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Effect of nicotine on the intimal hyperplasia after endothelial removal of the rabbit carotid artery
ISSN 0306-3623/97 $17.00+.00 PII S0306-3623 (96)00369-2 All rights reserved
Gen. Pharmac. Vol. 28, No. 5, pp. 653-659, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Effect of Nicotine on the Intimal Hyperplasia after Endothelial Removal of the Rabbit Carotid Artery Hidehisa Hamasaki,1 Jun Sato,1 Hiroshi Masuda,1 Satoru Tamaoki,1 Eiji Isotani, 2 Satoshi Obayashi, 3 Tomoyuki Udagawa4 and Hiroshi Azuma 1. 1DEPARTMENTOF MEDICINALCHEMISTRY,INSTITUTEFOR MEDICALAND DENTAL ENGINEERING,TOKYOMEDICALAND DENTALUNIVERSITY,2-3-10 SURUGADAI, KANDA, CHIYODA-KU,TOKYO 101, JAPAN [TEL: 81-3-5280-8034; Fax: 81-3-5280-8005]; DEPARTMENTS OF 2NEuROSURGERY,3OBSTETRICSAND GYNECOLOGY,AND 4ANESTHESIOLOGY, FACULTYOF MEDICINE, TOKYO MEDICALAND DENTALUNIVERSITY,TOKYO, JAPAN
ABSTRACT. 1. The present experiments were designed to investigate the effect of long-term oral nicotine (10mg/200ml/kg/day for 7 weeks) on the intimal hyperplasia after endothelial removal of the rabbit carotid artery. 2. The plasma concentrations of nicotine were determined to be 11.7-12.5ng/ml during the term of administration and corresponded to the plasma levels in human smokers. 3. Six weeks after the endothelial removal, light microscopy revealed a marked intimal hyperplasia. Administration of nicotine tended to accelerate the intimal hyperplasia, which was estimated by comparing the histological findings, D N A content and wet weight of the vessel wall. 4. Acetylcholine- and A23187-induced endothelium-dependent relaxations were greatly impaired in the hyperplastic artery strips. The impairment of relaxations tended to be accelerated in the nicotine group. Sodium nitroprusside-induced relaxation was not different between the control and the hyperplastic artery strips and remained unaffected in the nicotine group. 5. The concentrations of endogenous nitric oxide (NO) synthesis inhibitors, N%monomethyl-Larginine (L.NMMA) and asymmetrical N~,N~-dimethyl-L-arginine (ADMA) were significantly more increased in the regenerated endothelial cells compared with those in the control endothelial cells. The concentrations of L-NMMA and A D M A in the regenerated endothelial cells were significantly increased by as much as 1.3 × 10 -6 and 5.6x 10-7M, respectively, in the nicotine group. 6. Immunoreactive endothelin-1 was significantly increased in the hyperplastic vessel wall (2.4 times that of the control) in 6 weeks. Administration of nicotine tended to increase the level. 7. It seems possible to assume from these results that, although, under the present experimental conditions, nicotine exhibited a tendency to accelerate the intimal hyperplasia after endothelial removal, the longer exposure to nicotine or a higher dose of the agent or both would significantly accelerate the intimal hyperplasia through the enhanced impairment of endothelium-derived relaxing factor/ NO production, which might be brought about by the enhanced increases in L-NMMA and ADMA concentrations, and the enhanced increase in endothelin-1 in the vessel wall. GEN PHARMAC28;5:653-659, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Intimal hyperplasia, endothelial cells, nitric oxide (NO), L-NMMA, ADMA, endothelin-1, chronic nicotine INTRODUCTION Smoking contributes to the development of atherosclerosis. In addition to their role in acute thrombus formation, platelets are also important in the development of atherosclerosis (Martin et al., 1991; Steinberg et al., 1989). When the arterial endothelium has been damaged through mechanical and chemical factors, platelets interact with or adhere to the subendothelial connective tissue and initiate a sequence that leads to formation of atherosclerotic plaque (Ross, 1986; Schwartz et al., 1981). Endothelial cells normally prevent platelet adherence and aggregation partly through endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) (Azuma et al., 1986; Radomski et al., 1987). When the endothelial cells have been damaged, the platelets are activated and release mitogens such as platelet-derived growth factor, which encourages migration and *To whom correspondence should be addressed. Received 2 August 1996; accepted 19 August 1996.
proliferation of smooth muscle cells in the region of the endothelial injury. Cigarette smoking causes endothelial injury including ultrastructural changes in aortic and pulmonary endothelial cells, endothelial cell turnover and endothelial cell function (Booyse et al., 1981; Lin et al., 1992; Zimmerman and McGeachie, 1987) and is associated with dose-related and potentially reversible impairment of endothelium-dependent arterial dilatation in clinically healthy young adults, consistent with endothelial dysfunction (Celermajer et al., 1993). Zhu et al. (1993) have reported that passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits. Further, Penn et al. (1993) described a significant acceleration in the growth of atherosclerotic plaques associated with passive smoking in young cockerels that were eating a normal, low-cholesterol diet. However, the mechanism by which smoking produces atherosclerosis is not fully elucidated. In previous papers (Azuma et al., 1990, 1992, 1994; Niimi et al., 1994), we have demonstrated the existence of a marked intimal hy-
H. Hamasaki et al.
654 perplasia after endothelial removal of the rabbit carotid artery and the involvement of both an increased level of endothelin-1 (ET-1) in the vessel wall and a decreased production of EDRF/NO in the occurrence of the intimal hyperplasia. The present experiments were designed to investigate the effect of oral nicotine on the intireal hyperplasia after endothelial removal of the rabbit carotid artery, in connections with changes in EDRF/NO production, concentrations of N°-monomethyl-L-arginine (L-NMMA) and asymmetrical N°,N°-dimethyl-t-arginine (ADMA) in the endothelial cells and endothelin-1 level in the vessel wall. MATERIALS AND METHODS
E x p e r i m e n t a l animals Thirty-two Japanese White male rabbits were used. These rabbits were purchased at 8 weeks of age, housed individually for 1 week before starting the administration of nicotine in a temperature-controlled (23+_1°C) room and fed regular chow (RC-4, Oriental Yeast) throughout the experimental periods. The body weight changes and general behavior of all rabbits before and after the endothelial removal and during the administration of nicotine were normal. Nicotine bitartrate was dissolved in distilled water immediately before administration in a concentration of 0.05mg/ml and given as a drinking water in a dose of 10 mg/200 ml/kg/day for 7 weeks (from 1 week before to 6 weeks after the endothelial denudation) to 16 rabbits. The dose of nicotine did not affect drinking behavior. Two hundred milliliters per kilogram of water was sufficient for intake of nicotine and was completely consumed in a day. Another 16 rabbits given distilled water (200 ml/kg/day) without nicotine served as controls. This study complied with the Animal Welfare Regulations of Tokyo Medical and Dental University and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society.
D e t e r m i n a t i o n of p l a s m a concentration of nicotine Six rabbits each of the control and nicotine groups were randomly selected to determine the concentration of plasma nicotine according to the method described by Feyerabend (1987). In brief, 3 ml of blood was withdrawn through the central ear artery by using a 5-ml syringe containing 200 U of heparin 1 day before and 1, 3 and 7 weeks after beginning the administration of nicotine. Every blood collection was performed 4 hr after giving the drinking water. Separated plasma was transferred into a glass tube and frozen at -40°C until required for analysis. An aliquot of the plasma spiked with the internal standard (N-ethylnornicotine) was made alkaline with 1 N NaOH and extracted once with diethyl ether. The organic layer was transferred to a second tube and 1 N HC1 was added for neutralization. The ethereal extract was evaporated (centrifugal evaporator: CV-100D, Eyela, Tokyo, Japan) and the tube vortexed and centrifuged. After the extract was washed with diethyl ether, the solution was transferred to a Dreyer tube and made alkaline, and then nicotine was extracted into butylacetate. An aliquot (3 bd) of the butylacetate solution was injected onto the gas chromatograph (Hewlett-Packard model 5730A), which is equipped with nitrogenselection detectors. The gas chromatography conditions employed were as follows: Column: 1.8 mxl.2 mm (i.d.) glass column, packed with 2% carbowax 20M-TPA/5% KOH on chromatosorb W-HP 80-100 mesh, conditioned overnight at 200°C before use. Column temperature: 150°C Injector port temperature: 300°C Carrier gas: Helium (30 ml/min)
Air flow rate: 50 ml/min Hydrogen flow rate: 3 ml/min.
