Ann Thorac Surg 1998;66:500-505
© 1998 The Society of Thoracic Surgeons
Original articles: cardiovascular
Adrenomedullin in patients at high risk for pulmonary hypertension
Palaniswamy Vijay, PhDa,
Laszlo Szekely, MDa,
Thomas G. Sharp, MDa,
Andrew Miller, BPha,
Ko Bando, MDa,
John W. Brown, MDa
a Section of Cardiothoracic Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA
Address reprint requests to Dr Vijay, Department of Surgery, Indiana University School of Medicine, 545 Barnhill Dr, EH 215, Indianapolis, IN 46202-5125
e-mail: (pvijay{at}iupui.edu)
Presented at the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.
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Abstract
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Background. Adrenomedullin is a newly identified peptide with profound hypotensive effects. We investigated perioperative adrenomedullin levels among patients with congenital heart disease with and without pulmonary hypertension.
Methods. Levels of plasma adrenomedullin, endothelin-1, and nitric oxide metabolites were measured in three groups: (1) low pulmonary flow (n = 11); (2) high flow/low pulmonary arterial pressure (less than 60% systemic pressure) (n = 9); and (3) high flow/high pressure (n = 10). Samples were obtained preoperatively, on and off pump, and 3, 6, and 12 hours after bypass.
Results. Adrenomedullin levels were highest in the low pulmonary flow group (189.7 ± 15 pg/mL low flow versus 103.1 ± 9.5 pg/mL high flow/low pulmonary and 139 ± 17.5 pg/mL high flow/high pressure at 12 hours; p 
0.05). The arterial pressure/systemic pressure re-mained significantly lower in the high flow/low pulmonary pressure compared with the high flow/high pressure group (0.37 ± 0.08 versus 0.62 ± 0.11; p < 0.005). Perioperative endothelin-1 and nitric oxide levels remained low in the low pulmonary flow group but increased progressively in both high flow groups.
Conclusions. Circulating plasma adrenomedullin appears to affect baseline vascular tone in patients with intact endothelial function. It may interact with nitric oxide and endothelin-1 to help regulate blood pressure perioperatively in patients with congenital heart disease.
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Introduction
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Development of pulmonary hypertension (PH) attributable to increased pulmonary vascular resistance is a major concern in children with congenital heart defects who undergo operation using cardiopulmonary bypass (CPB). Postoperative PH has led to a significant number of deaths after corrective procedures [1].
Increased pulmonary vascular resistance, which is responsible for PH, has been documented as the result of damage to vascular endothelial cells during CPB [2, 3]. An intact endothelial cell lining is essential for the secretion of the majority of vasoactive mediators that regulate vascular tone. Vasoconstrictors such as the endothelins, endoperoxides, and superoxide anions, and vasodilators like nitric oxide (NO) and prostacyclins are released from intact endothelial cells to modulate the responsiveness of the underlying smooth muscle cells and thereby alter vascular tone.
Recently, adrenomedullin (ADM), a peptide of molecular mass of 6,047 Da, was isolated from human pheochromocytoma cells and shown to have a relatively rapid onset of action and long-lasting hypotensive effect. Human ADM consists of 52 amino acids, one disulfide cysteine-to-cysteine bond that forms a six-member ring structure, and a carboxy-terminal amide group [4]. Adrenomedullin has 27% homology to calcitonin gene-related protein, suggesting that ADM may belong to the calcitonin gene-related protein superfamily. Plasma ADM levels have been found to be elevated in patients with hypertension and renal disease [5]. It is also present in high concentrations in the right atrium of the heart, where it reaches a 5- to 50-fold excess over the concentrations found in the left atrium and the ventricles, respectively [6]. Adrenomedullin exerts its vasodilatory activity in a number of vascular beds including the lungs, suggesting that it may play an important role in modulating PH [7]. In the present study, we investigated the time course of plasma endothelin–1 (ET-1), nitric oxide (measured as nitrites [NO2] and nitrates [NO3]), and ADM and examined their influence in the development of postoperative PH in patients undergoing open heart operations using CPB with and without high preoperative pulmonary pressures.
