Preexisting rudimentary intercoronary anastomoses undergo remarkable growth and development in response to coronary artery occlusion. Proliferation of smooth muscle cells (SMC) and endothelial cells (EC) results in gradual transformation of the thin-walled native collateral channels into vessels that are similar in appearance to normal coronary arteries and as large as 1,000 μm in diameter when fully mature (1,2). Vasodilation of normal coronary arteries in response to agents such as acetylcholine (ACh), bradykinin (BK), and substance P is dependent on production of an endothelium-derived relaxing factor (EDRF), which is believed to be nitric oxide (NO) or a related compound (3). Several investigators have reported that NO-mediated responses are impaired in coronary arteries with newly regenerated endothelium after balloon denudation (4-6). In contrast, studies performed 12-24 weeks after coronary occlusion demonstrated that the newly developed endothelium of partially transformed collateral vessels 250-500 μm in diameter exhibited intact responses to ACh and BK (7,8). The present study was performed to characterize endothelium-dependent and endothelium-independent vasodilator responses in fully mature coronary collateral vessels that develop in response to prolonged coronary artery occlusion. Collateral vessel segments were isolated from canine hearts 6 months after a gradually occluding plug was introduced into a coronary artery to stimulate collateral development. Responses to the endothelium-dependent vasodilators, ACh, BK, and substance P, as well as the endothelium-independent vasodilator nitroglycerin (NTG), were examined in coronary collateral vessels as well as in normal epicardial arterial segments of similar size.
Induction of collateral vessel growth
Studies were performed on 8 adult mongrel dogs in which collateral vessel development was induced by embolization of the left anterior descending artery (LAD) with a hollow intraarterial plug. Animals were anesthetized with sodium pentobarbital (25-30 mg/kg intravenously, i.v.), intubated, and ventilated with a respirator. A cutdown was performed over the right carotid artery under sterile conditions. After administration of heparin sodium (6,000 U i.v.), an 8F Judkins right coronary catheter was introduced into the right carotid artery. Under fluoroscopic guidance, the catheter was advanced into the left coronary ostium and directed toward the LAD. An angioplasty guidewire (0.014 inch, US Catheter Instruments) was advanced through the catheter into the distal LAD. The coronary catheter was then removed without disturbing the guidewire, and a bullet-shaped hollow Teflon plug (2.5 mm OD, 1.2 mm ID, 4 mm long) with a stainless-steel radiopaque rim was pushed along the guidewire with a length of flanged PE-90 polyethylene tubing until the plug was firmly wedged in the coronary artery. The guidewire was then removed, and coronary angiographic images were obtained by injection of 5-10 ml 60% diatrizoate meglumine to visualize the position and patency of the plug. The catheter was then removed and the carotid artery repaired. Animals were treated with aspirin (80 mg/day) and dipyridamole (25 mg three times daily) for 2 days beginning on the morning of the embolization procedure to retard thrombotic occlusion of the plug while some initial increase in collateral flow occurred.
The animals were returned to the laboratory ≈6 months after coronary embolization, premedicated with morphine sulfate (1 mg/kg s.c.), anesthetized with α-chloralose (100 mg/kg i.v. followed by 10 mg/kg/h), intubated, and ventilated with a respirator. Supplemental oxygen was used to maintain arterial pO2 in the physiologic range. A 7F NIH catheter was introduced into the femoral artery and positioned in the ascending aorta for pressure monitoring. A left thoracotomy was performed in the fifth intercostal space, and the heart was suspended in a pericardial cradle. The coronary artery plug was located by palpation, and the LAD was dissected free for 1.0-1.5 cm proximal and distal to the plug. After administration of sodium heparin (200 U/kg, i.v., followed by 1,000 u/h), the artery was occluded proximally and a longitudinal arteriotomy was performed. The plug was extracted, and the artery was allowed to bleed freely to dislodge any residual thrombus. The artery was then cannulated with a thin-walled stainless-steel cannula (4.0 mm OD). Pressure at the cannula tip was measured with a 23-gauge tube incorporated into the wall of the cannula.
