Plasma endothelin-1 levels in patients with left-to-right shunt with or without pulmonary hypertension

International Journal of Cardiology 70 (1999) 57–62

Plasma endothelin-1 levels in patients with left-to-right shunt with or without pulmonary hypertension ¨ ¨ Nejat Akar H. Ercan Tutar*, Ayten Imamoglu, Semra Atalay, Halil Gumus, Department of Pediatric Cardiology, Ankara University Faculty of Medicine, Ankara, Turkey Accepted 1 March 1999

Abstract The aim of this study was to evaluate the role of endothelin-1 (ET-1) in pathophysiology of pulmonary hypertension (PH) secondary to congenital heart disease with left-to-right shunt. Twenty-three children (12 male, 11 female) aged 0.58–13 years were enrolled the study. Blood samples were drawn from superior vena cava, right atrium, right ventricle, pulmonary artery and pulmonary wedge or pulmonary vein during cardiac catheterization. Plasma ET-1 levels were assayed by ELISA. Patients were divided into two groups according to the presence or absence of PH. Plasma ET-1 levels of the study group were compared to the peripheral venous and arterial ET-1 levels of 11 healthy infants and children (aged 0.75–13 years). Plasma ET-1 levels in patients with left-to-right shunt were found significantly higher than those of controls. However, plasma ET-1 levels were similar between the two groups of the patients. Pulmonary venous ET-1 levels were higher than the levels of superior vena cava, this suggested an increased production of ET-1 in pulmonary vascular bed in patients with PH. No correlations were found between plasma ET-1 levels and pulmonary arterial pressure, pulmonary vascular resistance and pulmonary blood flow in the patients. Plasma ET-1 levels of the patients with left-to-right shunt were increased independently from pulmonary arterial pressure and pulmonary vascular resistance. This increase was related to the production of ET-1 in pulmonary vascular bed in patients with PH. ET-1 could not be found to be directly related to the development of PH in the patients with left-to-right shunt.  1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Congenital heart disease; Endothelin-1; Left-to-right shunt; Pulmonary hypertension

1. Introduction Patients with congenital heart disease (CHD) with left-to-right shunt have a risk of pulmonary hypertension (PH) during the course of the disease [1]. Pulmonary hypertension associated with progressive pulmonary vascular changes whose mechanisms are still poorly understood [2–4]. Endothelin-1 (ET-1) is

*Corresponding author. Tel.: 190-312-419-4094; fax: 190-312-3620581. E-mail address: [email protected] (H. . Tutar)

a 21-amino acid peptide produced by pulmonary vascular endothelium, is the most potent endogenous vasoconstrictor and has mitogenic activities on vascular smooth muscle cells and fibroblasts [5–7]. Both vasoconstriction and myofibroblast proliferation are thought to be responsible in the genesis of PH [1,8]. These observations indicate the possibility that ET-1 plays a role in the pathophysiology of PH. In this study, plasma ET-1 levels of the patients with left-to-right shunt with or without PH were measured. In order to evaluate the effect of pulmonary blood flow on plasma ET-1 levels and the role of ET-1 in the pathophysiology of PH, blood samples

0167-5273 / 99 / $ – see front matter  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 99 )00062-5

58

H.E. Tutar et al. / International Journal of Cardiology 70 (1999) 57 – 62

from various sites during cardiac catheterization were taken.

2. Methods

2.1. Subjects We studied 23 patients (12 males, 11 females) with left-to-right shunt. Their ages ranged from 0.58 to 13 years (5.864.4 years). Eleven healthy infants and children (five males, six females) aged between 0.75 and 13 years (6.0264.4 years) served as controls. No patient or control subject had known causes of increased ET-1, such as diabetes mellitus, systemic hypertension, sepsis, renal failure and cardiogenic shock. Written informed consent was obtained from the parent(s) of each child enrolled for the study. The study protocol was approved by the ethical committee of our institution.

2.2. Cardiac catheterization Routine sedation with intravenous midazolam for catheterization was used. Pressure measurements were done using fluid-filled catheters connected to pressure transducers. Oxygen consumption was estimated using age, sex and heart rate according to the method of La Farge and Miettinen [9]. Pulmonary (Q p ) and systemic (Q s ) blood flows were calculated using the standard Fick method and indexed for body surface area. The ratio of pulmonary / systemic blood flow (Q p /Q s ), pulmonary (PVR) and systemic (SVR) vascular resistance, and the ratio (PVR / SVR) were calculated according to the standard formula [10]. When Q p /Q s was .1.5 patients were assigned to had ‘high flow’.

finished within 5 min. All drugs were discontinued at least 24 h before catheterization. Paired samples of arterial blood from the radial artery and venous blood from the antecubital vein of contralateral extremity were obtained with minimal delay (less than 1 min) from control subjects in the supine position at rest. Blood samples were transferred to cooled polypropylene tubes containing EDTA. After centrifugation was done at 30003g for 15 min at 48C, the plasma was separated and stored at 2208C until ET-1 assay.

