Balloon Dilatation of Porcine Pulmonary Arteries Decreases Endothelium-Dependent Relaxations and Increases Vasoconstriction to Aggregating Platelets
Background Balloon dilatation of muscular coronary arteries disrupts endothelium and smooth muscle and allows platelet aggregation and adherence and cell proliferation, which can lead to restenosis. Balloon dilatation of the more distensible pulmonary artery is commonly performed, but the extent of damage to endothelium and its effect on the release of endothelium-derived nitric oxide (EDNO) and prostacyclin has not been studied.
Methods and Results We dilated distal pulmonary arteries of intact ex vivo porcine lungs (n=20; balloon-dilated [BD] group) using similar-sized adjacent vessels as controls with (E+ group) and without (E− group) endothelium. Isolated rings were studied in vitro. Aggregating platelets caused constrictions of quiescent rings from the BD (27.4±8% of constriction to 80 mmol/L KCl) and E− groups (24±5%), which were inhibited by pyrilamine, a histamine blocker (11±4%; P<.05), and an intact endothelium (8±5%; P<.05). Constrictions to histamine and KCl were similar in the BD and E− groups. In rings with increased tone, platelets caused endothelium-dependent relaxations in the E+ group (70±6% relaxation), which were significantly (P<.05) inhibited in the BD group (20±7%), by l-nitro-arginine (EDNO blocker, 17±7%) and in the E− group (21±9%). Balloon dilatation markedly reduced endothelium-dependent relaxations to 5-hydroxytryptamine, thrombin, acetylcholine, and the calcium ionophore A23187, but relaxations to sodium nitroprusside were unaffected.
Conclusions Despite the distensibility of the pulmonary artery, balloon dilatation significantly damages the pulmonary endothelium, decreases EDNO production, impairs vasodilation, and favors platelet-induced vasoconstriction.
Balloon angioplasty of coronary artery stenosis is a useful clinical tool, but restenosis and early thrombosis with occlusion are not uncommon complications.1 In small-diameter, muscular, poorly distensible arteries, balloon dilatation is known to disrupt the endothelium extensively, leaving the underlying vascular smooth muscle unprotected against circulating platelets and clot-forming blood elements.2 This allows platelet aggregation, thrombin deposition with further platelet adherence, and clot formation, which can then expand and cause vascular occlusion. Stimulation of vascular smooth muscle by circulating mitogens can also cause smooth muscle proliferation with subsequent restenosis. An intact endothelial layer appears to protect against this process.2
The endothelium can produce vasodilators and vasoconstrictors that regulate vascular tone.3 4 5 Two of these vasoactive substances, endothelium-derived nitric oxide (EDNO), an l-arginine–dependent nitrovasodilator,3 6 7 8 and prostacyclin, an endogenous prostaglandin produced from arachidonic acid, inhibit platelet aggregation and clot formation in vivo and in vitro.9 Platelet aggregation causes the release of platelet-derived vasoactive substances such as 5-hydroxytryptamine (5HT, serotonin), ADP, and catecholamines that may play a role in the vascular reactivity that occurs during platelet aggregation and thrombus formation.10 An intact pulmonary endothelium inhibits platelet aggregation and the effect of the vasoactive mediators released.9 10 Therefore, an intact endothelium may play a significant role in preventing platelet aggregation and clot formation. In addition, nitric oxide is released from an endothelium that is stimulated by the vasoactive substances released by platelets.10 Nitric oxide is also known to inhibit smooth muscle proliferation in vitro.11 This may be secondary to its activation of guanylate cyclase and the subsequent rise of cGMP. The loss of an intact endothelial layer would prevent this inhibitory effect and thus may play a role in the healing and remodeling process of the vessel wall.
