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The influence of ciclosporine A on the vasoactive effects of serotonin in in vitro perfused human umbilical arteries
Early Human Development 67 (2002) 69 – 77 www.elsevier.com/locate/earlhumdev
The inf luence of ciclosporine A on the vasoactive effects of serotonin in in vitro perfused human umbilical arteries Guttorm Haugen * Department of Obstetrics and Gynecology, The National Hospital, University of Oslo, 0027 Oslo, Norway Received 28 June 2001; received in revised form 29 November 2001; accepted 5 December 2001
Abstract Background: Pregnancy is feasible in organ-transplanted women, but little is known about possible effects of ciclosporine A on the circulation in the fetus and placenta. Aim: To investigate the influence of ciclosporine A (CsA) on the vasoactive effects of serotonin in human umbilical arteries. Study design and subjects: In vitro perfusion was performed in umbilical cord segments from seven organ-transplanted patients on CsA based immunosuppression and in 17 cords from uncomplicated pregnancies. Serotonin was administered in stepwise increasing concentrations from 10 10 to 10 5 M. In preparations from normal pregnancies, serotonin 10 7 M, was administered before and 30 min after the start of a continuous CsA infusion (1.0 mg/l). The influence of CsA 0.1 or 1.0 mg/l on the basal, unstimulated perfusion pressure was investigated in separate experiments. Outcome measures: Changes in perfusion pressure due to constrictory or dilatatory responses. Results: In all preparations from the organ-transplanted patients, serotonin induced a constrictory response that was non-significantly lower than that observed in the control group. The frequency of a dilatatory response preceding the vasoconstriction was 3/7 and 12/17 (non-significant) in the CsA-treated and control groups, respectively. In the experiments with CsA administration, a non-significant increase in the constrictory serotonin response was observed as compared to the control experiments. CsA did not alter the basal, unstimulated perfusion pressure. Conclusion: CsA did not have any significant influence on the vasoactive effect of serotonin in human umbilical arteries perfused in vitro. D 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ciclosporine A; Perfusion; Serotonin; Umbilical artery
* University Department of Obstetrics and Gynecology, Level F. Princess Anne Hospital, Southampton SO 16 5YA, UK. Tel.: +44-8079-8421; fax: +44-8078-6933. E-mail address: [email protected] (G. Haugen).
0378-3782/02/$ - see front matter D 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 2 5 5 - 9
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G. Haugen / Early Human Development 67 (2002) 69–77
1. Introduction Modern immunosuppressive drug regimens based on ciclosporine A (CsA) have greatly improved the life expectancy and given a favourable quality to the life of organtransplanted patients. For fertile women, a successful pregnancy might be a reality [1], and at least for kidney recipients, pregnancy does not seem to be deleterious to graft function nor long-term morbidity [2]. CsA is nephrotoxic and has been shown to induce haemodynamic alterations in the kidney [3]. As concerns the fetus, earlier reports indicated an increased frequency of growth retardation in pregnancies of kidney recipients on CsA based immunosuppressive regimen [4,5], but this has not been confirmed in later reports and review articles [6]. This does not exclude a possible effect of CsA on haemodynamic regulatory mechanisms in the fetus nor in the umbilicoplacental circulation. The human umbilical vessels lack innervation [7]. The regulation of the vascular tension is thus dependent on local autocrine mechanisms and vasoactive substances present in the blood stream, e.g. serotonin. In in vitro perfused umbilical arteries from uncomplicated term pregnancies, serotonin induces a dominating pressure increase usually preceded by a transient vasodilatation [8]. The present study was performed to investigate the influence of CsA on the effect of serotonin in such preparations. The study includes seven umbilical cords from pregnancies of organ-transplanted patients on CsA based immunosuppressive regimen.
2. Material and methods 2.1. Patients 2.1.1. Dose –response curves The transplanted group comprised three patients with kidney allografts, one patient with a liver allograft, one patient with combined kidney and liver allografts, and two hearttransplanted patients (Table 1). They all had single pregnancies 1 1/2 – 11 years following transplantation. As immunosuppressive drug regimen, they received azathioprine 25– 50 mg/day and/or prednisolone 5– 12.5 mg/day, and CsA 150 –400 mg/day. As additional drugs, one of the kidney-transplanted patients (pat. no. 1) received propranolol 20 mg/day and acetylsalicylic acid 75 mg/day. The patient with combined kidney and liver allografts (pat. no. 2) received furosemide 80 mg/day and acetylsalicylic acid 160 mg/day. Before pregnancy, both patients were normotensive with serum creatinine values about 140 Amol/ l. They remained normotensive throughout pregnancy, but serum creatinine values increased to above 200 Amol/l, and they were both delivered by Cesarean section due to deteriorating kidney function. Patient no. 6 received labetolol 100 mg/day due to nonproteinuric hypertension. Three patients (pat. no. 4, 5 and 7) developed moderate preeclampsia (hypertension and proteinuria 0.6– 1 g per 24 h). Due to increasing blood pressure (200/120 mm Hg), patient no. 5 received nifedipine 20 mg during delivery. Mean gestational age was 37 weeks + 4 days and mean birth weight 2924 g. As shown in Table 1, the length of gestation was heterogeneous with both pre-term and post-term deliveries.