2.4. ET-1 assay Plasma ET-1 concentration was measured by ELISA. The ET-1 ELISA kit was obtained from R&D Systems, (Minneapolis, USA) to perform the assay. Cross-reactivity with ET-2, ET-3, and human big ET-1 is 45, 14 and ,1%, respectively. The plasma samples (1 ml) and 1.5 ml of extraction solvent (acetone:1 M HCl:water (40:1:5)) were added to polypropylene tubes and centrifuged for 20 min at 30003g at 48C. Supernatants were evaporated to dryness at 378C under dry nitrogen gas. The dried extracts were reconstituted by adding 0.25 ml of sample diluent to each. Anti-ET-1-HRP conjugate (0.1 ml) were added to standards, samples and control extracts. After incubation at room temperature for 1 h, aspiration and washing six times by adding 0.3 ml of wash buffer were done. Substrate solution (tetramethylbenzidine) were added to each well. After incubation for 30 min 0.1 ml of stop solution were added each well and optical density of each well were read at 450 nm with correction at 620 nm. Sample concentrations were determined by comparison with the corresponding standard curve.

2.3. Blood sampling

2.5. Statistics

Blood samples of 5 ml were obtained from the patients at the following sites during cardiac catheterization before angiography: systemic venous blood (SV) from superior vena cava, right atrium, right ventricle, left or right pulmonary artery, and pulmonary vein or pulmonary arterial wedge position (pulmonary venous blood (PV)). The blood sampling was

All results were expressed as mean6SD. Comparison of mean values between the groups was performed with the unpaired two-tailed t-test. The gender distribution among the groups was compared using x 2 analysis. Correlations were analyzed with the Pearson’s correlation coefficient. A P value ,0.05 was considered statistically significant.

H.E. Tutar et al. / International Journal of Cardiology 70 (1999) 57 – 62

59

Table 1 Patient profile a

n Age (years) Sex (male:female) Pp (mmHg) Ps (mmHg) Q p (1 / min / m 2 ) Q s (1 / min / m 2 ) Q p /Q s ratio PVR (Wood units / m 2 ) SVR (Wood units / m 2 ) PVR / SVR ratio Heart rate (bpm) Diagnosis

Medication

PH group

Non-PH group

9 5.364.4 5:4 53.168.0 70.3611.8 8.662.5 3.760.9 2.561.2 5.261.2 18.165.1 0.3160.10 130.3625.2 VSD, 4; AVSD,1; APW, 1; VSD1ASD, 1; VSD1PDA, 1; VSD1ASD1PDA, 1 D1F, 2; D1F1C, 7

14 6.163.9 7:7 20.465.1 78.868.5 7.562.8 3.460.7 2.260.7 1.961.1 22.165.4 0.0960.05 109.8622.4 ASD, 5; PDA, 3; VSD, 1; AVSD, 2; VSD1PDA, 2; VSD1ASD, 1 D, 3; D1F, 3; no treatment, 8

NS NS P,0.001 NS NS NS NS P,0.001 NS P,0.001 NS

a APW, aortopulmonary window; ASD, atrial septal defect; AVSD, atrioventricular septal defect; C, captopril; D, digoxin; F, furosemide; PDA, patent ductus artenosus; Pp , mean pulmonary artery pressure; Ps , mean aortic pressure; PVR, pulmonary vascular resistance; Q p , pulmonary blood flow; Q s , systemic blood flow; SVR, systemic vascular resistance; VSD, ventricular septal defect.

3. Results Patients were divided into two groups according to the ratio of simultaneous pulmonary arterial to aortic mean pressure (Pp /Ps ). In nine patients, Pp /Ps was greater than 0.5 (PH group), and remaining 14 patients Pp /Ps was less than 0.5 (non-PH group). Table 1 shows the patient characteristics and hemodynamic data of each group. All patients had one or more defect that caused left-to-right shunt. All patients in the PH group had received digoxin and furosemide, except two of nine treated with captopril;

whereas in the non-PH group, none of the patients received captopril and eight patients in this group had been taking no medications at all. There were no significant differences between the two groups in parameters except those related to the PH. Table 2 shows plasma ET-1 levels at sequential sampling sites in the patient groups and in the peripheral venous and arterial samples of the control subjects. Systemic venous and PV ET-1 concentrations of the study groups were compared to the peripheral venous and arterial ET-1 levels of the controls, respectively. Systemic venous and PV ET-1

