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Cardiovascular Research 1998 38(3):772-781; doi:10.1016/S0008-6363(98)00053-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Morphological and functional changes in coronary vessel evoked by repeated endothelial injury in pigs

Shu-ichi Saitoh, Tomiyoshi Saito, Takayuki Ohwada, Atsushi Ohtake, Futoshi Onogi, Kazuhiko Aikawa, Kazuhira Maehara and Yukio Maruyama*

First Department of Internal Medicine, Fukushima Medical College, Hikari-ga-oka 1, Fukushima 960-12, Japan

* Corresponding author. Tel.: +81 (245) 48 21 11, ext. 2300; Fax: +81 (245) 48 18 21; E-mail: maruyama@cc.fmu.ac.jp

Received 13 March 1997; accepted 22 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We examined the morphological changes induced by repeated endothelial denudation in coronary artery (CA), as well as functional changes in the endothelium-dependent and smooth muscle responses to various vasoactive agents during the process of intimal thickening. Methods: We observed vascular responses in denuded and non-denuded portions of pig CA while being fed a normal diet (n=11, N group) or 2% cholesterol diet (n=25, C group) to intracoronary acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP), and isosorbide dinitrate (ISDN) with and without the nitric oxide synthesis inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg i.v.) over a period of 8 weeks. Balloon endothelial denudation of the left anterior descending CA was carried out every 2 weeks. Results: In N group, maximum vasoconstriction was obtained with ACh 2 weeks after the first denudation [26±5% vs. 1±1% pre-denudation, p<0.05]. L-NAME did not affect ACh-induced CA diameter changes. Thereafter, the response to ACh was attenuated by repeated denudation in N groups. However, the degree of 5-HT-induced CA narrowing at the denuded portion increased from 7±4% (0 week) to 88±8% (8 weeks) (p<0.05). The changes resulted in severe myocardial ischaemia, and suggested that endothelium-dependent vasodilation was progressively attenuated while hyperreactivity of vascular smooth muscle simultaneously increased. Vasodilation induced by SP was attenuated somewhat, but ISDN-induced vasodilation was preserved. Although mild hypercholesterolaemia was induced in C group, the vascular responses to these vasoactive agents did not differ from those of N group. Conclusions: Repeated CA endothelial injury and regeneration induce the change of morphology and vascular reactivity in the denuded portion regardless of atherogenic diet. This study strongly suggests that intimal thickening caused by repeated endothelial injury and regeneration induces specific vascular responses to vasoactive agents. Moreover, it is also suggested that during the progression of intimal thickening, increased vascular smooth muscle contraction and decreased endothelium-dependent dilation appear in a stimulus-dependent manner, often leading to severe coronary vasoconstriction accompanied with definitive ECG ST change.

KEYWORDS Repeated endothelial injury and regeneration; Pig; Coronary artery; Intimal thickening; Acetylcholine; 5-HT; Substance P; L-NAME


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Recent studies have indicated that endothelial injury plays an important role in coronary artery vasoconstriction, due to endothelium-dependent vasodilatory dysfunction [1, 2]. Under various pathologic conditions such as hyperlipidemia [3], hypertension [4], and atherosclerosis [5], intimal thickening is induced during the regenerative process of injured endothelium [6]. Endothelial injury and subsequent intimal proliferation may enhance the reactivity of the coronary artery to vasoactive agents such as acetylcholine, resulting in severe vasoconstriction [7]. On the other hand, coronary artery vasoconstriction has been related not only to endothelial dysfunction but also to vascular smooth muscle hyperreactivity [8, 9]. Thus, it is still controversial whether attenuation of endothelium-dependent vasodilation or smooth muscle hyperreactivity is more important in the pathogenesis of coronary artery vasospasm. Another unsettled problem is whether repeated vascular endothelial injury promotes the progression of neointima formation and could easily lead to atherosclerosis, and if so, how the vascular responses induced by various vasoactive agents depend on the process or degree of intimal thickening. The reason why repeated vascular endothelial injury must be taken into account in considering the genesis of atherosclerosis, and process of vascular remodeling and abnormal vascular reactivity, is because injury of this type is likely to occur repeatedly over the course of a lifetime as a result of local shear forces and various risk factors (e.g. hypercholesterolaemia, circulating vasoactive amines, immunocomplexes, chemical irritants in tobacco smoke, and infection [10, 11]) or as a result of repeated percutaneous transluminal coronary angioplasty (PTCA) [12].

