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Reduced endothelium-dependent relaxation of cystic fibrosis pulmonary arteries
European Journal of Pharmacology, 163 (1989) 401-403
401
Elsevier EJP 0190R
Rapid communication
Reduced endothelium-dependent relaxation of cystic fibrosis pulmonary arteries A.T. D i n h X u a n , T.W. H i g e n b o t t a m *, J. P e p k e - Z a b a , C. C l e l l a n d a n d J. W a l l w o r k Department of Respiratory Physiology, Papworth Hospital, Cambridge, U.K. Received 29 March 1989, accepted 30 March 1989
Endothelium-derived relaxing factors (EDRF), released by endothelial cells (Furchgott and Zawadzki, 1980), are potent local vasodilators in a variety of vascular beds. Initially described in canine pulmonary arteries (Chand and Altura, 1981), endothelium-dependent relaxation has been recently found in human pulmonary arteries (Greenberg et al., 1987). Cystic fibrosis (CF) is an autosomal recessive disease causing morbidity and mortality from pulmonary complications (Wood et al., 1976). The progressive loss of pulmonary function and resulting hypoxernia lead to cor pulmonale and secondary pulmonary hypertension. Combined heart-lung transplantation (HLT), a treatment for CF patients with respiratory failure (Scott et al., 1988), allows in vitro study of isolated human pulmonary arteries from the explanted lungs. Segments of proximal pulmonary arteries were obtained at H L T from 3 CF patients (2 women and 1 man, aged 24, 28 and 31). All were in respiratory failure, PaO2 = 7.02 + 0.91 kPa and PaCO 2 = 5.94 __+0.47 kPa (means _+ S.D.) and had reduced forced expiratory volume in one second (FEV 1 --- 21 + 11% predicted; mean _ S.D.). Control segments of similar sized pulmonary arteries were obtained from lobectomy in 6 non-CF patients (3 women and 3 men, 6 4 + 7 years old;
* To whom all correspondence should be addressed: Department of Respiratory Physiology, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE, U.K.
mean _+ S.D.) with bronchial carcinoma and normal FEV 1 (103 + 12% predicted, mean _+ S.D.) prior to operation. Immediately after excision, tissue was placed in pregassed (95% 02 and 5% CO2), and cold (4°C) Krebs-Ringer bicarbonate solution. Pair of tings (3-5 mm in length and 2-4 mm outer diameter) were cut from the pulmonary arteries, and the endothelium was carefully removed from one of each pair, using a pipe cleaner. Changes in isometric tension were recorded in organ baths (filled with 20 ml of K R buffer and bubbled with 95% O 2 and 5% CO 2 at 37°C) using a force transducer (Harvard Bioscience, MA, USA). After full relaxation, on an initial tension of 1.5 g, with repeated washes over 90 rain, the rings were submaximally precontracted with phenylephrine dichloride ( 1 0 - 6 t o 10 - 5 M ) t o obtain a stable plateau of contraction. Acetylcholine dichloride (ACh) or adenosine diphosphate (ADP) were then added to produce a cumulative doserelaxation response (10 -~° to 10 -5 M) (fig. 1) of pairs of rings with and without endothelium. Seven and 5 paired rings obtained from the 3 CF patients were tested with ACh and ADP, respectively. The 6 pairs of control rings came from the 6 non-CF patients. All rings from CF patients and half the rings from non-CF patients were pretreated with indomethacin (5 x 10 - 6 M ) to inhibit production of prostacyclin. Indomethacin pretreatment had no effect on relaxation of tings with and without endothelium. For example, the ECs0 values ( - l o g M, means ± S.E.M.) in non-CF pa-
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
402 I1)
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150
100
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I
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10
g
8
7
6
5
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ACh (-log M ) ¢.-.
g
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150
-
100 -
8
ID t-
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;
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ADP (-log M ) Fig. 1. Endothelium-dependent relaxation responses in rings without (open symbols) and with (closed symbols) endothelium to cumulative doses of acetylcholine (ACh) and adenosine diphosphate (ADP) in control (e) and cystic fibrosis (11) patients. Results are expressed as means + S.E.M. Statistical analysis was done by comparisons of m e a n s for unpaired values between rings obtained from CF and control patients. * P < 0.05; * * * P < 0.001.
