Vasomotor Response to Different Endothelium-Dependent Vasodilators in an Animal Model

Abstract: Background and Objectives. Incomplete re-endothelialization of stents can be revealed as paradoxical vasoconstriction with endothelium-dependent vasodilators. As no consensus exists about the best method or agent, our objective is to analyze the response to different drugs in a coronary swine model. Methods. Twenty-seven stents were implanted in 9 domestic swine. The vessel diameter of proximal and distal segments (≥5 mm) was assessed immediately post implantation. Different endothelium-dependent vasodilators were used: intracoronary (IC) acetylcholine, 20 μg (A2) and 40 μg (A4), IC serotonin (S), 100 μg, and isoproterenol (I), intravenous infusion. The results are presented as constriction (%) compared with maximal vasodilation with IC nitroglycerin (N, 200 μg). Results. In 10 vessels (37%), A4 provoked an occlusive spasm. Acetylcholine induced a higher degree of vasoconstriction (A4, 42 ± 39%; A2, 16 ± 14%) than the rest of the agonists (S, 6 ± 12%; I, 6 ± 11%; P<.01). The constriction rate was not related to the induced hemodynamic changes. Conclusions. After focal endothelial denudation in a coronary swine model, the constriction rate induced by different endothelium-dependent vasodilators is highly variable. The highest value is observed after IC acetylcholine bolus. The constriction rate does not correlate with the observed hemodynamic changes.

J INVASIVE CARDIOL 2012;24(7):320-323

Key words: stent endothelialization, vasomotor response, animal model

_________________________________________________

Delayed healing and impaired re-endothelialization are distinctive findings of the vascular response to the implantation of drug-eluting stents (DES), both in animal models^1-7 and in the clinical field.^8-10 Nevertheless, an acceptable endothelialization rate for DESs has been reported in the swine model at 28 days.^2,7,11-16 A common feature in many of these studies is the immaturity of the observed endothelial cells. The main in vivo hallmark of dysfunctional endothelium is abnormal vasomotor function,^13,17-22 showing paradoxical vessel vasoconstriction in response to endothelium-dependent vasodilators. This discriminant behavior could be used in preclinical research to look for DESs with better vascular healing profiles, but also in the clinical scenario to help assess the endothelialization of implanted DESs.^19,20

Togni et al first reported in humans that exercise, a physiological endothelium-dependent vasodilator, induced paradoxical vessel vasoconstriction 6 months after DES implantation.²³ Subsequently, preclinical studies with different DES have replicated this response.^{13,18,21,24-26} Different endothelium-dependent vasodilators have been used in preclinical research to evaluate vasomotor response: acetylcholine (ACh),^26,27 substance P,^25,28 bradykinin,^21,29-31 and serotonin.^29-33 Exercise and rapid atrial pacing were also used in human studies.^23,34-36 The wide variability in the methodology prompted us to compare the vasomotor responses of vessels with denuded endothelium after stent implantation to different endothelium-dependent vasodilators.

Methods

Animal model and procedures. Nine animals (domestic pigs, Large White race) were selected from the experimental farm of our university. They were 2 months old with a mean weight of 25 ± 3 kg. All experimental procedures and animal handling were conducted according to the European and local general directives for the protection of experimental animals (Directive 86/609/CE, R.D. 223/1988) and under the supervision of the bioethics committee of our university. The animals were pretreated with oral clopidogrel (300 mg) and aspirin (325 mg) mixed with food 1 day before the procedure. Anesthesia and surgical procedures were performed as described previously.^12,14,37 A 7 Fr introducer sheath was inserted into a carotid artery by surgical cut-down. Heparin (200 IU/kg) was administered intravenously. A 6 Fr, 40-cm long, modified AL1 guiding catheter (Iberhospitex S.A.) allowed the selective catheterization of both coronary ostia. Heart rate, blood pressure, and electrocardiogram (ECG) were monitored throughout the procedure.

