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Ann Thorac Surg 1998;66:1372-1377
© 1998 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Increased pulmonary blood flow produces endothelial cell dysfunction in neonatal swine

^a Pediatric Cardiothoracic Surgery, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
^b Department of Pathology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Address reprint requests to Dr Gaynor, Pediatric Cardiothoracic Surgery, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104
e-mail: (gaynor{at}email.chop.edu)

Presented at the Poster Session of the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.

	Abstract

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Background. The mechanisms by which increased pulmonary blood flow results in pulmonary hypertension have not been determined.

Methods. To determine if increased pulmonary blood flow produces endothelial dysfunction that precedes vascular remodeling and smooth muscle proliferation, neonatal swine (n = 12) (age, 6.1 ± 0.5 days) underwent ligation of the left pulmonary artery (LPA) to increase blood flow to the right lung. At 12 weeks of age, endothelium-dependent vasodilatation was assessed by acetylcholine infusion and endothelium-independent vasodilatation by inhaled nitric oxide (NO) in the LPA group and age-matched controls (CON) (n = 11).

Results. Mean pulmonary artery pressure was 24.1 ± 3.0 mm Hg in the LPA group and 20.8 ± 1.9 mm Hg in the CON group (p < 0.1). Pulmonary vascular resistance was 13.2 ± 2.2 Wood units in the LPA group and 5.8 ± 0.8 Wood units in the CON group (p = 0.001). Acute occlusion of the left pulmonary artery in the CON group increased pulmonary vascular resistance to 6.9 ± 3.9 Wood units (p = 0.04). Administration of acetylcholine in the CON group after preconstriction with the thromboxane A₂ analogue U46619 resulted in a 30.6% ± 5.4% decrease in pulmonary vascular resistance. In the LPA group, acetylcholine produced paradoxical vasoconstriction and a 15.4% ± 4.1% increase in pulmonary vascular resistance (p < 0.001 versus CON) indicating loss of endothelium-dependent vasodilatation. Nitric oxide decreased pulmonary vascular resistance by 41.9% ± 3.3% in the CON group and 30.8% ± 2.7% in the LPA group (p = 0.04 versus CON), indicating preserved endothelium-independent vasodilatation in both groups. Morphometric analysis was performed in 4 animals from each group. Medial wall thickness as percent of external diameter of small arteries (<100 µm) was the same in both groups (6.4% ± 0.4% in the LPA group versus 6.6% ± 0.4% in the CON animals; p > 0.1).

Conclusions. Increased pulmonary blood flow in immature animals produces endothelial cell dysfunction with loss of endothelium-dependent vasodilatation before the onset of pulmonary vascular remodeling. Subsequent smooth muscle proliferation may be mediated by endothelium-derived factors.

	Introduction

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Many forms of congenital heart disease result in left-to-right shunts with increased pulmonary blood flow [1, 2]. If surgical correction is not performed at an early age, increased pulmonary blood flow may lead to irreversible pulmonary vascular disease [1]. Even when repair is performed in infancy, children with increased pulmonary blood flow may be at increased risk for postoperative pulmonary hypertensive crises [2]. Progressive pulmonary hypertension occurs in some children despite successful early repair. There is no satisfactory medical therapy for pulmonary hypertension. Lung transplantation provides excellent palliation; however, the long-term results have been disappointing.

The cellular and molecular mechanisms underlying pulmonary vascular remodeling in response to the mechanical stimulus of increased flow have not been fully delineated. The hemodynamic consequences of increased pulmonary blood flow are more severe in immature than mature animals [3–5]. Alterations in endothelial cell morphology are among the earliest findings in children with increased pulmonary blood flow, and appear as early as 2 months of age [1]. Endothelial cells are elongated and thickened with evidence of cytoskeletal remodeling and increased microfilaments. Smooth muscle cells shift from a contractile phenotype to a synthetic phenotype, leading to an increased muscularization and increased extracellular matrix protein synthesis. There is increasing evidence that the vascular endothelium functions as a signal transducer for hemodynamic forces and modulates the vascular response to both acute and chronic alterations in flow [6, 7].