tients were 7.63 + 0.37 versus 7.58 + 0.43 with ACh and 7.74_ 0.37 versus 7.77 + 0.31 with ADP, in rings pretreated and untreated with indomethacin, respectively. There was no difference in response to ACh or to ADP between duplicate or triplicate CF rings from the same patient (one way analysis of variance). Dose-dependent relaxation occurred with either ACh or ADP in tings with endothelium
from both CF and non-CF patients, whereas rings without endothelium showed no response (fig. 1; P < 0.05 and 0.001 by paired t-test, in CF and non-CF patients, respectively). However, endothelium-dependent relaxation to ACh in CF patients was markedly reduced compared wtih nonCF patients (fig. 1); maximal relaxation (means _+ S.E.M.) = 33.1 + 3% versus 72.8 4- 7% in CF and non-CF patients, respectively (P < 0.001 by comparisons of means for unpaired values). A similar, but smaller, response was seen to ADP (fig. 1); maximal relaxation (means + S.E.M.) = 59 + 9% versus 84.2 + 2% (P < 0.01) in CF and non-CF patients, respectively. In the CF patients only, higher concentration of ACh were associated with contraction of the vascular tings (fig. 1). EDRF-mediated pulmonary vasorelaxation in patients with CF appears to be impaired by comparison with patients without CF. The mechanism of this pathophysiological response is unclear. It is not known whether this is unique to CF or a general phenomenon of respiratory failure. The latter seems, however, more likely. All our CF patients have evidence of cor pulmonale at the time of H L T (Scott et al., 1988). Histological examination of the explanted lungs of these 3 patients showed marked hypertrophy of the arteriolar medial smooth muscle, a hallmark of pulmonary hypertension. Furthermore, dilatation lesions, indicating grade IV pulmonary hypertension, were seen in one patient. The rings that were actually tested (2-4 mm outer diameter) also showed signs of atheroma, another feature of pulmonary hypertension, whereas none of the control vascular rings showed such pathological changes. Further experimentation is required to resolve the question as to whether impaired endothelium-dependent relaxation is a cause or a result of secondary pulmonary vascular disease.
Acknowledgements This study was supported by Grants No. 1871066 from the British Heart Foundation and No. 88 B / 1 from the Comit6 National contre les Maladies Respiratoires et la Tuberculose. We are grateful to Mr Ben Milstein for his editorial advice.
403
References Chand, N. and B. Altura, 1981, Acetylcholine and bradykinin relax intra-pulmonary arteries by acting on endothelial cells, Science 213, 1376. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Greenberg, B., K. Rhoden and P.J. Barnes, 1987, Endothe-
lium-dependent relaxation of human pulmonary arteries, Am. J. Physiol. 252, H434. Scott, J., T.W. Higenbottam, J. Hutter, M. Hodson, S. Stewart, A. Penketh and J. Wallwork, 1988, Heart-lung transplantation for cystic fibrosis, Lancet ii, 192. Wood, R.E., T.F. Boat and C.F. Doershuk, 1976, State of the art: cystic fibrosis, Am. Rev. Respir. Dis. 113, 833.
401
Elsevier EJP 0190R
Rapid communication
Reduced endothelium-dependent relaxation of cystic fibrosis pulmonary arteries A.T. D i n h X u a n , T.W. H i g e n b o t t a m *, J. P e p k e - Z a b a , C. C l e l l a n d a n d J. W a l l w o r k Department of Respiratory Physiology, Papworth Hospital, Cambridge, U.K. Received 29 March 1989, accepted 30 March 1989
Endothelium-derived relaxing factors (EDRF), released by endothelial cells (Furchgott and Zawadzki, 1980), are potent local vasodilators in a variety of vascular beds. Initially described in canine pulmonary arteries (Chand and Altura, 1981), endothelium-dependent relaxation has been recently found in human pulmonary arteries (Greenberg et al., 1987). Cystic fibrosis (CF) is an autosomal recessive disease causing morbidity and mortality from pulmonary complications (Wood et al., 1976). The progressive loss of pulmonary function and resulting hypoxernia lead to cor pulmonale and secondary pulmonary hypertension. Combined heart-lung transplantation (HLT), a treatment for CF patients with respiratory failure (Scott et al., 1988), allows in vitro study of isolated human pulmonary arteries from the explanted lungs. Segments of proximal pulmonary arteries were obtained at H L T from 3 CF patients (2 women and 1 man, aged 24, 28 and 31). All were in respiratory failure, PaO2 = 7.02 + 0.91 kPa and PaCO 2 = 5.94 __+0.47 kPa (means _+ S.D.) and had reduced forced expiratory volume in one second (FEV 1 --- 21 + 11% predicted; mean _ S.D.). Control segments of similar sized pulmonary arteries were obtained from lobectomy in 6 non-CF patients (3 women and 3 men, 6 4 + 7 years old;
* To whom all correspondence should be addressed: Department of Respiratory Physiology, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE, U.K.