Aortic and coronary cannula pressures were measured with Statham P23ID pressure transducers. Data were recorded on an eight-channel direct writing recorder (Coulbourne Instruments, Lehigh Valley, PA, U.S.A.). Interarterial collateral blood flow was measured by collecting retrograde flow from the coronary artery cannula into a graduated cylinder for 30-s intervals while the cannula tip was maintained at the level of the heart. The level of the cannula tip was maintained constant throughout the study. Control measurements of retrograde blood flow were repeated until consistent measured collections were obtained; three measurements were then obtained, and the results were averaged.
After hemodynamic measurements were made, the heart was rapidly excised and placed in cold, physiologic Krebs-Henseleit buffer solution with a composition of (in mM): NaCl 118, KCl 4.7, NaHCO3 25, CaCl2 3.0, MgSO4 1.22, KH2PO4 1.22, EDTA 0.5, and glucose 10.0. Segments were then removed from the midportions of the collateral vessels and from size-matched control vessels from branches of a left circumflex obtuse marginal coronary artery (LCX) distant from the collateralized region. Collateral vessels were easily identified on the epicardial surface of the left ventricle as tortuous interarterial anastomoses extending from the marginal branches of the LCX to the diagonal branches of the LAD descending artery, as well as from the posterior descending artery to the LAD near the apex of the left ventricle. Vessels were carefully dissected free under dissecting microscope (×40, Bausch and Lomb) and divided into 0.5-cm segments. The external diameters of the vessel segments were determined with the aid of a micrometer eyepiece mounted on the dissecting microscope. The control and collateral vessels ranged from 500 to 1,000 μm in diameter.
After the vessels had been dissected free, one to two collateral and one to two control segments were chosen for study. The vessels were suspended in 20-cc organ chambers containing Krebs-Henseleit buffer solution at 37°C oxygenated with a mixture of 95% O2/5% CO2. Each ring was suspended by two stirrups constructed of 100-μm tungsten wire that were passed through the vessel lumen. One stirrup was anchored inside the organ chamber, and the other was connected by 5-0 silk suture to a force transducer (model T43-05, Coulbourne Instruments). Isometric force was recorded on a direct writing recorder (Hewlett-Packard 7758A). The vessels were allowed to equilibrate for 45-60 min at minimal tension and were then placed at the optimal point on their length-tension curve, as determined by repeated exposures to 40 mM KCl. Basal tension of the rings was increased gradually until the contractions to 40 mM KCl were maximized. This length was then maintained throughout the remainder of the experiment.
After optimal basal tension had been achieved, vascular rings were preconstricted with prostaglandin F2α (PGF2α) (10-6-10-4.5M), so that >30% of the KCl contraction was attained. After preconstriction, responses to increasing concentrations of the endothelium-dependent vasodilators ACh, substance P, and BK, as well as the endothelium-independent vasodilator NTG, were observed. The response to the concentration of each agent was allowed to reach a plateau before the subsequent dose was added. Responses are reported as percent relaxation of the PGF2α-induced constriction for each concentration of agonist. The order of administration of vasoactive agents was randomized. Vessels were washed two to three times with Krebs-Henseleit buffer and allowed to equilibrate for 10-15 min between interventions. Collateral and normal arterial vessels from three more animals were used to examine the effect of inhibition of NO synthase on the response to the endothelium-dependent vasodilator ACh. Vessel rings were preconstricted with PGF2α (10-6-10-4.5M), and dose-response curves to ACh were observed during control conditions and in the presence of NG-monomethyl-L-arginine (L-NMMA 10-4M). All interventions were performed in the presence of indomethacin (10-5M) to exclude prostanoid effects.
ACh chloride, BK, substance P, L-NMMA, and indomethacin were obtained from Sigma Chemical (St. Louis, MO, U.S.A.). NTG was obtained from Lilly Pharmaceuticals. PGF2α was obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.). Stock solutions of BK (2 × 10-3M) and substance P (2 × 10-4M) were made in distilled water and stored at -20°C. ACh L-NMMA and NTG were prepared fresh on the day of the experiment with distilled water. All subsequent drug dilutions were performed with Krebs-Henseleit buffer on the day of the study.