The purpose of this study was first to clarify in a pig model how intimal thickening, including morphologic changes in endothelial cells, occurs after repeated endothelial injury. Our second goal was to determine the effects of vascular smooth muscle hyperreactivity and defective endothelial function on vasoconstriction in coronary arteries with repeatedly regenerated endothelium. To determine the relative influences of these two mechanisms on coronary vasoconstriction after repeated endothelial injury and endothelial regeneration, we measured coronary artery diameter changes every 2 weeks in denuded and non-denuded portions of pig coronary arteries that were stimulated with acetylcholine and 5-hydroxytryptamine (5-HT), with and without administration of N{omega}-nitro-L-arginine methyl ester (L-NAME), which inhibits nitric oxide biosynthesis [13]. We also examined the coronary artery diameter changes evoked by substance P, an endothelium-dependent vasodilator [14], and isosorbide dinitrate, an endothelium-independent vasodilator [15], with and without L-NAME. These studies were done using 2% cholesterol feeding pigs as well as normal chow pigs, because hypercholesterolaemia has been known to promote atherogenesis [16]or change vascular reactivity [8, 17].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials and surgical preparation
Disease-free pigs (mixture of Landrace and Deroc breeds), weighing 15 to 24 kg (19±0.8 kg), 3.2±0.4 months old, were housed individually under controlled room temperature conditions. Pigs were fed in normal chow (n=13) or containing 2% cholesterol at 30 g/kg per day (n=27) (Nihon Crea., Sendai, Japan) from one week before the first experiment to the final day of each experiment. The concentration of serum cholesterol in the pigs was measured enzymatically at the first experiment and at the end of the series. Following intramuscular injection of ketamine hydrochloride (10 mg/kg) followed by intravenous infusion of 2 mg/kg sodium thiopental, pigs were intubated and connected to a ventilator through which an oxygen mixture was administered. Anaesthesia was maintained with 1% to 2% halothane. Arterial levels of pO2 and pCO2 were kept within physiologic ranges (pH 7.35 to 7.45; pO2, 90 to 120 mmHg; pCO2,35 to 45 mmHg) by adjusting the volume or frequency of ventilation, use of supplemental oxygen, and intravenous infusion of sodium bicarbonate as necessary. Body temperature was maintained at 37°C with a heated blanket. Under aseptic conditions, an incision was made to expose the left (for the first to the third denudation procedure) or the right (for the fourth denudation to the final experiment) carotid artery. After isolation of the carotid artery, an 8 Fr sheath introducer (Termo, Tokyo, Japan) was inserted. Aortic pressure from the side line of the introducer and electrocardiograms from limb (II, aVF) and precordial leads (V1,5) were continuously monitored on a multichannel thermal recorder (Nihon Koden, Polygraph System, Tokyo, Japan) and stored on a tape with the use of a data recorder (PC-108M, SONY) for subsequent analysis. After the end of each experimental procedure, the carotid artery incision was repaired, and 1 g flomoxef sodium (Shionogi, Osaka, Japan) and 600 mg clindamycin phosphate (Nihon-Upjohn, Tokyo, Japan) were administered intravenously and intramuscularly, respectively, daily for 3 days from the day of each experiment to prevent infection.

2.2 Compound dose selection
In a preliminary study, we determined the optimal dose of acetylcholine. Acetylcholine (Ovisot, Daiichi, Tokyo, Japan) at doses of 0.1, 1, and 5 µg/kg was administered by intracoronary infusion in anaesthetized, intubated pigs, and changes in coronary diameter (Table 1), heart rate, and aortic blood pressure were measured. The results showed that there was no vasoconstriction by 0.1 µg/kg acetylcholine. When acetylcholine 5 µg/kg was administered before denudation on the first experimental day, a severe delay in filling by contrast medium was observed on coronary angiography at 1 min following drug infusion; it was therefore impossible to determine diameter changes in either the denuded or non-denuded portion from the angiogram. Furthermore, the mean aortic blood pressure dropped from 101±8 mmHg to 56±4 mmHg (n=6, p<0.05). We then defined an acetylcholine dose of 1 µg/kg as suitable for observing vascular responses since this dose freed us from concerns regarding haemodynamic changes. Doses of 5-HT, substance P, and isosorbide dinitrate were used as in previous reports [2, 18–21], in which the expected effect of each drug following intracoronary administration was attained without any changes in systemic haemodynamics. The dose of L-NAME was also derived from previous reports [13, 22], in which the inhibition effect of nitric oxide synthesis was maintained over 90 min.


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  		Table 1 Percentages of dose dependent vasoconstrictive changes in coronary artery diameters induced by acetylcholine with denudation repeated every 2 weeks for 6 weeks (i.e. maximum of four of denudations)