Table 2 Plasma endothelin-1 levels in patients with left-to-right shunt and controls

PH group (n:9) Non-PH group (n:14) All patients (n:23) Controls (n:11) a

SV (pg / ml))

RA (pg / ml)

RV (pg / ml)

PA (pg / ml)

PV (pg / ml)

PV/ SV ET-1 ratio

1.2960.23 * jNS 1.3360.39 *

1.3560.29 jNS 1.3060.56

1.3260.39 jNS 1.3460.52

1.2860.38 jNS 1.2860.52

1.4360.35 ** jNS 1.2060.45 ****

1.1060.15 *** jP,0.01 0.9060.16 ****

1.3260.34 jP,0.01 1.1060.09 a

1.3210.47

1.3360.47

1.2860.47

1.3060.42 jP,0.01 0.9660.10 b

0.9860.18 jP,0.05 0.8860.06 c

Peripheral venous sample. Peripheral arterial sample. c Arterial / venous ET-1 ratio. * P,0.05 vs. controls; ** P,0.01 vs. controls; *** P,0.001 vs. controls; **** NS vs. controls. PA, pulmonary artery; PV, pulmonary venous; RA, right atrium; RV, right ventricle; SV, systemic venous. b

60

H.E. Tutar et al. / International Journal of Cardiology 70 (1999) 57 – 62

levels in patients with left-to-right shunt were found significantly higher than those of corresponding values of controls. Plasma ET-1 levels, however, were not different between the patients with or without PH at all sampling sites. In the PH group, both SV and PV ET-1 levels were significantly higher than those of controls. In the non-PH group, although SV and PV ET-1 levels were higher than those of controls, the difference was not reached the statistical significance for PV ET-1 levels. In the PH group, PV/ SV ET-1 ratio was significantly higher than both the non-PH group and the controls. This ratio was not different between the non-PH group and the control subjects. In control subjects the arterial ET-1 concentration was less than the venous concentration in all cases. In the PH group, PV ET-1 concentration was higher than the SV ET-1 concentration except in two of nine cases. In the non-PH group, PV ET-1 concentration was higher than SV ET-1 concentration in only four of 14 cases. Plasma ET-1 levels were not correlated with Q p / Q s ratio, mean pulmonary artery pressure, PVR and PVR / SVR ratio. Significant positive correlation was found between PV/ SV ET-1 ratio and mean pulmonary artery pressure (r50.57, P,0.005), and PVR / SVR ratio (r50.62, P,0.005). But, PV/ SV ET-1 ratio was not correlated with Q p /Q s . Only two patients’ Q p /Q s ratio was below 1.5 and both had lower plasma ET-1 levels than those of patients with high flow, but statistical analysis was not carried out.

4. Discussion Increased plasma ET-1 levels have been found in most patients with primary and secondary PH [11– 14]. An increased plasma levels of ET-1 has already been reported in children with PH secondary to CHD [15]. However, we do not know yet whether an increase in plasma ET-1 is important in the pathogenesis of PH or just reflects the reaction of the pulmonary vascular bed to the underlying disease process. In patients with left-to-right shunt, conflicting results were reported in the literature about plasma ET-1 levels. Some found that plasma ET-1 levels were raised and correlated with increased pulmonary blood flow [16]. This finding, however, was not confirmed by the others [17,18].

The results of our study showed that in patients with left-to-right shunt plasma ET-1 levels were increased. Experimental data revealed increased production and release of endothelin from cultured endothelial cells exposed to increased flow [19]. Increased production of ET-1 was also documented after in utero placement of aortopulmonary shunt in lambs [20]. Our results suggest that increased pulmonary blood flow may be a stimulatory factor of increased ET-1 production in children with left-toright shunt which is consistent with the results of Vincent et al. [16]. In contrast, Gorenflo et al. [18] did not show a difference between patients who had Q p /Q s ,1.5 and Q p /Q s .1.5. They concluded that no increase in ET-1 levels in patients with left-to-right shunt was found. Their results, however, were depended on only two control subjects’ ET-1 values. Adatia and Haworth [17] also did not show an increase in ET-1 concentrations in patients with increased pulmonary blood flow. However, another study of the same group showed that plasma ET-1 levels were significantly higher after cardiopulmonary by-pass in those who had had high pulmonary blood flow before the operation [21]. They suggested that cardiopulmonary by-pass had a more injurious effect on pulmonary vascular bed with preexisting endothelial damage. Our results showed that plasma ET-1 concentrations were increased in patients with PH due to left-to-right shunt who had both volume and pressure overload to pulmonary circulation. This increment, however, was not different from patients without PH, who had only volume overload to pulmonary circulation. Furthermore, we did not show any correlation between ET-1 levels and parameters of PH (pulmonary artery pressure, PVR, PVR / SVR). As a result, the present study indicates that increased pulmonary blood flow raises plasma ET-1 levels independent of pulmonary artery pressure or PVR Vincent et al. [16], also indicated that plasma ET-1 concentrations had been raised in patients with increased pulmonary blood flow independent of pulmonary artery pressure. In the work of Gorenflo et al. [18] only a minority of their patients with left-to-right shunt had PH, so they could not reveal the effect of PH on plasma ET-1 levels. Yoshibayashi et al. [15] demonstrated that plasma ET-1 levels were significantly elevated in patients with CHD and PH compared with those of a

