INTRODUCTION

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MUCH EFFORT has been put into the evaluation of underlying pathophysiological processes leading to coronary restenosis after percutaneous transluminal coronary angioplasty (PTCA) procedures. However, the experimental data on vascular remodeling are mainly based on experimental studies using "overstretch" models to create vascular injuries and endothelial disintegrity followed by neointima formation (13, 24). In contrast, there is no information on the long-term impact of coronary devices with low-pressure balloons on vascular morphology and function. Nevertheless, these devices are increasingly used in angioscopy, coronary brachytherapy, and coronary embolization containment devices or as intracoronary shunts in minimally invasive bypass surgery (16, 19, 20, 27). There are two potential mechanisms by which these devices could lead to vascular injury, first, by mechanical vessel trauma and second, by inducing endothelial dysfunction due to endothelial ischemia during the balloon inflation or endothelial denudation during manipulation with the balloon. The aim of the present study was to evaluate whether low-pressure balloon placement in a coronary artery leads to long-term alterations of the vessel wall comparable to those seen after PTCA procedures.

	METHODS

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All animal studies including animal care and experiments complied with the guidelines of the National Institutes of Health for the use and care of laboratory animals. All animal studies were approved by the local ethical committee (Tierschutzkommission der Bezirksregierung Rheinhessen-Pfalz).

Animal model. Twelve German landrace pigs (mean weight 34.6 ± 0.2 kg) were included in the study. After premedication with azaperone (2 mg/kg body wt im; Stresnil, Janssen) general anesthesia was performed with metomidate (5 mg/kg body wt iv; Hypnodil, Janssen) and piritramid (0.5 mg/kg body wt iv; Dipidolor, Janssen). Muscle relaxation was achieved with carbacholine (0.05 mg/kg body wt). The pigs were mechanically ventilated with a mixture of 25% O₂-75% N₂O (Servo 900; Siemens) to provide normal blood gas readings (ABL 500; Radiometer, Copenhagen, Denmark). The right carotid and femoral arteries and the right jugular vein were dissected, and 8-F sheaths were put into each vessel. Afterwards, each animal received a bolus injection of heparin (5,000 IU iv). An 8-F guiding catheter (SHJL 3.5, Tourguide; ACS) was inserted through the right carotid artery for an angiography of the left coronary artery. Another 8-F catheter (Sherpa MB1; Medtronic) was positioned with its tip in the medial part of the great cardiac vein, and the position was checked by fluoroscopy. This catheter was used to withdraw blood to measure plasma creatine kinase, lactate, and endothelin-1 concentration in the coronary sinus. In addition, the same parameters were measured from blood samples taken in the ascending aorta to calculate the arteriovenous difference of each parameter. Endothelin-1 was measured as previously described (7). All blood samples were taken at baseline before, after 30 min of active coronary perfusion, and after 30 min of native reperfusion of the coronary vessel.

Histological studies. Frozen segments were cut into four slices of 10-µm thickness taken every 1 mm from the coronary segments. Afterwards, two of the four samples were stained with elastica-van Gieson and hematoxylin-eosin staining, respectively. The photomicrographs of each sample were digitized (Windigi 1.0; Rosenberg, Rosenheim, Germany) and calibrated with a micrometer slide. The area surrounded by the external elastic lamina (EEL), the area surrounded by the internal elastic lamina (IEL), and the luminal area were assessed by planimetry. The media area was then calculated by subtracting the IEL area from the EEL area. The neointima area was calculated by subtracting the lumen area from the IEL area (22). Because the vessels had no calcification or plaque burden, the neointima area (as assessed by the calculation) exclusively contained neointima. The media-to-lumen ratio was calculated by dividing the media area by the lumen area. We measured the proximal one-third of the ballooned segments to assess the luminal diameter (with the IEL as the border). To evaluate the accuracy of the method, 48 coronary segments without neointima were taken, the endothelial border to the vessel lumen was traced two times, and the difference between the measurements was calculated. Afterwards, the coefficient of variation between those differences was calculated.

Vascular reactivity studies. Vessels were cleaned of connective tissue and cut into rings (3 mm in length), which were mounted in an organ bath (Föhr Medical Instruments, Seeheim, Germany) for isometric force measurement as described previously (2). Rings were equilibrated for 30 min under a resting tension of 3 g in oxygenated (95% O₂-5% CO₂) Krebs-Henseleit solution (pH 7.4, 37°C) of the following composition (mmol/l): 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.6 CaCl₂, 1.2 K₂HPO₄, 25 NaHCO₃, and 12 glucose, with the cyclooxygenase inhibitor diclofenac (1 µmol/l). Rings were repeatedly contracted by KCl (50 mmol/l) until reproducible responses were obtained. Thereafter, the rings were preconstricted with the thromboxane mimetic U-46619 (10-100 nmol/l) to comparable constriction levels and the relaxant response to cumulative doses of bradykinin and to sodium nitroprusside was assessed with or without prior inhibition of endothelial nitric oxide synthase (eNOS) with N^G-nitro-L-arginine (L-NNA; 0.3 mmol/l, 30 min).