Just as coronary artery stenosis presents a difficult clinical situation for adult cardiologists, stenotic pulmonary arteries present a difficult clinical situation for the pediatric cardiologist and the pediatric cardiovascular surgeon because few interventions yield high success rates with this lesion.12 Balloon angioplasty of the stenotic proximal pulmonary arteries, using the standard 4- to 6-atm-burst-pressure balloons, is successful, however, in 60% of the cases, but restenosis and dilation failure are not uncommon.13 Similar to what is seen pathologically in coronary balloon dilatation, this method produces tears in the pulmonary vascular intima and media, which then undergo vascular remodeling.14 The majority of intimal tears are isolated, however, and thrombus formation with occlusion in large vessels is not a major complication in the distensible pulmonary arteries. However, the extent of thrombus formation and restenosis in smaller, distal pulmonary arteries has not been systematically addressed. These smaller vessels may be at higher risk for restenosis and occlusion. As pediatric cardiologists extend the usefulness of balloon angioplasty to smaller and more distal vessels, the importance of this information intensifies. The extent of endothelial injury or disruption after pulmonary balloon angioplasty and its effect on local vascular tone have not been studied previously. Therefore, unlike the coronary artery data, the impact of balloon dilatation on pulmonary arterial endothelial cell function and protection of the underlying vascular smooth muscle is untested. We previously demonstrated that mechanically disrupting the integrity of porcine pulmonary endothelium from large and small vessels in vitro with a stainless steel wire or cotton swab completely removes the endothelial layer from the vascular lumen, leaving the smooth muscle intact. This procedure allows platelet-induced vasoconstriction of isolated pulmonary arteries.10 Because balloon angioplasty is known to tear the coronary endothelial layer, we hypothesized that some degree of endothelial disruption would also occur after pulmonary arterial balloon dilatation despite the distensibility of the pulmonary arteries, with subsequent loss or decrease of EDNO and prostacyclin effect. If endothelial disruption were significant, this milieu would favor platelet aggregation and release of vasoactive substances, with the balance of vascular tone shifted toward vasoconstriction. We tested this hypothesis in vitro by performing balloon dilatation in porcine lungs. We studied the effects of dilatation on endothelial function in isolated pulmonary arteries exposed to platelets and the substances released from the platelets, specifically measuring changes in vascular tone and the release of EDNO and prostacyclin.
Porcine lungs from adult animals (n=20) obtained from a local slaughterhouse were transported to the laboratory (<30 minutes) in cold modified Krebs-Ringer bicarbonate (Krebs’) solution of the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 · 7H2O 1.2, KH2PO4 1.2, NaHCO3 25, CaEDTA 0.026, and glucose 11. One lung was removed at the level of the main bronchus, and a 7F balloon-dilating catheter (8 mm in diameter, 3 cm long) was introduced into a small distal pulmonary artery (2 to 3 mm in diameter) in the intact lung. The balloon was inflated twice to 4 atm for 20 seconds per inflation (balloon-dilated [BD] group). The lung parenchyma was dissected away from the pulmonary artery, and the dilated section of artery was then removed. The pulmonary artery was removed with the catheter still in place to ensure that the section removed was indeed the dilated section of artery. A similar-sized pulmonary artery that was not dilated by balloon located adjacent to the BD artery was removed to serve as the control artery.
Isolated Ring Preparations
The vessels were cleaned of excess lung tissue and adventitia and were cut into rings (4 to 5 mm long). We denuded some control rings by placing a thin (0.25-mm) stainless steel wire within the lumen of the ring. The ring was gently rolled 15 to 20 times on paper soaked with Krebs’ solution. The rings from BD and control arteries (with and without endothelium) were then suspended between a fixed stirrup and a force transducer (FT03, Grass Instruments) for isometric tension recordings (Gould 8000S, Gould Inc). The rings were lowered into 25-mL organ baths filled with Krebs’ solution maintained at 37°C and bubbled with a 95% O2/5% CO2 gas mixture. The optimal relation between length and tension was then determined by the maximal response to histamine (10 μmol/L) in each ring. Indomethacin (10 μmol/L) was instilled into the organ bath, and the vessels were incubated in this solution for 30 minutes. The rings were then studied in the quiescent state or under conditions of increased tone. For those rings studied under increased tone, histamine (0.3 to 1 μmol/L) was added to the bath to cause a contraction equal to 50% of the maximal response to 10 μmol/L histamine. The rings were then exposed to the agonists in a cumulative manner (see the “Protocols” section). Contractions are expressed as a percentage of maximal contraction for that agonist or as a percent of an 80 mmol/L KCl contraction (platelet-induced contractions only). Relaxations are expressed as a percentage of the submaximal contraction to histamine. The total experiment time was <6 hours.