 
2.3 Experimental protocol
Prior to selective coronary angiography, lidocaine (20 mg, Fujisawa Pharm., Osaka, Japan) was administered intravenously to prevent ventricular fibrillation, in addition to heparin (150 IU/kg). Heparin, 50 IU/kg, was also administered hourly to prevent blood coagulation. After making a control recording of the coronary diameter, vasoactive agents [acetylcholine, 1 µg/kg, 5-HT, 10 µg/kg (Wako Pure Chemical, Tokyo, Japan), substance P, 2 ng/kg (Sigma Chemical, USA), and isosorbide dinitrate, 25 µg/kg (Eisai, Tokyo, Japan)] were infused over a period of 30 s into the left main coronary artery. The order of administration of acetylcholine, 5-HT, and substance P was randomized. Isosorbide dinitrate was administered last because of its long half-life. All drugs were diluted with physiologic 0.9% NaCl solution to a volume of 1 ml. The same amount of 0.9% NaCl was used to flush residual drug from the Judkins-type catheter. During the administration of each drug, the catheter position was fixed in the left main coronary artery. Serial coronary angiography was performed 1 min after beginning administration of acetylcholine, substance P, and isosorbide dinitrate and 3 min after 5-HT [1]. There was at least a 15 min interval between completion of infusion of one drug and administration of the next. After administration of each drug alone, L-NAME (10 mg/kg, Sigma Chemical, USA) dissolved in 100 ml 0.9% NaCl solution was administered for 30 min intravenously [13], and acetylcholine, 5-HT, substance P, and isosorbide dinitrate were administered again. Next, balloon endothelial denudation was performed in the left anterior descending coronary artery using a 2 Fr Fogarty catheter (Baxter, USA) after intravenous administration of 0.5 mg diltiazem (Tanabe, Osaka, Japan) and 10 mg procainamide (Daiichi, Tokyo, Japan) to prevent lethal coronary spasm. Denudation was performed by inserting the Fogarty catheter into the left anterior descending coronary artery through a 6 Fr with 50 cm length hand-crafted Judkins-type catheter under fluoroscopic guidance. The catheter balloon was then inflated with 0.05 ml of Hexabrix (Tanabe, Osaka, Japan) and 0.05 ml of 0.9% NaCl solution, taking care not to distend the artery with the balloon. After balloon inflation, three denudations were performed over a 2 cm length at the same portion of the artery. The pressure of the inflated balloon in these procedures was less than 0.5 atm, resulting in mild injury damage to the subendothelium [23, 24]. Thirteen pigs fed a normal chow (castrated male=6, female=7) and twenty-seven pigs (castrated male=12, female=15) in 2% cholesterol feeding were used in the repeated denudation study. Two of the 13 pigs in normal chow and two of the 27 pigs in 2% cholesterol feeding were killed immediately after endothelial denudation and their coronary vessels were examined histologically. The effectiveness of the denudation technique and lack of apparent vascular smooth muscle damage in the vascular wall were confirmed in this examination. In the remaining 36 pigs, the denudation protocol was repeated every 2 weeks from week 0 until week 6. Coronary denudation, which was usually carried out after examining the vasoactive response to the various drugs mentioned above, was not performed at week 8.

2.4 Analysis of coronary angiograms
Left coronary angiograms were obtained in a left lateral projection by manually injecting 3 ml of Hexabrix 320 contrast medium through a 6 Fr right Judkins-type catheter. To obtain this angiogram, the catheter was inserted into the orifice of the left coronary artery through the sheath introducer under fluoroscopic guidance. Data were recorded on S-VHS video tape. The posture of the pig and the distance between the pig and image intensifier were kept constant throughout the experiment. Coronary artery diameter (CoD) changes following intracoronary administration of vasoactive agents were estimated prior to endothelial denudation at the 0 week, 2 weeks, 4 weeks, and 6 weeks timepoints and 2 weeks after the 6 weeks denudation. CoD analysis was performed by projecting the videotape of each coronary angiogram onto a viewing monitor (Ikegami Picture Monitor PM14-3H, Ikegami Tsushinki, Tokyo, Japan), and measuring CoD in the denuded and non-denuded portions in end-diastole using an automated edge contour detection computer system (Densitometric Analyzer CAD-98 ELK Angio System, Elk Medical Products, Tokyo, Japan). The criterion for selecting a segment of the left anterior descending coronary artery for measurement was that focal hyperconstriction or dilation in response to the drugs was at a maximum; a region from the left circumflex coronary artery with a similar baseline diameter, was selected as a control portion. Changes in the CoD induced by vasoactive agents were calculated as follows: percentage change in diameter=(1–diameter after AA/diameter before AA)x100, where AA is intracoronary administration of the agents mentioned above.

The absolute value of the diameter in mm was obtained using a 6 Fr catheter as a reference. To determine whether there was interobserver or intraobserver variability, the readings were performed by uniformed observers or by providing video tapes to two of three cardiologists conducting this study (S. S., T. O., A. O.). We found excellent correlations between repeated measurements (r=.98, p<0.001) and between different observers (r=.96, p<0.001). Difference in percent changes determined by two observers was 0.8±1.2% (NS) and 2.2±1.4% (NS) at the denuded and non-denuded portions.