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Material and methods
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Thirty patients with congenital heart disease who underwent surgical repair using CPB at Riley Hospital for Children in Indianapolis, Indiana, were included in this study. On the basis of preoperative pulmonary flow and the systolic pulmonary to systemic pressure ratio (Pp/Ps), patients were divided into three groups: low pulmonary flow (LF; n = 11), high flow but low pulmonary arterial pressure (less than 60% systemic pressure) (HF-LP; n = 9), and high flow and high pressure (HF-HP; n = 10). The patient demographics are given in Tables 1 and 2 . There were no significant differences between the groups of patients except for their ages and weight. The protocol was approved by the Indiana University School of Medicine Institutional Review Board.
Study protocol
Preoperative evaluation was performed primarily by echocardiography. Increased pulmonary flow was defined as a preoperative pulmonary/systemic flow (Qp/Qs) more than 1.5 by catheterization or by a combination of at least two of the following clinical findings: (1) cardiomegaly on roentgenogram, (2) increased pulmonary vascularity on roentgenogram, and (3) dilated right heart on echocardiography. Elevated pulmonary arterial pressure was defined as (1) pulmonary pressure more than 60% of systemic by catheterization, (2) tricuspid regurgitant velocity more than 3 m/s, (3) pulmonary regurgitant end-diastolic velocity more than 1.5 m/s, or (4) nonrestrictive ventricular septal defect with a velocity across pulmonary valve of more than 3.5 m/s by echocardiography. Our protocols for operative management, intraoperative and postoperative monitoring and strategy for prevention of postoperative PH have been published previously [8]. A 4-mL blood sample was collected in 7.5 mmol/L ethylenediaminetetraacetic acid and 500 KU aprotinin from the radial artery to measure ADM, ET-1, and NO metabolites as NO2 and NO3 at six time points: preoperatively, on CPB, off CPB, and 3, 6, and 12 hours after CPB.
Biochemical assays
Plasma ADM level was measured by a radioimmunoassay kit (Peninsula Laboratories, Inc, Belmont, CA). This assay is based on the competition of labeled iodine-125 ADM and unlabeled ADM binding to a limited quantity of specific antibodies. The amount of ADM is given as picograms per milliliters.
Plasma ET-1 level was measured by a two-site immunoassay (Amersham Life Science, Arlington Heights, IL). The ET-1 was extracted by column chromatography and incubated with an antiendothelin antibody overnight at 4°C. Bound ET-1 was detected using a peroxidase conjugated anti-ET-1 Fab' fragment. The resultant substrate was measured at 450 nm and compared to a standard curve of known amounts of synthetic ET-1 expressed as picograms per milliliters.
Nitric oxide was measured with a commercially available kit (Boehringer Mannheim, Indianapolis, IN). Nitrate present in the sample was reduced to nitrite by the reduced form of nicotinamide-adenine dinucleotide phosphate and nitrate reductase. The red-violet diazo dye formed by the addition of sulfanilamide, N-1-naphthlyl-ethylenediamine dihydrochloride was measured at 550 nm, and NO metabolites were calculated from a calibration curve ranging from 0.8 to 80 mmol/L. Nitric oxide level was expressed as millimoles per liter of NO metabolites.
Statistical analysis
Data were analyzed with a commercially available statistical program (Statistica for Windows, Statsoft, Tulsa, OK). Values were expressed as mean ± standard deviation. All assays were performed in duplicate. Differences between levels of ADM, ET-1, and NO metabolites from baseline values at different time points were analyzed by paired t tests. Between-group comparisons were performed using a two-tailed t test for independent samples. A p value of less than 5% was considered statistically significant.
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Results
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Plasma adrenomedullin levels
The perioperative changes in plasma ADM levels are given in Figure 1. Plasma ADM levels were measured for up to 12 hours after CPB. The ADM levels were significantly higher in LF groups compared to HF groups (p = 0.0001). Upon initiating CPB ADM levels in the LF group declined from their preoperative value of 227.6 ± 21.5 pg/mL to 87.8 ± 5.0 pg/mL, a level similar to that seen in both of the HF groups. The levels of ADM gradually increased during the first 12 hours postoperatively in all three groups with 12-hour levels of 190 ± 15 pg/mL in LF, 103 ± 10 pg/mL in HF-LP, and 139 ± 18 pg/mL in the HF-HP group (p 0.05).