Platelet suspensions (see the “Platelet Preparation” section) were instilled into organ chambers containing rings in the quiescent state and in chambers containing rings contracted with histamine. Final platelet concentration was 100 000 platelets/μL. Rings from the BD arteries were studied in parallel with control rings with and without endothelium.
Platelets were instilled into organ chambers containing BD and control rings. The platelet-induced change in tension was followed by exposure to 80 mmol/L KCl. The KCl contraction was used as a reference contraction for each individual ring because it is a non–receptor-mediated contractile agonist. Previous studies with KCl have shown that the contractions are not influenced by the presence or absence of the endothelium.10 In some control rings without endothelium, pyrilamine (5 μmol/L), a histamine antagonist, was present (30-minute incubation) before platelet exposure in an attempt to block the effect of histamine released from aggregating platelets.10 Dose-response curves to histamine (receptor-mediated agonist) and KCl (non–receptor-mediated agonist) were established for BD and control rings with and without endothelium to determine the responsiveness of the vascular smooth muscle to vasoconstrictors and to exclude damage to the vascular smooth muscle contractile apparatus.
Rings With Tone
In rings contracted with histamine to 50% of the maximal histamine contraction, platelet suspensions of 100 000/μL were instilled as a single concentration, and changes in tension were recorded. In some experiments, l-nitro-arginine (LNA), a nitric oxide synthase inhibitor, was present to prevent the synthesis and effect of EDNO. The relaxations were compared with a relaxation elicited to sodium nitroprusside (10 μmol/L) and expressed as a percent inhibition of the histamine contraction. Rings contracted with histamine were also exposed to increasing concentrations of 5HT (serotonin) and ADP (receptor-mediated, vasoactive substances released from platelets), acetylcholine (the classic receptor-mediated agonist for the release of endothelium-derived relaxation factor/EDNO),3 thrombin (a receptor-mediated agonist involved in clot formation and platelet aggregation),15 and the calcium ionophore A23187 (a non–receptor-mediated agonist that increases endothelial concentrations of calcium) with subsequent EDNO release. Rings from the BD group and control group without endothelium were also exposed to a full concentration-response curve to sodium nitroprusside (a nitrovasodilator that releases nitric oxide). This agonist was used to determine the responsiveness of the vascular smooth muscle to a direct vasodilator that stimulates cGMP formation.
Autogenous blood (250 to 500 mL) was collected from the aortas of the animals at the time of death. The blood was collected into a plastic container containing citrate anticoagulant adjusted to yield final concentrations (mmol/L) of sodium citrate 9.3, citric acid 0.7, and dextrose 14 (ACD 1). The blood was transferred to plastic centrifuge tubes (50 mL) and centrifuged at 500g at 22°C. The platelet-rich plasma was pipetted into plastic centrifuge tubes, and an equal volume of cold citrate anticoagulant solution containing (mmol/L) sodium citrate 93, citric acid 7, dextrose 105, and KCl 5 (ACD 2) was added to the platelet-rich plasma and centrifuged at 1600g at 4°C. The supernatant was discarded, and the remaining pellet was resuspended in ACD 2 (volume equal to 1/40th of the original blood volume). A platelet count of this suspension was obtained (Coulter Electronics, Inc), and the volume of suspension was adjusted so that when it was added to the organ chamber, the resulting platelet concentration within the organ chamber was 100 000/μL. Platelet aggregation on exposure to collagen within the vessel wall and the calcium-containing Krebs’ solution was evidenced by clearing of the initial turbid solution and formation of visible platelet clumps.