The current study was undertaken to test the hypothesis that increased blood flow in the developing lung produces endothelial dysfunction before the onset of significant vascular remodeling. A neonatal swine model of increased pulmonary blood flow secondary to ligation of the left pulmonary artery (LPA) was used [8, 9]. Endothelial function was assessed by the response to endothelial-dependent and -independent vasodilators. Smooth muscle proliferation and vascular remodeling were assessed by morphometric analysis.

	Material and methods

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All animals received humane care in compliance with the "Principles of Laboratory Animal Care" by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" by the National Academy of Sciences and National Institutes of Health (NIH publication 85-23, 1985).

Left pulmonary artery ligation
Neonatal swine (LPA, n = 12) underwent LPA ligation at a mean age of 6.1 ± 0.1 days and mean weight of 2.2 ± 0.2 kg. Anesthesia was induced with intramuscular ketamine (20 mg/kg) and acepromazine (1 mg/kg). An intravenous catheter was inserted in an ear vein. Anesthesia was maintained with fentanyl (100 µg/kg bolus and 50 to 100 µg/h continuous infusion) and pancuronium (0.3 mg/kg). Under sterile conditions a left thoracotomy was performed. The pericardium was opened and the LPA was doubly ligated at its origin. The ductus arteriosus was occluded with a hemostatic clip. The thoracotomy was repaired in layers and the animal was allowed to recover. Three additional animals underwent a sham operation in which the LPA was mobilized but not ligated.

Assessment of endothelial function
At 12 weeks of age, each animal underwent hemodynamic study and assessment of endothelial function along with age-matched controls that did not undergo operation (CON, n = 11). Anesthesia was induced with intramuscular ketamine (20 mg/kg) and acepromazine (1 mg/kg). An intravenous catheter was inserted in an ear vein. The animals were intubated mechanically ventilated. Anesthesia was maintained with fentanyl (100 µg/kg bolus and 50 to 100 µg · kg^-1 · h^-1 continuous infusion) and pancuronium (0.3 mg/kg). Ventilator settings were adjusted to maintain the arterial pH at 7.35 to 7.45, the carbon dioxide tension at 35 to 45 mm Hg, and the oxygen tension between 100 and 250 mm Hg.

A femoral arterial line was placed for blood gas sampling. A median sternotomy was performed. The pericardium was opened and an ultrasonic flow probe was placed on the main pulmonary artery (Transonic Systems, Inc, Ithaca, NY). Micromanometers (3F; Millar Instruments Inc, Houston, TX) were placed in the pulmonary artery and left atrium. An infusion catheter was placed in the pulmonary artery for drug administration. The animals were allowed to stabilize for 30 minutes before data acquisition.

Baseline data were collected including pulmonary artery and left atrial pressures, pulmonary artery flow (cardiac output), and heart rate. Pressure and flow data were collected at 200 Hz for 10 seconds with the ventilator placed at 3 mm Hg continuous positive pressure. After baseline data acquisition, the LPA was acutely occluded in the CON and sham-operated animals and data acquisition was repeated. Subsequent assessment of endothelial function in these animals was performed with the LPA occluded. Pulmonary vascular resistance was calculated using the following formula: .