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Table 1 Clinical data for the transplanted patients No.
Organ transplanted
Primary disease
CsA dose (mg/day)
Gestational age (weeks + days)
Blood pressure (mm Hg)
Birth weight (g)
1 2 3 4 5 6 7
kidney kidney/liver heart heart kidney liver kidney
glomerulonefritis primary hyperoxaluria dilated cardiomyopathy dilated cardiomyopathy glomerulonefritis unknown etiology glomerulonefritis
300 400 220 300 200 150 300
35 + 4 34 + 0 39 + 0 37 + 0 38 + 3 37 + 2 42 + 1
130/80 130/70 135/85 130/90 150/100 150/110 140/90
2470 2035 3790 3290 2900 2460 3520
CsA = ciclosporine A.
None of the infants were growth retarded [9] and they all had 5 min Apgar scores of 9 or above. The control group consisted of 17 women with uncomplicated term pregnancies. Mean gestational age at delivery was 39 weeks + 5 days (range 37 weeks + 5 days to 42 weeks + 0 days), and mean birth weight was 3641 g (range 2640 –4350 g). None of the infants were growth retarded, and they all had 5 min Apgar scores of 8 or above. 2.1.2. Experimental ciclosporine A exposure The umbilical cords were sampled from women with uncomplicated term pregnancies. 2.2. Perfusion studies The umbilical cords were collected immediately after delivery and immersed in Ringer’s solution at + 4 jC. A segment with length of about 10 cm was cut from the median part of the cord. In each segment, one artery was cannulated at both ends. The preparation was then mounted in a perfusion chamber and connected to a peristaltic pump delivering at constant flow. The vessels were perfused in the antegrade direction. Following an equilibration period of about 30 –40 min, the flow was adjusted to 15 –25 ml/min. A perfusion pressure of about 60– 70 mm Hg was achieved at this flow rate. The perfusion was carried out in a recirculating fashion until the start of drug infusion after which a non-recirculating perfusion was employed. As perfusate and cord immersion fluid was employed, an electrolyte solution with the following composition (in mM): NaCl 125, KCl 4, CaCl2 2, MgCl2 1, NaHCO3 22, NaH2PO4 0.3, Na2HPO4 1.7 and glucose 5.6. Both solutions were bubbled with a gas mixture of 5% O2, 5% CO2 and 90% N2, keeping a constant pH of about 7.40. For a more detailed description of the perfusion procedure, see Ref. [10]. 2.3. Drug administration In all experiments, serotonin was administered at the input side of the preparation through a side-infusion with non-pulsatile constant flow of about 1% of the total flow rate.
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In the dose – response studies, all preparations were first tested with serotonin 10 7 M. Thereafter, serotonin was administered at stepwise increasing concentrations from 10 10 to 10 5 M, quoted as final concentrations in the perfusate. In the studies with experimental CsA exposure, three to seven repeated serotonin infusions at a final concentration of 10 7 M were performed until three subsequent responses with almost equal pressure amplitudes were established. CsA at a final concentration of 1 mg/l (n = 7) or a blank electrolyte solution (control group, n = 7) was administered as a separate side-infusion at the input side. This side-infusion had a flow rate of about 1% of the total flow rate. After 30 min of continuous administration, three repeated serotonin infusions (10 7 M) were performed. The mean value of the latter pressure responses were compared to the mean of the three last serotonin responses before administration of CsA or the blank electrolyte solution. A possible effect of CsA on the basal perfusion pressure was investigated in separate experiments on preparations with proven serotonin sensitivity (n = 7). A continuous infusion of CsA at a final concentration of 0.1 mg/l and thereafter at a final concentration of 1 mg/l for about 60 min each was performed. This was preceded or succeeded by a continuous infusion of the blank electrolyte solution for about 60 min. In all experiments, serotonin exposure lasted until maximum response was achieved. After each exposure, the perfusion pressure had to become stabilised at a level close to that observed initially before the next dose was administered. The response was measured as the pressure change recorded when the response had reached a maximum. In the dose – response studies, the difference between the basal perfusion pressure and that observed at maximum response is presented as a percentage of the basal unstimulated level before each drug administration. In the experimental CsA studies the results are presented as absolute values (mm Hg) before serotonin exposure and at maximum response. 2.4. Ethics The study was approved by the Regional Ethics Committee and informed consent was obtained from the patients. 2.5. Drugs Serotonin creatinine sulfate was purchased from Sigma (St. Louis, MO) and dissolved directly into the electrolyte solution. Ciclosporine A was purchased from Sandoz Pharma (Basel, Switzerland). The drug was dissolved in polyoxyethylated castor oil and ethanol, giving final perfusate concentrations of 13 mg/l and about 0.01 vol.%, respectively. Such an ethanol concentration has been shown not to influence perfusion pressure (unpublished observations). 2.6. Statistics Analysis of variance (ANOVA) was used to analyse the dose –response curves, and Fisher’s exact test to analyse the frequency of biphasic pressure responses. Student’s t-test for paired and unpaired samples were used for statistical evaluation of the serotonin
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responses in the studies with experimental CsA administration. A p value < 0.05 was considered significant. The values are expressed as mean values F SEM.
