INTRODUCTION

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Heart-lung transplantation is a potential therapeutic modality for selected patients with irreversible pulmonary hypertension caused by Eisenmenger's syndrome with end-stage symptoms of heart failure. We have recently reported the results of a long-term study of the airway reactivity of tissue from patients with other conditions undergoing transplantation in which we found consistent concentration-related relaxation responses which were independent of disease state (1). While undertaking that study, we had the opportunity to study the airways of a single patient undergoing transplantation as a treatment for Eisenmenger's syndrome, and in doing so, observed the absence of a relaxation response to isoproterenol and levocromakalim.

There are previous reports of abnormal function in tissue from patients with Eisenmenger's syndrome. Absence of endothelium-dependent relaxation responses in isolated pulmonary arteries from Eisenmenger's patients has been reported (2). This study affirmed the previous observation of Wood (3) who was responsible for the description of the various cardiac anomalies associated with this condition. These findings, coupled with our observations in the airways of a single patient, led us to investigate the response to relaxant agonists in airways from other patients with Eisenmenger's syndrome. We now report the results of our studies, conducted over a 4-yr period, examining the relaxant as well as contractile responsiveness of airways from five patients with Eisenmenger's syndrome undergoing pulmonary transplantation. In addition, the dimensions and structure of the airway wall were examined morphometrically to investigate any relationship between airway wall structure and in vitro function in tissues from patients with Eisenmenger's syndrome.

	METHODS

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Lungs were obtained from patients undergoing heart-lung transplantation as a treatment for Eisenmenger's syndrome (with end stage heart failure, New York Heart Association  Class III) and over the same time period, nondiseased lungs from organ donors. The donor lungs were obtained when no suitable recipient was available, or when the donors had suffered trauma of a nature precluding the use of their lungs for transplantation. The protocol was approved by the Human Ethical Review Committee of the University of Sydney and permission for use of donor lungs for research was obtained from the next of kin by the transplant coordinators. The Eisenmenger's patients were all male and had an average age of 27.2 yr (SD ± 11.6 yr) while all but one of the organ donors were male and had an average age of 35.1 yr (SD ± 13.2 yr). Details of each patient are included in Table 1 which also displays spirometric data for the recipients obtained at assessment for transplant. Expiratory flow-volume loop testing was performed on a Medical Graphics System 1070/85 (St. Paul, MN). In all cases, the lung function testing had been performed not more than 14 mo prior to surgery and the predicted values used were those of Knudson and colleagues (4).

Airways were dissected from the surrounding parenchyma and cut into rings measuring approximately 4 to 5 mm in length and 3 to 6 mm in internal diameter. The rings were mounted under 1 to 2 g preload on stainless steel hooks in water-jacketed tissue baths which contained Krebs-Henseleit solution maintained at 37° C and continuously aerated with 5% CO₂ in oxygen. Stainless steel electrodes were inserted in some baths to allow electrical field stimulation to be performed. Between eight and 14 bronchial rings from each patient were used and the rings were studied in pairs for each treatment. The rings were allowed to equilibrate over a 90-min period during which the bath fluid was exchanged at 15 to 20 min intervals and the preload adjusted to that originally applied. All changes in isometric force were measured using Grass FT03 transducers (Quincy, MA) and recorded on Grass 7P polygraphs; electrical field stimulation was performed using Grass SD9 stimulators (at constant settings of voltage 40 V, duration 1 ms, applied for 20 s).

After the equilibration period, and when the tissues had attained a stable baseline, pairs of tissues were used to generate stimulation response curves to cumulative concentrations of acetylcholine (10 nM to 3 mM; Sigma, St. Louis, MO), carbachol (10 nM to 1 mM [carbamyl choline]; Sigma), histamine (10 nM to 1 mM; Sigma), isoproterenol (0.1 nM to 10 µM; Sigma), and levocromakalim (0.1 nM to 10 µM, [BRL 38227]; Smith Kline Beecham Pharmaceuticals, Great Burgh, UK), and to increasing frequencies of electrical field stimulation (0.5 to 64 Hz, first in the absence, then in the presence of 1 µM atropine [Sigma]). Only one relaxant or contractile intervention was studied in each tissue. At the conclusion of the experiment, the tissue was washed, removed from the bath, blotted and placed in neutral buffered formalin for fixation and later processing.