H.E. Tutar et al. / International Journal of Cardiology 70 (1999) 57 – 62

non-PH group. Their non-PH group, however, was not homogeneous, consisting of patients with decreased pulmonary blood flow (e.g. tetralogy of Fallot, pulmonary stenosis). Furthermore, the relationship between ET-1 and pulmonary blood flow has not been determined despite their patients in the PH group had significantly higher pulmonary blood flow. Increased plasma concentrations of ET-1 in our patients with left-to-right shunt could result from either an increase in the production of ET-1 or a decrease in its clearance, or both in pulmonary vascular bed. We determined the PV/ SV ET-1 ratio as an indication of net pulmonary production or clearance. In all healthy control subjects, the arterial levels of ET-1 were less than the corresponding venous levels. This is consistent with a net clearance by the lung of ET-1, as has been found in animal experiments [22]. In this study, nearly all patients with PH had elevation of ET-1 in the pulmonary venous plasma compared with systemic venous plasma, and PV/ SV ET-1 ratio was significantly higher than both in controls and in non-PH group, suggesting local pulmonary production of ET-1. We also found significant positive correlation between PV/ SV ET-1 ratio and parameters of PH. In the non-PH group, we could not demonstrate whether increased plasma ET-1 levels reflected an increase in the production of ET-1 or a decrease in its clearance, because their PV/ SV ET-1 ratio was not different from controls. Increased plasma ET-1 levels have been found in patients with primary PH in whom systemic arterial plasma levels were higher than systemic venous levels, reflecting pulmonary production of ET-1 [23]. An increased production of ET-1 in the pulmonary vascular bed has also been reported in children with PH secondary to CHD [15]. In a recent study by Giaid et al. [24], direct evidence of increased local pulmonary production of ET-1 in primary and secondary PH was provided by using immunocytochemical analysis and in situ hybridization. Most patients in this study had been administered digoxin and furosemide. At present, we do not know whether these drugs have any effect on the ET-1 production. However, we are aware that ACE inhibitors inhibit ET-1 secretion from cultured endothelial cells [25]. There were controversial results in clinical studies. Some [26] found that ACE inhibition

61

reduce the elevated plasma endothelin levels; however, that finding was not confirmed by others [27]. In our study, seven of nine patients in the PH group, and none of the patients in the non-PH group had received captopril. As a result, we conclude that captopril might cause less pronounced ET-1 increase in the PH group. Further work is needed to determine the effect of ACE inhibition on ET-1 in patients with CHD. This study did not show whether an increase in plasma ET-1 is important in the pathogenesis of PH or just reflects the reaction of the pulmonary vascular bed to the underlying disease process. In another study, serial measurements before and after surgery would be more useful to observe changes in ET-1 as pulmonary blood flow and parameters of PH vary.

Acknowledgements This study was supported by a grant (SBAG-AYD133) from the Scientific and Technical Research ¨ Council of Turkey. We specially thank Omer Uzunali for his technical assistance in determining plasma ET-1 levels.

References [1] Kulik TJ. Pulmonary hypertension. In: Fyler DC, editor, Nadas’ pediatric cardiology, Hanley & Belfus, Philadelphia, 1992, pp. 83–100. [2] Ilkiw RL, Miller-Hance W, Nihill MR. The pulmonary circulation. In: Garson A, Bricker JT, McNamara DG, editors, The science and practice of pediatric cardiology, Lea & Febiger, Philadelphia, 1990, pp. 360–85. [3] Heath D, Edwards JE. The pathology of hypertensive pulmonary vascular disease. A description of six grades of structural changes in the pulmonary arteries with special reference to congenital cardiac septal defects. Circulation 1958;18:533–47. [4] Rabinovitch M, Haworth SG, Castaneda AR, Nadas AS, Reid LM. Lung biopsy in congenital heart disease: a morphometric approach to pulmonary vascular disease. Circulation 1978;58:1107–22. [5] Yanagisawa M, Kurihara H, Kimura S et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5. [6] Nakaki T, Nakayano M, Yamamoto S, Kato R. Endothelin mediated stimulation of DNA synthesis in vascular smooth muscle. Biochem Biophys Res Commun 1989;158:880–3. [7] Peacock AJ, Dawes KE, Shock A, Gray AJ, Reeves JT, Laurent GJ. Endothelin-1 and endothelin-3 induce chemotaxis and replication of pulmonary artery fibroblasts. Am J Respir Cell Mol Biol 1992;7:492–9.