3. Results 3.1. Dose –response curves Serotonin induced a significant dose-dependent constrictory response (Fig. 1) in both groups ( p < 0.0001), which was non-significantly lower in the organ-transplanted group. At the doses 10 8 and 10 7 M, all responses in the CsA-treated group were below the mean values in the control group, and at the doses 10 6 and 10 5 M, six out of seven
Fig. 1. The response to serotonin 10 and end of drug infusion.
7
M showing a monophasic vasoconstriction. The arrows indicate the start
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Fig. 2. Dose – response curves for the constrictory response following serotonin infusion. The difference between the prestimulatory perfusion pressure and that observed at maximum response is presented as a percentage of the prestimulatory perfusion pressure (mean values F SEM). The transplanted group and the control group are presented by closed and open circles, respectively.
preparations showed a response below the mean values in the control group (Fig. 2). A dilatatory response preceding the constrictory response was observed in three of the seven preparations in the organ-transplanted group and 12 of the 17 control preparations (nonsignificant). 3.2. Experimental ciclosporine A exposure In the experiments with CsA administration, the maximum pressure response of serotonin was significantly increased ( p < 0.05) following CsA treatment as compared to
Table 2 The effect of ciclosporine A or blank electrolyte solution (control group) on the response to serotonin 10 the umbilical artery
Prestimulatory pressure Constrictory response
7
M in
Ciclosporine A group (n = 7)
Control group (n = 7)
Before treatment (mm Hg)
After treatment (mm Hg)
Before treatment (mm Hg)
After treatment (mm Hg)
70.1 F 8.9 152.9 F 9.8
75.0 F 12.2 201.9 F 14.7a
70.8 F 4.8 128.0 F 18.1
67.7 F 3.7 145.6 F 18.3
The values are presented as the perfusion pressure (mm Hg) before serotonin exposure (prestimulatory pressure) and at maximally developed constrictory responses before and after treatment (mean values F SEM). a p < 0.05 as compared to before treatment.
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the values before treatment, whereas a non-significant increase was observed after infusion of the blank electrolyte solution (Table 2). This pressure increase was nonsignificantly larger in the CsA-treated preparations as compared to the control segments (48.9 F 17.7 vs. 17.6 F 10.4 mm Hg). The frequency of dilatatory responses preceding the vasoconstrictory serotonin response was not altered after infusion of CsA nor the blank electrolyte solution (five out of seven preparations in both groups both before and after infusion). In the separate experiments performed to investigate the effect of CsA on the basal perfusion pressure, only small pressure changes were observed with small non-significant pressure increases of 3.8 F 2.5, 3.3 F 1.9 and 3.3 F 1.3 mm Hg following CsA 0.1 and 1.0 mg/l and the blank electrolyte solution, respectively.
4. Discussion In the dose –response experiments, serotonin induced a constrictory response in all preparations. In three of the seven preparations from organ-transplanted women, this response was preceded by vasodilatation and the frequency of biphasic pressure responses was non-significantly smaller compared to that obtained in the control group. In the organtransplanted group, the size of the constrictory response to serotonin was reduced as compared to the control group. This difference did not reach a statistical significance due to a relatively large variation within each group. The results of the present dose – response studies should be interpreted with caution. First, the number of preparations is small. Second, in addition to CsA, the patients received other drugs. The patients received prednisolone and azathioprine and two of them received acetylsalicylic acid in addition to propranolol, furosemide or nifedipine. One patient received labetolol. Prednisolone and acetylsalicylic acid interfere with prostanoid production. This is probably of minor importance since the vasoactive response to serotonin in human umbilical arteries is not altered by indomethacin treatment [11]. The influence of the other drugs is difficult to evaluate, and an effect on the serotonin response cannot be excluded. The organ-transplanted women also represent a heterogeneous group with different background diseases. Four of them developed pre-eclampsia or hypertension during pregnancy. In an earlier study using the same perfusion model, however, preeclampsia or non-proteinuric hypertension did not show to induce any significant alterations in the serotonin response [8]. In the studies with experimental CsA administration, the constrictory serotonin response following CsA exposure was increased from that in the control group, i.e. the results of these experiments may indicate an influence of CsA opposite of that obtained in the experiments on preparations from CsA-treated women. The contrary results may be caused by different experimental designs. The experiments with CsA administration represent short time exposure of a large CsA dose, whereas the other experiments represent long time exposure of a lower dose. Thus, the results cannot be directly compared. In the experiments with CsA administration, only one serotonin concentration of 10 7 M was employed. At this concentration in the dose – response experiments, the constrictory serotonin response in all preparations from the organ-transplanted group was below the mean value observed in
