Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration

Atherosclerosis 178 (2005) 271–277

Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration Gabriele Di Luozzo, Sanjeev Pradhan, Ajay K. Dhadwal, Alan Chen, Hirokazu Ueno, Bauer E. Sumpio∗ Section of Vascular Surgery, Department of Surgery, Yale University School of Medicine and Veterans’ Administration, 333 Cedar Street FMB-137, New Haven, CT 06520, USA Received 25 May 2004; received in revised form 27 August 2004; accepted 10 September 2004 Available online 11 November 2004

Abstract Cigarette smoke, specifically the nicotine contained within, has been shown to cause ultrastructural changes in vascular endothelium resulting in the development of atherosclerosis. Our study examines the effects of nicotine on vascular smooth muscle cell (VSMC) migration and attempts to eludicidate the cellular mechanisms governing those effects. Bovine aortic VSMC were cultured in 10% fetal bovine serum (FBS) growth media and exposed to 10−8 nicotine for varying periods of time. Boyden chamber chemotaxis assays and a scrape injury model using confluent cells were used to assess cell motility. Activation of the mitogen-activated protein kinases (MAPK), p38 and p44/42, was assessed using Western blotting methods. Nicotine, itself, did not cause significant VSMC migration. However, augmented migration was seen in nicotine-treated VSMCs (16.6 ± 3fold) and media (17.0 ± 4-fold) with 10% FBS as chemoattractant. Inhibitors of p38 and p44/42 diminished this migration by 48.5 ± 6% and 29.4 ± 2%, respectively. Immunoblotting verified p38 and p44/42 activation with nicotine and inhibition with inhibitors of p38 and p44/42. Nicotine-treated endothelial cell (EC) conditioned media (CM) was shown to increase migration 20.3 ± l.l-fold. This chemotactic effect was diminished both with heat treatment and serial dilution. In conclusion, nicotine enhances the chemotactiveness of VSMC. This migration is mediated via the MAPKs p38 and p44/42. Nicotine causes EC production of a chemoattractant molecule that enhances VSMC migration. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: MAP kinase; Migration; Nicotine; Atherosclerosis; Atherogenesis

1. Introduction Smoking is a preventable risk factor for the development of cardiovascular disease, chronic obstructive pulmonary disease and neoplasm formation [1]. Smoking is responsible for an estimated one in six deaths in the United States [2]. Epidemiological studies have shown a clear causal association between tobacco consumption and atherosclerotic vascular disease [3] and cessation of smoking results in significant declines in mortality due to cardiovascular disease [1]. Un-

∗

Corresponding author. Tel.: +1 203 785 2561; fax: +1 203 785 7609. E-mail address: [email protected] (B.E. Sumpio).

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.09.017

fortunately, the molecular mechanisms underlying smokinginduced atherosclerosis are not well defined. Cigarette smoke is a complex mixture of gases and solid particles. [1,4] Both the gaseous and particulate phases have been implicated in perturbing the normal vascular biology, specifically, by causing injury to the endothelium [5–8]. Ultrastructural changes ranging from endothelial cell (EC) vacuolation and subendothelial edema to desquamation have been documented following nicotine administration. However, there is a relative paucity of information on the effects of nicotine or other cigarette smoke constituents on vascular smooth muscle cells (VSMC). VSMC migration and proliferation leading to increased vessel wall thickness are central to the pathogenesis of atherosclerosis [9]. Endothelial injury,

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or dysfunction, creates an environment that exposes VSMCs to mitogens and chemoattractants, which appear to facilitate the growth of the atheromatous plaque [10,11]. Cellular migration is a complex process initiated by external stimuli that activates intracellular signaling pathways resulting in cell movement. The proteins p42/44 (also known as ERK 1/2) and p38 are two members of the mitogen-activated protein kinase (MAPK) family of signal transducers and have been reported to play pivotal roles in cytoskeletal reorganization, cellular growth, and migration in response to growth and inflammatory factors such as TNF-␣ and various other cytokines [12–14]. Overexpression of p38-␣ MAPK dominant negative or use of the specific p38 inhibitor SB203580 inhibited VSMC migration in response to a variety of stimuli [15]. Thrombospondin-1 (TSP-1), an extracellular matrix protein, has been shown to induce VSMC chemotaxis mediated by ERK1/2 [16]. The purpose of this study was to investigate the effects of nicotine on VSMC migration. We sought to elucidate and differentiate the role of nicotine as both a chemoattractant for VSMC as well as a modulator of the migration of VSMC. We postulated that nicotine exposure stimulates cultured VSMC to migrate by activating p38 and ERK 1/2. In addition, since nicotine exposure affects the vascular endothelium, we investigated the effect of nicotine on EC production of substances that might influence VSMC migration.

