependent mechanism. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, this peptide may play a role as a local antimigration factor in certain pathological conditions.
The migration of coronary artery medial SMCs into the intima and the proliferation of migrated cells are proposed to be key processes of intimal thickening in coronary atherosclerotic lesions.1 2 Previous reports suggest the importance of the RAS in SMC proliferation and migration after balloon injury.3 4 5 6 7 Actually, both angiotensin-converting enzyme inhibitors and AT1 receptor antagonists reduce intimal lesion formation after balloon injury.3 4 Furthermore, Dubey and coworkers8 have recently demonstrated with Boyden’s chamber method that Ang II, the major effector peptide of the RAS, stimulates migration of cultured rat aortic SMCs.
Adrenomedullin, a potent vasorelaxant peptide, has recently been isolated from the acid extract of human pheochromocytoma.9 10 This peptide, consisting of 52 amino acids, has one intracellular disulfide bond and shows homology with calcitonin gene–related peptide.9 10 This peptide stimulates cAMP formation in cultured rat vascular SMCs via specific receptors.11 12 13 Recently, we showed that rat adrenomedullin potently inhibits fetal calf serum–induced or platelet-derived growth factor–induced migration in cultured rat aortic SMCs.14 Subsequently, we showed that adrenomedullin modestly but significantly suppresses fetal calf serum–induced proliferation in cultured rat aortic SMCs.15 However, the role of adrenomedullin in the regulation of human coronary artery SMC migration and the interaction of this peptide and the RAS remain to be fully clarified.
Accordingly, the objectives of the current study were to determine whether Ang II stimulates migration of cultured SMCs derived from human coronary artery and, if so, to examine the possible effect of human adrenomedullin on Ang II–induced migration in these cells. In addition, we examined the mechanism of the interaction of adrenomedullin and Ang II on human coronary artery SMC migration.
Smooth Muscle Cell Basal Medium (SmBM) and human coronary artery SMCs were purchased from Clonetics Corp. Fetal calf serum, trypsin, and Versine were purchased from GIBCO Laboratories. Synthetic human adrenomedullin-(1-52) was purchased from Peptide Institute. 8-Bromo-cAMP, 3-isobutyl-1-methylxanthine (IBMX), Ang II, and bovine serum albumin were purchased from Sigma Chemical Co. Flasks and multiwell plates were purchased from Becton Dickinson & Co. The cAMP assay kit was purchased from Yamasa Shoyu Co, Ltd. Diff-Quick staining solution was purchased from Green-cross Corp. Forskolin was a gift from Nihon Kayaku Co, Ltd. The selective AT1 receptor antagonist losartan was donated by DuPont Merck Pharmaceutical Co. The AT2 receptor antagonist PD 123319 was donated by Parke Davis.
Culture of Human Coronary Artery 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% fetal bovine serum, 50 μg/mL gentamicin sulfate, and 50 μg/mL amphotericin-B. Cells were identified as SMCs according to their morphological and growth characteristics.16 17 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 passages 4 and 8 were used for the experiments.
We assayed SMC migration with a modification of Boyden’s chamber method using microchemotaxis chambers (Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp), as previously reported.14 In this experiment, polycarbonate filters with 12-μm-diameter pores were used. Cultured SMCs were trypsinized and suspended at a concentration of approximately 5.0×105 cells/mL in SmBM supplemented with 0.5% fetal calf serum. Cell number was counted with an electronic cell counter (model ZB1, Coulter Electronics). A 200-μL volume of SMC suspension was placed in the upper chamber, and 40 μL of medium and 0.4% bovine serum albumin containing 10−9, 10−8, 10−7, and 10−6 mol/L Ang II 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 (×400 magnification) for quantification of SMC migration. Migration activity was calculated as the mean number of migrated cells observed in four high-power fields and given as the mean value of four measurements.
Ang II–induced migration may be separated into chemotactic and chemokinetic components. The chemotactic component was determined by the addition of 10−7 mol/L Ang II to the lower chamber only, and the chemokinetic component was determined with 10−7 mol/L Ang II added to either the upper chamber only or to both the upper and lower chambers.18
To determine the effects of Ang II receptor antagonists on SMC migration, 10−7 mol/L Ang II with or without 10−6 and 10−7 mol/L of the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD 123319 was added to the lower chamber. To determine the effects of human adrenomedullin on Ang II–induced SMC migration, various concentrations (10−9, 10−8, 10−7, and 10−6 mol/L) of adrenomedullin were added to the lower chamber in addition to 10−7 or 10−8 mol/L Ang II. In separate experiments, to determine the effects of 8-bromo-cAMP or forskolin on Ang II–stimulated SMC migration, these agents were added to the lower chamber in addition to 10−7 mol/L Ang II.