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the control group. Thus, the interpretation of the results was not altered when only the responses at the serotonin concentration 10 7 M were considered. The mechanism of a possible interference of CsA on the present serotonin response has not been investigated. The drug passes the placental barrier and Venkataramanan et al. [12] observed an accumulation of CsA in umbilical cord tissue. The drug has been shown to induce a dose- and time-dependent cytotoxic effect on bovine endothelial cells in culture [13] and a reduction in prostacyclin production in cultured human umbilical vein endothelial cells [14]. However, the constrictory serotonin response in human umbilical arteries does not seem to be modulated by the endothelium and the endogenous prostanoid production [11,15]. CsA has been shown to stimulate the transmembrane Ca2 + influx in primary cultures of vascular smooth muscle cells [16], which may be involved in an altered serotonin responsiveness. However, most in vitro investigations have employed short time drug exposure and it cannot be excluded that the effect of CsA on vascular function might change during long time immunosuppressive treatment. In a study by Amorena et al. [17] the vehicle used for intravenous or oral CsA administration caused direct vascular effects. In the present series, the vehicle has not been tested separately and a possible effect of the vehicle both in the short- and long-time exposure experiments cannot be excluded. Amorena et al. [17] observed an influence of the vehicle on the synthesis of endothelium-derived relaxing factor, i.e. nitric oxide. However, nitric oxide does not seem to have an influence on the serotonin response in human umbilical arteries [15] except for a possible influence on the dilatatory response in the few preparations displaying a dominating dilatatory serotonin response as compared to the constrictory response [18]. None of the preparations in the present study showed such a response. In the present series, CsA did not have any influence on the basal perfusion pressure of human umbilical arteries. In other vessels from different species, the drug has been shown to cause an increase in smooth muscle vascular tension. In the femoral artery of the dog [19] and the rat aorta [20] this effect seems to be dependent on an increase in adrenergic activity. The lack of an influence of CsA on the basal perfusion pressure in the present study might thus be due to the absence of innervation and the scarcity of adrenergic receptors. Adrenalin and noradrenalin have only a minor effect on vascular tension in the human umbilical vessels [21]. In conclusion, CsA did not have a direct effect on vascular tension in human umbilical arteries and did not have any significant effects on the serotonin responsiveness as compared to a control group. Acknowledgement ˚ se Strutz for her technical assistance. The author wishes to acknowledge A References [1] Armenti VT, Ahlswede KM, Ahlswede BA, Jarrell BE, Moritz MJ, Burke JF. National transplantation pregnancy registry — outcomes of 154 pregnancies in cyclosporine-treated female kidney-transplant recipients. Transplantation 1994;57:502 – 6.
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[2] Sturgiss SN, Davison JM. Effect of pregnancy on long-term function of renal allografts. Am J Kidney Dis 1992;19:167 – 72. [3] Mihatsch MJ, Thiel G, Ryffel B. Cyclosporine A: action and side-effects. Toxicol Lett 1989;46:125 – 39. [4] Pickrell MD, Sawers R, Michael J. Pregnancy after renal transplantation: severe intra-uterine growth retardation during treatment with cyclosporin A. Br Med J 1988;296:825. [5] Haugen G, Fauchald P, Sødal G, Halvorsen S, Oldereid N, Moe N. Pregnancy outcome in renal allograft recipients: influence of cyclosporine A. Eur J Obstet Gynecol Reprod Biol 1991;39:25 – 9. [6] Armenti VT, Moritz MJ, Davison JM. Drug safety issues in pregnancy following transplantation and immunosuppression: effects and outcomes. Drug Saf 1998;19:219 – 32. [7] Reilly FD, Russell PT. Neurohistochemical evidence supporting an absence of adrenergic and cholinergic innervation in the human placenta and umbilical cord. Anat Rec 1977;188:277 – 86. [8] Haugen G. The vasoactive effects of serotonin in normal and single umbilical artery cords in normotensive and hypertensive pregnancies. Hypertens Pregnancy 1996;15:39 – 50. [9] Skjærven R, Gjessing HK, Bakketeig LS. Birthweight by gestational age in Norway. Acta Obstet Gynecol Scand 2000;79:440 – 9. [10] Bjøro K, Stray-Pedersen S. In vitro perfusion studies on human umbilical arteries: I. Vasoactive effects of serotonin, PGF2a and PGE2. Acta Obstet Gynecol Scand 1986;65:351 – 5. [11] Bjøro K, Stray-Pedersen S. Characterization of the responses to serotonin and prostanoids in human umbilical arteries perfused in vitro. Scand J Clin Lab Invest 1986;46:85 – 90. [12] Venkataramanan R, Koneru B, Wang CP, Burckart GJ, Caritis SN, Starzl TE. Cyclosporine and its metabolites in mother and baby. Transplantation 1988;46:468 – 9. [13] Zoja C, Furci L, Ghilardi F, Zilio P, Benigni A, Remuzzi G. Cyclosporine-induced