Influence of Postangioplasty β-Irradiation on Endothelial Function in Porcine Coronary Arteries

Intracoronary radiation therapy (ICRT) after angioplasty has recently been implemented to prevent and treat restenosis. The anatomic antirestenotic effects of these interventions have been reported for the short and medium term.¹²³⁴ Longer-term consequences of these interventions on the patency of the coronary vessels are currently under investigation. Animal studies have shown that irradiation produces immediate endothelial damage, with uniform impairment of response to acetylcholine, serotonin, and nitroglycerin demonstrated immediately after radiation damage.^567891011 In vitro follow-up at 6 months of irradiated rat aorta revealed impairment of the response to nitroglycerin and acetylcholine,⁵ whereas in vivo follow-up of irradiated porcine coronary arteries demonstrated an impaired response to nitroglycerin with a preserved response to acetylcholine at 4 weeks.⁶

Prolonged impairment of endothelial function may reflect an increased risk of delayed thrombosis and accelerated atherosclerosis beyond that expected after standard percutaneous transluminal coronary angioplasty (PTCA). Conversely, the rapid return of normal endothelial function would be supportive of the doses used in clinical trials with an associated low restenosis rate. Furthermore, reports of aneurysm and pseudoaneurysm formation in the first postangioplasty radiation study,¹² in which higher-than-expected doses were delivered to certain aspects of the arterial wall, raises the possibility that significant vessel wall death took place at those sites. No studies thus far have reported the physiological response of vessels after the combined injury of angioplasty and irradiation.

The aim of this study was to investigate the consequences of ICRT after PTCA on endothelial function and the reactivity of normal pig coronary arteries isolated immediately and 6 weeks after the intervention. This study was performed on isolated coronary vessels to allow for a precise pharmacological characterization of the endothelium-derived factors involved in the regulation of vascular reactivity post PTCA with or without ICRT.

Results

Twenty-two left anterior descending (LAD) and 21 left circumflex (Cx) arteries were used in this study, for a total of 43 separate porcine coronary arteries. Six arteries were studied as controls from animals that had undergone no irradiation, and another 5 vessels were from animals having undergone treatment in another vessel. There was no difference between these 2 groups, and they are subsequently considered as 1 control group (n=11). The remaining arms were the immediate and 6-week post-PTCA, 5 vessels each; immediate and 6-week post-ICRT, 5 vessels each; and immediate and 6-week post–combined PTCA+ICRT, 5 and 7 vessels, respectively. No difference between the response of the LAD and Cx arteries was noted.

Histology

In the balloon-injured arteries, at the site of internal elastic membrane (IEM) rupture, there was maximal intimal proliferation, as shown in Figure 1. This was composed of predominantly smooth muscle cells. Twenty percent to 50% of the IEM circumference was ruptured. Conversely, in the vessels that underwent angioplasty followed by irradiation, there was limited neointima and evidence of vessel wall thinning despite equivalent IEM rupture. These results are consistent with those previously reported describing and quantifying the effects of both β- and γ-irradiation after balloon injury in a porcine coronary model.¹³¹⁴¹⁵

Reactivity Study

Baseline Tension

Figure 2 represents the resting baseline tension of isolated coronary arterial rings. This is defined as the passive tension that promotes a maximal contractile response to a depolarizing solution containing 40 mmol/L KCl.

After all immediate interventions, baseline tension was significantly increased (P<0.05). Resting baseline tension of isolated rings 6 weeks after PTCA or ICRT alone had returned to normal levels; however, it was significantly reduced (P<0.05) at 6 weeks after their combination.

Contractile Responses

Immediate PTCA alone decreased the amplitude of the contraction induced by 40 mmol/L KCl, with no improvement at 6 weeks (Figure 3). ICRT alone potentiated this response in rings with an intact endothelium immediately (P<0.05) but not after 6 weeks. PTCA+ICRT had no effect on this response.

Contraction induced by prostaglandin F_2α (PGF_2α) (40 μmol/L) was potentiated by endothelial denudation (Figure 4). This response was maintained after immediate ICRT or PTCA alone. Immediately after combination of the 2 therapies, however, potentiation of PGF_2α-induced contraction occurred irrespective of the presence of the endothelium. After 6 weeks, PGF_2α contraction potentiation was limited to the endothelium-denuded rings only and thus was similar to the control response.