2.5 Morphology
Pigs were sacrificed after the in vivo studies by a lethal dose of intravenously administered pentobarbital, and tissue samples of denuded and non-denuded portions obtained from five of 10 pigs at 4 weeks and 5 pigs at 8 weeks in a normal chow and five of 21 pigs at 4 weeks and 5 of 13 pigs at 8 weeks in 2% cholesterol feeding were used for histological examination. Tissue samples were fixed with 10% formaldehyde, processed with paraffin, then cut into 3 µm thick sections and stained using the Elastica–van Gieson procedure for differential staining of elastic fibers and cells. Areas of intima and media were measured with a computer image analyzing system (PAB Mitsubishi-Kaisei, Japan). The intima to media area ratio was calculated as follows: intima area (mm2)/media area (mm2). In addition, at 8 weeks after experiment denuded and non-denuded tissue samples obtained from 5 pigs in normal chow and 2% cholesterol feeding respectively were fixed with 2% glutaraldehyde and dried, longitudinally bisected, mounted on aluminum studs, coated with gold, and examined using scanning electron microscope (Hitachi H-430, Japan).

2.6 Statistical analysis
All results were expressed as means±SEM. The statistical significance of differences between groups was evaluated by Student's t test for paired or unpaired data. Comparisons of haemodynamic parameters and coronary vascular responses prior to and following administration of drugs were carried out using two-way ANOVA, followed by Scheffe's multiple comparison test. Differences between values were considered to be statistically significant at p<0.05. All experiments using animals were performed according to the guidelines of Fukushima Medical College, the Japanese Government Animal Protection and Management Law (No. 105), and the Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Mortality
Eight of 36 pigs used in the repeated denudation study died before completion of the entire protocol, all deaths were due to ventricular fibrillation. Two death occurred immediately after the first coronary artery denudation, three at 2 weeks, one at 4 weeks, and two at 6 weeks.

3.2 Body weight, serum cholesterol, and coronary artery diameter
Mean body weight increased from 19±0.8 kg at the first denudation experiment to 26±2.8 kg (p<0.05) (normal chow pigs) and to 30±3.2 kg (p<0.05) (2% cholesterol fed pigs) after 8 weeks. In 2% cholesterol fed pigs, mean serum cholesterol concentration increased from 1.43±0.15 to 4.75±0.51 mmol/l (p<0.05) after the same period. The baseline mean coronary diameters of the denuded and non-denuded control portions were 2.1±0.2 and 2.2±0.3 mm before intervention and 2.3±0.4 and 2.5±0.3 mm after 8 weeks, respectively. Intravenous administration of L-NAME did not change the basal coronary diameters of the denuded and non-denuded control portions throughout the experimental period. Angiography failed to reveal organic coronary stenosis in the denuded portions.

3.3 Systemic haemodynamic responses to vasoactive agents
The mean aortic pressure and heart rate after intracoronary administration of each agent did not change throughout the experimental period not only in 2% cholesterol fed pigs but also in normal chow pigs (data not shown); pretreatment with intravenous administration of L-NAME tended to increase aortic pressure and to decrease heart rate, but not significantly.

3.4 Vascular reactivity studies using vasoactive agents in normal chow pigs
3.4.1 Acetylcholine study (Table 2)
In the baseline study prior to denudation at week 0, with and without L-NAME, neither vasodilation nor vasoconstriction was observed in the left anterior descending coronary artery or left circumflex coronary artery after intracoronary administration of acetylcholine (1 µg/kg). Two weeks after the first denudation in the left anterior descending coronary artery, the mean extent of vasoconstriction was found to be greater in the denuded portion than in the non-denuded portion (26±5% vs. 8±3%, p<0.05). However, 8 weeks after the first denudation, there was no difference in the mean extent of vasoconstriction between the repeatedly denuded portion and the non-denuded portion (6±4% vs. 9±2%, NS). With regard to the time course of changes in acetylcholine-induced vasoconstriction, however, the pattern was different between the denuded and non-denuded portions. In the denuded portion, maximum vasoconstriction appeared at 2 weeks, and thereafter this response was gradually attenuated with time during repeated endothelial denudation (8 weeks: 6±4%, p<0.05 vs. 2 weeks). On the other hand, vasoconstriction in the non-denuded portion of the left circumflex coronary artery was not different between at 2 weeks and at 8 weeks (2 weeks:8±3% vs. 8 weeks: 9±2%, NS). L-NAME did not change this acetylcholine-induced vasoconstriction both in the repeatedly denuded and non-denuded portions at all experimental timepoints.


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  		Table 2 Changes in coronary artery diameter evoked by vasoactive agents before and after intravenous administration of 10mg/kg L-NAME in normal chow pigs

 
3.4.2 5-HT study (Table 2, Fig. 1)
In the baseline study without any endothelial denudation (0 week), there was no vasoconstriction or vasodilation induced by 5-HT. 5-HT-induced vasoconstriction was augmented along the repeatedly denuded portion of the left anterior descending coronary artery (88±8%) after 8 weeks (p<0.05 vs. 0 week). Thus, coronary spasm was induced by 5-HT following four denudation procedures and this was associated with ST segment elevation in lead V5 (data not shown). In the repeatedly denuded portion, L-NAME augmented 5-HT-induced vasoconstriction at 0 and 2 weeks, but the degree of augmentation gradually decreased with time. At 8 weeks, L-NAME did not enhance 5-HT-induced vasoconstriction (88±8% without L-NAME vs. 90±6% with L-NAME). With regard to the non-denuded portion of the left circumflex coronary artery, 5-HT-induced vasoconstriction was also increased at 8 weeks (20±4%, p<0.05 vs. 0 week), and L-NAME enhanced 5-HT-induced vasoconstriction until the final weeks. To estimate CoD changes due separately to L-NAME-induced decreases in endothelium (nitric oxide)-dependent vasodilation or to an increase of vascular smooth muscle hyperreactivity, the following calculations were done using the data of 5-HT induced changes in coronary artery inner diameter (Table 2).