endothelial cell injury. Lab Invest 1986;55:455 – 62. [14] Voss BL, Hamilton KK, Samara ENS, McKee PA. Cyclosporine suppression of endothelial prostacyclin generation. Transplantation 1988;45:793 – 6. [15] Haugen G, Hovig T. Studies of autacoid responsiveness and endothelium dependency in human umbilical arteries. Scand J Clin Lab Invest 1992;52:141 – 9. [16] Meyer-Lehnert H, Schrier RW. Potential mechanism of cyclosporine A-induced vascular smooth muscle contraction. Hypertension 1989;13:352 – 60. [17] Amorena C, Castro A, Mu¨ller A, Villamil MF. Direct vascular effects in the rat of the vehicles used for the intravenous and oral administration of cyclosporine A. Clin Sci 1990;79:149 – 54. [18] Haugen G, Mellembakken J, Stray-Pedersen S. Characterization of the vasodilatatory response to serotonin in human umbilical arteries perfused in vitro. The influence of the endothelium. Early Hum Dev 1997;47: 185 – 93. [19] Tronc F, Carrier M, Pelletier CL. Mechanism of hind limb vasoconstriction due to cyclosporine A in the dog. Circ Res 1992;71:1159 – 64. [20] Xue H, Bukoski RD, McCarron DA, Bennett WM. Induction of contraction in isolated rat aorta by cyclosporine. Transplantation 1987;43:715 – 8. [21] Altura BM, Malaviya D, Reich CF, Orkin LR. Effects of vasoactive agents on isolated human umbilical arteries and veins. Am J Physiol 1972;222:345 – 55.
The inf luence of ciclosporine A on the vasoactive effects of serotonin in in vitro perfused human umbilical arteries Guttorm Haugen * Department of Obstetrics and Gynecology, The National Hospital, University of Oslo, 0027 Oslo, Norway Received 28 June 2001; received in revised form 29 November 2001; accepted 5 December 2001
Abstract Background: Pregnancy is feasible in organ-transplanted women, but little is known about possible effects of ciclosporine A on the circulation in the fetus and placenta. Aim: To investigate the influence of ciclosporine A (CsA) on the vasoactive effects of serotonin in human umbilical arteries. Study design and subjects: In vitro perfusion was performed in umbilical cord segments from seven organ-transplanted patients on CsA based immunosuppression and in 17 cords from uncomplicated pregnancies. Serotonin was administered in stepwise increasing concentrations from 10 10 to 10 5 M. In preparations from normal pregnancies, serotonin 10 7 M, was administered before and 30 min after the start of a continuous CsA infusion (1.0 mg/l). The influence of CsA 0.1 or 1.0 mg/l on the basal, unstimulated perfusion pressure was investigated in separate experiments. Outcome measures: Changes in perfusion pressure due to constrictory or dilatatory responses. Results: In all preparations from the organ-transplanted patients, serotonin induced a constrictory response that was non-significantly lower than that observed in the control group. The frequency of a dilatatory response preceding the vasoconstriction was 3/7 and 12/17 (non-significant) in the CsA-treated and control groups, respectively. In the experiments with CsA administration, a non-significant increase in the constrictory serotonin response was observed as compared to the control experiments. CsA did not alter the basal, unstimulated perfusion pressure. Conclusion: CsA did not have any significant influence on the vasoactive effect of serotonin in human umbilical arteries perfused in vitro. D 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ciclosporine A; Perfusion; Serotonin; Umbilical artery
* University Department of Obstetrics and Gynecology, Level F. Princess Anne Hospital, Southampton SO 16 5YA, UK. Tel.: +44-8079-8421; fax: +44-8078-6933. E-mail address: [email protected] (G. Haugen).
0378-3782/02/$ - see front matter D 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 2 5 5 - 9
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G. Haugen / Early Human Development 67 (2002) 69–77
1. Introduction Modern immunosuppressive drug regimens based on ciclosporine A (CsA) have greatly improved the life expectancy and given a favourable quality to the life of organtransplanted patients. For fertile women, a successful pregnancy might be a reality [1], and at least for kidney recipients, pregnancy does not seem to be deleterious to graft function nor long-term morbidity [2]. CsA is nephrotoxic and has been shown to induce haemodynamic alterations in the kidney [3]. As concerns the fetus, earlier reports indicated an increased frequency of growth retardation in pregnancies of kidney recipients on CsA based immunosuppressive regimen [4,5], but this has not been confirmed in later reports and review articles [6]. This does not exclude a possible effect of CsA on haemodynamic regulatory mechanisms in the fetus nor in the umbilicoplacental circulation. The human umbilical vessels lack innervation [7]. The regulation of the vascular tension is thus dependent on local autocrine mechanisms and vasoactive substances present in the blood stream, e.g. serotonin. In in vitro perfused umbilical arteries from uncomplicated term pregnancies, serotonin induces a dominating pressure increase usually preceded by a transient vasodilatation [8]. The present study was performed to investigate the influence of CsA on the effect of serotonin in such preparations. The study includes seven umbilical cords from pregnancies of organ-transplanted patients on CsA based immunosuppressive regimen.