Vascular relaxation was expressed as percent change from the preconstricted tension. Mean responses for each drug at each dose were compared by analysis of variance (ANOVA) for repeated measures; p < 0.05 was considered statistically significant. When a significant difference was noted, individual comparisons were made by the Scheffé test. The effective concentration for 50% response (EC50) was calculated for each dose-response curve by nonlinear regression analysis with an inhibitory sigmoid Emax model (9) (PCNONLIN; Lexington, Kentucky). Collateral and control vessel EC50 results were compared by Student's t test for paired data; p < 0.05 was considered statistically significant.
Embolization of the anterior descending coronary artery resulted in epicardial collateral vessel development with minimal infarct of the dependent myocardium. At the time of study, mean heart rate (HR) was 131 ± 14 beats/min. Mean distal coronary pressure (collateral pressure) with the cannula occluded was 88 ± 4 mm Hg, whereas simultaneously measured mean aortic pressure was 102 ± 5 mm Hg. Mean retrograde blood flow from the cannulated LAD was 39 ± 8 ml/min.
Isolated vascular ring studies
Characteristics of collateral and control vessel segments are shown in Table 1. Collateral and control vessel external diameters were comparable at 900 ± 39 and 907 ± 37 μm, respectively. The basal tension that resulted in optimal force development in response to 40 mM KCl was similar for control arteries and collateral vessels. At optimal basal tension, force development in response to 40 mM KCl was similar in collateral and control vessels. However, maximum collateral constriction in response to PGF2α (2 × 10-5M) was significantly less in collateral vessels than in control vessels (p < 0.05).
Endothelium-dependent responses were agonist dependent. As shown in Fig. 1, ACh caused significantly greater dose-dependent relaxation in collateral vessels than in control vessels (p < 0.001, ANOVA), so that the maximum relaxation achieved was greater in collateral vessels (129 ± 3.6% of preconstricted tension) than in normal vessels (110 ± 6.2% of preconstricted tension; p < 0.02). However, the EC50 value for ACh was similar in collateral vessels (2.2 × 10-7M) and in normal vessels (3.4 × 10-7M).
BK resulted in dose-dependent relaxation in collateral and control vessels (Fig. 2). The EC50 value tended to be lower for control vessels (1.06 × 10-8M) than for collateral vessels (1.82 × 10-8M), but this was not statistically significant. However, at the highest doses, collateral vessels relaxed significantly more than normal vessels, with maximal responses of 138 ± 4.6 vs. 108 ± 5.0% of initial constriction, respectively (p < 0.05).
Substance P resulted in dose-dependent relaxation in collateral and control vessels (Fig. 3). The EC50 value was similar in collateral (1.36 × 10-9M) and normal vessels (1.15 × 10-9M). In addition, maximal relaxation was similar for collateral vessels and normal vessels at 119 ± 5.1 and 113 ± 3.8% of preconstricted tension, respectively. Therefore, the dose-response curves for substance P were not different between collateral and normal vessels.
Endothelium-independent relaxation in response to NTG was not significantly different between collateral and control vessels (Fig. 4). The EC50 value for NTG was not significantly different between collateral and control vessels (1.37 × 10-7 and 0.68 × 10-7M, respectively). In addition, the maximum relaxation was similar for the two vessel types.
Inhibition of NO synthase
The effect of L-NMMA on ACh-induced relaxation was examined in normal and collateral vessels from 3 animals. L-NMMA caused a rightward shift of the dose-response curve to ACh; during control conditions, preconstricted collateral vessels underwent 125 ± 12% relaxation in response to the highest dose of ACh (10-5M), whereas after L-NMMA administration, collateral vessels underwent only 40 ± 10% relaxation in response to this dose of ACh (p < 0.01). During control conditions, normal vessels underwent 84 ± 7% relaxation in response to the maximum ACh dose, whereas after L-NMMA administration, ACh caused only 12 ± 6% relaxation (p < 0.01). Therefore, L-NMMA inhibited the ACh-induced relaxation in both normal and collateral vessels.