Immunohistochemistry. Ki67 was assessed as a marker of proliferation (21). The coronary samples were defrosted, dried for 10 min, and fixed with acetone. Immunostaining of the LAD and RCA samples was performed with monoclonal mouse anti-Ki67 (clone MIB-5; Dianova, Hamburg, Germany) and polyclonal rabbit anti-eNOS (Alexis/ABR, San Diego, CA) as primary antibodies, respectively. The Super Sensitive Detection Kit-Alkaline Phosphatase (BioGenex, San Ramon, CA) was then used as a secondary antibody. All immunostaining procedures were performed in parallel. Two investigators analyzed the slices independently by counting Ki67-positive nuclei per slice and by assessing the eNOS expression semiquantitatively using a score for the staining intensity from 0 to 3 in steps of 0.5 (0 = no staining; 1 = mild, 2 = intermediate, and 3 = maximal staining). From these two investigators the mean of both measurements was taken as the result per slice, and the final data are given as the mean of all analyzed slices.

Statistical analysis. Data are given as means ± SE. Comparisons between different groups were done with an unpaired two-tailed t-test; comparisons within one group were done with a paired two-tailed t-test. For multiple comparison a Bonferroni correction was applied. eNOS expression was compared with a Mann-Whitney U-test. Ki67 expression (assessed as Ki67-positive nuclei per analyzed sample) was compared with an unpaired two tailed t-test. The correlation between bradykinin-induced vasorelaxation in the presence of L-NNA and the thickness of the neointima formation was tested with a Pearson's correlation test with a significance level of 0.05. The coefficient of variation between measurements was calculated as the ratio of the standard deviation and the mean value (in %).

	RESULTS

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Morphology. The ballooned coronary segments were subdivided into group A, which showed no neointima formation (n = 13), and group B, which did show neointima formation (n = 20) (Fig. 1). Group C contained the coronary control segments taken from the RCA (n = 12). The area surrounded by the EEL and the IEL showed no statistically significant difference among the three groups. The lumen area of group B was significantly smaller than that of group C. The area of the media in groups B and C was significantly bigger than in group A. In contrast, the media-to-lumen ratio in group C did not differ from that in group A. However, the media-to-lumen ratio was significantly higher in group B than in groups A and C. In group B, we found a mean neointima area of 0.11 ± 0.003 mm² compared with no neointima in groups A and C (P < 0.005; Table 1). The mean diameter (measured from the IEL) of the proximal one-third of the ballooned LAD segments without neointima formation was significantly higher than the diameter of those segments with neointima formation [2.1 ± 0.07 mm (range 1.8-2.5 mm) vs. 1.6 ± 0.04 mm (range 1.4-2.2 mm) (P < 0.03); Figs. 1 and 2]. In all coronary segments containing neointima proliferation, a rupture of the IEL either in the histological sample itself or in one of the neighboring samples could be documented. The neointima itself mainly consisted of smooth muscle cells (assessed by light microscopy). The coefficient of variation of the control measurements was 5%. In all analyzed segments of each group, no endothelial damage could be detected with light microscopy.

View larger version (105K): [in this window] [in a new window]   Fig. 1.   Representative examples of coronary segments of group A (A), group B (B), and group C (C), all stained with elastica-van Gieson. Note the neointima proliferation in B (internal elastic lamina marked with white arrows). LAD, left anterior descending coronary artery; RCA, right coronary artery.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Representative examples (hematoxylin-eosin staining) of balloon-treated coronary segments without neointima formation (A) and a coronary segment from group B with neointima formation (B).

Vascular reactivity. A total of 14 LAD segments (5 from group A and 9 from group B) and 15 RCA control segments (group C) was studied. Sodium nitroprusside-induced relaxation of the coronary segments was identical in all three groups. Endothelium-dependent, bradykinin-induced relaxation was significantly attenuated in group B compared with group C (75 ± 3% vs. 98 ± 1%; P < 0.001). After administration of L-NNA, bradykinin-induced relaxation was again significantly attenuated compared with both groups A and C [20 ± 3% vs. 76 ± 9% and 68 ± 6%, respectively (P < 0.0003); Fig. 3].