Endothelium-Dependent Relaxation

In rings preconstricted with PGF_2α (40 μmol/L), substance P (0.1 μmol/L) induced a complete endothelium-dependent relaxation (Figure 5). This relaxation was decreased after all interventions immediately and did not recover after 6 weeks.

To identify the respective contributions of release of an endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide (NO) in the alteration of this endothelium-derived relaxing factors (EDRF)-dependent effect, we investigated the relaxant effects of substance P in 2 different experimental conditions. In depolarized conditions (tone induced by 40 mmol/L KCl and in the presence of indomethacin), substance P induced an NO-dependent relaxation that was significantly decreased only after immediate PTCA+ICRT compared with control. After 6 weeks, this NO-dependent relaxation was only trivial.

Although unchanged immediately, 6 weeks after ICRT, substance P–induced NO-dependent relaxation was reduced. PTCA, both immediate and long term, did not significantly modify the NO-dependent relaxation to substance P.

A potent endothelium-dependent, EDHF-mediated relaxation was induced by substance P (Figure 5). This relaxation was observed in control rings preconstricted with PGF_2α (40 μmol/L) combined with NO synthase inhibition by N^ω-nitro-l-arginine (100 μmol/L) and in the presence of indomethacin. This relaxation was reduced immediately after all interventions. After 6 weeks, the EDHF-dependent relaxation mediated by substance P was not significantly reduced by ICRT alone. It remained lower after PTCA alone and was eliminated after the combination of PTCA+ICRT.

Endothelium-Independent Relaxation

The sensitivity (pD₂) to sodium nitroprusside (SNP) (1 nmol/L to 10 μmol/L) of isolated rings preconstricted with a depolarizing solution (40 mmol/L KCl) was measured (Figure 6). Contrary to control, the sensitivity to SNP of treated rings was unaffected by endothelial denudation except immediately after PTCA alone, in which the pD₂ value to SNP decreased. This opposite effect was absent 6 weeks after PTCA. Finally, the sensitivity to SNP was higher after long-term PTCA+ICRT.

Discussion

The objectives of this study were to investigate in vitro the consequences of ICRT after PTCA on coronary reactivity and its endothelium-dependent regulation. The results, summarized in the Table, reveal that PTCA+ICRT has 2 important consequences: (1) it alters the passive mechanical properties of normal porcine coronary arterial wall, and (2) the relaxation mediated by endothelium-derived NO and EDHF are prevented without affecting smooth muscle contractility.

Intracoronary radiation therapy has been used in patients in an attempt to prevent restenosis. At doses similar to that used in this study, significant reduction of neointimal formation was attained.⁴ However, reports of aneurysm and pseudoaneurysm formation in the first postangioplasty radiation study,¹² in which higher-than-expected doses were used (up to a theoretical maximum of 92.5 Gy), raise the possibility that significant vessel wall death occurred when the combination of PTCA and ICRT was used. Despite the use of significantly lower doses as in this study, previously reported quantitative histomorphometric analyses showed a thinning of the arterial wall at the site of IEM rupture.¹³¹⁴¹⁵ In distinction to PTCA alone, its combination with ICRT reduced the width of the media at the IEM rupture site. These morphological changes were associated with a decrease in the passive mechanical properties of the coronary arterial wall (Figure 2). Arterial rings were stretched sequentially to a level that sustained a maximal contractile response induced by high K⁺ solution. As shown in Figure 3, PTCA+ICRT did not alter the amplitude of the contractile response. However, resting basal tension was severely reduced in this group (Figure 2). The baseline tension is normally independent of smooth muscle cell contraction in isometric myographs. Indeed, addition of a supramaximal concentration of SNP in Ca²⁺-free physiological solution does not decrease the tension of arterial rings below the level of the resting basal tension. Rather, it is the elastic component of the arterial wall that is responsible for this tension.¹⁶ Dissection after PTCA alone, either immediately or long term, had no effect on the resting basal tension, suggesting that the dissection of the intimal layer does not weaken the arterial wall. Thus, after PTCA, ICRT has multiple effects on the arterial wall structure, including a reduction in smooth muscle mass and a loss of elasticity not evidenced when these treatments are delivered in isolation or immediately.