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Fig 1. The dynamic change of arterial plasma adrenomedullin (ADM) concentration at different times during perioperative period. (CPB = cardiopulmonary bypass; HF-HP = high flow, high pressure; HF-LP = high flow, low pressure; LF = low flow; +p = 0.001 versus preoperatively; **p = 0.0001 versus preoperatively.)
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Changes in plasma endothelin-1 levels
The ET-1 levels in plasma measured from preoperative to 12 hours after bypass are given in Figure 2. There was a steady increase in ET-1 level in all three groups, with the highest release in the HF-HP patients (5.0 ± 0.5 pg/mL to 16.5 ± 1.9 pg/mL) and a peak value 6 hours after bypass (17.5 ± 1.9 pg/mL, HF-HP). The ET-1 levels were significantly higher at 3 hours after CPB in all groups when compared to baseline levels (p = 0.0001).
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Fig 2. The dynamic change of arterial plasma endothelin-1 (ET-1) concentration at different times during perioperative period. (CPB = cardiopulmonary bypass; HF-HP = high flow, high pressure; HF-LP = high flow, low pressure; LF = low flow; **p = 0.0001 versus preoperatively.)
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Plasma nitric oxide levels
The NO levels, measured as their metabolites, NO2 and NO3, are given in Figure 3. Preoperative NO levels were not different between any of the groups examined. The level of NO released into the blood increased over time, with higher levels seen in the two high flow groups (HF-LP and HF-HP) when compared to the LF group (p = 0.0001). Treatment with exogenous NO donors (nitroglycerin and nitroprusside, Table 3), did not influence the blood NO levels seen in the study.
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Fig 3. The dynamic change of arterial plasma nitric oxide (NO2/NO3) concentration at different times during perioperative period. (CPB = cardiopulmonary bypass; HF-HP = high flow, high pressure; HF-LP = high flow, low pressure; LF = low flow; **p = 0.0001 versus preoperatively.)
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Perioperative changes in blood pressure and flow in the pulmonary and systemic circulations
Blood pressures and pulmonary flow ratios measured before and after the operation are given in Table 4. The preoperative pulmonary to systemic flow ratios were 1.4 ± 0.3, 3.3 ± 1.3, and 2.7 ± 0.7 in the LF, HF-LP, and HF-HP groups, respectively. The pulmonary to systemic flow ratios in both of the high flow groups were significantly different from those of the LF group. The preoperative pulmonary to systemic pressure ratio was higher for the HP group (0.8 ± 0.1) when compared with either the LF or the HF-LP group (0.4 ± 0.2 and 0.4 ± 0.1). The postoperative pulmonary to systemic pressure ratio remained higher in the HP group than in either of the other two groups.
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Comment
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Adrenomedullin is a recently identified peptide that was initially isolated from adrenal medulla but has been found in other organs such as the heart, lungs, and kidneys [9]. It is synthesized and secreted by the endothelial cells and smooth muscle cells of the pulmonary vasculature [10–12]. It has a potent vasodilatory effect in the vascular beds of various organs such as the heart, brain, and kidneys [13, 14]. It has been shown that patients with essential hypertension and renal failure have elevated levels of ADM [15]. The plasma level of ADM would be expected to be the sum of synthesis and secretion from the various compartments in which it originates and its degradation in serum. In this study, we have demonstrated that preoperative plasma ADM levels are lower in patients who are at higher risk for the development of postoperative PH. Conversely, higher levels of ADM are seen in those patients who are at lowest risk for PH. This suggests that impaired ability to synthesize or secrete ADM in the pulmonary circulation may contribute to the risk of the development of pulmonary hypertensive events in pediatric patients undergoing open heart operations.
The use of CPB during operative procedures on the heart reduces blood flow through the lung to minimal levels, which may impede flow-dependent secretion of ADM from pulmonary endothelial cells while the contact of blood with the foreign surface of the circuit triggers the release of various cytokines such as interleukins 1 and 8, TNF-
, and INF-
. These cytokines have been shown to stimulate the release of ADM [16, 17]. Because the amount of ADM released by various compartments would be expected to depend on the structural and functional integrity of their endothelial cells, vascular beds with preexisting damage may be unable to respond appropriately to the stimulation of CPB. In those conditions in which the pulmonary vasculature is exposed to chronic high flow states, as seen in HF-LP and HF-HP patients in our study, the result may be a decrease in the ability of this vascular bed to synthesize or release ADM. Those patients who presented for operation with relatively low pulmonary flow states, may have suffered little or no damage to endothelial cells and thus maintained their ability to synthesize and secrete higher levels of ADM.