The following drugs were used: acetylcholine, ADP, the calcium ionophore A23187, histamine, indomethacin, LNA, pyrilamine maleate, 5HT creatinine sulfate (serotonin), sodium nitroprusside, and thrombin (all from Sigma Chemical Co). All drugs were prepared daily with distilled water. Indomethacin was prepared by dissolving it in an equimolar solution of Na2CO3 (10 μmol/L; solution pH 7.8; Krebs’ solution pH after instillation 7.4) and agitating it in a 37°C water bath. LNA was first dissolved in 100 μL of 1N HCl and then diluted with Krebs’ solution (pH 7.4).
Prostacyclin Concentration Determinations
Rings of pulmonary artery from the control group with endothelium and from the BD group were placed in 2 mL Krebs’ solution and incubated for 2 minutes (n=5 for each group). At the beginning of the incubation, the calcium ionophore A23187 was added to some rings to stimulate the release of prostacyclin. The incubation medium was then assayed in duplicate for the stable metabolite of prostacyclin, 6-keto-prostaglandin F1α (6-keto-PGF1α) by radioimmunoassay using methods previously described and validated.16 The direct assay procedure used standard (0 to 200 pg) and unknown quantities of 6-keto-PGF1α mixed in PBS plus Krebs’ buffer (1:1) prepared in duplicate 0.2-mL aliquots. The 0.1 mol/L PBS (pH 7.4) contained 0.1% polyvinylpyrrolidone (Sigma), which maintained a nonspecific binding of ≤1.5% of the total counts. Antiserum (0.1 mL; 1:4000 titer) and 0.1 mL 6-keto-(5,8,11,12,14,15-3H)PGF1α (150 Ci/mmol; New England Nuclear; 12 000 disintegrations per minute) were added successively, and tubes were incubated at 4°C for 8 to 12 hours. Bound and free ligands were separated with dextran-coated charcoal mixed in assay buffer. The sensitivity of the assay was consistently 2 pg or less. The Krebs’ buffer had a blank value across all assays of 2.7±1.4 pg/mL. Intra-assay and interassay coefficients of variation at 250 and 1000 pg/mL were 6.7% and 11.3%, and 8.7% and 12.5%, respectively. Recovered standard amounts of 6-keto-PGF1α and parallelism of diluted unknown samples yielded correlation coefficients of 0.995 and 0.999, respectively. Accuracy and precision were determined by adding known amounts of 6-keto-PGF1α to Krebs’ solution on a picogram-per-milliliter basis and assaying the samples. The antibody for 6-keto-PGF1α was a kind gift of William B. Campbell, PhD, Department of Pharmacology, University of Texas Southwestern Medical Center. Cross-reactivities of the antibody with PGA2, PGD2, PGE1, PGE2, PGF1α, PGF2α, 6,15-keto-PGF1α, and thromboxane B2 are 0.2%, 0.88%, 1.14%, 0.59%, 0.88%, 0.62%, 2.8%, and 0.001%, respectively. All values are reported as picograms per milligram of protein determined by a modified Lowry method.
The specimens (n=5) were fixed in 3% glutaraldehyde in PBS for 1 hour, placed in baskets to keep them separated, and loaded in an automatic specimen processor. The specimens were then moved through three 5-minute changes of PBS to rinse out the fixative, followed by two 5-minute changes of distilled water to rinse out phosphates that may have precipitated with the alcohols in the dehydration series. The specimens were then rinsed twice for 1 hour in 70% alcohol, followed by two 1-hour rinses each in 95% and 100% alcohol. While in 100% alcohol, the specimens were moved into a critical-point dryer apparatus (DCP-1, Denton Vacuum Inc). This allowed the alcohol to be exchanged for 100% liquid CO2 (three 10-minute rinses) followed by a 3-minute rinse at a pressure of 1470 psi at 55°C. This critical pressure and temperature caused a phase exchange from liquid to gas CO2, which was then slowly vented to allow the specimen to dehydrate completely but retain its original size and shape.