As preparation for morphometry, after fixation, the tissues were embedded in paraffin wax, cut into 6-µm sections (at 1-mm intervals) and stained with Gomori elastin trichrome stain (Probing and Structure Ltd., Queensland, Australia). Light microscopic images of each section were digitally captured using a Fujix HC1000 digital video camera (Fujix Corp., Japan) coupled to a Power Macintosh 8600 computer (Apple Corp., Cupertino, CA). Morphometric measurement of the dimensions of the airway [basement membrane perimeter (P_bm), lumenal perimeter (P_i), outer muscle perimeter (P_mo), outer perimeter (P_o), and the length of the basement membrane covered in epithelium (P_epi)] were performed using computer software (IPLab Spectrum; Signal Analytics, Vienna, VA) and a graphics tablet (Digitizer II, Wacom, Japan). The areas enclosed by each of these perimeters were also measured. The structural components of the airway [smooth muscle area (WA_m), cartilage area (WA_cart), and lumenal area (A_i)] were measured by point counting performed by overlay of a grid of known density over the digital image. All dimensions and areas measured were as defined by Bai and coworkers (5).

The contraction or relaxation to each concentration of agonist or each frequency of stimulation was measured in milligrams and the two values obtained for each pair of tissues undergoing the same treatment were averaged to give a single value. Mean values for the Eisenmenger's and organ donor groups were obtained for each point (with standard error of the mean values) along the cumulative concentration-response or frequency-response curves using the single averaged value from each patient, and mean curves were drawn for each stimulant for both groups. The resulting mean cumulative response curves for each agonist and for electrical field stimulation were subjected to between-group (Eisenmenger's syndrome and organ donor) comparison by repeated measures analysis of variance.

For the morphometric analysis the following areas were derived: inner wall area [WA_i = area enclosed by P_mo (A_mo)  area enclosed by P_bm (A_bm)], outer wall area [WA_o = area enclosed by P_o (A_o)  A_mo], total wall area [WA_t = WA_i + WA_o]. A square root transformation of all airway wall and component areas was performed resulting in a normal distribution. The relationship between each airway wall dimension and airway size (P_bm) was assessed using linear regression analysis. The intercept and slope of the line for airways from patients with Eisenmenger's syndrome and donors were calculated. The percentage of P_bm covered by epithelium was also calculated (P_epi/ P_bm). The area of smooth muscle and cartilage was calculated by conversion of the number of points falling over the structures to area by division of the number of points by the point density of the grid (points/mm²) which was determined by measurement of the lumenal area (A_i). Internal diameter was also calculated by dividing the basement membrane perimeter by . The average value for each dimension for each airway was calculated by deriving the mean for all the sections measured from each airway ring, and a mean and standard error of the mean value for Eisenmenger's syndrome and donor airways were derived. Comparison of the morphometric measurements obtained in airways from patients with Eisenmenger's syndrome and from organ donors was performed using analysis of variance (ANOVA) and multiple regression analysis.

	RESULTS

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Cumulative concentration-response curves to acetylcholine, carbachol, and histamine were obtained in tissues from both patient groups. In tissues from patients with Eisenmenger's syndrome, the magnitude of the response to the cholinergic agonists carbachol and acetylcholine was significantly greater than that obtained in tissues from the organ donors (Table 2, Figures 1A and 1B). Increased cholinergic responsiveness was also illustrated by a greater response to electrical field stimulation in tissues from patients with Eisenmenger's syndrome (Table 2, Figure 1C). In contrast to the increased cholinergic responsiveness in tissues from patients with Eisenmenger's syndrome, there was no significant difference in the cumulative concentration-response curves to histamine obtained in tissues from both groups (Figure 1D).

