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Final Discussion
Final Discussion E. Kenneth Weir, M.D., F.C.C.P.
initial discussion concerned the possible interaction T hebetween the pulmonary vascular endothelium and smooth muscle in both the normal control of vessel tone and in response to endothelial injury. It was suggested that the smooth muscle should be studied alone and in conjunction with the endothelium, in terms of contractility, calcium kinetics, membrane depolarization, and metabolism of highenergy substances. While it is possible to co-culture endothelial and smooth muscle cells, it is not clear that the data obtained from these cells (as a result of electrical, biochemical, or pharmacologic stimulation) can be extrapolated to the intact vessel. For example, they cannot be exposed to varying transmural pressures. Studies which compare cultures of systemic and pulmonary vascular smooth muscle might, however, reveal important differences between the two and their responses to contact with endothelium. The importance of examining cell-cell interactions at points of contact, in contrast to studying only potential mediator substances, was stressed. Work with muscle cells in culture might provide the opportunity not only to determine the amino acid sequence of individual enzymes, such as the light-chain kinase, but also the potential to alter the sequence and thus to alter cell function. A suggestion was made that investigators working on the mechanism of hypoxic pulmonary vasoconstriction should stop thinking about a single agonist or antagonist. Could there be a number of vasodilator and constrictor influences active at any time, possibly coordinated by the endothelial cell? The net effect might be altered by endothelial injury or under abnormal conditions, such as high flow. The next topic Was the possibility that hypoxic pulmonary vasoconstriction might be the result of a reduction in the formation or release of the endothelial-dependent relaxing factor (EDRF). Absolute anoxia is necessary to prevent all relaxation by this mechanism. As hemoglobin prevents EDRF action, the addition of hemoglobin in a perfused lung should initiate constriction during normoxia if EDRF plays a role in maintaining normoxic vasodilatation. Hemoglobin can shift the dose-response curve for the relaxation caused in smooth muscle by glyceryl trinitrate. It also interferes with photorelaxation of smooth muscle. Several observations have been made which may be relevant to the actions of hemoglobin. As reported by Don Will, the IV injection of a few milliliters of hemolyzed blood in cattle precipitates a marked increase in pulmonary arterial pressure. In the isolated perfused rat lung, the presence of RBCs (and presumably hemoglobin) in the perfusate prolongs the length of time that the lung will respond to hypoxic challenges. Although hemoglobin is the same molecular Reprint flIquem: Dr. Weir, Veterans Administration, Bldg 222, Fort SneUlng, St. Paul 55111
weight as albumin, because of differences in charge and shape, it is able to pass through the endothelium very rapidly and appears in the lung lymph. A brief discussion followed on techniques to cause endothelial injury or ablation in the lungs in order to study the consequent changes in pulmonary vascular reactivity. Endothelial damage can be produced by unilateral pulmonary arterial occlusion for two to three hours or by breathing 60 percent oxygen for a few days. One discussant reported that while hyperoxia or a-naphthylthiourea administration resulted in lung endothelial damage visible on electron microscopy, relaxation induced by acetylcholine was unchanged. Pulmonary arterial endothelium can be removed selectively by embolizing the lungs with beads of different sizes and reversing flow to recover the beads . The mechanisms by which hypoxia might stimulate pulmonary vasoconstriction were considered next. Some of the possible mechanisms include changes in membrane electrical activity, oxidative phosphorylation, sulfhydryl redoit status and mediators (eg, leukotrienes). Several participants stressed the importance of trying to elucidate the components of the mechanism, such as the sensor, transducer, and effector. In different tissues the sensor might be the same, but changes in transduction might alter the response. For example, in the myocardium (\Vl1son) and the lungs (MeMurtry), oxygen sensing is closely related to the ratio [ATP]/[ADP] [Pi]. However,hypoxiacaused vasodilatation in the coronary bed, in contrast to pulmonary vasoconstriction. (See discussion of Wilson and Erecinska). One discussant urged that the effect of oxygen binding on the physiochemical properties of the cell membrane be considered. Could the structure or function of calcium channels be altered by oxygen binding? A related possibility is that oxygen radicals change sulfhydryl redox status in membrane proteins. Lahirihas shown in the carotid body that sulfhydryl agents augment the neural response to hypoxia. Investigators were urged to compare the reactivity of pulmonary and systemic arteries in different species and at all levels of organization, down to enzyme and receptor specificity. The intrinsic difference between small pulmonary and systemic arteries is illustrated by an experiment reported by Daviss group. Lung tissue was transplanted to the hamster cheek pouch, and the pulmonary and systemic arteries reacted oppositely to changes in oxygen tension . An analogous situation exists in the fetus, where the ductus arteriosus and the pulmonary arteries respond differently to oxygen in the environment. These observations suggest that even in the same environment, pulmonary arteries have different responses than other arteries, and consequently studies should be directed at the vessels themselves. It was pointed out that the pulmonary and systemic circulations do not alwaysbehave differently. In the systemic circulation the CHEST I 88 I 4 I OCTOBER. 1985 I SUpplement