Our results confirm previous reports that endothelium-dependent responses to ACh and BK are intact in well-developed coronary collateral vessels (7,8). Our present findings extend the previous reports and demonstrate that the maximum responses to these agents are actually slightly enhanced in the larger collateral vessels examined in the present study. In addition, our results demonstrate intact vasodilation in coronary collateral vessels in response to substance P. These new findings are discussed in detail.
Response to NTG
Collateral vessel growth and development has been well described in the canine model (2,10). Coronary artery occlusion initially results in proliferation of both longitudinal and circumferential smooth muscle, but the longitudinal muscle fibers almost entirely regress ≤6 months after coronary occlusion, resulting in a muscular media similar in appearance to a normal artery (10). Our results show that this smooth muscle transformation is associated with normal relaxation in response to NTG. The vasodilator effect of NTG results from vascular NO generation. Schror and colleagues (2) reported that when NTG was added to the coronary perfusate of rabbit hearts, NO could be detected only after passage through the heart, indicating that nitric acid generation from NTG is not a spontaneous event, but instead requires exposure to the coronary vasculature. NO generation was not dependent on the endothelium, since pretreatment with L-NG-monomethylarginine or removal of the endothelium with trypsin did not impair NTG-induced NO release. Using bovine coronary artery subcellular fractions, Chung and Fung (12) demonstrated that production of NO from NTG is dependent on an enzyme associated with the plasma membrane of the SMC. The availability of thiols (cysteine) is necessary to facilitate conversion of NTG to the unstable intermediate nitrite (NO2), and subsequently to NO. Our present findings demonstrate that the enzymatic mechanism for generation of nitric acid from NTG is intact in well-developed coronary collateral vessels.
Response to vasoconstrictors
In the present study, vessel segments were subjected to progressively increasing stretch while repeated exposure to 40 mM KCl was used to determine the length at which maximum force development occurred. At this optimal point on the length-tension relation, the contractile response to 40 mM KCl was similar in collateral vessels and in normal arterial vessels of similar size. This finding is in agreement with the report of Flynn and associates (8) who examined segments of epicardial coronary collateral vessels from dogs 12 weeks after placement of an ameroid constrictor on the LAD. In contrast to the report of Flynn and associates (8) and to our results, those of Angus and co-workers (7) showed that the contraction in response to a higher concentration of KCl (124 mM) was significantly less in collateral vessels than in normal arteries of similar size. Morphologic studies by Angus and co-workers demonstrated that the collateral vessels had a thinner muscular media as compared with normal arterial vessels of similar size. The decreased smooth muscle in the collateral vessels may have transformed into an attenuated contraction in response to the maximal constricting dose of KCl used in the study of Angus and co-workers (7). In the present study, we used 40 mM KCl to assess changes in active tension at increasing vessel length. In this way, each vessel was placed optimally on its length-tension curve. At the lower KCl concentration used in the present study, differences in maximal contraction between collateral and normal vessels were not apparent. Therefore, differences in experimental design may explain the differences in the KCl constrictor response.
In the present study, preconstriction with PGF2α resulted in significantly less force development in collateral vessels than in normal vessels. Angus and co-workers (7) reported that responses to the thromboxane A2 analogue U46619 were also decreased in collateral vessels as compared with normal arterial vessels. Similarly, Parker and colleagues (13) reported that collateral vessels were less responsive to endothelin-1 than were size-matched normal vessels. The attenuated collateral PGF2α constrictor response we noted could be a result of the thinner muscular media in collateral vessels, although differences in receptor number or function could also lead to agonist-dependent differences in constrictor responses.