View larger version (23K): [in this window] [in a new window]   Figure 1.   Cumulative stimulation-response curves to increasing concentrations of carbachol (A), acetylcholine (B), histamine (D), isoproterenol (E ), levocromakalim (F ) and increasing frequency of electrical stimulation (C ) in tissues from patients with Eisenmenger's syndrome (open circles) and organ donors (closed circles). Mean changes in contraction or relaxation are expressed as mg of force; vertical bars indicate values for SEM. The number of patients from whom tissue was obtained for each stimulant is shown in Table 2. Significant differences in stimulation-response curves between the groups were assessed using ANOVA and are indicated as follows: *p < 0.01, p < 0.001, p = 0.0001.

The most striking finding in this study was a significantly impaired relaxation to differing stimuli in tissues from patients with Eisenmenger's syndrome (Table 2, Figures 1E and 1F). While slight relaxation responses were seen in some tissues from patients with Eisenmenger's syndrome (the mean maximal response to isoproterenol in these tissues was equal to 23% of the mean maximal response in donor tissue), in tissues from two of the five patients, there was a complete absence of relaxation to any concentration of isoproterenol or levocromakalim despite large contractile responses to carbachol elicited in the same tissues at the conclusion of the experiment. In these tissues, the addition of vasoactive intestinal peptide (1 µM), verapamil (0.1 µM), papaverine (10 µM), and sodium nitroprusside (10 µM) also failed to elicit any relaxation of the tissues. Moreover, even in the presence of atropine, there was no relaxation response to electrical field stimulation in any of the tissues from patients with Eisenmenger's syndrome, while a marked relaxant response was seen in tissues from the organ donors (see Figure 2).

View larger version (53K): [in this window] [in a new window]   Figure 2.   Absence of relaxation response to increasing stimulation frequencies in the presence of 1 µM atropine in airway tissue from a patient with Eisenmenger's syndrome (A). The frequency-related increases in relaxation responses in the presence of atropine observed in tissues from organ donors are exemplified by trace B.

Additional tissues from one patient with Eisenmenger's syndrome were set up as described previously but in this case, the addition of cumulative concentrations of isoproterenol or levocromakalim was performed in the presence of increased smooth muscle tone induced by the prior addition of 10 µM carbachol to the baths. Under these conditions, concentration-dependent relaxation responses (Figure 3) were obtained to both of these relaxant agonists. A complete reversal of the contraction induced by carbachol was attained at maximal concentrations of isoproterenol and levocromakalim.

View larger version (12K): [in this window] [in a new window]   Figure 3.   Relaxation responses to increasing concentrations of isoproterenol (A) and levocromakalim (B) in airway tissues from a patient with Eisenmenger's syndrome in the presence (closed symbols) and absence (open symbols) of carbachol-induced tone. Relaxation responses are expressed as mg of relaxation generated, and each point represents the average of the response obtained in two tissues.

Morphometric analysis of the tissues used for the functional studies allowed comparison of the sizes and structural composition of the airway wall in patients with Eisenmenger's syndrome and organ donors (Table 3, Figure 4). The airways from both groups of patients were not different in size as the mean internal airway diameter was 3.694 ± 0.130 mm and 3.544 ± 0.120 mm from patients with Eisenmenger's syndrome and organ donors respectively, and the total airway wall and areas were also equivalent (Table 3). There was also no difference in the amount of airway smooth muscle or cartilage in both groups. Despite this finding, the mean inner wall area of airways from patients with Eisenmenger's syndrome was slightly greater than that from organ donors (Table 3) indicating that these airways have slightly thicker airway walls interior to the smooth muscle layer. Although the mean inner wall area was greater, there was no difference between the groups in the slope or intercepts of the regression lines relating basement membrane perimeter and square root inner wall area, although the regression lines tend to diverge at larger values of basement membrane perimeter, suggesting that the larger airways may contribute to this finding (Figure 4A). Morphometry also revealed that there were no differences in the epithelial integrity in airways from each group, as the percentage of the basement membrane covered by epithelium was equivalent for both groups (84.63 ± 2.07% for donor airways and 83.10 ± 2.37% in airways from patients with Eisenmenger's syndrome).

