Introduction Material and methods

Atherosclerosis 154 2001 61 – 69 Chronic endothelial dysfunction after oversized coronary balloon angioplasty in pigs: a 12-week follow-up of coronary vasoreactivity in vivo and in vitro Francisco A.H. Fonseca a,b , Maria C.O. Izar a,b , Valentin Fuster a,b , Richard Gallo a,b , Adrian Padurean a,b , John T. Fallon a,c , E. Neil Schachter d , James H. Chesebro a,b , Juan J. Badimon a,b, a Cardio6ascular Biology Research Laboratory, Cardio6ascular Institute Box 1030 , Annenberg Building 24 , Mount Sinai School of Medicine, One Gusta6e Le6y Place, New York, NY 10029 , USA b The Zena and Michael A. Wiener Cardio6ascular Institute, Mount Sinai School of Medicine, New York, NY, USA c Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA d Pulmonary Di6ision, Mount Sinai School of Medicine, New York, NY, USA Received 7 June 1999; received in revised form 14 February 2000; accepted 2 March 2000 Abstract Previous studies have reported the development of vasoconstriction immediately after invasive coronary interventions. Other studies in animals have demonstrated that using oversized balloon angioplasty, vasospasm can be suppressed, even in the presence of endothelial denudation due to important structural alteration in vascular smooth muscle. The regenerated endothelium also appears to be impaired chronically by selective attenuation of in vitro endothelial dependent relaxation related to pertussis toxin-sensitive G proteins. The purpose of this investigation was to verify in vivo and in vitro vasoreactivity to bradykinin BK and serotonin 5-hydroxytryptamine; 5-HT endothelial dependent agonists as well as to nitroglycerin NTG exogenous nitric oxide donor at different times after oversized balloon angioplasty intervention ranging from 1 h to 12 weeks, in normal porcine coronary arteries. BK-induced vasodilatation in vivo was impaired acutely, but it was restored after 4 weeks. Serotonin caused vasoconstriction in vivo that was significantly augmented after 12 weeks. Conversely, endothelium-dependent vasodilatation in vitro to BK and 5-HT remained attenuated during the whole period of follow-up. Finally, relaxation elicited by NTG was reduced in the in vivo experiment until the first week after the procedure. Histological analysis showed severe arterial injury, and complete recovery of endothelial coverage after 4 weeks. In conclusion, this experiment supports evidence for the occurrence of the acute attenuation of vasoresponsiveness and chronic endothelial dysfunction following overstretching coronary balloon angioplasty. Abnormal remodeling associated with the severity of injury may contribute to chronic endothelial dysfunction. Differences found between in vivo and in vitro studies also suggest that multiple endogenous influences present in the former can attenuate the greater endothelial dysfunction demonstrated by endothelial assessment in vitro. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Vasoconstriction; Invasive coronary inteerventions; Balloon angioplasty www.elsevier.comlocateatherosclerosis

1. Introduction

Abnormal vasoreactivity has been observed in the presence of impaired endothelial function [1 – 3]. Vascu- lar interventions such as coronary balloon angioplasty promote a cascade of cellular injuries that include wide endothelial denudation and damage to the underlying vascular tissue as well [4,5]. Early in vitro experiments examining the recovery of endothelial function showed that regenerated endothelial cells selectively lose some G protein-coupled responses [6,7]. The lack of the protective role of the endothelium against vasoconstric- tor products released from platelets may contribute to vasospasm and thrombosis [8]. The importance of en- Corresponding author. Tel.: + 1-212-2418483; fax: + 1-212- 4266962. E-mail address : juan.badimonmssm.com J.J. Badimon. 0021-915001 - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0021-91500000458-5 dothelial integrity also includes the inhibitory effects on cells and molecule adhesion, smooth muscle cell prolif- eration, and matrix synthesis which appear to be crucial during vascular repair and restenosis [9 – 11]. Some in vivo human studies have also demonstrated endothelial dysfunction following vascular interventions, however, the presence of concomitant atherosclerosis, hyperten- sion, dyslipidemia, tobacco or diabetes are variables that can lead to paradoxical vasoresponses [12 – 15]. The aim of this study was the simultaneous assess- ment of vasoreactivity in vivo and in vitro using two different pathways for the endothelium-dependent va- sodilatation after oversized balloon angioplasty in nor- mal porcine coronary arteries.