2. Methods 2.1. Cell culture Cells were attained from the thoracic aorta of slaughtered calves. EC were obtained by the cell scrape method [17] and VSMC by the explant method [18]. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Chemical Company, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT) and 1% antibiotic–antimycotic solution (Gibco Laboratories, Grand Island, NY) comprised of 10,000 ␮g/ml penicillin G sodium, 10,000 ␮g/ml streptomycin sulfate, and 25 ␮g/ml amphotericin B in an incubator containing a humidified 5% CO2 /95% O2 at 37 ◦ C. Cells between passages 3 and 8 were used in all the experiments. 2.2. Chemotaxis assay Details of the assays using Boyden chambers have been previously reported [16]. Prior to experimentation, VSMCs were made quiescent by the use of serum-free medium (SFM) containing selenium, insulin and transferring [19] for 48 h. The control group remained in SFM and the experimental group was exposed to 10−8 M nicotine, which was added to SFM, for 48 h. Cells were trypsinized, suspended at a concentration of 1.0 × 106 cells/ml in SFM and 50 ␮l of either nicotine untreated or nicotine-treated VSMC suspension

(5.0 × 105 cells) was placed on filter of the upper Boyden chamber. Twenty-eight microliters each of SFM, 10% FBS, 10−8 M nicotine, or endothelial cell conditioned media (EC CM) were used as chemoattractants and placed in the bottom Boyden chamber. After 37 ◦ C for 4 h, migrated VSMC on the underside of the filter were counted in five random fields per well using 400× magnification. To assess the role of p38 MAPK and ERK 1/2 MAPK in cell migration, a subset of each group of suspended cells were treated with either the p38-specific inhibitor SB203580 (10 ␮M) or the MAPK kinase (MEKl)-specific inhibitor PD98059 (10 nM) for 30 min at 37 ◦ C. To study the release of chemoattractants by EC, EC were preconditioned for 48 h with either SFM or SFM treated with 10−8 M nicotine and the conditioned media (CM) collected. In order to characterize the EC CM, in some experiments, the CM was heated to 95 ◦ C for 10 min. In other experiments, the CM was diluted 1:1 and 1:2 with SFM. VSMC chemotaxis was determined utilizing the modified Boyden chamber described above. In both series of experiments, the nicotinetreated media was placed in the lower chamber wells to serve as a chemoattractant. 2.3. Injury model assay VSMC grown to confluency in 6-well culture dishes were preconditioned with SFM, 10−8 M nicotine-containing SFM, or 10% FBS for 48 h. A cell scraper was used to carefully remove half of the cells from the bottom of the well guided by a marker line. Media was changed to SFM except for one of the groups treated with nicotine; this media was changed to 10% FBS. A subset of cells was treated with 20 ␮g/ml mitomycin C to suppress SMC proliferation without affecting cell migration [20]. Pilot experiments had confirmed lack of SMC proliferation for up to 7 days (data not shown). Every 48 h the media was changed for all groups. Distance of migration was assessed on day 7 by digital photos taken of the migrating front. Microsoft Photo EditorTM software measured the distance of the migrating front from the scrape line. Data were normalized to the non-nicotine-treated group and presented as fold-change ± standard error of the mean (S.E.M.). 2.4. Western blot analysis VSMC were lysed with buffer containing 25 mmol/1 HEPES pH 7.4, 500 mmol/1 sodium chloride, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 5 mmol/1 EDTA, 50 mmol/1 sodium fluoride, 1 mmol/1 phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 mmol/1 sodium orthovanadate. Protein amounts were quantified using the modified Bradford method [21]. Samples containing equal amounts of protein were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. To detect the active (phosphorylated) form of each MAPK, phospho-specific p38 MAPK and ERK 1/2 MAPK antibodies purchased from Cell Signaling (Beverly, MA), were used ac-