View larger version (22K): [in this window] [in a new window]   Figure 4.   Relationships between size (measured as basement membrane perimeter) and wall thickness and areas in the airways used in the functional studies. The relationship between size and (A) inner wall area, (B) outer wall area, (C ) total wall area, and (D) smooth muscle area are shown for tissues from organ donors (open symbols) and patients with Eisenmenger's syndrome (closed symbols). The regression lines for each of the relationships are also shown with the finer regression line in each case being that from airways from organ donors, and the thicker regression line that computed for airways from patients with Eisenmenger's syndrome.

	DISCUSSION

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This study was generated following the observation that the airways taken from a patient with Eisenmenger's syndrome did not produce relaxation responses to either a high concentration of isoproterenol nor a high concentration of levocromakalim. This finding, coupled with the previous report of an absence of in vitro relaxation responses in the pulmonary arteries of Eisenmenger's patients, prompted us to carry out a more comprehensive study of the relaxant and contractile responsiveness of patients with this condition. We now report that the airways from these patients exhibit markedly reduced or absent relaxation responses as well as significantly enhanced contractile responses to cholinergic stimulation. Despite these great differences in function in airways from patients with Eisenmenger's syndrome, morphometric analysis of the airways used for the functional studies revealed that there are no differences in airway wall structure in these patients.

It is possible that cholinergic responses may be enhanced via a decrease in cholinergic metabolism due to deficiencies in the amount or activity of acetylcholinesterase. Our observed increase in response to acetylcholine and electrical field stimulation would support the notion of decreased cholinesterase function in the airways of patients with Eisenmenger's syndrome. Our simultaneous observation of significantly enhanced responses to carbachol in these airways would at first glance tend to preclude this conclusion however, as carbachol is insensitive to the actions of acetylcholinesterase. The enhanced response to carbachol was however, not as great as that to acetylcholine, so it is possible that there is some alteration in this enzyme in this disease. The enhanced contractile responsiveness of airways from patients with Eisenmenger's syndrome is not, however, likely to be due solely to any decrease in the amount or activity of acetylcholinesterase and would involve an additional mechanism for the observed cholinergic hyperresponsiveness.

Although we did not formally determine the optimal preload for each tissue from each patient in this study and instead elected to apply a consistent preload for each tissue (based on tissue size), we are of the opinion that the differences we observed between tissues in the two patient groups are not explained on this basis. While Mitchell and coworkers (6) have reported that optimal contractile responsiveness is dependent upon optimal resting length, which is dependent upon airway size, these same investigators have shown that spontaneous tone in isolated human bronchi is inherent and not a manifestation of an imposed preload (7). We therefore believe that the absence of relaxation in tissues from patients from Eisenmenger's syndrome is not dependent upon the preload applied, but rather deficiency of inherent smooth muscle tone.