mean _+ S.D.) with bronchial carcinoma and normal FEV 1 (103 + 12% predicted, mean _+ S.D.) prior to operation. Immediately after excision, tissue was placed in pregassed (95% 02 and 5% CO2), and cold (4°C) Krebs-Ringer bicarbonate solution. Pair of tings (3-5 mm in length and 2-4 mm outer diameter) were cut from the pulmonary arteries, and the endothelium was carefully removed from one of each pair, using a pipe cleaner. Changes in isometric tension were recorded in organ baths (filled with 20 ml of K R buffer and bubbled with 95% O 2 and 5% CO 2 at 37°C) using a force transducer (Harvard Bioscience, MA, USA). After full relaxation, on an initial tension of 1.5 g, with repeated washes over 90 rain, the rings were submaximally precontracted with phenylephrine dichloride ( 1 0 - 6 t o 10 - 5 M ) t o obtain a stable plateau of contraction. Acetylcholine dichloride (ACh) or adenosine diphosphate (ADP) were then added to produce a cumulative doserelaxation response (10 -~° to 10 -5 M) (fig. 1) of pairs of rings with and without endothelium. Seven and 5 paired rings obtained from the 3 CF patients were tested with ACh and ADP, respectively. The 6 pairs of control rings came from the 6 non-CF patients. All rings from CF patients and half the rings from non-CF patients were pretreated with indomethacin (5 x 10 - 6 M ) to inhibit production of prostacyclin. Indomethacin pretreatment had no effect on relaxation of tings with and without endothelium. For example, the ECs0 values ( - l o g M, means ± S.E.M.) in non-CF pa-
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
402 I1)
'!
150
100
5o
I--
I
I
I
I
I
I
I
10
g
8
7
6
5
4
ACh (-log M ) ¢.-.
g
"=
150
-
100 -
8
ID t-
I-
I
lo;8-
I
;
I
s
ADP (-log M ) Fig. 1. Endothelium-dependent relaxation responses in rings without (open symbols) and with (closed symbols) endothelium to cumulative doses of acetylcholine (ACh) and adenosine diphosphate (ADP) in control (e) and cystic fibrosis (11) patients. Results are expressed as means + S.E.M. Statistical analysis was done by comparisons of m e a n s for unpaired values between rings obtained from CF and control patients. * P < 0.05; * * * P < 0.001.
tients were 7.63 + 0.37 versus 7.58 + 0.43 with ACh and 7.74_ 0.37 versus 7.77 + 0.31 with ADP, in rings pretreated and untreated with indomethacin, respectively. There was no difference in response to ACh or to ADP between duplicate or triplicate CF rings from the same patient (one way analysis of variance). Dose-dependent relaxation occurred with either ACh or ADP in tings with endothelium
from both CF and non-CF patients, whereas rings without endothelium showed no response (fig. 1; P < 0.05 and 0.001 by paired t-test, in CF and non-CF patients, respectively). However, endothelium-dependent relaxation to ACh in CF patients was markedly reduced compared wtih nonCF patients (fig. 1); maximal relaxation (means _+ S.E.M.) = 33.1 + 3% versus 72.8 4- 7% in CF and non-CF patients, respectively (P < 0.001 by comparisons of means for unpaired values). A similar, but smaller, response was seen to ADP (fig. 1); maximal relaxation (means + S.E.M.) = 59 + 9% versus 84.2 + 2% (P < 0.01) in CF and non-CF patients, respectively. In the CF patients only, higher concentration of ACh were associated with contraction of the vascular tings (fig. 1). EDRF-mediated pulmonary vasorelaxation in patients with CF appears to be impaired by comparison with patients without CF. The mechanism of this pathophysiological response is unclear. It is not known whether this is unique to CF or a general phenomenon of respiratory failure. The latter seems, however, more likely. All our CF patients have evidence of cor pulmonale at the time of H L T (Scott et al., 1988). Histological examination of the explanted lungs of these 3 patients showed marked hypertrophy of the arteriolar medial smooth muscle, a hallmark of pulmonary hypertension. Furthermore, dilatation lesions, indicating grade IV pulmonary hypertension, were seen in one patient. The rings that were actually tested (2-4 mm outer diameter) also showed signs of atheroma, another feature of pulmonary hypertension, whereas none of the control vascular rings showed such pathological changes. Further experimentation is required to resolve the question as to whether impaired endothelium-dependent relaxation is a cause or a result of secondary pulmonary vascular disease.
Acknowledgements This study was supported by Grants No. 1871066 from the British Heart Foundation and No. 88 B / 1 from the Comit6 National contre les Maladies Respiratoires et la Tuberculose. We are grateful to Mr Ben Milstein for his editorial advice.
403
References Chand, N. and B. Altura, 1981, Acetylcholine and bradykinin relax intra-pulmonary arteries by acting on endothelial cells, Science 213, 1376. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Greenberg, B., K. Rhoden and P.J. Barnes, 1987, Endothe-
lium-dependent relaxation of human pulmonary arteries, Am. J. Physiol. 252, H434. Scott, J., T.W. Higenbottam, J. Hutter, M. Hodson, S. Stewart, A. Penketh and J. Wallwork, 1988, Heart-lung transplantation for cystic fibrosis, Lancet ii, 192. Wood, R.E., T.F. Boat and C.F. Doershuk, 1976, State of the art: cystic fibrosis, Am. Rev. Respir. Dis. 113, 833.