Contractile responses to high K⁺ and PGF_2α were maintained 6 weeks after PTCA+ICRT. Only PTCA alone significantly reduced the contraction induced by high external K⁺. There is therefore a discrepancy between the results obtained with 2 different stimuli after PTCA that was not modified after 6 weeks of recovery. This discrepancy may be explained by the difference in amplitude of the contraction induced by the 2 stimuli: PTCA alone reduced the ability of the isolated vessels to fully constrict (high K⁺), whereas a lower contraction induced by PGF_2α was maintained. This suggests that PTCA alone reduces the maximal contractile response. However, acute ICRT prevented the decreased response to high K⁺ after PTCA. This is most likely due to the direct effect of ICRT on the endothelium. Indeed, as seen in Figure 3, there is a strong potentiation of the contractile response to high K⁺, suggesting that ICRT immediately reduces the endothelium-dependent inhibitory effect on coronary contractility.

Interestingly, PGF_2α-induced contractions of denuded arterial rings were not affected by the different procedures (Figure 4). Removal of the endothelium potentiated the contraction mediated by PGF_2α, although this potentiation was not significant 6 weeks after ICRT or PTCA alone. This suggests that the basal release of EDRFs significantly prevented PGF_2α-induced contractions.

The contractile response induced by PGF_2α was increased after acute PTCA+ICRT irrespective of the presence or absence of endothelium. Thus the endothelium-dependent regulation of this contractile response was obliterated. This may be due either to the release of vasoconstricting factors counterbalancing the relaxant effects of EDRF or by a lack of basal EDRF production. This effect was transitory, suggesting recovery of some endothelium-dependent regulation after 6 weeks. The endothelium had not been entirely removed by the PTCA+ICRT because substance P still induced a relaxation immediately, albeit significantly reduced (Figure 5).

Substance P stimulated the release of EDRF that produced a maximal relaxation of control coronary arterial rings precontracted by PGF_2α in the presence of indomethacin (Figure 5). The contribution of NO appears to be less than that of EDHF. A similar observation has been recently reported for acetylcholine-induced dilation of large epicardial coronary arteries in conscious dogs.¹⁷ However, in the context of our experimental conditions, it is important to consider that NO has been shown to prevent EDHF production.¹⁸¹⁹ Thus, despite the fact that numeric addition of the NO-dependent (≈30%) and EDHF-dependent (≈60%) responses corresponds to the relaxation mediated by substance P (≈90%), the contribution of EDHF to the combined response cannot be ascertained.

Endothelium-dependent relaxation was reduced by the different in vivo interventions used in this study (Figure 5). Immediately, ICRT or PTCA alone did not affect NO-dependent relaxation, but it was reduced when these 2 procedures were combined. After 6 weeks, ICRT alone reduced substance P–mediated NO-dependent relaxation of the vessels. In combination with PTCA, this response was absent. Thus ICRT damaged the endothelium-dependent release or availability of NO, which was exacerbated when combined with PTCA.

EDHF-dependent relaxation mediated by substance P was decreased immediately after ICRT and PTCA, individually and in combination. This relaxation partly recovered 6 weeks after ICRT but not after PTCA and combined PTCA+ICRT. Several reasons may explain this result. There are functional signs of regeneration of the endothelium 6 weeks after PTCA and ICRT alone that are clearly absent after the double procedure. Second, even if physical endothelial regeneration is complete, it has been shown that the new endothelium may be functionally deficient.²⁰ Surprisingly, however, whereas substance P–induced relaxation was trivial 6 weeks after PTCA+ICRT, removal of the endothelium potentiated the contraction induced by PGF_2α. This implies that receptor-operated release of EDRFs is absent 6 weeks after PTCA+ICRT, but the endothelium still prevents contraction and thus constitutively produces EDRFs.

Removal of the endothelium potentiated the relaxant effect of SNP in control arteries (Figure 6), reflecting an increased guanylate cyclase sensitivity associated with the loss of NO.²¹ This supersensitivity to nitrovasodilators has also been described in vivo after inhibition of vascular NO synthase.²²

The potentiating effect of endothelial removal on SNP-induced relaxation was blunted in all groups, suggesting a lack of endothelium-dependent regulation of smooth muscle sensitivity to SNP. The origin of this alteration is unknown because it is not specific for 1 type of procedure (ICRT or PTCA). The only link between these changes is the endothelial dysfunction, since in denuded vessels SNP-induced relaxation is similar in all groups except immediately after PTCA. In this latter case, it is possible that rubbing the endothelium immediately after the PTCA but not after healing damaged the smooth muscle, as shown by the decrease in sensitivity to SNP. Also, a clear increase in SNP-induced relaxation was observed after long-term PTCA+ICRT. This is most likely to be associated with the decreased NO production and/or release observed under stimulated conditions (Figure 5). This would be in agreement with previously published data showing that NO synthase inhibition increases guanylate cyclase sensitivity both in vitro and in vivo.²¹²²

It is important to note that our results differ from a recent in vivo study⁶ in which SNP-induced dilation was absent immediately and in the long term after ICRT, whereas acetylcholine-induced relaxation was restored after 5 weeks. However, we have no explanation for this discrepancy other than the different methodological approach.