Endothelial function in collateral vessels
Vascular remodeling during collateral development is associated with a prominent increase in the number and size of the EC. The EC in the newly transformed collateral vessels are oriented in the direction of blood flow, with prominent ridges bulging into the vascular lumen (14). With continued maturation, endothelial bulges become less prominent and EC density tends to decrease; however, even 1 year after coronary occlusion, histologic abnormalities of the endothelium persist (14). Despite these morphologic abnormalities, our present data confirm that endothelium-dependent vascular relaxation is intact, in contrast to the impairment of endothelial function reported in regenerated endothelium after mechanical endothelial denudation of canine and porcine coronary arteries (4-6). Differences in function between newly developed coronary artery endothelium and transformed collateral endothelium may be related to differences in the stimuli for EC growth. Schaper and associates (14) suggested that increased shear stress secondary to turbulent flow stimulates EC proliferation in collateral vessels. Increased shear stress may also alter basal and stimulated NO release. Using isolated rings of canine femoral artery, Miller and co-workers (15) demonstrated that persistently increased blood flow produced by a surgically created arteriovenous fistula caused augmented relaxation to ACh, suggesting that chronic increases in blood flow can enhance receptor-stimulated production of NO. Similarly, Sessa and colleagues (11) recently reported that daily exercise enhances ACh-induced coronary artery vasodilation. They concluded that chronic increases in shear result in enhanced NO synthase gene expression. Chronically increased flow across the coronary collateral vessels might similarly augment NO release. Differences in the nature and degree of vascular injury could also influence function of the newly developed endothelium in collateral and denuded arterial vessels. Medial damage occurs with mechanical denudation (6), which might result in a greater inflammatory reaction and release of mitogenic factors that could alter the function of the newly regenerated EC.
In contrast to our present findings, in the study of Angus and co-workers (7) similar endothelium-dependent relaxation occurred collateral and control vessels in response to ACh. The augmented maximum responses to ACh and BK in the present study could be due to synergistic effects between NO and endothelium-dependent hyperpolarizing factor (EDHF). ACh and BK stimulate production of both NO and EDHF in normal coronary arteries (16-18). EDHF causes hyperpolarization and relaxation of vascular smooth muscle, whereas NO results in relaxation only (17). In the study of Angus and co-workers (7), vessels were preconstricted with KCl rather than PGF2α. The use of KCl as a constrictor would suppress hyperpolarization and subsequent vascular relaxation in response to endothelial production of EDHF. In the present study, L-NMMA, which acts as a competitive inhibitor of NO synthase, caused greater inhibition of ACh-induced relaxation in normal vessels than in collateral vessels. In contrast to responses to ACh, responses to substance P were not significantly different between normal and collateral vessels. Substance P is a known endothelium-dependent vasodilator that does not result in smooth muscle hyperpolarization (19). These findings could be explained by enhanced EDHF production in collateral vessels. If collateral vessels produce more EDHF in response to ACh and BK than do normal vessels, preconstriction with PGF2α rather than KCl could account for the enhanced responses to ACh and BK in the present study. Increased EDHF production in collateral vessels could also explain the differing responses to ACh after NO synthase blockade in the present study. Therefore, after L-NMMA administration, ACh still caused 40% relaxation in collateral vessel segments but only 12% relaxation of normal coronary artery segments. The greater residual relaxation in collateral vessel segments induced by ACh after NO synthase blockade suggests that another vasodilator was present. Because the vessels were studied in the presence of indomethacin, this dilator cannot be a prostaglandin but could be EDHF. Additional studies are needed to determine whether hyperpolarization in response to muscarinic receptor stimulation is augmented in well-developed coronary collateral vessels as compared with normal coronary arteries.
Mature coronary collateral vessels preconstricted with PGF2α demonstrated similar sensitivity and equal or greater relaxation in response to NO-dependent vasodilators than did normal arterial vessels of similar size. These findings demonstrate that the endothelium in collateral vessels that develops in response to coronary artery occlusion has intact NO-dependent vasodilator mechanisms.
Acknowledgment: This work was supported by U.S. Public Health Service Grants No. HL20598 and HL32427 from the National Heart, Lung and Blood Institute, a grant from the Minnesota Medical Foundation; and a Medical Student Research Fellowship from the American Heart Association (J.A.). We thank Todd Pavek and Melanie Crampton for expert technical assistance.
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