2. Material and methods

2 . 1 . Animals This project was approved by the Mount Sinai School of Medicine animal management program which is accredited by the American Association for the Accreditation of Laboratory Animal Care AAALAC and meets NIH standards as set forth in the ‘Guide for the Care and Use of Laboratory Animals’. Thirty-two Yorkshire albino pigs of both sexes, 9 – 12 weeks of age, weighing 23 – 29 kg, were used. The animals were housed individually and fed a regular chow. During the coronary interventions, the animals were initially sedated with ketamine hydrochloride 15 mgkg, i.m. and then deeply anesthetized with sodium pentobarbital 25 mgkg, i.v.. They were intubated and ventilated. Additional intermittent boluses of pentobarbital were intravenously administered to avoid any unnecessary discomfort during the procedure. 2 . 2 . Coronary angioplasty Coronary angioplasty was performed as previously described [16]. Briefly, arterial access was obtained via either right or left femoral artery. All the animals received a 100 IUkg of initial heparin bolus followed by another bolus of 50 IUkg per h after 1 h of experiment. Under fluoroscopic guidance, coronary bal- loon angioplasty was performed in two of the three major epicardial coronary arteries using a 0.014 angio- plasty guide wire Advanced Cardiovascular System, CA and the balloon catheter 20 mm length and 4 mm maximal diameter, Cordis was placed at the proximal site of the respective coronary artery. Angioplasty was performed by three inflations, the first at 8 atm for 15 s, and the second and third at 10 atm for 20 s, with 60-s rest periods between inflations. Angiograms were taken before, during and after balloon angioplasty. Bal- loon:vessel ratio was measured and was comparable among these coronaries 1.26 9 0.03. After the angio- plasty procedure, the arterial sheath was removed and the skin wound closed with sutures. The animals were allowed to recover, returned to their pens and were then reanesthetized at 1, 2, 4, 8 or 12 weeks post-PTCA for vasoreactivity studies. 2 . 3 . Coronary 6asoreacti6ity in 6i6o A 3F infusion catheter was placed at the proximal site of the coronary artery and angiograms were ob- tained using nonionic contrast medium Omnipaque before and after each drug infusion. The protocol of drugs included in sequence: dextrose 5, bradykinin BK 10 − 9 – 10 − 7 M, serotonin 10 − 8 – 10 − 6 M and nitroglycerin NTG 10 − 6 M. Each drug was infused by a Harvard syringe pump with 4 ml of total volume at a rate of 2 mlmin, and there was a 15-min interval between each class of drug infusion. Pictures were taken at the same phase of cardiac cycle and changes in vessel diameter compared to baseline angiogram at the site of coronary angioplasty were measured blindly by two independent observers using an electronic digi- tal caliper [17,18]. 2 . 4 . Coronary 6asoreacti6ity in 6itro The animals were euthanized by an overdose of Sleepway Fort Dodge Laboratories, NJ. The heart was removed and the ascending aorta was immediately cannulated. The heart was flushed with one liter of cold Krebs – Henseleit solution. The coronary arteries were carefully dissected with the heart immersed in the same solution and the region of injury was macroscopically identified and cut into four sections of 4 mm length. The same anatomic portion of untouched coronary arteries served as control. Therefore, twelve coronary rings obtained from the three major coronaries were studied simultaneously. Four coronary rings were tested for each agonist. The coronary rings were suspended horizontally between two stainless steel stirrups, in organ chambers filled with 20 ml Krebs – Henseleit solu- tion of the following composition mM: NaCl 110.0, KCl 4.8, CaCl 2 2.3, K s PO 4 1.2, MgSO 4 1.2, NaHCO 3 25, and glucose 11.0 at pH 7.4, 37°C, and gassed with 95 O 2 and 5 CO 2 . Isometric tension was simulta- neously recorded on a 12-channel polygraph Model 7E, Grass Instrument Co, MA. Injured and control coronary rings were studied simultaneously. The tissues were allowed to equilibrate for 60 min under a tension of 5 g that had been determined previously to be the optimal tension for contraction of the porcine coronary arteries in our model. After this period the tension was readjusted and the tissues were incubated for 1 h with indomethacin 10 − 5 M to avoid the production of endogenous prostaglandins. In those specimens used to determine relaxations by 5-HT, the rings were also incubated with ketanserin 3 × 10 − 6 M to avoid the direct activation of 5-HT 2 receptors on vascular smooth muscle. To examine vascular relaxations, the rings were precontracted with prostaglandin F2a 6 × 10 − 6 M. After the maximal prostaglandin F2a-induced contrac- tion was achieved, relaxation responses were deter- mined to increasing doses of BK 10 − 10 – 10 − 7 M, 5-HT 10 − 9 – 10 − 6 M, and NTG 10 − 9 – 10 − 6 M given in log increments. Relaxations were expressed as the percent of the maximal tension of contracted tissue by prostaglandin F2a. Concentration-response curves Fig. 1. Plot of percent lumen diameter change from base line mean 9 SEM at the proximal site of the control coronary arteries after intracoronary administration of dextrose 5, increasing concentrations of BK and serotonin, and after NTG. §, P B 0.05 versus base line Wilcoxon signed rank sum test; , P B 0.05 vs base line Friedman test Table 1 Assessment of coronary diameter changes after the infusion of 5 dextrose, BK, serotonin, and NTG at the site of intervention mean 9 SEM a,b 2 weeks 4 weeks 8 weeks 12 weeks 1 h Drugs 1 week Control 1.8 9 1.1 0.6 9 1.0 1.2 9 1.2 0.5 9 0.8 D 5 0.1 9 1.6 − 0.4 9 0.9 − 2.5 9 3.8 4.9 9 2.3 3.7 9 4.5 0.4 9 1.6 − 0.3 9 1.3 BK 10−9 M 0.3 9 0.4 1.0 9 1.8 3.9 9 1.5 − 3.3 9 2.7 3.2 9 3.4 6.5 9 2.3 10.7 9 2.6 BK 10−8 M 0.9 9 2.6 7.1 9 2.0 0.9 9 2.5 9.7 9 1.6 − 1.4 9 1.5 2.4 9 0.6 BK 10−7 M 3.2 9 2.2 8.3 9 2.6 6.3 9 3.7 13.4 9 2.0 − 0.8 9 3.3 − 1.8 9 1.4 0.4 9 3.2 − 1.6 9 2.3 5-HT 10−8 M − 3.7 9 4.5 0.6 9 2.0 1.8 9 1.6 − 4.6 9 4.4 − 1.0 9 2.6 0.4 9 2.6 − 5.4 9 3.7 5-HT 10−7 M − 1.2 9 2.7 − 3.2 9 2.3 − 7.7 9 3.2 − 15.3 9 3.8 − 18.9 9 3.0 5-HT 10−6 M − 10.0 9 1.8 − 0.8 9 0.9 − 11.8 9 5.0 − 13.0 9 3.1 − 11.2 9 2.2 10.7 9 1.4 16.5 9 2.1 NTG 10−6 M 10.2 9 2.8 2.0 9 1.0 19.4 9 2.8 13.4 9 2.8 8.2 9 3.4 15 5 10 5 14 9 8 n a Values are percent changes from base line and n refers to the number of arteries. b Lower mean rank , P = 0.023; , P = 0.0001; , P = 0.0017; , P = 0.0002 Kruskal–Wallis test for comparison of the same drug concentration between time-points. Table 2 Assessment of heart rate, systolic SBP and diastolic blood pressure DBP changes after the infusion of 5 dextrose, BK, serotonin, and NTG, mean 9 SD 5-HT before Group 5-HT after Dextrose before NTG before NTG after Dextrose after BK before BK after 117 9 24 125 9 27 117 9 28 125 9 30 115 9 24 118 9 28 Heart rate 115 9 25 115 9 25 156 9 21 148 9 27 155 9 27 152 9 25 156 9 22 156 9 22 159 9 21 SBP 158 9 23 87 9 22 87 9 20 88 9 19 82 9 23 85 9 23 80 9 22 88 9 18 89 9 19 DBP PB0.05 after versus before, t-test correlated groups. Fig. 2. a: dose-response curve to BK in rings precontracted by PGF2a 6 × 10 − 6 M. Vasorelaxation to BK was impaired after injury , P B 0.05 control vs. all time-points, Kruskal – Wallis test. b: endothelium-dependent vasodilatation elicited by 5-HT was also attenuated after injury. P B 0.05, control vs. all time-points, § control vs. 1 week, 12 week, † control weeks 1 day, 1 week, 2 weeks, 8 weeks, 12 weeks. were plotted using the Kaleidograph software version 3.04 for the Power Macintosh 8200 Cupertino, CA. Data points were fitted by iteration to the logistic function: E = E max 1 + IC 50 [A] n , where E, observed muscle tension grams above baseline; [A], the concen- tration of the agonist; IC 50 , the [A] eliciting one half of the maximal response; n, slope of the curve. 2 . 5 . Morphometric analysis Injured and control coronary rings were fixed by immersion in 4 paraformaldehyde in 0.1 M PBS at pH 7.4. Coronary segments were cross-sectioned at 2-mm intervals, paraffin embedded, sectioned 5 mm, and stained with combined Masson elastin CME. Immunohistochemical staining using biotinylated Doli- chos Biflorus Agglutinin DBA, B-1035, Vector Labo- ratories, Barlingame, CA was used to identify endothelial coverage [5,19]. Histomorphometric analy- sis was performed blindly to quantify the endothelial coverage and neointima and media layers. Images from slides were acquired using Adobe Photoshop™ 4.0 program and computer-assisted planimetry was made using NIH Image 1.60. 2 . 6 . Drugs Bradykinin acetate, and 5-HT creatine sulfate, in- domethacin and prostaglandin F2a were obtained from Sigma Chemical Co St Louis, MO; ketanserin bitar- trate from Janssen Pharmaceutica, Beerse, Belgium; and NTG from Abbott Laboratories, IL. Bradykinin, 5-HT, NTG and prostaglandin F2a were dissolved in distilled water, indomethacin in supersatured TRIS- base, and ketanserin in dimethyl sulfoxide DMSO, Sigma. All agonists are reported as molar concentra- tion present in the organ chamber. For in 6i6o studies the drugs were infused diluted in 5 dextrose, and the final molar concentration reported was estimated on basis of a 80 mlmin coronary blood flow. 2 . 7 . Statistical analysis The data were expressed as mean 9 SEM. Unless otherwise stated, n refers to the number of arteries. As a normal distribution cannot be assumed for both morphologic parameters and percent changes in vasore- activity, the Kruskal – Wallis, and Friedman or Wilcoxon signed rank sum tests were used to compare mean group values StatView 512™. For comparison of heart rate and blood pressure changes after drug infusion, t-test correlated groups was used. A P value less than 0.05 was considered significant.

3. Results