The identical response of the 2 groups of controls from irradiated and nonirradiated animals suggests that the effects are entirely local and that a significant systemic or distant effect, as has been previously suggested,⁵ can be excluded.

Clinical Implications

The implications of endothelial dysfunction over a short vessel segment are unclear. First, a patent arterial segment that has impaired reactivity is a scenario not dissimilar to a stented segment. This is clearly preferable to a restenosed segment. This may have different implications if the treated segment is long. Second, the reduction of vessel wall integrity, as shown by a reduction in baseline tension, mandates longer-term follow-up than usual in the clinical situation. Angiographic and intravascular ultrasound studies can provide bountiful information regarding the stability of these vessel segments.⁴ Finally, because of the severity of endothelial dysfunction demonstrated, the possibility of very delayed regeneration must be entertained. Although this study was performed on normal porcine arterial rings, its findings have significant implications for a treatment currently under extensive clinical investigation.

Limitation of the Study

Because this study was performed immediately and at 6 weeks after intervention, no conclusion can be drawn regarding the progression of this process. Furthermore, the effects demonstrated are limited to the site of the intervention and the response of proximal and distal vessel segments were not assessed, although the reactivity of nontreated coronary arteries from animals in which another coronary vessel had been irradiated was unaltered. Finally, atherosclerosis being the background of this clinical intervention, we do not know what the impact would be of PTCA+ICRT in this context. This study was designed to investigate the effects of PTCA+ICRT and ICRT alone on coronary reactivity and endothelial function; our data cannot predict the outcome of PTCA+ICRT in a hypercholesterolemic/atherosclerotic clinical background.

Conclusions

PTCA+ICRT altered the passive mechanical properties of porcine coronary arterial wall. Furthermore, receptor-operated release of endothelium-derived NO and EDHF were reduced by ICRT and PTCA alone and prevented by the combination of PTCA+ICRT. The ongoing impairment of endothelial function after 6 weeks in this model further supports the longer-term follow-up of all patients undergoing post-PTCA irradiation.

Methods

Animals

Juvenile Yorkshire pigs (weight 25 to 30 kg, obtained from a local supplier) were treated with 650 mg aspirin and 30 mg nifedipine (to prevent coronary spasm) 24 hours before the procedure. The animals received an intramuscular injection of ketamine (12 mg/kg), xylazine (8 mg/kg), and atropine (1.2 mg/kg) as premedication. After intubation, anesthesia was induced and maintained with 1% halothane in a 2:1 mixture of oxygen and air. After percutaneous puncture of the right or left femoral artery, an 8F femoral sheath was placed and heparin (150 U/kg) and xylocaine (100 mg) were administered as intra-arterial boluses. An 8F right Amplatz guiding catheter (AR2, Cordis Corp)was advanced to the base of the aorta. Under fluoroscopic visualization, the left main coronary ostium was engaged for digital arteriography (View System, Electromed).

Angioplasty and Radiation Therapy

Two vessels, the left anterior descending (LAD) and circumflex (Cx) coronary arteries, were treated in each animal. Arteries were randomly assigned to be treated with balloon angioplasty (PTCA), ICRT, or PTCA+ICRT or were left untouched. Arterial diameters at treated segment sites were 2.5 to 3.8 mm as assessed by quantitative coronary angiography. The balloon (Viva, Boston Scientific) was sized to achieve a balloon-to-artery ratio of ≈1.2. Balloon diameter was 2.5 to 4.0 mm and length was 20 mm. Arterial injury was performed by a single 60-second inflation at 6 atm.

ICRT was performed with a β-emitter (Sr/Y⁹⁰, Novoste Corp) through a noncentered, 5F, closed-end catheter to the LAD or Cx coronary artery. A dose of 16 Gy to a depth of 2 mm from the source center was delivered with a 3-cm source train to adequately cover the balloon-injured segments.

Immediately after these procedures or after 6 weeks, the pigs were killed and the hearts were explanted.