2. Material and methods 2.1. Patients 2.1.1. Dose –response curves The transplanted group comprised three patients with kidney allografts, one patient with a liver allograft, one patient with combined kidney and liver allografts, and two hearttransplanted patients (Table 1). They all had single pregnancies 1 1/2 – 11 years following transplantation. As immunosuppressive drug regimen, they received azathioprine 25– 50 mg/day and/or prednisolone 5– 12.5 mg/day, and CsA 150 –400 mg/day. As additional drugs, one of the kidney-transplanted patients (pat. no. 1) received propranolol 20 mg/day and acetylsalicylic acid 75 mg/day. The patient with combined kidney and liver allografts (pat. no. 2) received furosemide 80 mg/day and acetylsalicylic acid 160 mg/day. Before pregnancy, both patients were normotensive with serum creatinine values about 140 Amol/ l. They remained normotensive throughout pregnancy, but serum creatinine values increased to above 200 Amol/l, and they were both delivered by Cesarean section due to deteriorating kidney function. Patient no. 6 received labetolol 100 mg/day due to nonproteinuric hypertension. Three patients (pat. no. 4, 5 and 7) developed moderate preeclampsia (hypertension and proteinuria 0.6– 1 g per 24 h). Due to increasing blood pressure (200/120 mm Hg), patient no. 5 received nifedipine 20 mg during delivery. Mean gestational age was 37 weeks + 4 days and mean birth weight 2924 g. As shown in Table 1, the length of gestation was heterogeneous with both pre-term and post-term deliveries.
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Table 1 Clinical data for the transplanted patients No.
Organ transplanted
Primary disease
CsA dose (mg/day)
Gestational age (weeks + days)
Blood pressure (mm Hg)
Birth weight (g)
1 2 3 4 5 6 7
kidney kidney/liver heart heart kidney liver kidney
glomerulonefritis primary hyperoxaluria dilated cardiomyopathy dilated cardiomyopathy glomerulonefritis unknown etiology glomerulonefritis
300 400 220 300 200 150 300
35 + 4 34 + 0 39 + 0 37 + 0 38 + 3 37 + 2 42 + 1
130/80 130/70 135/85 130/90 150/100 150/110 140/90
2470 2035 3790 3290 2900 2460 3520
CsA = ciclosporine A.
None of the infants were growth retarded [9] and they all had 5 min Apgar scores of 9 or above. The control group consisted of 17 women with uncomplicated term pregnancies. Mean gestational age at delivery was 39 weeks + 5 days (range 37 weeks + 5 days to 42 weeks + 0 days), and mean birth weight was 3641 g (range 2640 –4350 g). None of the infants were growth retarded, and they all had 5 min Apgar scores of 8 or above. 2.1.2. Experimental ciclosporine A exposure The umbilical cords were sampled from women with uncomplicated term pregnancies. 2.2. Perfusion studies The umbilical cords were collected immediately after delivery and immersed in Ringer’s solution at + 4 jC. A segment with length of about 10 cm was cut from the median part of the cord. In each segment, one artery was cannulated at both ends. The preparation was then mounted in a perfusion chamber and connected to a peristaltic pump delivering at constant flow. The vessels were perfused in the antegrade direction. Following an equilibration period of about 30 –40 min, the flow was adjusted to 15 –25 ml/min. A perfusion pressure of about 60– 70 mm Hg was achieved at this flow rate. The perfusion was carried out in a recirculating fashion until the start of drug infusion after which a non-recirculating perfusion was employed. As perfusate and cord immersion fluid was employed, an electrolyte solution with the following composition (in mM): NaCl 125, KCl 4, CaCl2 2, MgCl2 1, NaHCO3 22, NaH2PO4 0.3, Na2HPO4 1.7 and glucose 5.6. Both solutions were bubbled with a gas mixture of 5% O2, 5% CO2 and 90% N2, keeping a constant pH of about 7.40. For a more detailed description of the perfusion procedure, see Ref. [10]. 2.3. Drug administration In all experiments, serotonin was administered at the input side of the preparation through a side-infusion with non-pulsatile constant flow of about 1% of the total flow rate.