The high perioperative plasma levels of ET-1 in HF-LP and HF-HP patients indicates that CPB stimulates a potent risk factor for the development of postoperative PH. Levels of ET-1 peaked at around 6 hours after bypass and had begun to decline by the 12-hour time point. Importantly the postoperative elevation of ET-1 was considerably higher in those patients known to be at highest risk for PH (HF-HP) as compared with LF and HF-LP patients, who have been shown to have a low perioperative pulmonary hypertensive event rate [8, 18].
Levels of NO continued to increase throughout the 12 hours of this study, a finding that is consistent with the hypothesis that NO is a major counterbalancing factor when ET-1 is released. The lack of a significant difference in NO levels at 12 hours between the two HF groups in spite of higher ET-1 release in the HF-HP group, suggests that limitations in the ability to synthesize or secrete NO could contribute to the risk of PH. Our findings in this study suggest that impaired endothelial cell function as manifested by lower release of ADM may exacerbate the imbalance between vasoconstriction and vasodilation in patients with PH.
The late increase in ADM seen in patients in the HF-HP group could be the result of improvement in pulmonary vascular endothelial cell function with a resultant increase in the ability to synthesize and secrete ADM. However, we have also demonstrated a correlation between ADM release in the coronary circulation and myocardial damage as assayed by troponin release from myocytes [19, 20]. Patients with longer, more complex operative repairs may experience minor degrees of myocardial damage that in turn stimulates the release of ADM from the coronary vasculature. Release of ADM from this source may be the explanation for the late increase in plasma levels seen in this study.
Circulating ADM can stimulate NO synthesis and release from endothelial cells, smooth muscle cells, and myocytes. The proportional increases seen in NO in the groups are much higher than the proportional increases in ADM when compared with baseline levels. Because ADM is a more potent vasodilator than NO, a relatively smaller amount of ADM may be sufficient to counteract the vasoconstriction caused by ET-1. Previously, we analyzed the release of ADM in adult patients with coronary artery disease undergoing CPB. In these patients, one would not expect to find damaged pulmonary endothelium from chronic high flow states, yet ADM increased immediately after bypass and remained elevated throughout the first 12 hours postoperatively. Endothelin-1 and NO levels also progressively increased over this time frame. These patients did not have significant or persistent postoperative hypertension [21]. This suggests a role for ADM in helping to regulate pulmonary artery pressure in the presence of elevated ET-1.
The mechanism of interaction between NO and ADM in regulating vascular tone in the presence of a potent vasoconstrictive stimulus is not known. Adrenomedullin released from endothelial cells may have endocrine effects throughout the systemic and pulmonary circulations. It may also have local or paracrine effects on the neighboring smooth muscle cells. Adrenomedullin secreted into the interstitium by endothelial cells may activate Gs-proteins present in smooth muscle cells and thereby increase adenylate cyclase activity. The resultant decrease in calcium sensitivity will result in vasorelaxation. In addition, locally released ADM can, in an autocrine fashion, cause the release of NO from endothelial cells through an indirect pathway using inositol 1,4,5-triphosphate as a second messenger [22].
Our findings are consistent with the hypothesis that ET-1 plays a role as a major culprit in the development of PH postoperatively in pediatric patients who undergo heart operations using CPB. The effects of ET-1 appear to be counteracted by the release of several vasodilatory mediators. Nitric oxide levels increase and remain elevated for at least 6 hours after operation, whereas ADM levels increase more gradually throughout the first 12 hours. This suggests that NO mechanisms are generally more able to respond acutely and that significant increases in ADM release may require the induction of additional synthetic mechanisms. We speculate that preexisting injury to pulmonary endothelial vasculature in different pulmonary flow states may influence ADM release, although our study does not directly evaluate the extent of endothelial injury. The balance between levels of vasoconstrictors and vasodilators at various time points may determine the propensity to develop pulmonary hypertensive events.
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Abstract
Introduction