After preincubation, cell monolayers were washed twice with phosphate-buffered saline and stimulated for 30 minutes with different concentrations of human adrenomedullin dissolved in SmBM that contained 5×10−4 mol/L IBMX. The reaction was stopped by rapid aspiration and addition of 2 mL ice-cold 65% ethanol, as previously described.19 After evaporation by a centrifugal evaporator, the dry residue was dissolved in an assay buffer. cAMP levels were determined by radioimmunoassay with a cAMP assay kit, as previously described.13
Calculations and Analysis
The statistical significance of differences was evaluated with an unpaired ANOVA, and probability values were calculated by Scheffé’s method.20 All values are expressed as mean±SD.
Effect of Ang II on SMC Migration
Fig 1⇓ shows the time-dependent effects of various concentrations (10−9, 10−8, 10−7, and 10−6 mol/L) of Ang II on human coronary artery SMC migration. Ang II stimulated cell migration in a concentration-dependent manner between 10−6 and 10−8 mol/L. SMC migration increased during the initial 6 hours of incubation, after which the rate of increase declined slightly. Therefore, subsequent experiments on SMC migration were done with cells incubated for 6 hours.
Ang II–induced migration can be separated into two components: chemotaxis and chemokinesis. The chemotactic component was determined by the addition of Ang II to the lower chamber only, and the chemokinetic effect was measured with Ang II added to either the upper chamber only or to both the upper and lower chambers. Evaluation of Ang II–induced SMC migration showed that the migration-stimulating effect of Ang II was chemotactic in nature for cultured human coronary artery SMCs (Fig 2⇓).
Effects of Ang II Receptor Antagonist on SMC Migration
Effects of losartan and PD 123319 on Ang II–stimulated SMC migration are shown in Fig 3⇓. The migration-stimulating effect of Ang II was clearly blocked by the AT1 receptor antagonist losartan, but it was not blocked by the AT2 receptor antagonist PD 123319. This suggests that the AT1 receptor is coupled to Ang II–induced migration in cultured human coronary artery SMCs.
Effect of Adrenomedullin on Ang II–Induced SMC Migration
Fig 4A⇓ shows effects of various concentrations (10−9, 10−8, 10−7, and 10−6 mol/L) of human adrenomedullin on SMC migration induced by 10−7 or 10−8 mol/L Ang II. Human adrenomedullin significantly inhibited Ang II–induced migration in a concentration-dependent manner between 10−8 and 10−6 mol/L.
Effects of adrenomedullin on cellular cAMP level in cells treated with 10−7 or 10−8 mol/L Ang II are shown in Fig 4B⇑. In parallel with the inhibition by adrenomedullin of Ang II–induced SMC migration, cellular cAMP increased after treatment with adrenomedullin (Fig 4A⇑ and 4B⇑).
Effects of 8-Bromo-cAMP and Forskolin on Ang II–Induced SMC Migration
To elucidate whether the inhibitory effect of adrenomedullin on Ang II–induced SMC migration is causally linked to the increase in cellular cAMP, we examined the effect of 8-bromo-cAMP, a cAMP analogue, on Ang II–induced SMC migration. Inhibition of Ang II–induced SMC migration by adrenomedullin could be reproduced by this analogue at concentrations of 10−5 and 10−4 mol/L (Fig 5A⇓). Furthermore, we examined the effect of forskolin, an activator of adenylate cyclase, on Ang II– induced SMC migration. The addition of forskolin also reduced Ang II–induced SMC migration at concentrations of 10−6 and 10−5 mol/L (Fig 5B⇓).
In the present study, we showed that Ang II stimulated migration of cultured human coronary artery SMCs in a concentration-dependent manner. Furthermore, we showed that Ang II–induced migration was markedly reduced by the selective AT1 receptor antagonist losartan. In contrast, this stimulation by Ang II was not affected by the selective AT2 receptor antagonist PD 123319. These results indicate that Ang II stimulates migration of human coronary artery SMCs via AT1 receptors.