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cording to company protocols. The ECL technique was used to visualize the proteins. The membranes were then stripped and probed with total p38 and ERK 1/2 primary antibodies (Cell Signaling) to verify equal protein loading. Band densities on autoradiograms were measured with a densitometer (Image Quant, Molecular Dynamics, Sunnyvale, CA) and normalized to total protein loaded. Data are presented as fold-change ± S.E.M.

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Migration studies utilizing the scrape injury model (Fig. 2A and B) demonstrate a 9.7 ± 2.0-fold (p < 0.001) and 6 ± 1.0-fold (p < 0.05) increase in VSMC migration in the group of cells exposed to 10% FBS and nicotine, respectively, when compared to the SFM group. In the group treated with nicotine added to 10% FBS there was a 17.0 ± 3.5-fold increase in VSMC migration versus control (p < 0.001). Thus, the addition of nicotine to 10% FBS resulted in an 8-fold increase in migration compared to 10% FBS alone.

2.5. Statistical analysis 3.2. Involvement of p38 and ERK 1/2 in VSMC migration The cell counts are represented as the mean ± S.E.M. The data was normalized to the cell counts for the untreated VSMC group (VSMC/SFM). All experiments were performed in at least triplicate. Statistical significance was determined using one-way ANOVA and post hoc test. Statistical significance was accepted at a p < 0.05.

3. Results 3.1. Effect of nicotine on VSMC migration Fig. 1 shows that there was an 11.9 ± 3-fold increase in VSMC migration with 10% FBS as chemoattractant compared to SFM (p < 0.05). Nicotine alone was not a chemoattractant and did not significantly alter VSMC migration. However, following 48 h of nicotine preconditioning, VSMC migration was significantly augmented. The migration of the preconditioned smooth muscle cells (PSMCs) towards SFM or 10% FBS was enhanced by 2.1 ± 0.8 and 16.6 ± 3.0-fold in comparison to the control group, respectively (p < 0.05). Moreover, preconditioning SMCs resulted in a 30% greater migration over unconditioned cells when 10% FBS was used as the chemoattractant (p < 0.05).

Fig. 1. SMC chemotaxis after exposure to nicotine. The y-axis represents fold increase in SMC migration compared to basal conditions (SFM, 1st column). The x-axis represents the experimental conditions on the top and bottom of the Boyden chamber (SFM: serum free media, NIC: nicotine). There was no significant change in SMC migration with nicotine (2nd column) as a chemoattractant. With 10% FBS as a chemoattractant, (4th column) SMC migration increased by 11.9 ± 3-fold (p < 0.05). When SMCs were preconditioned with nicotine (PSMC) there was a 2.1 ± 0.8 and 16.6 ± 3-fold increase in migration with SFM (3rd column) and 10% FBS (last column) used as a chemoattractants, respectively (p < 0.05).