2718
vascular response to hypoxia varies from organ to organ. Even when considered as a whole, the systemic circulation does not dilate in response to hypoxia in long-term highaltitude dwellers. Twoother possible mechanisms concerning the etiology of acute hypoxic pulmonary vasoconstriction were briefly discussed . The sympathetic nervous system is probably not very important, as 6-hydroxydopamine does not affect the hypoxic pressor response in sheep. Because mediators are likely to be locally acting substances, one discussant suggested that they be studied at tissue level as well as in the blood, lymph, alveolar lavage, etc. The last section of the discussion involved the pathophysiology and potential reversibility ofchronic pulmonary hypertension . A discussant suggested that the platelets may playa role, possibly causing vasoconstriction through the release of thromboxane or structural changes perhaps through the release of smooth muscle growth factor. One group reported no changes in platelet function during chronic hypoxia, and another group found no changes in ~-thromboglobulin levels in eight patients with primary pulmonary hypertension. It is not clear whether vasoconstriction leads to "work hypertrophy" of medial smooth muscle or whether in some instances a separate stimulus causes the hypertrophy and permits stronger vasoconstriction. Both inhibition of vasoconstriction and of smooth muscle proliferation might theoretically be beneficial. An inflammatory cell infiltrate
272S
has been noted in some models of pulmonary hypertension. Further studies are needed to determine whether changes in this infiltrate would alter the hemodynamic or pathologic course of the pulmonary hypertension. Several participants discussed the potential for regression of the pathologic changes in experimental or clinical pulmonary hypertension. After an animal is removed from chronic hypoxia, the vessels never completely return to normal despite return to normal pressures. This is also true of highaltitude residents who have been at sea level for several years. Lung biopsies have been studied in a series of patients with congenital heart disease before and several years after banding of the pulmonary artery. In some cases there was complete reversal of histologic changes (especially medial hypertrophy), but in others there was only partial reversal. Patients with plexiform lesions did not show recovery. The critical feature is probably the type and extent of intimal fibrosis. Experimentally intimal fibrosis can be studied in high-flow models. One discussant reported that in their experience the appearance of vessels seen in lung biopsies did not help in the prediction of responsiveness of the pulmonary vascular bed to vasodilators such as prostacyclin. Another participant speculated that the defect in primary pulmonary hypertension involves depressed endothelial production of prostacyclin. Progress in this area requires correlation of changes in function with changes in the pathology.
initial discussion concerned the possible interaction T hebetween the pulmonary vascular endothelium and smooth muscle in both the normal control of vessel tone and in response to endothelial injury. It was suggested that the smooth muscle should be studied alone and in conjunction with the endothelium, in terms of contractility, calcium kinetics, membrane depolarization, and metabolism of highenergy substances. While it is possible to co-culture endothelial and smooth muscle cells, it is not clear that the data obtained from these cells (as a result of electrical, biochemical, or pharmacologic stimulation) can be extrapolated to the intact vessel. For example, they cannot be exposed to varying transmural pressures. Studies which compare cultures of systemic and pulmonary vascular smooth muscle might, however, reveal important differences between the two and their responses to contact with endothelium. The importance of examining cell-cell interactions at points of contact, in contrast to studying only potential mediator substances, was stressed. Work with muscle cells in culture might provide the opportunity not only to determine the amino acid sequence of individual enzymes, such as the light-chain kinase, but also the potential to alter the sequence and thus to alter cell function. A suggestion was made that investigators working on the mechanism of hypoxic pulmonary vasoconstriction should stop thinking about a single agonist or antagonist. Could there be a number of vasodilator and constrictor influences active at any time, possibly coordinated by the endothelial cell? The net effect might be altered by endothelial injury or under abnormal conditions, such as high flow. The next topic Was the possibility that hypoxic pulmonary vasoconstriction might be the result of a reduction in the formation or release of the endothelial-dependent relaxing factor (EDRF). Absolute anoxia is necessary to prevent all relaxation by this mechanism. As hemoglobin prevents EDRF action, the addition of hemoglobin in a perfused lung should initiate constriction during normoxia if EDRF plays a role in maintaining normoxic vasodilatation. Hemoglobin can shift the dose-response curve for the relaxation caused in smooth muscle by glyceryl trinitrate. It also interferes with photorelaxation of smooth muscle. Several observations have been made which may be relevant to the actions of hemoglobin. As reported by Don Will, the IV injection of a few milliliters of hemolyzed blood in cattle precipitates a marked increase in pulmonary arterial pressure. In the isolated perfused rat lung, the presence of RBCs (and presumably hemoglobin) in the perfusate prolongs the length of time that the lung will respond to hypoxic challenges. Although hemoglobin is the same molecular Reprint flIquem: Dr. Weir, Veterans Administration, Bldg 222, Fort SneUlng, St. Paul 55111