The specimens were mounted on platforms/planchettes with a conductive paste (colloidal silver). Once mounted, the specimens were placed in a vacuum evaporator (DV-502, Denton Vacuum Inc) where, under vacuum, a sputter coat (argon gas plus a charge released across a thin foil of gold and palladium so that a metal “plasma” was allowed to fall evenly onto all surfaces of the specimens) was applied. The specimens were then placed into the scanning electron microscope (JEOL 840A, JEOL U S A, Inc) for viewing. These data were used to determine the extent of endothelial disruption present with and without balloon dilatation.
Results are expressed as mean±SEM. For platelet-induced contractions in quiescent rings, the maximal contraction is expressed as a percentage of the KCl (80 mmol/L) contraction for each ring. Platelet-induced relaxations in rings with tone are expressed as percent inhibition of the submaximal histamine contraction. Dose-response curves to histamine and KCl are expressed as a percentage of the maximal tension achieved to each agonist. Relaxations to endothelium-dependent and endothelium-independent agonists are expressed as percent inhibition of the submaximal histamine contraction for each ring. Unless otherwise stated, n indicates the number of animals used and the number of lungs from which the arteries were dilated. Student’s t tests for paired and unpaired observations were used when appropriate. ANOVA was used for comparisons of multiple means, with Scheffé’s test as an adjunct when statistical significance was found between multiple observations. Results were considered statistically significant when probability values were <.05.
In quiescent rings, platelet suspensions caused a significant increase in tension in control rings without endothelium (24±5% of an 80 mmol/L KCl contraction) and in BD rings (27±8%). In control rings with endothelium, aggregating platelets caused a significantly smaller change in tension (8±5%) compared with the other two groups. The platelet-induced contractions in the BD group were comparable to those observed in control rings without endothelium (Fig 1⇓). Pyrilamine, a histamine antagonist, significantly (P<.05) attenuated the platelet-induced contraction in control rings without endothelium (11±4% of 80 mmol/L KCl contraction).
In rings with tone induced by histamine, aggregating platelets caused significant relaxations only in control rings with endothelium (70±6%, P<.05 compared with rings without endothelium). The relaxations were inhibited by LNA, a competitive antagonist for nitric oxide synthase (17±7%, P<.05). In rings purposely denuded of their endothelium and in BD rings, relaxations (21±9% and 20±7%, respectively) were significantly attenuated and were similar to the changes in tension observed in rings incubated with LNA, the nitric oxide synthase inhibitor (Fig 2⇓).
Histamine (1 nmol/L to 10 μmol/L) caused dose-dependent increases in tension in each group. Rings without endothelium and BD rings contracted to a similar degree. Maximal contractions in rings with endothelium (0.75±0.18 g) were statistically similar (P=.08) to those from the other two groups (BD, 1.29±0.16 g; E−, 1.25±0.21 g), but the dose-response curve was shifted to the right significantly (P<.01) (Fig 3⇓). Potassium chloride (0 to 80 mmol/L) caused dose-dependent contractions that were similar between rings without endothelium and BD rings, confirming that contractility of the vascular smooth muscle was not altered by balloon dilatation or angioplasty (Fig 3⇓).
Acetylcholine (1 nmol/L to 1 μmol/L), the calcium ionophore A23187 (1 nmol/L to 1 μmol/L), 5HT (1 nmol/L to 10 μmol/L), and thrombin (0.01 to 1 U/mL) caused concentration-dependent relaxations in control rings with endothelium. These relaxation responses were abolished or attenuated in rings from the BD group and in control rings without endothelium. (Fig 4⇓). However, ADP (10 nmol/L to 100 μmol/L) caused relaxations in rings from all three groups that were statistically largest in the control group with endothelium and intermediate in the BD group, both of which were larger (P=.02) than in the control group without endothelium (Fig 5⇓).
Rings from the BD group and the control group without endothelium exhibited similar relaxation responses to increasing concentrations of sodium nitroprusside (1 nmol/L to 10 μmol/L), a donor of nitric oxide (Fig 6⇓).