Endothelial removal Endothelial removal of the rabbit carotid artery was performed according to a method described previously (Azuma et al., 1990, 1992, 1994, 1995a,b; Niimi et al., 1994). In brief, the right common carotid artery was exposed under anesthesia with sodium pentobarbital (25 mg/kg, i.v.) by aseptic surgery. An embolectomy catheter (12040-3F/40 cm/3F; Baxter Healthcare Corporation, Santa Ana, CA, USA) was then inserted into the proximal portion of the artery in retrograde fashion through a small nick made near the carotid bifurcation. The luminal surface of the artery from the origin to the balloon entrance was then gently rubbed back and forth with the balloon filled with 0.15 ml of air. This method of removing endothelial cells did not cause deep injury of the vascular wall (Azuma et al., 1990, 1992, 1994, 1995a,b; Niimi et al., 1994). After the balloon was removed, the arterial incision was sutured with 10-0 nylon thread, keeping patency of the artery. The left carotid artery underwent a sham operation and served as a control. At 6 weeks after the balloon denudation, animals were anesthetized with sodium pentobarbital (25 mg/kg, i.v.) and exsanguinated through the femoral arteries.
Morphological examination Wet weight and the isolated entire length of the control left and the previously denuded right carotid arteries that had been harvested from six rabbits each of the control and nicotine groups were determined. Results were given as mg wet weight/50-mm length. Approximately 20 mg wet weight of the carotid arteries of which endothelial cells had been removed was used for determining DNA and protein contents by the fluorometric method described by Kissane and Robins (1958) and by using a micro-BCA kit (Pierce, Rockford), respectively. Approximately 5-mm pieces of the control left and hyperplastic right carotid arteries were fixed in a 10% neutral solution of formaldehyde and were embedded in paraffin after dehydration with ethanol. Thin sections were stained by the ElasticaVan Gieson method for microscopic analysis. To provide a semiquantitative parameter for assessing intimal hyperplasia, the intimal area and medial area were determined by means of an image analyzer (Quadra-950, Macintosh) and image scanner (Scanjet IIc, Hewlett Packard). The ratio (%) of the intimal area to the intimal area plus medial area was calculated.
Organ chamber experiments The left and hyperplastic right carotid arteries of the rabbits just described were used for determining the mechanical responses. A transverse strip, 2 mm in width, was cut off with a razor blade and mounted vertically in an organ chamber containing 5 ml of modified Krebs solution continuously bubbled with 95% 02 and 5% CO2 at 37 +-0.5°C. Special care was taken to avoid unintentional rubbing of the intraluminal surface. The mechanical responses were determined according to a method described elsewhere (Azuma et al., 1990, 1992; Niimi et al., 1994). The composition of the modified Krebs solution was as follows (raM): NaCI 115.0, KC1 4.7, MgSO~ 7 H20 1.2, CaClz 2 H20 2.5, KH2PO4 1.2, NaHCO3 25.0 and glucose 10.0. After 60 min of equilibration, 10 6 M acetylcholine (ACh) was given to all strips during the contraction caused by 10 6 M noradrenaline to test for the presence or absence of functioning endothelial cells. After this, relaxation responses to ACh ( 10-9-10 -5 M), sodium nitroprusside (SNP, 10 9-3x10-s M) and A23187 (10 9_
Effect of Nicotine on the Intimal Hyperplasia 3x10 -6 M) were examined during a contraction caused by 10 6 M noradrenaline in this order. Relaxation responses to these agonists were given as a percentage of the 10-6 M noradrenaline-induced contraction. In another setting, the involvement of EDRFfNO in ACh(10 -6 M)- and A23187 (10 -6 M)-induced relaxations was confirmed by repeating these responses with the endothelium removed with a cotton swab or in the presence of methylene blue (10 5 M) or NO-nitro-L-arginine (NOARG; 10 -4 M). Any effect of prostanoid formation during endothelium-dependent relaxation was ruled out by comparing responses with and without the addition of indomethacin (10 s M) to the organ chamber. Specimens were incubated with these antagonists for 20 min before adding 10 -6 M noradrenaline.
Determination of L, N M M A and A D M A in endothelial cells Contents of L-NMMA and ADMA in the regenerated and control endothelial cells were determined by means of automated high-performance liquid chromatography (HPLC) according to a method described elsewhere (Azuma et al., 1995b). In brief, the automated analytical system was equipped with a solvent selection system (LV980-03), 2 HPLC pumps (PU-980), an autosampler (AS-950), a column oven (CO-960), a fluorescence detector (FP-920) and an integrator (807-1T; all from Jasco, Tokyo, Japan). As described previously (Azuma et al., 1995b), endothelial cells were collected from ten rabbits each of the control and nicotine groups by gently rubbing the innermost surface of the previously denuded right and sham-operated left carotid arteries with a microspatula in 5-M icecold HEPES buffer (pH 7.4) and then sonicated at 20 W for 15 sec (Sonifier 250, Branson), followed by an addition of trichloroacetic acid at a final concentration of 5%. Arterial strips from which endothelium had been deliberately removed were devoid of endothelial cells on the luminal surface, and the intact subendothelial layer was identified by scanning electron microscopy (Azuma et al., 1990). The homogenate was centrifuged at 1300g for 10 rain. The supernatant was separated and a 100-1xl aliquot of the supernatant was applied for HPLC. The composition of the eluting buffers and the elution time were the same as those described in a previous paper (Azuma et al., 1995b). The buffers were prepared from freshly distilled and deionized water, adjusted to the required pH and filtered through a Millipore filter (Type HA, 0.45-~m pore size, Millipore, Waters). The flow rates were 0.55 ml/min for the buffers and 0.33 ml/min for the orthophthalaldehyde fluorogenic reagent (Nakalai Chemicals Co., Kyoto, Japan). One column (AApak Li, 6.0 mm i.d.xl00 mm, Jasco) temperature, 60°C, was used during the analysis. Regeneration of the column was achieved in 15 rain with a solution of 0.6 M lithium hydroxide and reequilibration was completed in 60 min with the first buffer (0.1 N lithium citrate, pH 6.06). The column back-pressure generated during the analysis was 28kg/cm 2. Paraphenylenediamine dihydrochloride (Wako Pure Chemicals) was used as an internal standard. The equations of the relation between the concentrations (y) of authentic compounds and the ratio (x: response factor) of the fluorescence peak area of the authentic compounds to that of the internal standard were determined as follows:
y=26.38x+O.137 for L-NMMA (r=0.9986),
655 above) was used for determining the DNA concentration (/*g/ml) in endothelial cells, according to the fluorometric method described by Kissane and Robins (1958).
Determination of the relations between number of endothelial cells and DNA concentration To estimate tentatively the intracellular water space of endothelial cells, the relations between the number of endothelial cells and the DNA concentration was determined. Twenty-five tubes containing different numbers of endothelial cells were prepared from 24 carotid arteries of 12 additional normal rabbits. Endothelial cells were collected as described above. The DNA concentration (Izg/ml) in endothelial cells was determined. The equation between the number of endothelial cells (y) and the DNA concentration (x) was obtained by the least-square method as follows: y=(14.3x+6)x 10a (r=0.9840). From a determination of the DNA concentration in endothelial cells, the number of endothelial cells is calculated by using the equation. If intracellular water space is assumed to be 2 pl/endothelial cell (Baydoun et al., 1990), intracellular concentrations of L-NMMA and A D M A can be tentatively calculated.
Determination of immunoreactive endothelin.1 Tissue samples were prepared from the carotid arteries of ten rabbits each of the control and nicotine groups according to a method described previously (Azuma et al., 1994, 1995a; Kitamura et al., 1989). In brief, the control and the previously denuded carotid arteries from which endothelial cells had been deliberately removed as described above were homogenized in a Polytron at maximum speed for 20 sec in 20 volumes of 1 M acetic acid containing 0.01% Triton X-100 and immediately boiled for 10 min to inactivate protease. The homogenate was chilled and then centrifuged at 40,O00g for 30 min at 4°C. The equivalent of 70 mg wet weight of the supernatant was loaded onto a Sep-Pak C18 cartridge (Waters), which had been preequilibrated with 1 M acetic acid, and the adsorbed materials were eluted with 3 ml of 60% acetonitrile in 0.1% trifluoroacetic acid. The eluate was obtained and lyophilized for the radioimmunoassay (RIA) of immunoreactive endothelin-1 (ir-ET-1). The RIA of ir-ET-1 was performed as described previously (Azuma et al., 1994, 1995a), and radioactivity was measured in an Aloca ARC-600 gamma counter. The cross reactivity of the rabbit antiET-1 antiserum (Peninsula) used in the present experiments has been estimated to be 100% against ET-1 and 7% each against ET2 and ET-3. Therefore, it seems likely that the ET-l-like immunoreactivity detected by this RIA system is mainly ir-ET-1.
Statistical analysis Values are expressed as mean_+S.E, mean. The statistical analysis was carried out by a two-way analysis of variance (ANOVA) or Student's t-test. When P was less than 0.05, the value was regarded as statistically significant. RESULTS
y=54.05x+0.235 for ADMA (r=0.9933).