Twenty-seven stents (3.5 mm diameter, 18 mm length) were implanted according to a predetermined scheme in each coronary artery. Every animal received 1 bare-metal stent (Apolo; Iberhospitex S.A.), 1 paclitaxel-eluting stent (Active; Iberhospitex S.A.), and 1 dual-drug eluting stent (simvastatin + paclitaxel, Irist-Duo; Iberhospitex S.A.) in the proximal segment of each coronary artery. All stents were deployed at nominal pressure (9 atm), allowing higher pressure in a second inflation if the relation to the vessel was less than 1.1 to obtain light-to-moderate overstretch (10%-20%). We have previously assessed that direct stent implantation in native swine coronary arteries only causes focal, partial endothelial denudation (Figure 1).

Assessment of vasomotor response. Angiographic images were obtained with a digital x-ray system (GE OEC 9900 Elite, GE Healthcare) and recorded as non-compressed DICOM films. The best angiographic view to avoid any overlapping of the stented segment, proximal, and distal reference segments (≥10 mm) was selected for each coronary artery. The stent-to-artery ratio was analyzed in these postimplantation angiographic images. The vasomotor tests were performed immediately after stent implantation in the right coronary artery and after implantation of both stents (left anterior descending and left circumflex) in the left coronary artery.

The endothelium-dependent vasodilators were: (1) A2 = intracoronary ACh, 20 µg infused during 2 minutes; (2) A4 = intracoronary ACh, 40 µg infused during 2 minutes; (3) S = intracoronary serotonin, 100 µg bolus; and (4) I = intravenous isoproterenol, incremental infusion rate until maximal heart rate is achieved without drop in mean blood pressure. Finally, maximal endothelium-independent vasodilation was induced with intracoronary nitroglycerin (N), 200 µg bolus. A washout period of ≥2 minutes (or until heart rate returns to basal values) was allowed between every drug phase. Angiographic series were recorded at the end of the infusion in the A2 and A4 tests, at the point of maximal heart rate change in the S and I tests, and 1 minute after the nitroglycerin bolus. The vessel diameter was measured both proximal and distal to the stent at the same point (separated at least 5 mm from the stent edges) in each angiographic run. Off-line quantitative coronary analysis was performed using validated software (Medis QCA-CMS 6.0). As baseline vasoreactivity status may vary between animals, we decided to use the maximal, endothelium-independent, vasodilation as the reference diameter to establish the comparisons. Changes in vessel diameter are presented, thus, as percent reduction as compared with the post-nitroglycerin vessel diameter.

Statistical methods. Continuous variables are presented as mean ± standard deviation. Vessel diameter values and percentage changes are compared with paired t-test and correlation r coefficient. The potential influence of other co-variables in these parameters is assessed with the analysis of variance (ANOVA) test. Probability values of P<.05 are considered significant.

Results

All stents were implanted as defined per protocol. The mean stent-to-artery ratio was 1.06 ± 0.23, without significant differences between stent types or coronary arteries. The vasomotor tests were performed as planned without complications. Of note, occlusive vessel spasm was induced in 10 vessels (37%) after the infusion of ACh 40 µg (A4). All these cases showed spontaneous flow recovery in the first minute after infusion, without further hemodynamic or arrhythmic consequences. In these cases, the washout phase was prolonged up to 5 minutes.

The animals showed the predicted hemodynamic changes after the vasodilators administration. ACh did not induce significant changes in heart rate and only a modest drop in mean blood pressure (16 ± 14 mm Hg with A4 dose; P<.05). Conversely, serotonin was associated with a significant increase both in heart rate (54 ± 42 bpm; P<.01) and mean blood pressure (36 ± 23 mm Hg; P<.01). Isoproterenol was also associated with a significant increase in heart rate (82 ± 33 bpm; P<.001) without significant change in blood pressure. Figure 2 illustrates these changes.