Evaluation of Ang II–induced migration showed that the migration-stimulatory effect of Ang II was chemotactic rather than chemokinetic in nature for cultured human coronary artery SMCs. This finding seems to be compatible with the report by Dubey and coworkers8 that Ang II stimulates the migration of rat aortic SMCs.
In the balloon injury model, Ang II has been found to contribute importantly to intimal lesion formation.3 4 Interestingly, in the rat intimal lesion, development can be reduced by inhibition of SMC migration alone, in the absence of an effect on SMC proliferation.21 22 Actually, angiotensin-converting enzyme inhibitors have been shown to reduce intimal lesion size by inhibiting SMC migration without affecting SMC proliferation in the balloon injury model.4 Although we have no direct evidence, these observations and our data raise the possibility that Ang II–induced SMC migration contributes to the development of restenotic lesions in humans after angioplasty.
Second, we showed for the first time that human adrenomedullin strongly inhibits Ang II–induced migration of human coronary artery SMCs in a concentration-dependent manner. In fact, SMC migration induced by 10−7 and 10−8 mol/L Ang II was significantly inhibited by human adrenomedullin-(1-52) at concentrations of 10−8 to 10−6 mol/L. Human adrenomedullin-(1-52) at 10−6 mol/L inhibited migration induced by 10−7 and 10−8 mol/L Ang II by approximately 65% and 55%, respectively. Although human adrenomedullin-(1-52) is a major circulating form in humans,23 24 25 the normal plasma concentrations (approximately 10−10 to 10−11 mol/L) are much lower than concentrations of synthetic adrenomedullin, which can inhibit SMC migration.23 24 25 However, plasma adrenomedullin concentrations are found to be high in hypertensive individuals with organ damage24 25 and in individuals with severe congestive heart failure.26 Furthermore, local levels of adrenomedullin in coronary vascular tissues may be much higher than plasma concentrations of adrenomedullin, because it has recently been shown that a considerable amount of adrenomedullin is synthesized in and secreted from vascular endothelial cells.27 With this matter taken into account, our results suggest that adrenomedullin, by acting locally as a paracrine factor, inhibits the migration of human coronary artery SMCs when the RAS is activated in coronary vascular tissues. Previously, we showed that adrenomedullin had a modest antiproliferative effect on rat aortic SMCs.15 Consequently, human adrenomedullin may antagonize the development of intimal thickening induced by Ang II in the coronary artery in certain pathological conditions, for example, after angioplasty. However, the present experiment was done on cultured SMCs; therefore, extrapolation to in vivo conditions should be made with caution.
We have obtained some evidence for a causal link between cAMP production and inhibition of Ang II–induced SMC migration. First, human adrenomedullin increased cAMP levels, and this effect paralleled the inhibition of migration. Second, a cAMP analogue, 8-bromo-cAMP, and an activator of adenylate cyclase, forskolin, significantly inhibited Ang II–induced migration. These results suggest that human adrenomedullin inhibits Ang II–induced migration of human coronary artery SMCs at least partly through a cAMP-dependent mechanism. However, further studies are necessary to elucidate the involvement of cAMP and its related systems in the inhibition by human adrenomedullin of Ang II–induced migration of human coronary artery SMCs.
Overall, the present work suggests that Ang II stimulates human coronary artery SMC migration via AT1 receptors and human adrenomedullin potently inhibits this stimulation. Taken together with the findings that adrenomedullin is synthesized in and secreted from vascular endothelial cells,27 possibly in coronary vascular tissues, this peptide may antagonize the development of intimal lesions in certain pathological conditions. However, more studies will be necessary to elucidate the exact role of endogenous adrenomedullin on intimal thickening during the process of RAS-related coronary atherosclerosis.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1, AT2||=||angiotensin II type 1, type 2 (receptor)|
|SMC||=||smooth muscle cells|
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan (572-690-231-646). The authors thank Atsumi Ohnishi and Yuka Inoshita (Division of Hypertension and Atherosclerosis, The First Department of Internal Medicine, Osaka City University Medical School) for their technical assistance.
- Received October 4, 1996.
- Revision received October 21, 1996.
- Accepted November 18, 1996.
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