Figure 1
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  		Fig. 1 CoD changes due to L-NAME on 5-HT-induced decreases in endothelium-dependent vasodilation, as well as CoD changes due to an increase of vascular smooth muscle hyperreactivity induced by 5-HT administration, are shown separately in the repeatedly denuded portion. Open circle ({circ}): Estimated CoD changes (percent) due to endothelium-dependent vasodilatory changes (indicated negative values on the y axis, left), Open square ({square}): Estimated CoD changes (percent) due to vascular smooth muscle hyperreactivity changes (indicated positive values on the y axis, right).

 
(1). Endothelium-dependent vasodilation; CoD value after intracoronary administration of 5-HT with L-NAME in the repeatedly denuded portion was subtracted from the corresponding value after intracoronary administration of 5-HT without L-NAME for each timepoint. (2). Vascular smooth muscle hyperreactivity; the CoD value after intracoronary administration of 5-HT with L-NAME in the non-denuded portion of the circumflex coronary artery was subtracted from the corresponding value obtained with L-NAME and 5-HT at the repeatedly denuded portion of the left anterior descending coronary artery for each timepoint. As shown in Fig. 1, endothelium-dependent vasodilation rapidly decreased after two denudations and became almost zero after four denudations, whereas smooth muscle hyperreactivity gradually increased until three denudations had been performed and thereafter showed no change.

3.4.3 Substance P study (Table 2)
Without L-NAME, in the repeatedly denuded portion of the left anterior descending coronary artery, substance P-induced vasodilation was gradually attenuated with time (0 week: –19±3% to 8 weeks: –9±2%, p<0.05). On the other hand, in the non-denuded portion of the left circumflex coronary artery, substance P-induced vasodilation was unchanged throughout the experiment. Substance P-induced vasodilation which was partially blocked by L-NAME decreased with time in the denuded portion. As a result, at 8 weeks, substance P-induced vasodilation in the denuded portion was not altered by L-NAME (–9±2% without L-NAME vs. –7±3% with L-NAME). However, in the non-denuded portion, L-NAME attenuated or tended to attenuate substance P-induced vasodilation at all experimental timepoints.

3.4.4 Isosorbide dinitrate study (Table 2)
Isosorbide dinitrate-induced vasodilation was not different between denuded and non-denuded portions throughout the experiment. L-NAME did not affect isosorbide dinitrate-induced vasodilation at the repeatedly denuded portion and non-denuded portions.

3.5 Vascular reactivity studies using vasoactive agents in 2% cholesterol fed pigs (Table 3, Fig. 2)
Changes in vascular responses induced by acetylcholine, 5-HT, substance P, and isosorbide dinitrate with and without L-NAME were not different between normal fed and 2% cholesterol fed pigs throughout the experiment. Coronary vasospasm, which was shown in normal diet pigs, was also induced by 5-HT following four denudation procedures (8 weeks) and this was associated with ST segment elevation in lead V5.


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  		Table 3 Changes in coronary artery diameter evoked by vasoactive agents before and after intravenous administration of 10mg/kg L-NAME in 2% cholesterol fed pigs

 

Figure 2
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  		Fig. 2 Coronary angiograms and electrocardiograms obtained after 5-HT (10 µg/kg) administration at week 0 (A) and 8 weeks (B) in 2% cholesterol fed pigs. Focal narrowing is obvious along the ST elevation on the electrocardiogram with the V5 lead (arrow indicates the site of spasm) (B).

 
3.6 Morphologic change
In the light microscopic examination, the intima to media area ratio in the repeatedly denuded portion increased both in normal chow pigs (4 weeks:0.14±0.05 vs. 8 weeks: 0.30±0.06, p<0.05) and in 2% cholesterol fed pigs (4 weeks:0.13±0.03 vs. 8 weeks: 0.28±0.04, p<0.05) (Fig. 3A, upper panel). In the non-denuded portion, intimal thickening had hardly appeared at 4 weeks, and a thin intimal layer had appeared at 8 weeks [intima to media ratio: 0.04±0.02 in normal chow pigs, 0.08±0.04 in 2% cholesterol fed pigs (Fig. 3B, upper panel)]. There was no difference in the intima to media ratio between pigs fed 2% cholesterol chow and pigs fed normal chow with respect to either the denuded or non-denuded portion. In scanning electron micrographs of luminal surfaces of coronary arteries at 2 weeks after the 6 weeks denudation in normal chow and 2% cholesterol fed pigs, endothelial cells covered the surface of the lumen in the denuded coronary artery, as was also the case with in the non-denuded one (Fig. 3A and 3B, lower panel). In the denuded portion (Fig. 3A, lower panel), however, abnormal multishaped endothelial cells of varying sizes without prominences appeared. On the other hand, in the non-denuded portion, regular spindle-shaped endothelial cells bearing the prominences were observed.