The observed diameters after each drug test are shown in Figure 3; all the measurements are separated by segment location (proximal and distal). Both ACh doses induced significant vasoconstriction: A2 induced 16 ± 14% constriction (vs post-N diameter; P<.01) and A4 induced 42 ± 39% constriction (P<.001). Serotonin and isoproterenol were also associated with diameter values slightly, but significantly, smaller than those observed after maximal vasodilation: S, 6 ± 12% (P<.05) and I, 6 ± 11% (P<.05). Correlations between maximal vasodilation values and the diameters measured after every endothelium-dependent agonist reveal similar findings: A2-N, r = 0.68; A4-N, r = 0.64; S-N, r = 0.82; I-N, r = 0.81. As expected, distal diameters are significantly smaller than the proximal values. However, the changes observed after the administration of the different drugs were alike. No relationships were observed between the constriction rate and type of stent, artery, or segment location.

Discussion

The main findings of this analysis are: (1) different endothelium-dependent vasodilators induce widely variable grades of paradoxical vasoconstriction in stented segments with denuded endothelium; (2) high doses of Ach frequently induce severe vasospasm; and (3) hemodynamic changes are not related with vessel diameter changes.

Vasomotor dysfunction and DESs. The abnormal vasomotor response to different endothelium-dependent vasodilators in animal models has been confirmed after sirolimus-^13,21,24 and paclitaxel-eluting stents.^{13,18,21,25,26} Zotarolimus-^35,38 and biolimus-eluting stents^35,36 might show better results in terms of endothelial-dependent vasomotor function than first-generation DESs. Coronary arteries previously treated with DES show both abnormal vasomotor responses and morphological features of endothelial dysfunction, as reduced endothelial nitric oxide synthase (eNOS) expression¹³ and markers of oxidative stress.²⁵ However, the link between morphological markers of mature endothelium, as eNOS expression, and functional assessment of vasomotor response to ACh is controversial.³⁹

Endothelium-dependent vasodilators. Different methods have been used to provoke this paradoxical vasoconstriction. ACh is one of the most used agonists in human research.^40-43 Some debate exists over the adequacy of using this drug in the swine model as porcine arteries seem to have limited presence of muscarinic receptors and ACh therefore would not induce sufficient vasodilation.^13,44,45 Conversely, other authors have demonstrated significant differences in vasomotor response between different DESs using ACh.^26,27 Our present results and previous data³⁹ support the value of ACh as a trigger for the paradoxical vasoconstriction related to endothelial disfunction. Altough the higher ACh dose (40 µg) is lower than that used in human protocols,^41,43 it was related to a significant incidence of occlusive vasospasm (37%), suggesting a potential overdosing in this animal model. A potential non-specific spastic response of ACh could also be interpreted from these data, at least in the normal coronary swine model. Extrapolation to the clinical human scenario should be done with caution, as some authors have used ACh to induce coronary spasm in coronary arteries “without significant lesions.”^46,47

Serotonin has also been used as endothelium-dependent vasodilator^29,31 or in vasospasm models.^32,33,47,48 However, some conflicting results remark the preferential action of serotonin causing smooth muscle cell hypercontraction over the endothelium-mediated vasodilation.³⁰ Our data show inconclusive results, with vessel diameters not significantly different from basal values and slightly lower than those observed after maximal vasodilation. In this case, a suboptimal dose may explain these findings.

Exercise is a physiologic endothelium-dependent vasodilator and, therefore, it was used in some clinical studies.^23,34 Rapid atrial pacing has been used by other authors to reproduce (some of) the exercise-induced hemodynamic changes.^35,36 Isoproterenol, a ß-mimetic agent, induces heart rate acceleration and endothelium-dependent vasodilation.^49,50 The observed results in our series are quite similar to those obtained after serotonin administration.