Reactivity Study

Isolation of Vessels

LAD and Cx arteries were isolated 15 to 30 minutes after the removal of the heart from the chest and placed in cold (4°C) solution. Approximately 15 minutes later, the surrounding tissues were removed under a binocular microscope. The blood vessels were cut into rings (2 mm in length). In some rings, the endothelium was removed mechanically by insertion of a wooden peg into the lumen and gentle rolling.²³ The functional removal of the endothelium was assessed by the disappearance of endothelium-dependent relaxation induced by substance P (0.1 μmol/L).

Solutions and Drugs

The tissues were incubated in modified Krebs-Ringer bicarbonate solution with the following composition (mmol/L): NaCl 130, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ 1.17, NaHCO₃ 14.9, CaCl₂ 1.6, EDTA 0.026, and glucose 10, and aerated with a 12% O₂/5% CO₂/83% N₂ gas mixture (pH 7.4). The solution contained indomethacin (10 μmol/L, inhibitor of cyclooxygenase). To prepare K⁺-rich solutions (40 mmol/L and 127 mmol/L), equimolar amounts of NaCl were replaced with KCl. The following drugs were used: indomethacin, N^ω-nitro-l-arginine, prostaglandin F_2α (PGF_2α), sodium nitroprusside (SNP) (Sigma), and substance P (American Peptide Co). Stock solutions were prepared in distilled water except for indomethacin (dissolved in ethanol) and substance P (30% dimethylsulfoxide/70% distilled water, vol/vol). Preliminary experiments indicated that none of the solvents used produced detectable changes in isometric force at the concentrations used (data not shown).

Organ Chamber Experiments

The rings were mounted on 20-μm tungsten wires in microvessel myographs (IMF, University of Vermont) filled with control solution (10 mL) and maintained at 37°C. One of the tissue holders was connected to a force transducer (Minebea UL-10) to record changes in isometric tension. The rings were stretched to the optimal point of their length-active tension relation, as determined by the contractile response to 40 mmol/L K⁺ solution at progressive levels of stretch as previously described.²³ More specifically in this study, rings were initially subjected to 2 tension loads of 1.5 g and 1 of 1 g at 20-minute intervals. After these 3 cumulative tension loads, rings were stimulated with high K⁺. Then, an additional tension load of 0.5 g was applied and rings stimulated with high K⁺ after 20 minutes. If the contraction was greater by >20% compared with the previous response, a second 0.5-g load was applied. A total force of 5.0±0.5 g was necessary to obtain a stable contractile response, with no difference between the 7 groups in this study.

Two rings of each vessel, one with and one without endothelium, were mounted in parallel on myographs. In denuded arteries, substance P (0.1 μmol/L) did not induce relaxation.

Histology

To confirm that the PTCA and irradiation protocol used produces anatomic results equivalent to those previously reported,¹³¹⁴¹⁵ in a separate series of experiments, 4 arteries were treated with balloon angioplasty alone as described above and a further 4 vessels were treated with ICRT immediately after balloon angioplasty. The pigs were killed 6 weeks after the procedure as described above. The treated arteries were rapidly cannulated and flushed with saline. Perfusion fixation was performed with 2.5% phosphate-buffered glutaraldehyde at 100 to 110 mm Hg for 30 minutes. Gross ischemic myocardial damage was excluded by macroscopic assessment. The treated vessel segments were localized and dissected from the epicardial surface and cut into 3- to 4-mm sections. These sections were processed and embedded in paraffin. Cross-sections (4 μm) were stained with Movat Pentachrome for histological examination.

Animal Care

The pigs were fed regular chow with no additive. They received no medication during the follow-up period; in particular, they did not receive aspirin or ticlopidine. All animals were cared for in accordance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences (NIH publication 85-23, revised 1985).

Statistical Analysis

Results are expressed as mean±SEM. In all experiments, n represents the number of arteries. Vasoconstrictions are expressed as percent of the maximal response (E_max) obtained in the presence of 40 mmol/L K⁺ at the beginning of each individual experiment; vasorelaxations are expressed as the percent inhibition of the preconstricting tone. The level of tone was measured at the plateau of constriction. The half-maximum effective concentrations (EC₅₀) of SNP were measured from each individual dose-response curve with a logistic curve-fitting program (Allfit, University of Montréal). The pD₂ value is the negative log of the EC₅₀. Statistical differences between means were determined by ANOVA followed by a Scheffé’s F test. A value of P<0.05 was accepted as significant for differences between groups of data.

The Methods section of this article can be found at http://www.circulationaha.org

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Footnotes