P l a s m a concentration of nicotine, general behaviour and body weight gain
The content (pmol/ml) of each compound in the endothelial cells was determined by the equation described above after estimating the response factor of the peak corresponding to the retention time of the authentic compound and obtained as pmol/txg of DNA. An appropriate amount of the homogenate after sonication (see
The plasma concentrations of nicotine, 1 day before and 1, 3 and 7 weeks after beginning the administration of nicotine (10 mg/200 ml/kg/day) was determined to be less than 5 (n=6), 12.2_+1.2 (n=6), 11.7+1.0 (n=6) and 12.5+1.5 (n=6) ng/ml, respectively. On the other hand, the plasma concentrations of nicotine in the
H. Hamasaki et al.
656 control group ( n = 6 ) was less than 5 ng/ml at 1 day before and 1, 3 and 7 weeks after the administration of the vehicle. The general behavior of the rabbits was normal throughout the 7-week administration periods (from 1 week before to 6 weeks after the endothelial removal). Body weight at 1 week before the endothelial removal was determined to be 1.53-+0.03 kg for the control group ( n = 1 6 ) and 1.60_+0.03 kg for the nicotine group (n=16). These values were not significantly different from each other. Changes in body weight gain, which is given as the difference between the weight at each week after the endothelial removal and the value at 1 week before the operation, are shown in Fig. h The body weight gain at 3, 4 and 5 weeks after the endothelial removal was significantly (P<0.05) lower than the corresponding value in the control group.
Intimal hyperplasia The effect of nicotine on the intimal hyperplasia was estimated by comparing three different parameters: histological findings, DNA content and wet weight of the vessel wall. Results are shown in Table 1. In the sham-operated left carotid artery, no noticeable histological changes could be detected and the ratio (%) of intimal area to intimal area plus medial area was calculated to be 4.6-+0.2% ( n = 6 ) . Six weeks after the endothelial removal, a marked intimal hyperplasia resulting from new accumulation of proliferating smooth muscle cells and connective tissue was observed. The histological parameter was significantly (P
(EIR) remained unaltered in all groups. The relaxation responses to the submaximal concentration of 10 -6 M A23187 and 10 5 M SNP and the effects of nicotine were compared between the control left and the previously denuded right carotid artery strips and are shown in Fig. 3.
L . N M M A and A D M A
Results are shown in Table 2. The L-NMMA and A D M A concentrations were determined to be (4.2_+0.2)× 10 7 and (2.1 -+0.1)x 10-7 M (n=4), respectively, in the control endothelial cells and significantly (P<0.01 and P
Immunoreactive endothelin. 1 contents in the vessel wall In the control left carotid artery from which endothelial cells had been deliberately removed before homogenate was prepared, the irET-1 content was determined to be 168.7-+22.4 pg/g wet weight ( n - 3 ) , which was significantly (P
DISCUSSION The plasma concentrations of nicotine at 1, 3 and 7 weeks after beginning the nicotine administration (10 mg/kg/200 ml/day for 7 weeks) ranged between 11.7 and 12.5 ng/ml, which nearly corre-
Endothelium.dependent and endothelium.independent relaxations In the control left and the previously denuded right carotid artery strips, 10 6 M ACh and 10 -6 M A23187 produced relaxations during the contraction induced by 10 -6 M noradrenaline. The relaxations were abolished after deliberate denudation of the endothelial cells [complete endothelial removal without damage to the smooth muscle layer was confirmed by scanning electron microscopy (Azuma et al., 1990)] or greatly reduced by pretreatment with 10 5 M methylene blue and 10 4 M NOARG, an inhibitor of NO synthesis (Kobayashi and Hattori, 1990) but unaffected by 10 -5 M indomethacin (data not shown), indicating that the ACh- and A23187-induced relaxations are caused, depending on the endothelimn, and mediated by EDRF/NO but not by prosranoids. ACh (10 9-10-5 M) produced a concentration-dependent relaxation of the sham-operated left carotid artery, in which nicotine did not significantly attenuate the magnitude of the relaxation response to ACh. In contrast, the ACh-induced endothelium-dependent relaxation (EDR) was greatly impaired in the previously denuded right carotid artery. The impairment of the ACh-induced EDR tended to be enhanced in the nicotine group (Fig. 2). In addition to the impairment of the ACh-induced EDR in the previously denuded right carotid artery, A23187 (10 9-3 x 10 6 M)induced EDR was also significantly decreased. The decrease was slightly enhanced in the nicotine group. In contrast, the SNP (10 9-3×10 5 M)-induced endothelium-independent relaxation
concentrations in endothelial cells
800
--
400
=o
I
I
I
j
0
2
4
6
Weeks after e n d o t h e l i a l r e m o v a l
F I G U R E 1. Changes in body weight gain of rabbit after the endothelial removal in the control (solid circles) and nicotine (open circles) groups. Body weight at 1 week before the endothelial removal was determined to be 1.53-+0.03 kg ( n = 16) for control group and 1.60_+0.03 kg ( n = 16) for nicotine group. Body weight gain was given as the difference of the value at each week after the endothelial removal from that at 1 week before the operation. Vertical bars show standard errors of the means. T h e asterisk (*) indicates significant difference vs. corresponding control at P
Effect of Nicotine on the Intimal Hyperplasia
657
TABLE 1. Effect of nicotine on the hyperplastic vascular responses to endothelial removal of rabbit carotid arteries Group
I/(I + M)(%)
1. Sham-operated left carotid artery + vehicle 2. Sham-operated left carotid artery + nicotine 3. Denuded right carotid artery + vehicle 4. Denuded right carotid artery + nicotine
DNA (~g/mg wet wt) a Wet wt (mg/50 mm)
4.6 -+ 0.2 (6)
7.0 +- 0.8 (3)
74.3 -+ 4.3 (6)
5.6 -+ 0.2 (6)
7.2 -+ 0.8 (3)
77.7 -+ 4.2 (6)
26.9 -+ 3.7 (6) c
10.8 _+ 0.9 (3) b
96.3 -4- 3.3 (6) c
28.2 +- 1.2 (6) c
11.2 -4- 0.4 (3) c
104.0 +- 5.6 (6) c
Results are given as mean -+ S.E. mean. Endothelial cells were removed before homogenates were prepared in experiment. ~.cSignificant difference vs. corresponding sham-operated control group at P < 0.05 and P < 0.005, respectively. Abbreviations: 1, intimal area; M, medial area. The number in parentheses indicates the number of determinations.
sponds to the plasma levels in human smokers (Itani et al., 1988). Although there is considerable interindividual variability in measured nicotine levels, the mean plasma concentrations in human smokers (10-30 cigarettes smoked per day) are reportedly determined to be 8.6-15.0 ng/ml (Itani et al., 1988). In this respect, we consequently observed the effect of the oral nicotine on the intimal hyperplasia after endothelial removal at concentrations nearly corresponding to those in human smokers. We have demonstrated that there was a strong inverse correlation between the intimal hyperplasia and the amount of EDRF/NO produced by endothelial cells (Niimi et al., 1994). It is well established that EDRF/NO is an antiaggregating substance (Azuma et al., 1986; Radomski et al., 1987) and that the activation of platelets is considered an initiating event for intimal hyperplasia (Ross, 1986; Schwartz et al., 1981). According to Garg and Hassid (1989), the endogenous EDRF/NO may act as an inhibitory modulator of the vascular smooth muscle cell mitogenesis and proliferation. Therefore, it is suggested that the reduced production of EDRF/NO from endothelial cells is one of the important factors causing the intimal
Concentration
of ACh (-log M)
9
8
7
6
5
I
I
I
I
I
SO
FIGURE 2. Comparison of endothelium-dependent relaxation response of control left (solid line) and previously denuded right (dotted line) carotid arteries to acetylcholine (ACh) between vehicle (solid circles) and nicotine (open circles) groups. Each point represents the mean of six determinations. Vertical bars show standard errors of the means. Relaxation is given as a percentage (percent relaxation) to the 10 -6 M noradrenaline-induced contraction. The EDR was significantly different (P<0.005) between control left and previously denuded right carotid arteries of both groups.