Study limitations. Although we have used validated methods, the drugs and doses used in this experiment do not cover the whole spectrum of possibilities that appear in the literature. We cannot rule out over- or under-dosing or missing the best drug. Direct stent implantation in normal swine coronary arteries is one of the most used animal models to test vasomotor responses to DESs. As described in our methodology, we have observed that only focal, partial endothelial denudation is caused. While it could be perceived as a limitation, this situation may also mimic the long-term status of vascular healing after DES implantation with incomplete re-endothelialization.

Conclusion

After focal endothelial denudation with direct stent implantation in a coronary swine model, the constriction rates induced by different endothelium-dependent vasodilators are highly variable. The highest vasoconstriction is obtained with IC ACh, especially with a 40 μg dose. The hemodynamic changes do not correlate with the constriction rate.

References

1. Farb A, Heller PF, Shroff S, et al. Pathological analysis of local delivery of paclitaxel via a polymer-coated stent. Circulation. 2001;104(4):473-479.
2. Suzuki T, Kopia G, Hayashi S, et al. Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation. 2001;104(10):1188-1193.
3. Finn AV, Kolodgie FD, Harnek J, et al. Differential response of delayed healing and persistent inflammation at sites of overlapping sirolimus- or paclitaxel-eluting stents. Circulation. 2005;112(2):270-278.
4. Finn AV, Nakazawa G, Joner M, et al. Vascular responses to drug eluting stents: importance of delayed healing. Arterioscler Thromb Vasc Biol. 2007;27(7):1500-1510.
5. van Beusekom HM, Saia F, Zindler JD, et al. Drug-eluting stents show delayed healing: paclitaxel more pronounced than sirolimus. Eur Heart J. 2007;28(8):974-979.
6. John MC, Wessely R, Kastrati A, et al. Differential healing responses in polymer- and nonpolymer-based sirolimus-eluting stents. JACC Cardiovasc Interv. 2008;1(5):535-544.
7. Joner M, Nakazawa G, Finn AV, et al. Endothelial cell recovery between comparator polymer-based drug-eluting stents. J Am Coll Cardiol. 2008;52(5):333-342.
8. Farb A, Burke AP, Kolodgie FD, Virmani R. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation. 2003;108(14):1701-1706.
9. Joner M, Finn AV, Farb A, et al. Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk. J Am Coll Cardiol. 2006;48(1):193-202.
10. Finn AV, Joner M, Nakazawa G, et al. Pathological correlates of late drug-eluting stent thrombosis: strut coverage as a marker of endothelialization. Circulation. 2007;115(18):2435-2441.
11. Wilson G, Polovick J, Huibregtse B, Poff B. Overlapping paclitaxel-eluting stents: long-term effects in a porcine coronary artery model. Cardiovasc Res. 2007;76(2):361-372.
12. Perez de Prado A, Perez Martinez C, Cuellas Ramon C, et al. Endothelialization of nonapposed stent struts located over the origin of a side branch. J Interv Cardiol. 2009;22(3):222-227.
13. van Beusekom HM, Sorop O, van den Heuvel M, et al. Endothelial function rather than endothelial restoration is altered in paclitaxel- as compared to bare-metal, sirolimus- and tacrolimus-eluting stents. EuroIntervention. 2010;6(1):117-125.
14. Perez de Prado A, Perez-Martinez C, Cuellas-Ramon C, et al. Time course of reendothelialization of stents in a normal coronary swine model: characterization and quantification. Vet Pathol. 2011;48(6):1109-1117.
15. Nakazawa G, Finn AV, John MC, et al. The significance of preclinical evaluation of sirolimus-, paclitaxel-, and zotarolimus-eluting stents. Am J Cardiol. 2007;100(8B):36M-44M.
16. Nakazawa G, Nakano M, Otsuka F, et al. Evaluation of polymer-based comparator drug-eluting stents using a rabbit model of iliac artery atherosclerosis. Circ Cardiovasc Interv. 2011;4(1):38-46.
17. Lerman A, Eeckhout E. Coronary endothelial dysfunction following sirolimus-eluting stent placement: should we worry about it? Eur Heart J. 2006;27(2):125-126.
18. van Beusekom HMM, Sorop O, van den Heuvel M, et al. Early endothelialization in paclitaxel eluting stents is similar to other drug eluting stents but shows transient endothelial dysfunction (Abstr). EuroIntervention. 2009;5:E65.