weight as albumin, because of differences in charge and shape, it is able to pass through the endothelium very rapidly and appears in the lung lymph. A brief discussion followed on techniques to cause endothelial injury or ablation in the lungs in order to study the consequent changes in pulmonary vascular reactivity. Endothelial damage can be produced by unilateral pulmonary arterial occlusion for two to three hours or by breathing 60 percent oxygen for a few days. One discussant reported that while hyperoxia or a-naphthylthiourea administration resulted in lung endothelial damage visible on electron microscopy, relaxation induced by acetylcholine was unchanged. Pulmonary arterial endothelium can be removed selectively by embolizing the lungs with beads of different sizes and reversing flow to recover the beads . The mechanisms by which hypoxia might stimulate pulmonary vasoconstriction were considered next. Some of the possible mechanisms include changes in membrane electrical activity, oxidative phosphorylation, sulfhydryl redoit status and mediators (eg, leukotrienes). Several participants stressed the importance of trying to elucidate the components of the mechanism, such as the sensor, transducer, and effector. In different tissues the sensor might be the same, but changes in transduction might alter the response. For example, in the myocardium (\Vl1son) and the lungs (MeMurtry), oxygen sensing is closely related to the ratio [ATP]/[ADP] [Pi]. However,hypoxiacaused vasodilatation in the coronary bed, in contrast to pulmonary vasoconstriction. (See discussion of Wilson and Erecinska). One discussant urged that the effect of oxygen binding on the physiochemical properties of the cell membrane be considered. Could the structure or function of calcium channels be altered by oxygen binding? A related possibility is that oxygen radicals change sulfhydryl redox status in membrane proteins. Lahirihas shown in the carotid body that sulfhydryl agents augment the neural response to hypoxia. Investigators were urged to compare the reactivity of pulmonary and systemic arteries in different species and at all levels of organization, down to enzyme and receptor specificity. The intrinsic difference between small pulmonary and systemic arteries is illustrated by an experiment reported by Daviss group. Lung tissue was transplanted to the hamster cheek pouch, and the pulmonary and systemic arteries reacted oppositely to changes in oxygen tension . An analogous situation exists in the fetus, where the ductus arteriosus and the pulmonary arteries respond differently to oxygen in the environment. These observations suggest that even in the same environment, pulmonary arteries have different responses than other arteries, and consequently studies should be directed at the vessels themselves. It was pointed out that the pulmonary and systemic circulations do not alwaysbehave differently. In the systemic circulation the CHEST I 88 I 4 I OCTOBER. 1985 I SUpplement
2718
vascular response to hypoxia varies from organ to organ. Even when considered as a whole, the systemic circulation does not dilate in response to hypoxia in long-term highaltitude dwellers. Twoother possible mechanisms concerning the etiology of acute hypoxic pulmonary vasoconstriction were briefly discussed . The sympathetic nervous system is probably not very important, as 6-hydroxydopamine does not affect the hypoxic pressor response in sheep. Because mediators are likely to be locally acting substances, one discussant suggested that they be studied at tissue level as well as in the blood, lymph, alveolar lavage, etc. The last section of the discussion involved the pathophysiology and potential reversibility ofchronic pulmonary hypertension . A discussant suggested that the platelets may playa role, possibly causing vasoconstriction through the release of thromboxane or structural changes perhaps through the release of smooth muscle growth factor. One group reported no changes in platelet function during chronic hypoxia, and another group found no changes in ~-thromboglobulin levels in eight patients with primary pulmonary hypertension. It is not clear whether vasoconstriction leads to "work hypertrophy" of medial smooth muscle or whether in some instances a separate stimulus causes the hypertrophy and permits stronger vasoconstriction. Both inhibition of vasoconstriction and of smooth muscle proliferation might theoretically be beneficial. An inflammatory cell infiltrate
272S
has been noted in some models of pulmonary hypertension. Further studies are needed to determine whether changes in this infiltrate would alter the hemodynamic or pathologic course of the pulmonary hypertension. Several participants discussed the potential for regression of the pathologic changes in experimental or clinical pulmonary hypertension. After an animal is removed from chronic hypoxia, the vessels never completely return to normal despite return to normal pressures. This is also true of highaltitude residents who have been at sea level for several years. Lung biopsies have been studied in a series of patients with congenital heart disease before and several years after banding of the pulmonary artery. In some cases there was complete reversal of histologic changes (especially medial hypertrophy), but in others there was only partial reversal. Patients with plexiform lesions did not show recovery. The critical feature is probably the type and extent of intimal fibrosis. Experimentally intimal fibrosis can be studied in high-flow models. One discussant reported that in their experience the appearance of vessels seen in lung biopsies did not help in the prediction of responsiveness of the pulmonary vascular bed to vasodilators such as prostacyclin. Another participant speculated that the defect in primary pulmonary hypertension involves depressed endothelial production of prostacyclin. Progress in this area requires correlation of changes in function with changes in the pathology.