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In the dose – response studies, all preparations were first tested with serotonin 10 7 M. Thereafter, serotonin was administered at stepwise increasing concentrations from 10 10 to 10 5 M, quoted as final concentrations in the perfusate. In the studies with experimental CsA exposure, three to seven repeated serotonin infusions at a final concentration of 10 7 M were performed until three subsequent responses with almost equal pressure amplitudes were established. CsA at a final concentration of 1 mg/l (n = 7) or a blank electrolyte solution (control group, n = 7) was administered as a separate side-infusion at the input side. This side-infusion had a flow rate of about 1% of the total flow rate. After 30 min of continuous administration, three repeated serotonin infusions (10 7 M) were performed. The mean value of the latter pressure responses were compared to the mean of the three last serotonin responses before administration of CsA or the blank electrolyte solution. A possible effect of CsA on the basal perfusion pressure was investigated in separate experiments on preparations with proven serotonin sensitivity (n = 7). A continuous infusion of CsA at a final concentration of 0.1 mg/l and thereafter at a final concentration of 1 mg/l for about 60 min each was performed. This was preceded or succeeded by a continuous infusion of the blank electrolyte solution for about 60 min. In all experiments, serotonin exposure lasted until maximum response was achieved. After each exposure, the perfusion pressure had to become stabilised at a level close to that observed initially before the next dose was administered. The response was measured as the pressure change recorded when the response had reached a maximum. In the dose – response studies, the difference between the basal perfusion pressure and that observed at maximum response is presented as a percentage of the basal unstimulated level before each drug administration. In the experimental CsA studies the results are presented as absolute values (mm Hg) before serotonin exposure and at maximum response. 2.4. Ethics The study was approved by the Regional Ethics Committee and informed consent was obtained from the patients. 2.5. Drugs Serotonin creatinine sulfate was purchased from Sigma (St. Louis, MO) and dissolved directly into the electrolyte solution. Ciclosporine A was purchased from Sandoz Pharma (Basel, Switzerland). The drug was dissolved in polyoxyethylated castor oil and ethanol, giving final perfusate concentrations of 13 mg/l and about 0.01 vol.%, respectively. Such an ethanol concentration has been shown not to influence perfusion pressure (unpublished observations). 2.6. Statistics Analysis of variance (ANOVA) was used to analyse the dose –response curves, and Fisher’s exact test to analyse the frequency of biphasic pressure responses. Student’s t-test for paired and unpaired samples were used for statistical evaluation of the serotonin
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responses in the studies with experimental CsA administration. A p value < 0.05 was considered significant. The values are expressed as mean values F SEM.
3. Results 3.1. Dose –response curves Serotonin induced a significant dose-dependent constrictory response (Fig. 1) in both groups ( p < 0.0001), which was non-significantly lower in the organ-transplanted group. At the doses 10 8 and 10 7 M, all responses in the CsA-treated group were below the mean values in the control group, and at the doses 10 6 and 10 5 M, six out of seven
Fig. 1. The response to serotonin 10 and end of drug infusion.
7
M showing a monophasic vasoconstriction. The arrows indicate the start
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Fig. 2. Dose – response curves for the constrictory response following serotonin infusion. The difference between the prestimulatory perfusion pressure and that observed at maximum response is presented as a percentage of the prestimulatory perfusion pressure (mean values F SEM). The transplanted group and the control group are presented by closed and open circles, respectively.
preparations showed a response below the mean values in the control group (Fig. 2). A dilatatory response preceding the constrictory response was observed in three of the seven preparations in the organ-transplanted group and 12 of the 17 control preparations (nonsignificant). 3.2. Experimental ciclosporine A exposure In the experiments with CsA administration, the maximum pressure response of serotonin was significantly increased ( p < 0.05) following CsA treatment as compared to
Table 2 The effect of ciclosporine A or blank electrolyte solution (control group) on the response to serotonin 10 the umbilical artery
Prestimulatory pressure Constrictory response
7
M in
Ciclosporine A group (n = 7)
Control group (n = 7)
Before treatment (mm Hg)
After treatment (mm Hg)
Before treatment (mm Hg)
After treatment (mm Hg)
70.1 F 8.9 152.9 F 9.8
75.0 F 12.2 201.9 F 14.7a
70.8 F 4.8 128.0 F 18.1
67.7 F 3.7 145.6 F 18.3
The values are presented as the perfusion pressure (mm Hg) before serotonin exposure (prestimulatory pressure) and at maximally developed constrictory responses before and after treatment (mean values F SEM). a p < 0.05 as compared to before treatment.
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the values before treatment, whereas a non-significant increase was observed after infusion of the blank electrolyte solution (Table 2). This pressure increase was nonsignificantly larger in the CsA-treated preparations as compared to the control segments (48.9 F 17.7 vs. 17.6 F 10.4 mm Hg). The frequency of dilatatory responses preceding the vasoconstrictory serotonin response was not altered after infusion of CsA nor the blank electrolyte solution (five out of seven preparations in both groups both before and after infusion). In the separate experiments performed to investigate the effect of CsA on the basal perfusion pressure, only small pressure changes were observed with small non-significant pressure increases of 3.8 F 2.5, 3.3 F 1.9 and 3.3 F 1.3 mm Hg following CsA 0.1 and 1.0 mg/l and the blank electrolyte solution, respectively.