We assessed the role of the MAPK family in VSMC migration by using selective inhibitors of p38 and MEK1 (Fig. 3A and B). The p38 inhibitor, SB203580 (SB), decreased SMC migration towards SFM and 10% FBS by 39 ± 1.3% and 38 ± 3% (p < 0.05), respectively. PSMC migration toward SFM and 10% FBS was decreased by 81.1 ± 3% (p < 0.001) and 62 ± 6% (p < 0.05), respectively. Furthermore, VSMC migration was inhibited by PD98059, a MEK1/ERK pathway inhibitor, in the SFM and 10% FBS groups by 37.4 ± 4% and 38 ± 3% (p < 0.05), respectively. Lastly, reductions of 74 ± 3% (p < 0.001) and 29.4 ± 2% (p < 0.05) were observed in PSMC migration when PD98059 was added to the media and SFM and 10% FBS were used as chemoattractants, respectively. Furthermore, p38 and ERK 1/2 activation following nicotine preconditioning was confirmed using Western blotting (Fig. 4). PD98059 significantly inhibited phosphorylation of ERK 1/2. 3.3. Effect of EC conditioned media on VSMC migration Forty-eight hour EC CM in the presence or absence of nicotine was used as the chemoattractant for the VSMC (Fig. 5). The positive control group (10% FBS as chemoattractant) demonstrated an 18.8 ± 1.8-fold increase in migration compared to the negative control (SFM as chemoattractant) (p < 0.001). As previously observed above, nicotine alone was a poor VSMC chemoattractant. EC CM used as a chemoattractant stimulated VSMC migration by 10.5 ± 1.2fold (p < 0.001). However, there was an even greater increase in migration observed (20.3 ± 1.1-fold) when nicotinetreated EC CM was the chemoattractant (p < 0.001). To characterize the chemoattractive activity of the conditioned media, the EC CM was diluted or heated and VSMC migration was assessed. Fig. 6A shows that there was a 14.5 ± 2.5% and 29 ± 1.3% (p < 0.05) decrease in migration when the untreated EC CM was diluted 2:1 and 1:1 compared to control, respectively. Dilution of the nicotine-treated EC CM caused an even greater reduction in SMC migration (24.1 ± 1.4% (p = NS) and 66 ± 3.3% (p < 0.001)). Fig. 6B demonstrates that exposing the EC CM to heat significantly abolished the activity of the chemoattractants in the EC CM. There was a 90 ± 5% and 92 ± 15% reduction in SMC migration in the untreated EC CM and nicotine-treated EC CM groups (p < 0.001), respectively.

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Fig. 2. SMC migration following scrape injury. (A) SMCs were grown to confluence and then treated with 20 ␮g/ul mitomycin C. The cells were scraped over an index mark and the amount of distance migrated was measured at 7 days. The SMCs were treated with SFM (panel A, ×40), 10% FBS (panel B, ×40), nicotine in SFM (panel C, ×40) and nicotine in 10% FBS (panel D, ×10). A lower magnification was used in photograph D to show the increased distance of migration from the scrape line and (B) The y-axis represents fold increase in SMC migration. The x-axis represents the various media the cells were maintained in. A 9.7 ± 2-fold increase in migration was observed in SMCs maintained in 10% FBS (2nd column) vs. control (1st column) (p < 0.001). SMCs treated with nicotine in serum free media had a 6 ± 1-fold increase in migration (p < 0.05). Nicotine added to 10% FBS resulted in a 17 ± 3.5-fold increase in migration (p < 0.001).

4. Discussion In the present study, we demonstrate that nicotine is not a VSMC chemoattractant. However, exposure of VSMC to 10−8 M nicotine not only enhanced cell migration but also increased VSMC chemotaxis towards FBS. The range of plasma concentrations of nicotine in smokers is between 10−5 and 10−8 M [1]. It is interesting to note that Carty et al. reported that this concentration of nicotine stimulated SMC DNA synthesis and proliferation [22]. This effect was more prominent when the CM was supplemented with serum. Taken together, our findings, and that of others, support the hypothesis that there may be a common intracellular signaling pathway for the two stimuli, which may be additive on the cell response. Our immunoblot results demonstrate that 10% FBS and nicotine, both independently, activated ERK 1/2

and p38, which have been implicated in cell motility. In addition, the cell injury model experiments clearly demonstrate that both 10% FBS and nicotine have a significant impact on the rate of VSMC migration; when 10% FBS and nicotine are combined, they have an additive effect. Furthermore, our preliminary experiments also demonstrated that the effect of nicotine was dose-dependent (data not shown). We do not have data on the length of preconditioning time with nicotine that is needed to affect VSMC migration. Previous reports indicate that nicotine can stimulate basic fibroblast growth factor and matrix metalloproteinase (MMP) production, important factors that enhance cellular motility. [23] However, the increase in VSMC MMP mRNA expression did not occur until after 12 h of incubation with nicotine. [23] This data may be consistent with the results of our pilot experiments that VSMC exposure to nicotine should be