Prostacyclin Concentration Determination
Prostacyclin concentrations were measured as the stable metabolite 6-keto-PGF1α. Concentrations of 6-keto-PGF1α were comparable between control and BD segments (387±59 versus 397±72 pg/mg protein). The levels were also similar between the groups after stimulation with the calcium ionophore A23187 (554±83 versus 459±72 pg/mg protein, respectively; P=.4). Exposure to the calcium ionophore A23187 did not significantly increase the concentration of 6-keto-PGF1α in control rings (387±59 versus 554±83 pg/mg protein; P=.1) or in BD rings (397±72 versus 459±72 pg/mg protein; P=.6).
Segments from control vessels with endothelium and from those following balloon dilatation were perfused with a platelet-rich solution (100 000/μL) immediately after removal from the lung. Scanning electron micrographs revealed an intact endothelium with a cobblestone appearance in control rings with endothelium. After balloon dilatation, the endothelium was disrupted with a sharp line of demarcation, and the underlying vascular smooth muscle was exposed. Platelet and fibrin aggregates were seen along the smooth muscle (Fig 7⇓).
Balloon dilatation of peripheral pulmonary arteries remains a useful tool in pediatric cardiology for patients with peripheral pulmonary artery stenoses. The angioplasty procedure is successful in 60% of cases but is associated with recurrent stenoses.13 Human pathological studies have revealed isolated disruption of the intimal and medial layers with subsequent remodeling of the intima.14 Our porcine studies confirm a significant disruption of the endothelial layer after balloon dilatation of normal peripheral pulmonary arteries. These findings support a similar morphological pathology after balloon angioplasty in porcine and human vessels.
Circulating platelets usually do not aggregate along a normal endoluminal surface because of the smooth surface of the endothelial cell and the antiaggregatory effects of EDNO and prostacyclin,9 17 both of which are synthesized and released locally at the surface of the endothelial cell. On disruption of the endothelial cell barrier, however, the antiaggregatory effects of EDNO and prostacyclin are no longer present locally, and platelets may aggregate and adhere to the underlying vascular smooth muscle. On aggregation, porcine platelets release a number of vasoactive substances in high concentrations. These substances include histamine (1 to 2 μmol/L), 5HT (5 to 6 μmol/L), thromboxane (150 to 200 pmol/L), and ADP.10 Platelet aggregation can also promote clot formation and thrombin deposition.15 These vasoactive substances can act locally to cause either vasoconstriction or vasodilation.10 The tone of a blood vessel is governed by the balance between these vasoconstrictors and vasodilators. The balance, however, is also dependent on the presence or absence of the endothelium.
We demonstrated previously that porcine platelets cause pulmonary arterial vasoconstriction in the absence of endothelium that is mediated by histamine.10 Thromboxane apparently plays a minor, if any, role in this vasoconstriction. In the presence of endothelium, vasodilation occurs when platelets aggregate secondary to the release of 5HT, ADP, and its by-products, with a possible contribution by thrombin if the coagulation cascade is activated.15 These substances—5HT, ADP, and thrombin—bind via receptors to the endothelial cell surface and stimulate the release of EDNO, which then causes vasodilation by activating guanylate cyclase with accumulation of cGMP within the vascular smooth muscle.18 19 In addition, ADP is also broken down into AMP and adenosine. Adenosine binds to the P2 receptor (blocked by theophylline) on the pulmonary arterial vascular smooth muscle and causes relaxations, even in the absence of endothelium. We used porcine platelets in this study because we wanted to mimic as closely as possible the scenario that might occur in vivo. From our previous studies, it was evident that histamine was a contractile agonist and serotonin was a dilating substance. From a clinical viewpoint, one would like to know how this relates to humans. The difference between the porcine and human responses lies in the substances released from aggregating platelets and their effects on the endothelium and smooth muscle. Human platelets also release serotonin and ADP in high concentrations similar to what is observed in the pig.20 Histamine is not found in significant quantities. In humans, however, serotonin is a contractile agonist; it is a vasodilator in pigs.21 22 23 Although the individual responses to each agonist may be different in our porcine model compared with the human situation, the fact remains that contractile and dilator substances are both released from aggregating platelets. The responses observed depend on the presence and absence of endothelium.10 20
In this model of balloon dilatation, endothelial cell disruption occurs after dilatation and allows platelet aggregation and release of vasoactive substances. Platelets cause comparable vasoconstriction in vitro in quiescent vessels purposely denuded of their endothelium and in vessels undergoing balloon dilatation. This vasoconstriction is attenuated by the histamine blocker pyrilamine, consistent with our previous studies that demonstrated release of histamine from platelets and blockade of the vasoconstriction with the histamine antagonist.10 Rings with an intact endothelium exhibited very small or no changes in tension in the quiescent state when exposed to aggregating platelets. This probably is due to the basal release of EDNO, which suppresses the contraction, and is further supported by the rightward shift of the histamine dose-response curve in rings with endothelium only.24 In fact, rings from the BD group contracted to a similar degree as control rings without endothelium. Based on our previous work, the amount of histamine released (1 to 3 μmol/L) and the statistically significant difference in the histamine-induced contractions between rings with and without endothelium (between 1 and 5 μmol/L) correlates fairly well.10 These data suggest that balloon dilatation causes significant endothelial denudation, disruption, or dysfunction, mimicking that seen with mechanical de-endothelialization in which >90% of the endothelial cells are removed.