hyperplasia after endothelial removal (Azuma et al., 1990, 1992, 1995b; Niimi et al., 1994). Furthermore, cigarette smoking causes endothelial injury including ultrastructural changes in aortic and pulmonary endothelial cells, endothelial cell turnover and endothelial cell function (Booyse et al., 1981; Linet al., 1992; Zimmerman and McGeachie, 1987) and is associated with an impairment of endothelium-dependent arterial dilatation in clinically healthy young adults, consistent with endothelial dysfunction (Celermajer et al., 1993). Long-term smoking reduces the NO-dependent component of basal vascular tone and impairs endothelium-dependentvasodilator response to low-dose endothelin-1 (Kiowski et al., 1994). From this point of view, it seems worthwhile to examine whether or not nicotine modifies the intimal hyperplasia and EDRF/NO production in endothelial cells. The intimal hyperplasia tended to be accelerated but not significantly by nicotine, compared with employing three different parameters--histological findings, DNA content and wet weight of the vessel wall--and the reduced production of EDRF/ NO in the regenerated endothelial cells also tended to be enhanced but not significantly by nicotine. According to Zhu et al. (1993), passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits. Further, Penn et al. (1993) described a significant acceleration in the growth of atherosclerotic plaques associated with passive smoking in young cockerels that were eating a normal, lowcholesterol diet. It seems likely, therefore, that greater enhancement of the impaired EDRF/NO production by endothelial cells after administration of nicotine may result in greater acceleration of the intimal hyperplasia. Recently, we suggested that the accumulation of endogenous inhibitors (NC-monomethyl-L-arginine and asymmetrical NS,N°-dimethyli.-arginine) for NO synthesis was associated with decreased EDRF/NO production from endothelial cells and with intimal hyperplasia (Azuma et al., 1995b). Thus, we determined the changes in concentrations of the methylarginine derivatives in the endothelial cells. The concentrations of L-NMMA and ADMA in the regenerated endothelial cells were significantly higher than those in the control endothelial cells. The accumulation of these methylarginine derivatives in the endothelial cells was significantly accelerated in the nicotine group. Vallance et al. (1992) reported that L-NMMA and ADMA at concentrations of 5 × 10-6 to 3 × 10 4 M inhibited NO biosynthesis by J774 macrophage cytosol in a concentration-dependentmanner. In addition, we have reported that c-NMMA and ADMA inhibit NO production at concentrations of 3 x 10 s to 3 x 10 -4 M in the rabbit carotid arteries (Azuma et al., 1995b). The highest increased concentration of L-NMMA and ADMA in the regenerated endothelial cells of the nicotine group was
658
H. Hamasaki et al. 10-6M A 2 3 1 8 7 0
I
C
D
C
T A B L E 3. Immunoreactive ET-1 (ir-ET-1) content in control left and hyperplastic right carotid arteries and effect of nicotine
10-5M SNP
i
f
D
I
C
D
C
D
Group
-~
1. Sham-operated left carotid artery + vehicle 2. Sham-operated left carotid artery + nicotine 3. Denuded right carotid artery + vehicle 4. Denuded right carotid artery + nicotine
sc
m0 I I Vehicle
I I Nicotine
I I Vehicle
I I Nicotine
F I G U R E 3. Comparison of the relaxation responses of control left (C) and previously denuded right (D) carotid arteries to A 2 3 1 8 7 and sodium nitroprusside (SNP) between vehicle and nicotine groups. Relaxation is given as a percentage (percent relaxation) to the 1 0 -6 M noradrenaline-induced contraction. Vertical bars show standard errors of the means. Each experiment consists of six determinations. T h e asterisks (* and ***) indicate significant difference vs. corresponding control at P < 0 . 0 5 and P < 0 . 0 0 5 , respectively.
tentatively estimated to be 1.3 × 10 -6 and 5.6× 10 7 M, respectively (Table 2), which seems insufficient to produce a complete inhibition of N O synthesis in endothelial cells, resulting in the slightly enhanced impairment of N O production in the nicotine group. T h e increase in plasma endothelin-l, a potent mitogen (Hirata et al., 1989), is thought to reflect a rise in vascular production of the peptide in response to the impaired production of EDRF/NO by endothelial cells (Boulanger and Liischer, 1990). W e have reported that the increased level of endothelin-1 in the vessel wall accompanied by the decreased production of EDRF/NO would be involved in the intimal hyperplasia (Azuma et al., 1994, 1995a; Niimi et al., 1994). In the present experiments, the administration of nicotine tended to augment the increase in endothelin-1 content in the hyperplastic vessel wall. T h e enhanced increase in the endothelin-1 content by nicotine may result from the enhanced impairment of
T A B L E 2. Comparison of L - N M M A and A D M A concentrations in control and regenerated endothelial cells (ECs) and effect of nicotine C o n c e n t r a t i o n ( x 10 7 M) Group 1. 2. 3. 4.
Control ECs Regenerated Control ECs Regenerated
n + vehicle ECs + vehicle + nicotine ECs + nicotine
4 4 4 4
L.NMMA 4.2 6.3 7.9 12.9
-+ 0.2 ± 0.2 ~' -+ 0.7" _+ 0.9 ~'~
ADMA 2.1 2.9 3.4 5.6
--+ -+ -+
0.1 0.2 ~1 0.3 '~ 0.6 ~'~
Results are given as mean +- S.E. mean. ~,.dSignificant difference vs. group 1 at P < 0.005 and P < 0.01, respectively. b.,.Significant difference vs. group 3 at P < 0.005 and P < 0.05, respectively. Significant difference vs. group 2 at P < 0.005. The concentrations of L-NMMA and ADMA were tentatively estimated from the equation between the DNA concentration and the number of endothelial cells and the intracellular water space reported by Baydoun et al. (1990) (see text).
ir-ET- 1 co n t e n t (pg/g wet weight)
168.7 -+ 22.4 (3) 187.5 -+ 3.6 (3) 407.3 -+ 65.1 (3) ~ 480.9 -+ 40.5 (3) b
Results are given as mean -+ S.E. mean. Endothelial cells were removed before homogenates were prepared. ~,.hSignificant difference vs. corresponding value in the sham-operated group at P < 0.05 and P < 0.005, respectively. The number in parentheses indicates the number of determinations.
the EDRF/NO production and may play a role in accelerating the intimal hyperplasia. In conclusion, it seems possible to assume that, although, under the present experimental conditions, nicotine exhibited a tendency to accelerate the intimal hyperplasia after endothelial removal, longer exposure to nicotine or higher doses of the agent or both would significantly accelerate the intimal hyperplasia through the enhanced impairment of EDRF/NO production, which might be brought about by the enhanced increases in L-NMMA and A D M A concentrations, as well as the enhanced increase in endothelin-1 in the vessel wall. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 08672604) and by the Smoking Research Foundation (SRF) for Biomedical Research in Japan. The authors thank Dr. A. Saito for preparation of histological specimens.
References Azuma H., Funayama N., Kubota T. and lshikawa M. (1990) Regeneration of endothelial cells after balloon denudation of the rabbit carotid artery and changes in responsiveness. Jpn. J. Pharmacol. 52, 541 552. Azuma H., Hamasaki H. and Niimi Y. (1994) Role of endothelin-1 in neointima formation after endothelial removal in rabbit carotid arteries. Am. J. Physiol. 267 (Hear Circ. Physiol. 36), H2259-H2267. Azuma H., Hamasaki H., Sato J., Isotani E., Obayashi S. and Matsubara, O. (1995a) Different localization of ETA and ETB receptors in the hyperplastic vascular wall. J. Cardiovasc. Pharmacol. 25,802-809. Azuma H., Ishikawa M. and Sekizaki S. (1986) Endothelium-dependent inhibition of platelet aggregation. Br. J. Pharmacol. 88, 411-415. Azuma H., Niimi Y. and Hamasaki H. (1992) Prevention of intimal thickening after endothelial removal by a nonpeptide angiotensin 11 receptor antagonist, losartan. Br. J. Pharmacol. 106, 665-67l. Azuma H., Sato J., Hamasaki H., Sugimoto A , Isotani E. and Obayashi S. (1995b) Accumulation of endogenous inhibitors for nitric oxide synthesis and decreased content of L-arginine in the regenerated endothelial cells. Br. J. Pharmacol. 115, 1001 1004. Baydoun A. R., Emery P.W., Pearson J. D. and Mann G. E. (1990) Substrate-dependent regulation of intracellular amino acid concentration in cultured bovine aortic endothelial ceils. Biochem. Biophys. Res. Commun. 173,940-948. Booyse F. M., Osikowicz G. and Quarfuot A. J. ( 1981 ) Effects of chronic oral consumption of nicotine on the rabbit aortic endothelium. Am. J. Pathol. 102, 229-238. Boulanger C. and Ltischer T. F. (1990) Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. J. Clin. Invest. 85,587-590. Celermajer D. S., Sorensen K. E., Georgakopoulos D., Bull C., Thomas O., Robinson J. and Deanfield J. E. (1993) Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-
Effect of Nicotine on the Intimal Hyperplasia dependent dilation in healthy young adults. Circulation 88 (Part 1), 2149-2155. Feyerabend C. (1987) Determination of nicotine in physiological fluid by gaschromatography. In Environmental Carcinogens: Methods of Analysis and Exposure Measurement. (Edited by O'Neil I. K., Brunneman K. D., Dodet B. and Hoffman D.) pp. 299-307. IARC Scientific Publications No. 81, Lyon. Garg U. C. and Hassid A. (1989) Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J. Clin. Invest. 83, 1774-1777. Hirata Y., Takagi Y., Fukuda Y. and Marumo F. (1989) Endothelin is a potent mitogen for rat vascular smooth muscle cells. Arteriosclerosis 78, 225-228. Itani S., Higashi E. and Shimizu Y. (1988) Simultaneous nicotine and cotinine levels in plasma and urine as an index of environmental tobacco smoke uptake. In Indoor and Ambient Air Quality. (Edited by Perry R. and Kirk P.) pp.259-266. Salper, London. Kissane J. M. and Robins E. (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissue with special reference to the central nervous system. J. Biol. Chem. 233, 184-188. Kitamura K., Tanaka T., Kato J., Ogawa T., Eto T. and Tanaka K. (1989) lmmunoreactive endothelin in rat kidney inner medulla: marked decrease in spontaneously hypertensive rats. Biochem. Biophys. Res. Commun. 162, 38-44. Kiowski W., Linder L., Stoschitzky K., Pfisterer M., Burckhardt D., Burkart K. and Btihler F.R. (1994) Diminished vascular response to inhibition of endothelium-derived nitric oxide and enhanced vasoconstriction to exogenously administered endothelin-1 in clinically healthy smokers. Circulation 90, 27-34. Kobayashi Y. and Hattori K. (1990) Nitroarginine inhibits endotheliumderived relaxation. Jpn. J. Pharmacol. 52, 167-169.