It is unlikely that the functional differences observed in this study can be explained on the basis of structural differences between airways from the two groups. Airway wall perimeter (8) and wall area (7) are conserved during relaxation and contraction and so comparisons between airways used for the functional studies allowed us to use morphometric analysis to investigate any possible connection between structure and function. The airways from both groups were found to be of the same caliber and to have the same average total airway wall area. There was no difference in the amount of smooth muscle in airways from both groups, indicating that the increased contractile response cannot be explained by an increased amount of airway smooth muscle. Moreover, there was no difference in the area of the airway wall occupied by cartilage, which suggests that there was no difference in preload imposed by the cartilage on smooth muscle contraction or relaxation in airways from both groups. Airways from patients with Eisenmenger's syndrome had slightly greater inner wall areas than airways from organ donors. As the average internal perimeters and smooth muscle areas of the airways were the same, this suggests that the lamina propria of patients with Eisenmenger's syndrome is slightly thicker than that of organ donors. It has been suggested that such an increase in the area lumenal to the smooth muscle can amplify the effects of smooth muscle contraction, but this is not likely to be the explanation for the enhanced contraction in this study, as there was no observed increase in histamine responsiveness and such an effect would be nonspecific in nature. We are therefore of the opinion that the functional characteristics of airways from patients with Eisenmenger's syndrome cannot be explained by the structure of the airway wall.

The absence of relaxation was not specific to agonists operating through certain receptors, as the response was absent when  receptors as well as peptidergic receptors were stimulated. In addition, agents acting on ion channels such as verapamil and levocromakalim produced no relaxation and this was also true of agents directly stimulating relaxant signaling pathways such as papaverine and the nitric oxide donor, sodium nitroprusside. Neurally stimulated relaxation was not seen in the airways of patients with Eisenmenger's syndrome despite being observed in donor tissues, indicating that relaxation appears to be absent in these tissues irrespective of the stimulant.

Perhaps the most plausible explanation for the findings in this study is a lower resting smooth muscle tone in airways from these patients. A lower endogenous intrinsic tone would result in an increased contractile capacity, as seen with the cholinergic agonists in this study. The lack of a significantly greater response to histamine in the airways of patients with Eisenmenger's syndrome would, at first instance, provide evidence against this mechanism, however histamine has multiple effects in the airways. Vincenc and colleagues (9) demonstrated that contraction to histamine in parenchymal strips was preceded by relaxation at lower concentrations, although this initial relaxation was not seen in tissues from all patients. The biphasic response to histamine was attributed in part to a mixed population of H₁ and H₂ histamine receptors mediating contraction and relaxation in airway smooth muscle, as well as the effect of histamine binding to receptors on the parenchymal vasculature. In the present study, it is therefore possible that in the airways of patients with Eisenmenger's syndrome which have an increase in the number and size of pulmonary vessels, some of the response to histamine may result from stimulation of histamine receptors on the vasculature. A lower intrinsic smooth muscle tone in these patients would be responsible for masking the relaxant phase which attenuates the maximal attainable contractile response.

A decrease in endogenous smooth muscle tone would also provide an explanation for our observation of absence of relaxant responses to agonists applied to the airways from patients with Eisenmenger's syndrome. In this study, experimentation with relaxant agonists was performed from baseline and not induced smooth muscle tone. Such a procedure resulted in marked relaxation responses to isoproterenol, levocromakalim, and electrical field stimulation (in the presence of atropine) in tissues from donors, but a complete absence of relaxation in the airways of two patients with Eisenmenger's syndrome and only a slight relaxation response in tissues from the three other patients. Perhaps the best evidence for a lower level of baseline tone comes from studies carried out on the airways obtained from the most recent patient undergoing transplantation. In this case, additional parallel tissues were set upin one set of tissues, relaxant agonists were applied to tissues at baseline tone, while in the other set, carbachol-induced tone was obtained before the addition of the relaxant agonists. In the tissues in which tone was induced, the magnitude of the relaxation response to isoproterenol was approximately 7 times as great as that in tissues in which relaxation was induced from baseline tone. Interestingly, pretransplant lung function testing of these patients indicated a complete absence of response to bronchodilators, despite baseline airflow limitation in some patients and the report of "asthma-like" symptoms including wheeze. This suggests that in vivo bronchodilatation may also be impaired, or that the airway obstruction which causes airflow limitation in these patients may not result from alterations in smooth muscle tone, but may be due to vascular engorgement with resultant airway lumenal obstruction caused by increased pulmonary pressures and increased anastomotic blood flow to the bronchial vasculature. Although we would like to further examine relaxation responses in the presence of augmented airway smooth muscle tone, this is impractical. This is an extremely rare condition and, in the last 7 yr, only 14 of 229 patients undergoing lung transplantation have had a diagnosis of Eisenmenger's syndrome.