In rings with increased tone, platelets cause only very small relaxations or constrictions in rings subjected to balloon dilatation and in control rings purposely de-endothelialized. The small relaxations observed might be secondary to a limited number of endothelial cells still present after angioplasty that still produce EDNO or to the direct vasodilating action of ADP and adenosine. Prostacyclin is an unlikely contributor to the relaxation because indomethacin was present in all the experiments and the amount of prostacyclin produced by rings after balloon dilatation was not significantly elevated or different from the basal release. In rings with intact endothelium, platelets cause large endothelium-dependent relaxations that are abolished by LNA.25 26 This strongly suggests that the platelet-induced relaxation in rings with endothelium is mediated by EDNO. Endothelium-dependent relaxations are also seen when rings are exposed to 5HT, the platelet-derived vasoactive mediator, to thrombin, and to acetylcholine. These relaxations are also abolished by balloon dilatation to a degree similar to that observed in control rings without endothelium. This further predicts that a diffuse endothelial dysfunction or disruption causes diminished relaxations after balloon dilatation, even in distensible pulmonary arteries, because the relaxations to multiple agonists were significantly diminished. All the aforementioned agonists mediate their relaxations through a receptor. However, experiments using the calcium ionophore A23187, a non–receptor-mediated agonist, produced results identical to those of acetylcholine and the other agonists, confirming that these observations were not receptor-dependent or secondary to loss or downregulation of receptors or to diminution in receptor availability or coupling.
Vascular smooth muscle function did not appear altered by this procedure, as evidenced by an intact vascular smooth muscle by electron microscopy, similar contraction curves with potassium chloride and histamine, and similar relaxation curves to sodium nitroprusside between control rings without endothelium and rings undergoing balloon dilatation. The activation of guanylate cyclase with the subsequent accumulation of smooth muscle cGMP is responsible for the relaxations observed to EDNO19 and to nitrovasodilators such as sodium nitroprusside. The similar relaxations to sodium nitroprusside in control rings without endothelium and in those after balloon dilatation suggest that the sensitivity and availability of the enzyme guanylate cyclase are not altered by balloon dilatation. It also suggests that an enzymatic alteration is not responsible for the differences in EDNO-induced relaxations to the various endothelium-dependent agonists observed between groups.