Figure 3
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  		Fig. 3 Upper panel: Results obtained at 8 weeks in 2% cholesterol fed pigs with Elastica–van Gieson staining of the repeatedly denuded (A) and non-denuded (B) portions of coronary arteries under light microscopy (x200). Lower panel: Scanning electron microscopy showing the luminal surface of the coronary artery (x1000). A: Repeatedly denuded portion; the intima is considerably thickened in the light microscope, and abnormal multishaped endothelial cells of varying sizes can be seen in the repeatedly denuded portion in the electron microscope. B: Non-denuded portion; the region is covered with normal endothelium in the electron microscopy.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study was undertaken to clarify how multiple endothelial injury and regeneration alter vascular morphology and affect coronary artery responses to various stimuli. The major findings of the present study are that repeated cycles of endothelial injury and regeneration accelerate intimal thickening which consisted of human {alpha}-actin positive smooth muscle cells and neutrophils (data not shown) and morphological changes in endothelial cells resulting in an alteration of the vascular response to various vasoactive agents in the repeated area. Specifically, we found that hypercontraction by acetylcholine was limited to the early stages of denudation, whereas that induced by 5-HT progressively increased in intensity with repeated denudations (Tables 2 and 3Go). The bimodal response to acetycholine with time is thought to be related to a decrease in vascular smooth muscle hypercontractility, and hyperreactivity to 5-HT is presumed to be due to acquisition of vascular smooth muscle hypercontractility, as well as to the development of endothelial dysfunction.

With respect to acetylcholine-induced responses, the most important difference from the previous single denudation model [21, 25]is that absolutely no vasoconstriction with acetylcholine was noted in the third or fourth denudation procedure, despite the appearance of significant intimal thickening. It has been suggested that endothelium-dependent vasodilation is largely mediated by nitric oxide, and its synthesis through nitric oxide synthase is competitively inhibited by L-arginine analogues such as L-NAME [26]. Accordingly, we examined acetylcholine-induced vascular responses in the presence and absence of L-NAME, even though acetylcholine does not dilate pig coronary arteries due to its weak ability to stimulate endothelium-dependent relaxation (Tables 1 and 2Go, 3) [27, 28]. As a result, L-NAME did not influence acetylcholine-induced vascular responses (Tables 2 and 3Go), suggesting that nitric oxide-dependent vasodilation is related neither to acetylcholine-induced narrowing in the denuded portion at 2 weeks nor to the attenuation of this narrowing with time. Moreover, isosorbide dinitrate-induced vasodilation was maintained until the final experiment, which suggests that the responsiveness of smooth muscle to exogenous nitric oxide is not altered with repeated endothelial injury. Accordingly, the attenuation of vessel narrowing following acetylcholine administration probably reflects changes in the expression of muscarinic receptor in the smooth muscle itself. It is unlikely that acetylcholine induces vasoconstriction via {alpha}-adrenergic transmission or the production of cyclooxygenase-dependent vasoconstricting substances [29], but the mechanism underlying its action in this process remains to be clarified.

5-HT has a dual effect on vascular smooth muscle; it causes direct constriction as well as indirect endothelium-dependent relaxation of this tissue [30]. In this study, in contrast to the non-denuded region, the augmentation of 5-HT-induced vasoconstriction by L-NAME was gradually attenuated with time in the portion which had been repeatedly denuded (Tables 2 and 3Go). Namely, 5-HT-induced nitric oxide-dependent vasodilation progressively decreased with repeated cycles of denudation and endothelial regeneration, and had almost disappeared by week 8 with the concomitant loss of substance P- induced nitric oxide- dependent vasodilation. In contrast, 5-HT-induced smooth muscle hyperreactivity increased until 6 weeks and thereafter reached near plateau (Fig. 1), assuming that the estimation of endothelium-dependent and -independent responses to 5-HT described in the Results section is valid, and that L-NAME inhibits endothelial nitric oxide synthesis alone and does not affect smooth muscle function in vivo [8, 31]. Thus, this investigation of the role played by the endothelium in the multiple endothelial injury model suggests that the attenuation of endothelium-dependent vasodilation may contribute greatly to the development of coronary vasospasm, compared to the contribution made by smooth muscle hypercontraction. This phenomenon may be partly related to the morphological changes in the regenerated endothelium, as shown in Fig. 3. However, the exact mechanism by which the restoration of nitric oxide-producing capacity is rendered less effective, in spite of the fact that the endothelial layer is repaired after each injury, remains to be clarified. In addition, it is of interest that maximum hypercontraction was obtained after approximately four denudations, demonstrating that the difference between single [8]and multiple denudation models is large with respect to the vascular constrictive response to 5-HT. In our model, 5-HT-induced vasoconstriction at 8 weeks after four denudation procedures was markedly suppressed by 10 µg/kg ketanserin (5HT2 antagonist) administered by the intracoronary route 10 min before 5-HT infusion (data not shown). These data suggest that 5-HT exerts its dominant vasoconstrictive action through 5HT2 receptors in this vasospasm model. However, there is some controversy as to the dominant 5-HT receptor subtype [32–34]. Moreover, it is needed to determine the contribution of receptor sensitivity in mediating changes in the 5-HT-induced vasomotor response in different types of smooth muscle cells in neointima.