659 Lin S.J., Hong C.Y., Chang M. S., Chiang B.N. and Chien S. (1992) Long-term nicotine exposure increases aortic endothelial cell death and enhances transendothelial macromolecular transport in rats. Arterioscler. Thromb. 12, 1305-1312. Martin J. F., Bath P. M. and Burr M. L. (1991) Influence of platelet size on outcome after myocardial infarction. Lancet 338, 1409-1411. Niimi Y., Azuma H. and Hirakawa K. (1994) Repeated endothelial removal augments intimal thickening and attenuates EDRF release. Am. J. Physiol. 266 (Heart Circ. Physiol. 35), H1348-H1356. Penn A. and Snyder C. A. (1993) Inhalation of sidestream cigarette smoke accelerates development of arteriosclerotic plaques. Circulation 88 (Part 1), 1820-1825. Radomski M. W., Palmer R. M. J. and Moncada S. (1987) The ant iaggregating properties of vascular endothelium: interaction between prostacyclin and nitric oxide. Br. J. Pharmaco/. 92, 639-646. Ross R. (1986) The pathogenesis of atherosclerosis: an update. N. Engl. J. Med. 314, 488-500. Schwartz S.M., Gajdusek C.M. and Selden S.C. (1981) Vascular wall growth control: the role of endothelium. Arteriosclerosis 1, 107-171. Steinberg D., Parthasarathy S., Carew T. E., Khoo J. C. and Witztum J. L. (1989) Beyond cholesterol: modification of low-density lipoprotein that increase its atherogenecity. N. Engl. J. Med. 320, 915-924. Vallance P., Leone A., Calver A., Collier J. and Moncada S. (1992) Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339, 572-575. Zhu B.Q., Sun Y.-P., Sievers R., lsenberg R., Glanz S.A. and Parmley W. W. (1993) Passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits. J. Am. Coll. Cardiol. 21,225-232. Zimmerman M. and McGeachie J. (1987) The effect of nicotine on aortic endothelium: a quantitative ultrastructural study. Atherosclerosis 63, 33-41.
Gen. Pharmac. Vol. 28, No. 5, pp. 653-659, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Effect of Nicotine on the Intimal Hyperplasia after Endothelial Removal of the Rabbit Carotid Artery Hidehisa Hamasaki,1 Jun Sato,1 Hiroshi Masuda,1 Satoru Tamaoki,1 Eiji Isotani, 2 Satoshi Obayashi, 3 Tomoyuki Udagawa4 and Hiroshi Azuma 1. 1DEPARTMENTOF MEDICINALCHEMISTRY,INSTITUTEFOR MEDICALAND DENTAL ENGINEERING,TOKYOMEDICALAND DENTALUNIVERSITY,2-3-10 SURUGADAI, KANDA, CHIYODA-KU,TOKYO 101, JAPAN [TEL: 81-3-5280-8034; Fax: 81-3-5280-8005]; DEPARTMENTS OF 2NEuROSURGERY,3OBSTETRICSAND GYNECOLOGY,AND 4ANESTHESIOLOGY, FACULTYOF MEDICINE, TOKYO MEDICALAND DENTALUNIVERSITY,TOKYO, JAPAN
ABSTRACT. 1. The present experiments were designed to investigate the effect of long-term oral nicotine (10mg/200ml/kg/day for 7 weeks) on the intimal hyperplasia after endothelial removal of the rabbit carotid artery. 2. The plasma concentrations of nicotine were determined to be 11.7-12.5ng/ml during the term of administration and corresponded to the plasma levels in human smokers. 3. Six weeks after the endothelial removal, light microscopy revealed a marked intimal hyperplasia. Administration of nicotine tended to accelerate the intimal hyperplasia, which was estimated by comparing the histological findings, D N A content and wet weight of the vessel wall. 4. Acetylcholine- and A23187-induced endothelium-dependent relaxations were greatly impaired in the hyperplastic artery strips. The impairment of relaxations tended to be accelerated in the nicotine group. Sodium nitroprusside-induced relaxation was not different between the control and the hyperplastic artery strips and remained unaffected in the nicotine group. 5. The concentrations of endogenous nitric oxide (NO) synthesis inhibitors, N%monomethyl-Larginine (L.NMMA) and asymmetrical N~,N~-dimethyl-L-arginine (ADMA) were significantly more increased in the regenerated endothelial cells compared with those in the control endothelial cells. The concentrations of L-NMMA and A D M A in the regenerated endothelial cells were significantly increased by as much as 1.3 × 10 -6 and 5.6x 10-7M, respectively, in the nicotine group. 6. Immunoreactive endothelin-1 was significantly increased in the hyperplastic vessel wall (2.4 times that of the control) in 6 weeks. Administration of nicotine tended to increase the level. 7. It seems possible to assume from these results that, although, under the present experimental conditions, nicotine exhibited a tendency to accelerate the intimal hyperplasia after endothelial removal, the longer exposure to nicotine or a higher dose of the agent or both would significantly accelerate the intimal hyperplasia through the enhanced impairment of endothelium-derived relaxing factor/ NO production, which might be brought about by the enhanced increases in L-NMMA and ADMA concentrations, and the enhanced increase in endothelin-1 in the vessel wall. GEN PHARMAC28;5:653-659, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Intimal hyperplasia, endothelial cells, nitric oxide (NO), L-NMMA, ADMA, endothelin-1, chronic nicotine INTRODUCTION Smoking contributes to the development of atherosclerosis. In addition to their role in acute thrombus formation, platelets are also important in the development of atherosclerosis (Martin et al., 1991; Steinberg et al., 1989). When the arterial endothelium has been damaged through mechanical and chemical factors, platelets interact with or adhere to the subendothelial connective tissue and initiate a sequence that leads to formation of atherosclerotic plaque (Ross, 1986; Schwartz et al., 1981). Endothelial cells normally prevent platelet adherence and aggregation partly through endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) (Azuma et al., 1986; Radomski et al., 1987). When the endothelial cells have been damaged, the platelets are activated and release mitogens such as platelet-derived growth factor, which encourages migration and *To whom correspondence should be addressed. Received 2 August 1996; accepted 19 August 1996.
proliferation of smooth muscle cells in the region of the endothelial injury. Cigarette smoking causes endothelial injury including ultrastructural changes in aortic and pulmonary endothelial cells, endothelial cell turnover and endothelial cell function (Booyse et al., 1981; Lin et al., 1992; Zimmerman and McGeachie, 1987) and is associated with dose-related and potentially reversible impairment of endothelium-dependent arterial dilatation in clinically healthy young adults, consistent with endothelial dysfunction (Celermajer et al., 1993). Zhu et al. (1993) have reported that passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits. Further, Penn et al. (1993) described a significant acceleration in the growth of atherosclerotic plaques associated with passive smoking in young cockerels that were eating a normal, low-cholesterol diet. However, the mechanism by which smoking produces atherosclerosis is not fully elucidated. In previous papers (Azuma et al., 1990, 1992, 1994; Niimi et al., 1994), we have demonstrated the existence of a marked intimal hy-
H. Hamasaki et al.
654 perplasia after endothelial removal of the rabbit carotid artery and the involvement of both an increased level of endothelin-1 (ET-1) in the vessel wall and a decreased production of EDRF/NO in the occurrence of the intimal hyperplasia. The present experiments were designed to investigate the effect of oral nicotine on the intireal hyperplasia after endothelial removal of the rabbit carotid artery, in connections with changes in EDRF/NO production, concentrations of N°-monomethyl-L-arginine (L-NMMA) and asymmetrical N°,N°-dimethyl-t-arginine (ADMA) in the endothelial cells and endothelin-1 level in the vessel wall. MATERIALS AND METHODS
E x p e r i m e n t a l animals Thirty-two Japanese White male rabbits were used. These rabbits were purchased at 8 weeks of age, housed individually for 1 week before starting the administration of nicotine in a temperature-controlled (23+_1°C) room and fed regular chow (RC-4, Oriental Yeast) throughout the experimental periods. The body weight changes and general behavior of all rabbits before and after the endothelial removal and during the administration of nicotine were normal. Nicotine bitartrate was dissolved in distilled water immediately before administration in a concentration of 0.05mg/ml and given as a drinking water in a dose of 10 mg/200 ml/kg/day for 7 weeks (from 1 week before to 6 weeks after the endothelial denudation) to 16 rabbits. The dose of nicotine did not affect drinking behavior. Two hundred milliliters per kilogram of water was sufficient for intake of nicotine and was completely consumed in a day. Another 16 rabbits given distilled water (200 ml/kg/day) without nicotine served as controls. This study complied with the Animal Welfare Regulations of Tokyo Medical and Dental University and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society.