4. Discussion In the dose –response experiments, serotonin induced a constrictory response in all preparations. In three of the seven preparations from organ-transplanted women, this response was preceded by vasodilatation and the frequency of biphasic pressure responses was non-significantly smaller compared to that obtained in the control group. In the organtransplanted group, the size of the constrictory response to serotonin was reduced as compared to the control group. This difference did not reach a statistical significance due to a relatively large variation within each group. The results of the present dose – response studies should be interpreted with caution. First, the number of preparations is small. Second, in addition to CsA, the patients received other drugs. The patients received prednisolone and azathioprine and two of them received acetylsalicylic acid in addition to propranolol, furosemide or nifedipine. One patient received labetolol. Prednisolone and acetylsalicylic acid interfere with prostanoid production. This is probably of minor importance since the vasoactive response to serotonin in human umbilical arteries is not altered by indomethacin treatment [11]. The influence of the other drugs is difficult to evaluate, and an effect on the serotonin response cannot be excluded. The organ-transplanted women also represent a heterogeneous group with different background diseases. Four of them developed pre-eclampsia or hypertension during pregnancy. In an earlier study using the same perfusion model, however, preeclampsia or non-proteinuric hypertension did not show to induce any significant alterations in the serotonin response [8]. In the studies with experimental CsA administration, the constrictory serotonin response following CsA exposure was increased from that in the control group, i.e. the results of these experiments may indicate an influence of CsA opposite of that obtained in the experiments on preparations from CsA-treated women. The contrary results may be caused by different experimental designs. The experiments with CsA administration represent short time exposure of a large CsA dose, whereas the other experiments represent long time exposure of a lower dose. Thus, the results cannot be directly compared. In the experiments with CsA administration, only one serotonin concentration of 10 7 M was employed. At this concentration in the dose – response experiments, the constrictory serotonin response in all preparations from the organ-transplanted group was below the mean value observed in
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the control group. Thus, the interpretation of the results was not altered when only the responses at the serotonin concentration 10 7 M were considered. The mechanism of a possible interference of CsA on the present serotonin response has not been investigated. The drug passes the placental barrier and Venkataramanan et al. [12] observed an accumulation of CsA in umbilical cord tissue. The drug has been shown to induce a dose- and time-dependent cytotoxic effect on bovine endothelial cells in culture [13] and a reduction in prostacyclin production in cultured human umbilical vein endothelial cells [14]. However, the constrictory serotonin response in human umbilical arteries does not seem to be modulated by the endothelium and the endogenous prostanoid production [11,15]. CsA has been shown to stimulate the transmembrane Ca2 + influx in primary cultures of vascular smooth muscle cells [16], which may be involved in an altered serotonin responsiveness. However, most in vitro investigations have employed short time drug exposure and it cannot be excluded that the effect of CsA on vascular function might change during long time immunosuppressive treatment. In a study by Amorena et al. [17] the vehicle used for intravenous or oral CsA administration caused direct vascular effects. In the present series, the vehicle has not been tested separately and a possible effect of the vehicle both in the short- and long-time exposure experiments cannot be excluded. Amorena et al. [17] observed an influence of the vehicle on the synthesis of endothelium-derived relaxing factor, i.e. nitric oxide. However, nitric oxide does not seem to have an influence on the serotonin response in human umbilical arteries [15] except for a possible influence on the dilatatory response in the few preparations displaying a dominating dilatatory serotonin response as compared to the constrictory response [18]. None of the preparations in the present study showed such a response. In the present series, CsA did not have any influence on the basal perfusion pressure of human umbilical arteries. In other vessels from different species, the drug has been shown to cause an increase in smooth muscle vascular tension. In the femoral artery of the dog [19] and the rat aorta [20] this effect seems to be dependent on an increase in adrenergic activity. The lack of an influence of CsA on the basal perfusion pressure in the present study might thus be due to the absence of innervation and the scarcity of adrenergic receptors. Adrenalin and noradrenalin have only a minor effect on vascular tension in the human umbilical vessels [21]. In conclusion, CsA did not have a direct effect on vascular tension in human umbilical arteries and did not have any significant effects on the serotonin responsiveness as compared to a control group. Acknowledgement ˚ se Strutz for her technical assistance. The author wishes to acknowledge A References [1] Armenti VT, Ahlswede KM, Ahlswede BA, Jarrell BE, Moritz MJ, Burke JF. National transplantation pregnancy registry — outcomes of 154 pregnancies in cyclosporine-treated female kidney-transplant recipients. Transplantation 1994;57:502 – 6.
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