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Fig. 3. (A) Effect of the selective p38 inhibitor, SB203580 (SB)on SMC chemotaxis. The y-axis represents fold increase in SMC migration compared to basal conditions (SFM, 1st column). The x-axis represents the experimental conditions on the top and bottom of the Boyden chamber. SB203580 decreased SMC migration towards SFM (3rd column) and 10% FBS (7th column) by 39.3 ± 1.3% and 38 ± 3% compared to the non-treated SMC controls (1st and 6th columns), respectively (p < 0.05). Migration of SMCs preconditioned with nicotine was decreased by 81.1 ± 3% (p < 0.05) (5th column) and 62 ± 6% (last column) (p < 0.05) by SB203580 compared to their respective controls (4th and 8th columns) and (B) effect of the selective ERK 1/2 inhibitor, PD98059 (PD) on SMC migration. The y-axis represents fold increase in SMC migration compared to SFM (1st column). The x-axis represents the experimental conditions on the top and bottom of the Boyden chamber. PD98059 decreased SMC migration towards SFM (3rd column) and 10% FBS (7th column) by 37.4 ± 4% and 38 ± 3% compared to the non-treated SMC controls (1st and 6th columns), respectively (p < 0.05). Migration of SMCs preconditioned (PSMC) with nicotine was decreased by 74 ± 3% (5th column) (p < 0.001) and 29.4 ± 2% (last column) (p < 0.05) by PD98059 compared to their respective controls (4th and 8th columns).

Fig. 4. Representative blots showing p38 and ERK 1/2 activation in SMCs stimulated with nicotine. The top panels demonstrate activation of each MAPK. SMC maintained in SFM was used as the negative control (lane 1) and 10% FBS stimulation was used as the positive control (lane 2). The third lane demonstrates nicotine activation of the respective MAPKs with inhibition of this activation in SMCs pre-treated with PD (lane 4). The bottom panels demonstrate loading control. These results were confirmed in three separate experiments.

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Fig. 5. Effect of EC conditioned media on SMC migration. The y-axis represents the number of migrated SMCs. The x-axis represents the chemoattractant used in the bottom of the Boyden chamber. The second column demonstrates an 18.8 ± 1.8-fold increase in SMC migration when 10% FBS is used as the chemoattractant (p < 0.001). The third column confirms that nicotine alone is a poor VSMC chemoattractant. When EC CM was used a chemoattractant (4th column), there was a 10.5 ± 1.2-fold increase in migration (p < 0.001). The use of nicotine-treated EC CM as a chemoattractant (last column) resulted in a 20.3 ± 1.1-fold increase in migration (p < 0.001).

at least 12 h to produce a significant effect (data not shown). This suggests that de novo protein synthesis may be involved and explains why unstimulated SMC do not migrate towards the nicotine chamber during the 4 h migration experiments in the Boyden chamber assays (Fig. 1). The MAPK signaling pathway appears to be involved in cellular movement in response to nicotine. Nicotine

stimulation of SMCs may have both transcriptional and non-transcriptional independent processes. Non-genomic dependent pathways that regulate cell motility have been demonstrated [12]. Our chemotaxis studies and protein phosphorylation immunoblots confirm that nicotine activates SMC p38 and ERK 1/2 pathways. The specific MAPK inhibitors, SB203580 and PD98059, used in this study reversed the effects of nicotine on SMC migration and the activity of p38 and MEK1, respectively. Despite the prominent effect of these inhibitors, VSMC migration did not return to baseline even when used in combination (data not shown). This may be explained by the fact that nicotine and 10% FBS may stimulate other intracellular signaling pathways or de novo synthesis of growth factors. For instance, it is reported that nicotine stimulates the production of endothelins, which, in turn, enhance both cellular proliferation and migration. [24,25] Further studies to delineate this and other potential pathways remain to be investigated. The interaction between EC and SMC is important in maintaining the normal architectural and mechanical properties of the arterial wall. Our study focused on the effects of nicotine on EC and any subsequent effects this process might have on VSMC migration. We demonstrated that EC CM served as a strong chemoattractant for VSMC and that stimulation of EC with nicotine further enhanced chemoattraction. This suggests that EC stimulated by nicotine are capable of producing a transferable chemoattractant. In support of