D e t e r m i n a t i o n of p l a s m a concentration of nicotine Six rabbits each of the control and nicotine groups were randomly selected to determine the concentration of plasma nicotine according to the method described by Feyerabend (1987). In brief, 3 ml of blood was withdrawn through the central ear artery by using a 5-ml syringe containing 200 U of heparin 1 day before and 1, 3 and 7 weeks after beginning the administration of nicotine. Every blood collection was performed 4 hr after giving the drinking water. Separated plasma was transferred into a glass tube and frozen at -40°C until required for analysis. An aliquot of the plasma spiked with the internal standard (N-ethylnornicotine) was made alkaline with 1 N NaOH and extracted once with diethyl ether. The organic layer was transferred to a second tube and 1 N HC1 was added for neutralization. The ethereal extract was evaporated (centrifugal evaporator: CV-100D, Eyela, Tokyo, Japan) and the tube vortexed and centrifuged. After the extract was washed with diethyl ether, the solution was transferred to a Dreyer tube and made alkaline, and then nicotine was extracted into butylacetate. An aliquot (3 bd) of the butylacetate solution was injected onto the gas chromatograph (Hewlett-Packard model 5730A), which is equipped with nitrogenselection detectors. The gas chromatography conditions employed were as follows: Column: 1.8 mxl.2 mm (i.d.) glass column, packed with 2% carbowax 20M-TPA/5% KOH on chromatosorb W-HP 80-100 mesh, conditioned overnight at 200°C before use. Column temperature: 150°C Injector port temperature: 300°C Carrier gas: Helium (30 ml/min)
Air flow rate: 50 ml/min Hydrogen flow rate: 3 ml/min.
Endothelial removal Endothelial removal of the rabbit carotid artery was performed according to a method described previously (Azuma et al., 1990, 1992, 1994, 1995a,b; Niimi et al., 1994). In brief, the right common carotid artery was exposed under anesthesia with sodium pentobarbital (25 mg/kg, i.v.) by aseptic surgery. An embolectomy catheter (12040-3F/40 cm/3F; Baxter Healthcare Corporation, Santa Ana, CA, USA) was then inserted into the proximal portion of the artery in retrograde fashion through a small nick made near the carotid bifurcation. The luminal surface of the artery from the origin to the balloon entrance was then gently rubbed back and forth with the balloon filled with 0.15 ml of air. This method of removing endothelial cells did not cause deep injury of the vascular wall (Azuma et al., 1990, 1992, 1994, 1995a,b; Niimi et al., 1994). After the balloon was removed, the arterial incision was sutured with 10-0 nylon thread, keeping patency of the artery. The left carotid artery underwent a sham operation and served as a control. At 6 weeks after the balloon denudation, animals were anesthetized with sodium pentobarbital (25 mg/kg, i.v.) and exsanguinated through the femoral arteries.
Morphological examination Wet weight and the isolated entire length of the control left and the previously denuded right carotid arteries that had been harvested from six rabbits each of the control and nicotine groups were determined. Results were given as mg wet weight/50-mm length. Approximately 20 mg wet weight of the carotid arteries of which endothelial cells had been removed was used for determining DNA and protein contents by the fluorometric method described by Kissane and Robins (1958) and by using a micro-BCA kit (Pierce, Rockford), respectively. Approximately 5-mm pieces of the control left and hyperplastic right carotid arteries were fixed in a 10% neutral solution of formaldehyde and were embedded in paraffin after dehydration with ethanol. Thin sections were stained by the ElasticaVan Gieson method for microscopic analysis. To provide a semiquantitative parameter for assessing intimal hyperplasia, the intimal area and medial area were determined by means of an image analyzer (Quadra-950, Macintosh) and image scanner (Scanjet IIc, Hewlett Packard). The ratio (%) of the intimal area to the intimal area plus medial area was calculated.
Organ chamber experiments The left and hyperplastic right carotid arteries of the rabbits just described were used for determining the mechanical responses. A transverse strip, 2 mm in width, was cut off with a razor blade and mounted vertically in an organ chamber containing 5 ml of modified Krebs solution continuously bubbled with 95% 02 and 5% CO2 at 37 +-0.5°C. Special care was taken to avoid unintentional rubbing of the intraluminal surface. The mechanical responses were determined according to a method described elsewhere (Azuma et al., 1990, 1992; Niimi et al., 1994). The composition of the modified Krebs solution was as follows (raM): NaCI 115.0, KC1 4.7, MgSO~ 7 H20 1.2, CaClz 2 H20 2.5, KH2PO4 1.2, NaHCO3 25.0 and glucose 10.0. After 60 min of equilibration, 10 6 M acetylcholine (ACh) was given to all strips during the contraction caused by 10 6 M noradrenaline to test for the presence or absence of functioning endothelial cells. After this, relaxation responses to ACh ( 10-9-10 -5 M), sodium nitroprusside (SNP, 10 9-3x10-s M) and A23187 (10 9_
Effect of Nicotine on the Intimal Hyperplasia 3x10 -6 M) were examined during a contraction caused by 10 6 M noradrenaline in this order. Relaxation responses to these agonists were given as a percentage of the 10-6 M noradrenaline-induced contraction. In another setting, the involvement of EDRFfNO in ACh(10 -6 M)- and A23187 (10 -6 M)-induced relaxations was confirmed by repeating these responses with the endothelium removed with a cotton swab or in the presence of methylene blue (10 5 M) or NO-nitro-L-arginine (NOARG; 10 -4 M). Any effect of prostanoid formation during endothelium-dependent relaxation was ruled out by comparing responses with and without the addition of indomethacin (10 s M) to the organ chamber. Specimens were incubated with these antagonists for 20 min before adding 10 -6 M noradrenaline.
Determination of L, N M M A and A D M A in endothelial cells Contents of L-NMMA and ADMA in the regenerated and control endothelial cells were determined by means of automated high-performance liquid chromatography (HPLC) according to a method described elsewhere (Azuma et al., 1995b). In brief, the automated analytical system was equipped with a solvent selection system (LV980-03), 2 HPLC pumps (PU-980), an autosampler (AS-950), a column oven (CO-960), a fluorescence detector (FP-920) and an integrator (807-1T; all from Jasco, Tokyo, Japan). As described previously (Azuma et al., 1995b), endothelial cells were collected from ten rabbits each of the control and nicotine groups by gently rubbing the innermost surface of the previously denuded right and sham-operated left carotid arteries with a microspatula in 5-M icecold HEPES buffer (pH 7.4) and then sonicated at 20 W for 15 sec (Sonifier 250, Branson), followed by an addition of trichloroacetic acid at a final concentration of 5%. Arterial strips from which endothelium had been deliberately removed were devoid of endothelial cells on the luminal surface, and the intact subendothelial layer was identified by scanning electron microscopy (Azuma et al., 1990). The homogenate was centrifuged at 1300g for 10 rain. The supernatant was separated and a 100-1xl aliquot of the supernatant was applied for HPLC. The composition of the eluting buffers and the elution time were the same as those described in a previous paper (Azuma et al., 1995b). The buffers were prepared from freshly distilled and deionized water, adjusted to the required pH and filtered through a Millipore filter (Type HA, 0.45-~m pore size, Millipore, Waters). The flow rates were 0.55 ml/min for the buffers and 0.33 ml/min for the orthophthalaldehyde fluorogenic reagent (Nakalai Chemicals Co., Kyoto, Japan). One column (AApak Li, 6.0 mm i.d.xl00 mm, Jasco) temperature, 60°C, was used during the analysis. Regeneration of the column was achieved in 15 rain with a solution of 0.6 M lithium hydroxide and reequilibration was completed in 60 min with the first buffer (0.1 N lithium citrate, pH 6.06). The column back-pressure generated during the analysis was 28kg/cm 2. Paraphenylenediamine dihydrochloride (Wako Pure Chemicals) was used as an internal standard. The equations of the relation between the concentrations (y) of authentic compounds and the ratio (x: response factor) of the fluorescence peak area of the authentic compounds to that of the internal standard were determined as follows:
y=26.38x+O.137 for L-NMMA (r=0.9986),
655 above) was used for determining the DNA concentration (/*g/ml) in endothelial cells, according to the fluorometric method described by Kissane and Robins (1958).