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Vasoreactivity was studied with sodium nitroprusside (SNP) and bradykinin (BKN) before and after NG-nitro-L-arginine (L-NNA) administration. SNP-induced relaxation was identical in all groups. Group B showed a significant impairment of BKN-induced vasorelaxation compared with group C before L-NNA administration. After L-NNA administration, group B showed a significantly reduced relaxation compared with both groups A and C. y-Axis shows maximum dose-dependent relaxation in %; x-axes shows the dosage of SNP and BKN. § Significant differences compared with group C; * significant differences compared with groups A and C.

Immunohistochemistry. A total of 90 coronary slices was assessed for the presence of Ki67 and eNOS (group A, 26 slices from 4 coronary arteries; group B, 40 slices from 8 coronary arteries; group C, 24 slices from 12 coronary arteries). There was no difference in Ki67-positive cells (evaluated separately for the coronary endothelium, the subendothelial intima/neointima, and media) among the three groups. The efficacy of the Ki67 detection kit was tested with highly proliferative tissue (human tonsillar tissue), which showed good staining of the nuclei.

Vascular relaxation and neointima thickness. There was a significant negative linear correlation between the coronary relaxation induced by bradykinin in the presence of L-NNA and the neointima thickness, with a coefficient of correlation of 0.65 and a significance level of 0.004 (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   There is a linear inverse correlation between the BKN-induced vasorelaxation in the presence of L-NNA and the neointimal thickness of the coronary segments.

Blood chemistry. For analysis of the blood samples the study group of 12 animals was subdivided into group I, which contained 3 animals without any sign of neointima, and group II, which comprised 9 animals presenting neointima formation. In group I there was no myocardial lactate production or increase of creatine kinase during and after active coronary perfusion. However, there was a significant increase of the arteriovenous endothelin-1 difference after 30 min of active perfusion, with a slight decay after 30 min of native reperfusion of the vessel. In group II there was no increase in creatine kinase or endothelin-1 during or after active perfusion. However, we saw a significant myocardial lactate production occurring after 30 min of active perfusion. After 30 min of native reperfusion there was still a myocardial lactate production, but this did not reach significance compared with baseline values (Table 3).

	DISCUSSION

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Multiple factors have been identified as promoting the proliferation process in different vessel layers, leading to neointima formation after coronary angioplasty and stenting (9, 23, 25). One factor in this cascade is the injury of the coronary endothelium (11). There are clear data indicating the impact of PTCA overstretch injury on endothelial function and vascular morphology in terms of profound neointima proliferation and impaired vascular relaxation (24). However, there is an increasing use of different coronary devices with low-pressure balloon placement that are supposed to be nontraumatic to the coronary artery (e.g., brachytherapy catheters, embolization containment devices, angioscopy catheters, intracoronary shunts). These balloons can affect the coronary vessel by mechanical vessel trauma and regional endothelial ischemia-denudation. Prior studies showed the effect of overstretch injury on the release of endothelin-1 accompanied by neointima proliferation (3, 4, 8). In our model we could not document an increase in endothelin-1 release from the coronary arteries during the inflation period of the balloons in group II animals presenting neointima formation. Therefore, endothelin-1 did not seem to contribute to the neointima proliferation in our model.

The acute impact of pure ischemia-reperfusion injury on endothelium-dependent relaxation in the absence of mechanical vessel trauma has been shown in both in vitro and in vivo experiments including long-term impairment of endothelium-dependent vasorelaxation evoked by aggregating platelets lasting up to 6 mo (12, 14, 17, 18). One hypothesis derived from these data was the potential development of regional thrombosis leading to vascular spasm or induction of proliferation. This would prohibit the widespread clinical use of the above-mentioned coronary devices, because they occlude the vessel at the site of the balloons, resulting at least in an endothelial ischemia of the coronary segment with balloon contact. Because we completely occluded the coronary vessels at the balloon site (with maintained distal perfusion), thus creating local endothelial ischemia, one should expect impaired vasorelaxation in all of the studied segments. However, endothelium-dependent, bradykinin-induced vascular relaxation showed a significant impairment only in group B and not in group A, despite the same endothelial "ischemic trauma." Therefore, it seems unlikely that a pure endothelial ischemia could be responsible for this impaired endothelium-dependent relaxation. Because mechanical denudation of balloon-treated segments with subsequent formation of regenerated endothelium does not necessarily lead to an impaired coronary relaxation induced by bradykinin, we cannot rule out endothelial denudation in group A (26, 29). However, there was no difference in eNOS expression of the endothelium between group A and the control group. In contrast, we saw a significantly reduced eNOS expression only in the coronary endothelium in group B (presenting neointima proliferation), supporting the hypothesis that a mechanical vessel trauma remains most likely a profounding factor leading to an impaired relaxation. A previous study reported findings of profound vessel damage induced by low-pressure balloon devices occurring at low inflation pressures between 1.5 and 2 atm in the rat aorta (1). In addition, Murray et al. (15) described ruptures in the IEL of human necropsy coronary segments after angioplasty with commercially available PTCA catheters (15). During the inflation process acute pressure drops could be documented at pressures <200 kPa ( 2 atm), indicating a rupture of vessel layers including the IEL. These studies support the hypothesis that low pressures <2 atm can lead to mechanical vessel trauma. However, we are not aware of any previous experiments that examined inflation pressures of only 1 atm. We can only speculate as to whether in our model the combination of low inflation pressures and prolonged inflation time may have aggravated the vessel trauma.