Fig. 6. (A) Effect of dilution on EC conditioned media as chemoattractants for SMCs. The y-axis represents the number of migrated SMCs. The x-axis represents the chemoattractant used in the bottom of the Boyden chamber. There was a 14.5 ± 2.5% (p = NS) and 29 ± 1.3% (p < 0.05) reduction in SMC migration observed when the nicotine-treated (4th column) and untreated (3rd column) EC CM was diluted 2:1. A 1:1 dilution of the nicotine-treated (last column) and untreated (5th column) EC CM reduced their stimulatory effect on migration by 24.1 ± 4% (p = NS) and 66 ± 3.3% (p < 0.001), respectively and (B) effect of adding heat to the EC conditioned media on SMC migration. The y-axis represents the number of migrated SMCs. The x-axis represents the chemoattractant used in the bottom of the Boyden chamber. There was a 90 ± 5% and 92 ± 15% reduction in SMC migration in the heat-treated groups, respectively (p < 0.001).

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this hypothesis, it has been previously reported that EC can stimulate VSMC proliferation via endothelin-1 in a bilayer co-culture system. [25] This finding has been confirmed by other groups that have characterized endothelin-1 on human atherosclerotic arteries [26] and its ability to sustain chemotactic properties. [27] However, there are other potential chemoattractants such as vascular endothelial growth factor, platelet derived growth factor, and transforming growth factor-␤. [10,28] The EC-derived chemotactic agent is present at low tonic levels in the un-stimulated state, which may be a response in part to the EC in vitro environment. Nicotine-exposed EC CM resulted in a greater stimulation of the SMC migration. This effect was more potent than 10% FBS as a chemoattractant. Whether the stimulatory effects of 10% FBS and EC CM are caused by a different chemotactic agent or through a different mechanism of action remains unknown. However, our studies indicate that the chemotactic agent(s) is likely a protein or a substance associated with a protein moiety because of our ability to abrogate the response by heat or dilution. In addition, we can infer that the molecule is water-soluble and smaller than 8 ␮m based on its ability to pass through the Boyden chamber filter. In summary, we show that nicotine, a constituent of cigarette smoke, is a potent stimulus for the migration of VSMC. Nicotine itself is not a chemoattractant for VSMC, but may “activate” intracellular pathways to make VSMC more amenable to migration. The studies indicate that the MAPK pathway, through p38 and ERK 1/2, may play a critical role in this phenomenon. In addition, ECs exposed to nicotine may augment SMC migration through the release or synthesis of chemoattractants. These studies may have potentially important implications in our understanding of the mechanism of the detrimental effect of nicotine on vascular wall pathology. References [1] Kilaru S, Frangos SG, Chen AH, et al. Nicotine: a review of its role in atherosclerosis. J Am Coll Surg 2001;193:538–46. [2] Ravenliolt RT. Tobacco’s impact on the 20th century US mortality patterns. Am J Prev Med 1985;1:4–17. [3] Hertzer NR, Young JR, Kramer JR, et al. Routine coronary angiography prior to elective aortic reconstruction; results of selective myocardial revascularization in patients with peripheral vascular disease. Arch Surg 1979;114:1336–44. [4] Friedman GD, Dales LG, Ury HK. Mortality in middle-aged smokers and non-smokers. New Eng J Med 1979;300:213–7. [5] Zimmerman M, McGeachie J. The effect of nicotine on aortic endothelium: a quantitative ultrastructural study. Atherosclerosis 1987;63:33–41. [6] Lin SJ, Hong CY, Chang MS, Chiang BN, Chien S. Long-term nicotine exposure increases aortic endothelial cell death and enhances transendothelial macromolecular transport in rats. Arterioscler Thromb 1992;12:1305–12. [7] Murohara T, Kugiyama K, Ohgushi M, Sugiyama S, Yasue H. Cigarette smoke extract contracts isolated porcine coronary arteries by superoxide anion-mediated degradation of EDRF. Am J Phyisol 1994;266:H874–80.

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