Determination of the relations between number of endothelial cells and DNA concentration To estimate tentatively the intracellular water space of endothelial cells, the relations between the number of endothelial cells and the DNA concentration was determined. Twenty-five tubes containing different numbers of endothelial cells were prepared from 24 carotid arteries of 12 additional normal rabbits. Endothelial cells were collected as described above. The DNA concentration (Izg/ml) in endothelial cells was determined. The equation between the number of endothelial cells (y) and the DNA concentration (x) was obtained by the least-square method as follows: y=(14.3x+6)x 10a (r=0.9840). From a determination of the DNA concentration in endothelial cells, the number of endothelial cells is calculated by using the equation. If intracellular water space is assumed to be 2 pl/endothelial cell (Baydoun et al., 1990), intracellular concentrations of L-NMMA and A D M A can be tentatively calculated.
Determination of immunoreactive endothelin.1 Tissue samples were prepared from the carotid arteries of ten rabbits each of the control and nicotine groups according to a method described previously (Azuma et al., 1994, 1995a; Kitamura et al., 1989). In brief, the control and the previously denuded carotid arteries from which endothelial cells had been deliberately removed as described above were homogenized in a Polytron at maximum speed for 20 sec in 20 volumes of 1 M acetic acid containing 0.01% Triton X-100 and immediately boiled for 10 min to inactivate protease. The homogenate was chilled and then centrifuged at 40,O00g for 30 min at 4°C. The equivalent of 70 mg wet weight of the supernatant was loaded onto a Sep-Pak C18 cartridge (Waters), which had been preequilibrated with 1 M acetic acid, and the adsorbed materials were eluted with 3 ml of 60% acetonitrile in 0.1% trifluoroacetic acid. The eluate was obtained and lyophilized for the radioimmunoassay (RIA) of immunoreactive endothelin-1 (ir-ET-1). The RIA of ir-ET-1 was performed as described previously (Azuma et al., 1994, 1995a), and radioactivity was measured in an Aloca ARC-600 gamma counter. The cross reactivity of the rabbit antiET-1 antiserum (Peninsula) used in the present experiments has been estimated to be 100% against ET-1 and 7% each against ET2 and ET-3. Therefore, it seems likely that the ET-l-like immunoreactivity detected by this RIA system is mainly ir-ET-1.
Statistical analysis Values are expressed as mean_+S.E, mean. The statistical analysis was carried out by a two-way analysis of variance (ANOVA) or Student's t-test. When P was less than 0.05, the value was regarded as statistically significant. RESULTS
y=54.05x+0.235 for ADMA (r=0.9933).
P l a s m a concentration of nicotine, general behaviour and body weight gain
The content (pmol/ml) of each compound in the endothelial cells was determined by the equation described above after estimating the response factor of the peak corresponding to the retention time of the authentic compound and obtained as pmol/txg of DNA. An appropriate amount of the homogenate after sonication (see
The plasma concentrations of nicotine, 1 day before and 1, 3 and 7 weeks after beginning the administration of nicotine (10 mg/200 ml/kg/day) was determined to be less than 5 (n=6), 12.2_+1.2 (n=6), 11.7+1.0 (n=6) and 12.5+1.5 (n=6) ng/ml, respectively. On the other hand, the plasma concentrations of nicotine in the
H. Hamasaki et al.
656 control group ( n = 6 ) was less than 5 ng/ml at 1 day before and 1, 3 and 7 weeks after the administration of the vehicle. The general behavior of the rabbits was normal throughout the 7-week administration periods (from 1 week before to 6 weeks after the endothelial removal). Body weight at 1 week before the endothelial removal was determined to be 1.53-+0.03 kg for the control group ( n = 1 6 ) and 1.60_+0.03 kg for the nicotine group (n=16). These values were not significantly different from each other. Changes in body weight gain, which is given as the difference between the weight at each week after the endothelial removal and the value at 1 week before the operation, are shown in Fig. h The body weight gain at 3, 4 and 5 weeks after the endothelial removal was significantly (P<0.05) lower than the corresponding value in the control group.
Intimal hyperplasia The effect of nicotine on the intimal hyperplasia was estimated by comparing three different parameters: histological findings, DNA content and wet weight of the vessel wall. Results are shown in Table 1. In the sham-operated left carotid artery, no noticeable histological changes could be detected and the ratio (%) of intimal area to intimal area plus medial area was calculated to be 4.6-+0.2% ( n = 6 ) . Six weeks after the endothelial removal, a marked intimal hyperplasia resulting from new accumulation of proliferating smooth muscle cells and connective tissue was observed. The histological parameter was significantly (P
(EIR) remained unaltered in all groups. The relaxation responses to the submaximal concentration of 10 -6 M A23187 and 10 5 M SNP and the effects of nicotine were compared between the control left and the previously denuded right carotid artery strips and are shown in Fig. 3.
L . N M M A and A D M A
Results are shown in Table 2. The L-NMMA and A D M A concentrations were determined to be (4.2_+0.2)× 10 7 and (2.1 -+0.1)x 10-7 M (n=4), respectively, in the control endothelial cells and significantly (P<0.01 and P
Immunoreactive endothelin. 1 contents in the vessel wall In the control left carotid artery from which endothelial cells had been deliberately removed before homogenate was prepared, the irET-1 content was determined to be 168.7-+22.4 pg/g wet weight ( n - 3 ) , which was significantly (P
DISCUSSION The plasma concentrations of nicotine at 1, 3 and 7 weeks after beginning the nicotine administration (10 mg/kg/200 ml/day for 7 weeks) ranged between 11.7 and 12.5 ng/ml, which nearly corre-
Endothelium.dependent and endothelium.independent relaxations In the control left and the previously denuded right carotid artery strips, 10 6 M ACh and 10 -6 M A23187 produced relaxations during the contraction induced by 10 -6 M noradrenaline. The relaxations were abolished after deliberate denudation of the endothelial cells [complete endothelial removal without damage to the smooth muscle layer was confirmed by scanning electron microscopy (Azuma et al., 1990)] or greatly reduced by pretreatment with 10 5 M methylene blue and 10 4 M NOARG, an inhibitor of NO synthesis (Kobayashi and Hattori, 1990) but unaffected by 10 -5 M indomethacin (data not shown), indicating that the ACh- and A23187-induced relaxations are caused, depending on the endothelimn, and mediated by EDRF/NO but not by prosranoids. ACh (10 9-10-5 M) produced a concentration-dependent relaxation of the sham-operated left carotid artery, in which nicotine did not significantly attenuate the magnitude of the relaxation response to ACh. In contrast, the ACh-induced endothelium-dependent relaxation (EDR) was greatly impaired in the previously denuded right carotid artery. The impairment of the ACh-induced EDR tended to be enhanced in the nicotine group (Fig. 2). In addition to the impairment of the ACh-induced EDR in the previously denuded right carotid artery, A23187 (10 9-3 x 10 6 M)induced EDR was also significantly decreased. The decrease was slightly enhanced in the nicotine group. In contrast, the SNP (10 9-3×10 5 M)-induced endothelium-independent relaxation
concentrations in endothelial cells
800
--
400
=o
I
I
I
j
0
2
4
6
Weeks after e n d o t h e l i a l r e m o v a l
F I G U R E 1. Changes in body weight gain of rabbit after the endothelial removal in the control (solid circles) and nicotine (open circles) groups. Body weight at 1 week before the endothelial removal was determined to be 1.53-+0.03 kg ( n = 16) for control group and 1.60_+0.03 kg ( n = 16) for nicotine group. Body weight gain was given as the difference of the value at each week after the endothelial removal from that at 1 week before the operation. Vertical bars show standard errors of the means. T h e asterisk (*) indicates significant difference vs. corresponding control at P
Effect of Nicotine on the Intimal Hyperplasia
657
TABLE 1. Effect of nicotine on the hyperplastic vascular responses to endothelial removal of rabbit carotid arteries Group
I/(I + M)(%)
1. Sham-operated left carotid artery + vehicle 2. Sham-operated left carotid artery + nicotine 3. Denuded right carotid artery + vehicle 4. Denuded right carotid artery + nicotine
DNA (~g/mg wet wt) a Wet wt (mg/50 mm)
4.6 -+ 0.2 (6)
7.0 +- 0.8 (3)
74.3 -+ 4.3 (6)
5.6 -+ 0.2 (6)
7.2 -+ 0.8 (3)
77.7 -+ 4.2 (6)
26.9 -+ 3.7 (6) c
10.8 _+ 0.9 (3) b
96.3 -4- 3.3 (6) c
28.2 +- 1.2 (6) c
11.2 -4- 0.4 (3) c
104.0 +- 5.6 (6) c
Results are given as mean -+ S.E. mean. Endothelial cells were removed before homogenates were prepared in experiment. ~.cSignificant difference vs. corresponding sham-operated control group at P < 0.05 and P < 0.005, respectively. Abbreviations: 1, intimal area; M, medial area. The number in parentheses indicates the number of determinations.