There are further several issues which should be discussed.

First, although there have been no data showing how multiple endothelial injury alters spontaneous vascular tone or responsiveness to vasoactive substances, we performed a total of four denudation procedures spaced two weeks apart. Because it is conceivable that in models of multiple endothelial injury and regeneration, the progression of intimal thickening might be enhanced since even a single endothelial injury has been reported to produce intimal thickening in the carotid artery [35].

Second, since vascular responsiveness to various vasoactive agents was not different between pigs fed normal chow and pigs fed a 2% cholesterol diet, in terms of either denuded or non-denuded portions, the level of hypercholesterolaemia in this study might not be high enough to affect the vascular reactivity or morphological changes, at least within the period observed. We must also consider that the duration of hypercholesterolaemia may be short. In this study, we mainly examined not the effect of hypercholesterolaemia on vascular responses but the effect of repeated endothelial injury on vascular responses.

Third, it was clearly shown in our study that the natural process of ageing causes time-dependent changes in vascular reactivity. As far as we know, there is little study concerning effects of ageing on vascular responsiveness to vasoactive agents. However, it should be noted that effects of ageing on vascular responses may differ depending on the generation tested, and therefore further study is needed to apply the present results obtained from the relatively young generation to the old one.

Finally, the present study clearly shows that intimal thickening in pig coronary artery following repeated endothelial injury and regeneration induced specific responses to different vasoactive agents over time. These results suggest that abnormal types of specific agonist-endothelium or -smooth muscle interactions, which may occur in various disease states throughout life and are associated with repeated endothelial regeneration, appear to play an important role in the genesis of changes in coronary arterial tone.

Time for primary review 28 days.