19. Pendyala LK, Yin X, Li J, et al. The first-generation drug-eluting stents and coronary endothelial dysfunction. JACC Cardiovasc Interv. 2009;2(12):1169-1177.
20. Muhlestein JB. Endothelial dysfunction associated with drug-eluting stents. J Am Coll Cardiol. 2008;51(22):2139-2140.
21. van den Heuvel M, Sorop O, Batenburg WW, et al. Specific coronary drug-eluting stents interfere with distal microvascular function after single stent implantation in pigs. JACC Cardiovasc Interv. 2010;3(7):723-730.
22. van Beusekom HM, Serruys PW. Drug-eluting stent endothelium: presence or dysfunction. JACC Cardiovasc Interv. 2010;3(1):76-77.
23. Togni M, Windecker S, Cocchia R, et al. Sirolimus-eluting stents associated with paradoxic coronary vasoconstriction. J Am Coll Cardiol. 2005;46(2):231-236.
24. Li J, Jabara R, Pendyala L, et al. Abnormal vasomotor function of porcine coronary arteries distal to sirolimus-eluting stents. JACC Cardiovasc Interv. 2008;1(3):279-285.
25. Pendyala LK, Li J, Shinke T, et al. Endothelium-dependent vasomotor dysfunction in pig coronary arteries with paclitaxel-eluting stents is associated with inflammation and oxidative stress. JACC Cardiovasc Interv. 2009;2(3):253-262.
26. Farhan S, Hemetsberger R, Matiasek J, et al. Implantation of paclitaxel-eluting stent impairs the vascular compliance of arteries in porcine coronary stenting model. Atherosclerosis. 2009;202(1):144-151.
27. Nakamura T, Brott BC, Brants I, et al. Vasomotor function after paclitaxel-coated balloon post-dilation in porcine coronary stent model. JACC Cardiovasc Interv. 2011;4(2):247-255.
28. Shinke T, Li J, Chen JP, et al. High incidence of intramural thrombus after overlapping paclitaxel-eluting stent implantation: angioscopic and histopathologic analysis in porcine coronary arteries. Circ Cardiovasc Interv. 2008;1(1):28-35.
29. Shimokawa H, Vanhoutte PM. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ Res. 1989;64(5):900-914.
30. Miyata K, Shimokawa H, Yamawaki T, et al. Endothelial vasodilator function is preserved at the spastic/inflammatory coronary lesions in pigs. Circulation. 1999;100(13):1432-1437.
31. Jeanmart H, Malo O, Carrier M, et al. Comparative study of cyclosporine and tacrolimus vs newer immunosuppressants mycophenolate mofetil and rapamycin on coronary endothelial function. J Heart Lung Transplant. 2002;21(9):990-998.
32. Shimokawa H, Seto M, Katsumata N, et al. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res. 1999;43(4):1029-1039.
33. Kandabashi T, Shimokawa H, Miyata K, et al. Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta. Circulation. 2000;101(11):1319-1323.
34. Togni M, Raber L, Cocchia R, et al. Local vascular dysfunction after coronary paclitaxel-eluting stent implantation. Int J Cardiol. 2007;120(2):212-220.
35. Hamilos M, Sarma J, Ostojic M, et al. Interference of drug-eluting stents with endothelium-dependent coronary vasomotion: evidence for device-specific responses. Circ Cardiovasc Interv. 2008;1(3):193-200.