sponds to the plasma levels in human smokers (Itani et al., 1988). Although there is considerable interindividual variability in measured nicotine levels, the mean plasma concentrations in human smokers (10-30 cigarettes smoked per day) are reportedly determined to be 8.6-15.0 ng/ml (Itani et al., 1988). In this respect, we consequently observed the effect of the oral nicotine on the intimal hyperplasia after endothelial removal at concentrations nearly corresponding to those in human smokers. We have demonstrated that there was a strong inverse correlation between the intimal hyperplasia and the amount of EDRF/NO produced by endothelial cells (Niimi et al., 1994). It is well established that EDRF/NO is an antiaggregating substance (Azuma et al., 1986; Radomski et al., 1987) and that the activation of platelets is considered an initiating event for intimal hyperplasia (Ross, 1986; Schwartz et al., 1981). According to Garg and Hassid (1989), the endogenous EDRF/NO may act as an inhibitory modulator of the vascular smooth muscle cell mitogenesis and proliferation. Therefore, it is suggested that the reduced production of EDRF/NO from endothelial cells is one of the important factors causing the intimal
Concentration
of ACh (-log M)
9
8
7
6
5
I
I
I
I
I
SO
FIGURE 2. Comparison of endothelium-dependent relaxation response of control left (solid line) and previously denuded right (dotted line) carotid arteries to acetylcholine (ACh) between vehicle (solid circles) and nicotine (open circles) groups. Each point represents the mean of six determinations. Vertical bars show standard errors of the means. Relaxation is given as a percentage (percent relaxation) to the 10 -6 M noradrenaline-induced contraction. The EDR was significantly different (P<0.005) between control left and previously denuded right carotid arteries of both groups.
hyperplasia after endothelial removal (Azuma et al., 1990, 1992, 1995b; Niimi et al., 1994). Furthermore, cigarette smoking causes endothelial injury including ultrastructural changes in aortic and pulmonary endothelial cells, endothelial cell turnover and endothelial cell function (Booyse et al., 1981; Linet al., 1992; Zimmerman and McGeachie, 1987) and is associated with an impairment of endothelium-dependent arterial dilatation in clinically healthy young adults, consistent with endothelial dysfunction (Celermajer et al., 1993). Long-term smoking reduces the NO-dependent component of basal vascular tone and impairs endothelium-dependentvasodilator response to low-dose endothelin-1 (Kiowski et al., 1994). From this point of view, it seems worthwhile to examine whether or not nicotine modifies the intimal hyperplasia and EDRF/NO production in endothelial cells. The intimal hyperplasia tended to be accelerated but not significantly by nicotine, compared with employing three different parameters--histological findings, DNA content and wet weight of the vessel wall--and the reduced production of EDRF/ NO in the regenerated endothelial cells also tended to be enhanced but not significantly by nicotine. According to Zhu et al. (1993), passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits. Further, Penn et al. (1993) described a significant acceleration in the growth of atherosclerotic plaques associated with passive smoking in young cockerels that were eating a normal, lowcholesterol diet. It seems likely, therefore, that greater enhancement of the impaired EDRF/NO production by endothelial cells after administration of nicotine may result in greater acceleration of the intimal hyperplasia. Recently, we suggested that the accumulation of endogenous inhibitors (NC-monomethyl-L-arginine and asymmetrical NS,N°-dimethyli.-arginine) for NO synthesis was associated with decreased EDRF/NO production from endothelial cells and with intimal hyperplasia (Azuma et al., 1995b). Thus, we determined the changes in concentrations of the methylarginine derivatives in the endothelial cells. The concentrations of L-NMMA and ADMA in the regenerated endothelial cells were significantly higher than those in the control endothelial cells. The accumulation of these methylarginine derivatives in the endothelial cells was significantly accelerated in the nicotine group. Vallance et al. (1992) reported that L-NMMA and ADMA at concentrations of 5 × 10-6 to 3 × 10 4 M inhibited NO biosynthesis by J774 macrophage cytosol in a concentration-dependentmanner. In addition, we have reported that c-NMMA and ADMA inhibit NO production at concentrations of 3 x 10 s to 3 x 10 -4 M in the rabbit carotid arteries (Azuma et al., 1995b). The highest increased concentration of L-NMMA and ADMA in the regenerated endothelial cells of the nicotine group was
658
H. Hamasaki et al. 10-6M A 2 3 1 8 7 0
I
C
D
C
T A B L E 3. Immunoreactive ET-1 (ir-ET-1) content in control left and hyperplastic right carotid arteries and effect of nicotine
10-5M SNP
i
f
D
I
C
D
C
D
Group
-~
1. Sham-operated left carotid artery + vehicle 2. Sham-operated left carotid artery + nicotine 3. Denuded right carotid artery + vehicle 4. Denuded right carotid artery + nicotine
sc
m0 I I Vehicle
I I Nicotine
I I Vehicle
I I Nicotine
F I G U R E 3. Comparison of the relaxation responses of control left (C) and previously denuded right (D) carotid arteries to A 2 3 1 8 7 and sodium nitroprusside (SNP) between vehicle and nicotine groups. Relaxation is given as a percentage (percent relaxation) to the 1 0 -6 M noradrenaline-induced contraction. Vertical bars show standard errors of the means. Each experiment consists of six determinations. T h e asterisks (* and ***) indicate significant difference vs. corresponding control at P < 0 . 0 5 and P < 0 . 0 0 5 , respectively.
tentatively estimated to be 1.3 × 10 -6 and 5.6× 10 7 M, respectively (Table 2), which seems insufficient to produce a complete inhibition of N O synthesis in endothelial cells, resulting in the slightly enhanced impairment of N O production in the nicotine group. T h e increase in plasma endothelin-l, a potent mitogen (Hirata et al., 1989), is thought to reflect a rise in vascular production of the peptide in response to the impaired production of EDRF/NO by endothelial cells (Boulanger and Liischer, 1990). W e have reported that the increased level of endothelin-1 in the vessel wall accompanied by the decreased production of EDRF/NO would be involved in the intimal hyperplasia (Azuma et al., 1994, 1995a; Niimi et al., 1994). In the present experiments, the administration of nicotine tended to augment the increase in endothelin-1 content in the hyperplastic vessel wall. T h e enhanced increase in the endothelin-1 content by nicotine may result from the enhanced impairment of
T A B L E 2. Comparison of L - N M M A and A D M A concentrations in control and regenerated endothelial cells (ECs) and effect of nicotine C o n c e n t r a t i o n ( x 10 7 M) Group 1. 2. 3. 4.
Control ECs Regenerated Control ECs Regenerated
n + vehicle ECs + vehicle + nicotine ECs + nicotine
4 4 4 4
L.NMMA 4.2 6.3 7.9 12.9
-+ 0.2 ± 0.2 ~' -+ 0.7" _+ 0.9 ~'~
ADMA 2.1 2.9 3.4 5.6
--+ -+ -+
0.1 0.2 ~1 0.3 '~ 0.6 ~'~
Results are given as mean +- S.E. mean. ~,.dSignificant difference vs. group 1 at P < 0.005 and P < 0.01, respectively. b.,.Significant difference vs. group 3 at P < 0.005 and P < 0.05, respectively. Significant difference vs. group 2 at P < 0.005. The concentrations of L-NMMA and ADMA were tentatively estimated from the equation between the DNA concentration and the number of endothelial cells and the intracellular water space reported by Baydoun et al. (1990) (see text).
ir-ET- 1 co n t e n t (pg/g wet weight)
168.7 -+ 22.4 (3) 187.5 -+ 3.6 (3) 407.3 -+ 65.1 (3) ~ 480.9 -+ 40.5 (3) b
Results are given as mean -+ S.E. mean. Endothelial cells were removed before homogenates were prepared. ~,.hSignificant difference vs. corresponding value in the sham-operated group at P < 0.05 and P < 0.005, respectively. The number in parentheses indicates the number of determinations.
the EDRF/NO production and may play a role in accelerating the intimal hyperplasia. In conclusion, it seems possible to assume that, although, under the present experimental conditions, nicotine exhibited a tendency to accelerate the intimal hyperplasia after endothelial removal, longer exposure to nicotine or higher doses of the agent or both would significantly accelerate the intimal hyperplasia through the enhanced impairment of EDRF/NO production, which might be brought about by the enhanced increases in L-NMMA and A D M A concentrations, as well as the enhanced increase in endothelin-1 in the vessel wall. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 08672604) and by the Smoking Research Foundation (SRF) for Biomedical Research in Japan. The authors thank Dr. A. Saito for preparation of histological specimens.
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