Physiologically, the scenario of balloon dilatation, endothelial cell disruption, loss of EDNO and prostacyclin, and platelet aggregation with histamine release would favor vasoconstriction that is unopposed by endothelium-derived vasodilators. These data represent the first study of this scenario in pulmonary arterial segments and suggest that balloon dilatation causes endothelial disruption that is significant even in distensible arteries. In the intact animal, the underlying smooth muscle would still be exposed to circulating mitogens, and proliferation of vascular smooth muscle cells may still occur. If the mechanism is similar to that observed in the coronary artery,2 the extent of the proliferation would likely govern the amount of obstruction produced, and the effect of circulating EDNO and prostacyclin from proximal segments may play a regulatory role in the proliferative effect. In large pulmonary arteries, endothelial disruption may not cause clinically significant thrombus formation or vascular occlusion as seen in peripheral arteries.2 However, in the smaller, more peripheral pulmonary arteries similar to those used in this study, endothelial disruption could certainly lead to a scenario of vasoconstriction, thrombus formation, and occlusion. As pediatric cardiologists continue to extend the usefulness of this procedure to even smaller vessels, this scenario will become clinically more important. In this model of balloon dilatation, the balloon inflation pressures used were similar to those used in clinical practice, mimicking as closely as possible the events that might take place after clinical angioplasty. The distensibility of the vessels should be similar in ex vivo lungs as in in vivo lungs, but a difference in distensibility characteristics, including those between the porcine model and humans, has not been systematically studied. Although we speculate that the mechanism of restenosis involving pulmonary arteries may be the same as that observed in the coronary and peripheral arteries, this was not addressed by this study. Future in vivo dilatation and recovery studies will be necessary to delineate any similarities or discrepancies in the mechanism of restenosis between pulmonary and systemic arteries.
This research was supported in part by grant 91G-083 from the American Heart Association, Texas Affiliate.
- Received June 21, 1994.
- Revision received August 29, 1994.
- Accepted September 23, 1994.
- Copyright © 1995 by American Heart Association
Brenner BM, Troy JL, Ballerman BJ. Endothelium-dependent vascular responses. J Clin Invest. 1989;84:1373-1378.
Moncada S, Radomski MW, Palmer RMJ. Endothelium-derived relaxing factor: identification as nitric oxide and role in the control of vascular tone and platelet function. Biochem Pharmacol. 1988;37: 2495-2501.
Moncada S, Palmer RMJ, Higgs EA. Relationship between prostacyclin and nitric oxide in the thrombotic process. Thromb Res. 1990;suppl 11:3-13.
Zellers TM, Shimokawa H, Yunginger J, Vanhoutte PM. Heterogeneity of endothelium-dependent and endothelium-independent responses to aggregating platelets in porcine pulmonary arteries. Circ Res. 1991;68:1437-1445.
Edwards BS, Lucas RV, Lock JE, Edwards JE. Morphologic changes in the pulmonary arteries after percutaneous balloon angioplasty for pulmonary arterial stenosis. Circulation. 1985;71: 195-201.
Kaplan JE, Moon DG, Weston LK, Minnear FL, Del Vecchio PJ, Shepard JM, Fenton JW II. Platelets adhere to thrombin-treated endothelial cells in vitro. Am J Physiol. 1989;257:H423-H433.
Magness RR, Osei-Boaten K, Mitchell MD, Rosenfeld CR. In vitro prostacyclin production by ovine uterine and systemic arteries: effects of angiotensin II. J Clin Invest. 1985;76:2206-2212.
Venturini CM, Weston LK, Kaplan JE. Platelet cGMP, but not cAMP, inhibits thrombin-induced platelet adhesion to pulmonary vascular endothelium. Am J Physiol. 1992;263:H606-H612.
Rapoport RM, Murad F. Agonist-induced endothelium-dependent relaxations in rat thoracic aorta may be mediated through cGMP. Circ Res. 1983;52:352-357.
Houston DS, Shepherd JT, Vanhoutte PM. Aggregating platelets cause direct contraction and endothelium-dependent relaxation of isolated canine coronary arteries: role of serotonin, thromboxane A2 and adenine nucleotides. J Clin Invest. 1986;78:539-544.
Fischell TA, Derby G, Tse TM, Stadius ML. Coronary artery vasoconstriction routinely occurs after percutaneous transluminal coronary angioplasty: a quantitative arteriographic analysis. Circulation. 1988;78:1323-1334.
Fischell TA, Bausback KN, McDonald TV. Evidence for altered epicardial coronary artery autoregulation as a cause of distal coronary vasoconstriction after successful percutaneous transluminal coronary angioplasty. J Clin Invest. 1990;86:575-584.
Martin W, Furchgott RF, Villani GM, Lothianandan D. Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther. 1986;237:529-538.