A decrease in baseline tone may occur in the airways from these patients by a number of mechanisms. The vasculature of patients with Eisenmenger's syndrome is dramatically altered in structure (10) and indeed when a slice is made through the lung of one of these patients and one examines the cut surface, it is obvious that there is a significant increase in the number, size, and wall thickness of the pulmonary vessels. It is therefore possible that the significant sheer stress which occurs in these vessels (and also probably in the bronchial vessels) owing to increased pulmonary pressures may induce the continued release from the endothelium of relaxant compounds within and around the airway wall, which in turn cause continued airway smooth muscle relaxation. It is unlikely that the epithelium, rather than the endothelium, could be responsible for a decreased endogenous tone, as the amount of epithelium present in each airway ring used was the same as that for the organ donors. It is however possible that the epithelium of these patients may produce a different profile of substances such as lipoxygenase products which influence basal muscle tone. This is unlikely to be the case in the present study, as Watson and colleagues have shown that in human airways, leukotriene-induced baseline tone is relatively independent of the epithelium and that cyclo-oxygenase products play little role in baseline tone (11). The methodology employed in this study would mitigate against substances derived from the endothelium or epithelium as being responsible for any decrease in baseline tone. After calibration, the tissues were equilibrated and washed over a 1-h period and the effect of such mediators would likely have been eliminated before the initiation of the concentration-response curves.

It is also possible that a decrease in the amount of extracellular matrix or an alteration in the matrix components in the airway walls of patients with Eisenmenger's syndrome may be responsible for a lower resting tone of the muscle. This proposition is supported by the findings of Bramley and colleagues (12) in their study of the effects of perturbations to airway wall collagen on the responsiveness of human airways. Incubation of the airways in collagenase, which induced a disruption of collagen in these tissues, was found to result in a lower passive tension, couple with a potentiation of subsequent contractile responsiveness. It is therefore possible that alterations in the matrix composition of the walls of patients with Eisenmenger's syndrome may lead to a similar decrease in baseline tone. This proposition is supported by the observed loss of normal elastic recoil at low lung volumes in a patient with Eisenmenger's syndrome undergoing plethysmography (13).

Reversed or bidirectional shunting through cardiac defects in Eisenmenger's syndrome results in severe hypoxemia and it is possible that such a decrease in oxygenation may be responsible for a decrease in baseline smooth muscle tone. Studies of the effects of hypoxemia on lung function in healthy volunteers have demonstrated dilatation of the main bronchus with an increase in cross sectional area of approximately 20% upon desaturation (to a Sa_O2 of 80 to 85%). These changes were shown to be independent of changes in the pattern of ventilation and vagal tone (14). In addition, hypoxemia has been shown to increase bronchial blood flow (15, 16) which may result in increased exposure of the airway wall to circulating relaxant factors and a decrease in intrinsic airway tone. Interestingly, hypoxic exposure has been shown to induce nonspecific increases in bronchial responsiveness to inhaled spasmogens in sheep, although in this study, there was no indication of the effect of hypoxia on the responsiveness to bronchodilators (17).

In summary therefore, we have shown that airways isolated from patients with Eisenmenger's syndrome exhibit, when compared with airways isolated from healthy organ donors, markedly increased contractile responses to cholinergic stimulation and markedly impaired relaxation responses to a range of relaxant agents. We believe that the most likely explanation for this pattern of responsiveness is a decrease in the intrinsic tone of the airway smooth muscle, although the mechanism responsible for this change is uncertain. It is therefore possible that patients with this condition may exhibit similar responses in vivo, particularly in the presence of severe hypoxemia in the advanced stages of the condition.