Although in our model the media-to-lumen ratio as a marker of media proliferation was significantly increased in group B compared with groups A and C, Ki67 expression did not differ among the three groups. This shows that the process of proliferation subsided at least after the 3-mo follow-up. However, we saw a completely normal endothelium-independent relaxation in the balloon-treated segments both with and without neointima formation, indicating a normal function of the coronary media. Therefore, the reduced endothelium-dependent relaxation in response to bradykinin might be explained by a reduced eNOS expression in group B (6). Furthermore, after administration of L-NNA, the endothelium-dependent, nitric oxide (NO)-independent relaxation was significantly attenuated in group B compared with both groups A and C. This indicates that, in addition to NO-dependent relaxation, the relaxation mediated by the endothelium-derived hyperpolarizing factor (EDHF) is impaired. Thollon and coworkers (28) recently described a decay of endothelium-dependent hyperpolarization in regenerated endothelium induced by serotonin, but bradykinin-induced hyperpolarization was preserved in their model. In contrast, we observed an impaired bradykinin-induced vasorelaxation in the presence of L-NNA. One possible explanation for this difference might be the presence of neointima proliferation in our model [unfortunately, Thollon et al. (28) did not report the morphological findings in their angioplasty model]. Our data showed a significant linear inverse correlation between the extent of neointimal thickness and the reduced relaxation, indicating a potentially reduced "propagation" of EDHF-mediated relaxation to the medial layers of the vessel. Kohler and colleagues (10) recently reported the finding of an impaired endothelial Ca²⁺-activated K⁺ (K_Ca) channel contributing to functional alterations of regenerated endothelium. Other studies showed that K_Ca channel-mediated endothelial hyperpolarization might be propagated via gap junctions to vascular smooth muscle cells (5). These data support the hypothesis that neointima proliferation leads to reduced propagation of vasorelaxing factors (such as EDHF) to the media layers of the vessel. In our study, morphological analysis of the vessel wall from balloon-treated segments in group B showed neointima formation exclusively containing smooth muscle cells. However, when compared with other experimental overstretch models, in which profound neointima proliferation led to significant endoluminal stenosis, in our model neointima formation did not induce endoluminal narrowing of the coronary vessel (29). Neointima formation occurred in LAD segments that had a rupture of the IEL. Mechanical trauma was only present in LAD segments in which the balloon of 2.5-mm diameter was oversized compared with the vessel diameter, indicating that the main effect of the balloon in our model might also have been the induction of an overstretch injury rather than an endothelial denudation. This happened even though care was taken to select vessel target areas that had diameters adequate to accommodate the fixed balloon size of 2.5 mm. Looking at the profound overlap in the range of vessel diameters in this study (range 1.8-2.5 mm in vessels without neointima formation vs. 1.4-2.2 mm in vessels with neointima formation), it becomes obvious that there is no clear cut-off value of oversizing that leads to neointima proliferation. Therefore, it might not be possible to avoid this kind of damage in every clinical case, even when adequate care is taken to select appropriately sized balloons.

In summary, after low-pressure balloon treatment of porcine coronary arteries, in a substantial number of experiments, morphological and functional impairment of the coronary segments involved in the procedure could be found. These changes could be the potential trigger leading to late development of coronary stenosis. However, pure coronary endothelial ischemia-denudation does not necessarily lead to morphological and functional changes if it is not accompanied by profound local vessel trauma. This highlights the importance of precisely matching low-pressure balloon devices to coronary diameters, which would be facilitated by on-line quantitative coronary angiography and the availability of a selection of intermediate-sized balloons.

Limitations of study. One limitation of this study is that we only had morphology data (including the luminal diameters) after the coronary intervention. Thus we have no quantitative information regarding baseline values for the media areas or vessel sizes, and therefore we can only speculate about the underlying pathophysiological mechanisms leading to neointima proliferation or changes in the size of the media area. In addition, in our study the prototype active perfusion catheter was only available with a fixed balloon size, thus limiting the chance for an exact adjustment of the catheter to the coronary anatomy of the study animals.