   Acknowledgements
 
We gratefully acknowledge the support of Dr. Kiichi Sato, Jinsenkai Medical Institute, Fukushima, in the care of our experimental animals. We also wish to thank Hitomi Nakamura for her assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Zeiher AM, Drexler H, Wollschlager H, Just H. Modulation of coronary vasomotor tone in humans: Progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation (1991) 84:319–340.
  2. Yasue H, Horio Y, Nakamura N, Fujii H. Induction of coronary spasm by acetylcholine in patients with variant angina. Circulation (1986) 74:955–963.[Abstract/Free Full Text]
  3. Florentin RA, Nam SC, Lee KT, Lee KJ, Thomas WA. Increased myotonic activity in aortas of swine. Arch Path Lab Med (1969) 88:463–469.
  4. Schwartz SM, Benditt EP. Aortic endothelial cell replication: Effects of age and hypertension in the rat. Circ Res (1977) 41:248–255.[Abstract/Free Full Text]
  5. Stary HC, Blankenhorn DH, Chandler AB, et al. A definition of the intima of human arteries and its atherosclerosis-prone regions. Circulation (1992) 85:391–405.[Free Full Text]
  6. Shimokawa H, Flavahan NA, Vanhoutte PM. Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries. Circ Res (1989) 65:740–753.[Abstract/Free Full Text]
  7. El-Tamimi H, Mansour M, Wargovich TJ, et al. Constrictor and dilator response to intracoronary acetylcholine in adjacent segments of the same coronary artery in patients with coronary artery disease. Circulation (1994) 89:45–51.[Abstract/Free Full Text]
  8. Fukai T, Egashira K, Hata H, et al. Serotonin-induced spasm in a swine model. Circulation (1993) 88:1922–1930.[Abstract/Free Full Text]
  9. Kuga T, Egashira K, Mohri M, et al. Bradykinin is impaired at the atherosclerotic site but is preserved at the spastic site of human coronary arteries in vivo. Circulation (1995) 92:183–189.[Abstract/Free Full Text]
  10. Fuster V, Lewis A. Mechanism leading to myocardial infarction: Insights from studies of vascular biology. Circulation (1994) 90:2126–2146.[Abstract/Free Full Text]
  11. Ross R. The pathogenesis of atherosclerosis. New Engl J Med (1986) 314:488–500.[Web of Science][Medline]
  12. Kitazume H, Kubo I, Iwama T, Ageishi Y. Repeat coronary angioplasty as the treatment of choice for restenosis. Am Heart J (1996) 132:711–712.[CrossRef][Web of Science][Medline]
  13. Canty JM Jr, Schwartz JS. Nitric oxide mediates flow-dependent epicardial coronary vasodilation to changes in pulse frequency but not mean flow in conscious dogs. Circulation (1994) 89:375–384.[Abstract/Free Full Text]
  14. Toda N, Okamura T. Endothelium-dependent and -independent responses to vasoactive substances of isolated human coronary arteries. Am J Physiol (1989) 257:988–995.
  15. Harrison DG, Bates JN. The vasodilators: new idea about old drugs. Circ Res (1993) 87:1461–1467.
  16. Florentin RA, Nam SC. Dietary-induced atherosclerosis in miniature swine: I. Gross and light microscopy observation: Time of development and morphologic characteristics of lesion. Exp Mol Pathol (1968) 8:263–301.[CrossRef][Web of Science][Medline]
  17. Egashira K, Hirooka Y, Kai H, et al. Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia. Circulation (1994) 89:2519–2524.[Abstract/Free Full Text]
  18. Faggiotto A, Ross R, Harker L. Studies of hypercholesterolemia in the nonhuman primates. Arteriosclerosis (1984) 4:323–340.[Abstract/Free Full Text]
  19. Chu A, Cobb FA. Vasoactive effects of serotonin on proximal coronary arteries in awake dogs. Circ Res (1987) 61:II81–II87. (Supp II).[Medline]
  20. Yamamoto H, Yoshimura H, Noma M, et al. Preservation of endothelium-dependent vasodilation in the spastic segment of the human epicardial coronary artery by substance P. Am Heart J (1992) 123:298–303.[CrossRef][Web of Science][Medline]
  21. Hayashi Y, Tomoike H, Nagasawa K, et al. Functional and anatomical recovery of endothelium after denudation of coronary artery. Am J Physiol (1988) 254(23):H1081–H1090.[Web of Science][Medline]
  22. Node K, Kitakaze M, Kosaka H, et al. Plasma nitric oxide endoproducts are increased in the ischemic canine heart. Biochem Biophys Res Commun (1995) 211:370–374.[CrossRef][Web of Science][Medline]
  23. Santoian EC, Gravanis MB, Schneider JE. Use of the porous balloon in porcine coronary arteries: Rationale for low pressure and volume delivery. Catheter Cardiovasc Diagn (1993) 30(4):348–354.[Web of Science][Medline]
  24. Webster MW, Chesebro JH, Hera SM, Mruk JS, Grill DE, Fuster V. Effect of balloon inflation on smooth muscle cell proliferation in the porcine carotid artery. J Am Coll Cardiol (1990) 15(2):165A.
  25. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature (1980) 288:373–376.[CrossRef][Medline]
  26. Rees DD, Palmer RMJ, Schulz R, Hodson JF, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol (1990) 101:746–752.[Web of Science][Medline]
  27. Kawamura A, Fujiwara H, Ishida M, et al. Protective effects of nipradiol, isosorbide dinitrate, and bunazosin on coronary artery constriction induced by intracoronary injection of acetylcholine in pigs. Cardiovasc Res (1990) 24:1013–1019.[Web of Science][Medline]
  28. Hata H, Egashira K, Fukai T, et al. The role of endothelium-derived nitric oxide in acetylcholine-induced coronary vasodilation in closed-chest pigs. Coron Artery Dis (1993) 4:891–898.[Web of Science][Medline]
  29. Nakayama K, Osol G, Halpern W. Reactivity of isolated resistance porcine coronary arteries to cholinergic and adrenergic drugs and transmural pressure changes. Circ Res (1988) 62:741–748.[Abstract/Free Full Text]
  30. Cappelli-Bigazzi M, Nuno DW, Lamping KG. Evidence of a role for compounds derived from arginine in coronary response to serotonin in vivo. Am J Physiol (1991) 261:H404–H409.[Web of Science][Medline]
  31. Nathan C. Nitric oxide as a secretary product of mammalian cells. FASEB J (1992) 6:3051–3064.[Abstract]
  32. Mc Fadden EP, Clarke JG, Davies GJ, Kaski JC, Haider AW, Maseri A. Effect of intracoronary serotonin on coronary vessels in patients with stable angina and patients with variant angina. New Engl J Med (1991) 324:649–655.
  33. Kaumann AJ, Frenken M, Posival H, Brown AM. Variable participation of 5-HT1-like receptors and 5-HT2 receptors in serotonin-induced contraction of human isolated coronary arteries. Circulation (1994) 90:1141–1153.[Abstract/Free Full Text]
  34. Simokawa H, Vanhoutte PM. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and in atherosclerosis. Circ Res (1989) 64:900–914.[Abstract/Free Full Text]
  35. Krasinski K, Spyridopoulos I, Asahara T, van der Zee R, Isner JM, Losordo DW. Estradiol accelerates functional endothelial recovery after arterial injury. Circulation (1997) 95:1768–1772.[Abstract/Free Full Text]

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