62

H.E. Tutar et al. / International Journal of Cardiology 70 (1999) 57 – 62

[8] Stewart DJ, Langleben D. Endothelin and pulmonary hypertension. In: Luscher TF, editor, The endothelium in cardiovascular disease, Springer, Berlin, 1995, pp. 184–94. [9] LaFarge CG, Miettinen OS. The estimation of oxygen consumption. Cardiovasc Res 1970;4:23–30. [10] Vargo TA. Cardiac catheterization-hemodynamic measurements. In: Garson A, Bricker JT, McNamara DG, editors, The science and practice of pediatric cardiology, Lea & Febiger, Philadelphia, 1990, pp. 913–45. [11] Cacoub P, Dorent R, Maistre G et al. Endothelin-1 in primary pulmonary hypertension and the Eisenmenger syndrome. Am J Cardiol 1993;71:448–50. [12] Rosenberg AA, Kennaugh J, Koppenhafer SL, Loomis M, Chatfield BA, Abman SH. Elevated immunoreactive endothelin-1 levels in newborn infants with persistent pulmonary hypertension. J Pediatr 1993;123:109–14. [13] Goerre S, Wenk M, Bartsch P et al. Endothelin-1 in pulmonary hypertension associated with high-altitude exposure. Circulation 1995;90:359–64. [14] Yamamoto K, Ikeda U, Mito H, Fujikawa H, Sekiguchi H, Shimada K. Endothelin production in pulmonary circulation of patients with mitral stenosis. Circulation 1994;89:2093–8. [15] Yoshibayashi M, Nishioka K, Nakao K et al. Plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. Evidence for increased production of endothelin in pulmonary circulation. Circulation 1991;84:2280–5. [16] Vincent JA, Ross RD, Kassab I, Hsu IM, Pinsky WW. Relation of elevated plasma endothelin in congenital heart disease to increased pulmonary blood flow. Am J Cardiol 1993;71:204–1207. [17] Adatia I, Haworth SG. Circulating endothelin in children with congenital heart disease. Br Heart J 1993;69:233–6. [18] Gorenflo M, Gross P, Bodey A et al. Plasma endothelin-1 in patients with left-to-right shunt. Am Heart J 1995;130:537–42.

[19] Yoshizumi M, Kurihara H, Sugiyama T et al. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun 1989;161:859–64. [20] Wong J, Reddy VM, Hendricks-Munoz K, Liddicoat IR, Gerrets R, Fineman JR. Endothelin-1 vasoactive responses in lambs with pulmonary hypertension and increased pulmonary blood flow. Am J Physiol 1995;269:H1965–72. [21] Komai H, Adatia IT, Elliott MJ, deLeval MR, Haworth SG. Increased plasma levels of endothelin-1 after cardiopulmonary bypass in patients with pulmonary hypertension and congenital heart disease. J Thorac Cardiovasc Surg 1993;106:473–8. [22] DeNucci G, Thomas R, D’Orleans-Juste P et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endotheliumderived relaxing factor. Proc Natl Acad Sci USA 1988;85:9797– 800. [23] Stewart DJ, Levy RD, Cernacek P, Langleben D. Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease? Ann Intern Med 1991;114:464–9. [24] Giaid A, Yanagisawa M. Langleben D et al., Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. New Engl J Med 1993;328:1732–9. [25] Momose N, Fukuo K, Morimoto S, Ogihara T. Captopril inhibits endothelin-1 secretion from endothelial cells through bradykinin. Hypertension 1993;2:921–4. [26] Galatius-Jensen S, Wroblewski H, Emmeluth C, Bie P, Haunsf S, Kastrup J. Plasma endothelin in congestive heart failure: effect of the ACE inhibitor, fosinopril. Cardiovasc Res 1996;32:1148–54. [27] Townend J, Doran J, Jones S, Davies M. Effect of angiotensin converting enzyme inhibition on plasma endothelin in congestive heart failure. Int J Cardiol 1994;43:299–304.