36. Hamilos MI, Ostojic M, Beleslin B, et al. Differential effects of drug-eluting stents on local endothelium-dependent coronary vasomotion. J Am Coll Cardiol. 2008;51(22):2123-2129.
37. Perez de Prado A, Perez-Martinez C, Cuellas-Ramon C, et al. Scanning electron microscopy analysis of luminal inflammation induced by different types of coronary stent in an animal model. Rev Esp Cardiol. 2011;64(2):159-162.
38. Kim JW, Seo HS, Park JH, et al. A prospective, randomized, 6-month comparison of the coronary vasomotor response associated with a zotarolimus- versus a sirolimus-eluting stent. J Am Coll Cardiol. 2009;53(18):1653-1659.
39. Perez de Prado A, Perez  C, Cuellas C, et al. Endothelial nitric oxide synthase (eNOS) activity and vasomotor dysfunction of coronary arteries after DES: link or chance? (Abstr). EuroIntervention. 2011;5:M25.
40. Hofma SH, van der Giessen WJ, van Dalen BM, et al. Indication of long-term endothelial dysfunction after sirolimus-eluting stent implantation. Eur Heart J. 2006;27(2):166-170.
41. Obata JE, Kitta Y, Takano H, et al. Sirolimus-eluting stent implantation aggravates endothelial vasomotor dysfunction in the infarct-related coronary artery in patients with acute myocardial infarction. J Am Coll Cardiol. 2007;50(14):1305-1309.
42. Shin DI, Kim PJ, Seung KB, et al. Drug-eluting stent implantation could be associated with long-term coronary endothelial dysfunction. Int Heart J. 2007;48(5):553-567.
43. Kim JW, Suh SY, Choi CU, et al. Six-month comparison of coronary endothelial dysfunction associated with sirolimus-eluting stent versus paclitaxel-eluting stent. JACC Cardiovasc Interv. 2008;1(1):65-71.
44. Cowan CL, McKenzie JE. Cholinergic regulation of resting coronary blood flow in domestic swine. Am J Physiol. 1990;259(1 Pt 2):H109-H115.
45. Hata H, Egashira K, Fukai T, et al. The role of endothelium-derived nitric oxide in acetylcholine-induced coronary vasoconstriction in closed-chest pigs. Coron Artery Dis. 1993;4(10):891-898.
46. Ong P, Athanasiadis A, Hill S, et al. Coronary artery spasm as a frequent cause of acute coronary syndrome. J Am Coll Cardiol. 2008;52(7):523-527.
47. Kanazawa K, Suematsu M, Ishida T, et al. Disparity between serotonin- and acetylcholine-provoked coronary artery spasm. Clin Cardiol. 1997;20(2):146-152.
48. Mongiardo R, Finocchiaro ML, Beltrame J, et al. Low incidence of serotonin-induced occlusive coronary artery spasm in patients with recent myocardial infarction. Am J Cardiol. 1996;78(1):84-87.
49. Dawes M, Chowienczyk PJ, Ritter JM. Effects of inhibition of the L-arginine/nitric oxide pathway on vasodilation caused by beta-adrenergic agonists in human forearm. Circulation. 1997;95(9):2293-2297.
50. Garovic VD, Joyner MJ, Dietz NM, Boerwinkle E, Turner ST. Beta(2)-adrenergic receptor polymorphism and nitric oxide-dependent forearm blood flow responses to isoproterenol in humans. Journal Physiol. 2002;546(Pt 2):583-589.

_________________________________________________

From the Fundación Investigacion Sanitaria en León – HemoLeon and Institute of Biomedicine (IBIOMED), León, Spain.
Funding: This study was supported by a Research grant (GRS 403/A/09) from Consejeria de Sanidad, Junta de Castilla y León, Spain.
Disclosure: The authors have completed and returned the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors report no conflicts of interest regarding the content herein.
Manuscript submitted January 16, 2012, provisional acceptance given February 14, 2012, final version accepted February 27, 2012.
Address for correspondence: Armando Pérez de Prado, MD, S. Cardiología Intervencionista – Hospital de León, Altos de Nava SN – 24008 León, Spain. Email: aperez@secardiologia.es