Receptor-mediated endothelial-dependent vasodilatation was assessed by the response to a continuous acetylcholine (ACH) infusion at 12.5 µg · kg^-1 · min^-1 into the pulmonary artery. Data were acquired before and 5 minutes after the ACH infusion was begun. Endothelial-independent vasodilatation was assessed by the response to inhaled nitric oxide (NO). Data were collected immediately before and 5 minutes after institution of inhaled NO at 20 parts per million (ppm) to the ventilator circuit. The NO concentration was monitored with a Chemiluminescence Nitric Oxide Analyzer (model 42H; Thermo Environmental Instruments, Franklin, MA). Nitrogen dioxide concentrations were also monitored and did not exceed 1 ppm. In the LPA ligation animals, the response to NO was tested before ACH in 6 cases and after ACH in 6 cases. In the control animals, 6 animals received NO before ACH and 5 received NO after ACH. The order in which the drugs were administered did not alter the response. Preliminary studies revealed elevated baseline PVR in the LPA ligation animals; therefore, endothelial-dependent and -independent vasodilatation were assessed without additional preconstriction. To determine if preconstriction altered the response to ACH and NO, endothelial function was also assessed in a subgroup of LPA animals after preconstriction with the thromboxane A₂ analogue U46619. Baseline PVR was lower in the control animals; therefore, endothelial-dependent and -independent vasodilatation were assessed only after preconstriction. U46619 was diluted in saline solution to a concentration of 2 µg/mL and infused through the pulmonary artery catheter. The dose was titrated to achieve a PVR between 9 and 16 WU, as preliminary experiments had demonstrated similar values in the LPA ligation animals.

Tissue preparation and morphometric analysis
After data acquisition, the animals were euthanized with a 40 mEq intravenous bolus of potassium chloride while under deep anesthesia. The main pulmonary artery and LPA distal to the ligature were cannulated and the pulmonary vessels injected with Barium-Micropaque (Picker Corp, Trevose, PA) at a pressure of 100 cm H₂O. Injection was continued until flow ceased. The pulmonary artery was ligated and the heart and lungs were removed from the chest. The trachea was cannulated and the lungs were fixed transtracheally with 10% buffered formalin at a pressure of 20 cm H₂O. Injection continued until the lungs appeared completely expanded and flow ceased. The pulmonary artery and trachea were ligated, and the lungs were removed and fixed in formalin for at least 24 hours.

At least one section from each lobe was taken to include hilum and distal pleura. Sections were processed and embedded in paraffin. Five-micrometer histologic sections were cut and stained with hematoxylin and eosin, trichromic, and elastin stains. Histologic sections were examined with an eyepiece graticle. Small arteries (less than 100 µm in diameter) were examined in these sections. The external diameter (distance from outer elastic lamina to outer elastic lamina) and medial thickness (distance between inner and outer elastic lamina) were measured. Muscularization was assessed by calculating medial wall thickness as a percent of the external diameter.

Statistical analysis
Hemodynamic data and morphometric data are presented as mean ± standard error of the mean. Endothelial-dependent and -independent vasodilatation are quantitated as the percent change in PVR in response to ACH and NO, respectively. For each intervention, the Wilcoxon signed-rank test was used to compare within groups and the Wilcoxon rank-sum test was used to compare data between groups. A p value less than 0.05 was considered significant.

	Results

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After LPA ligation, 12 animals underwent hemodynamic study and evaluation of endothelial function at 12 weeks of age (mean weight, 12.4 ± 0.8 kg). Eleven age-matched controls that did not undergo operation were evaluated (mean weight, 27.6 ± 1.4 kg; p < 0.001). The animals that underwent sham operation weighed 30.5 ± 2.6 kg at the time of study. Mean baseline pulmonary artery pressure was 24.1 ± 3.0 mm Hg in the LPA group and 20.8 ± 1.9 mm Hg in the CON group (p > 0.05) (Table 1). Cardiac output was 1.7 ± 0.1 L/min in the LPA animals and 3.11 ± 0.3 L/min in the CON animals (p < 0.001). Pulmonary vascular resistance was 13.2 ± 2.2 WU in the LPA animals and 5.8 ± 0.8 WU in the CON animals (p = 0.001) (Fig 1). Acute occlusion of the LPA in the CON animals resulted in an increase in pulmonary artery pressure to 23.4 ± 2.4 mm Hg (p = 0.05) and an increase in PVR to 6.9 ± 1.2 WU (p = 0.04) (see Table 1). The baseline pulmonary artery pressure in the animals that underwent sham operation was 19.2 ± 2.6 mm Hg and the baseline PVR was 6.0 ± 1.5 WU.

View larger version (12K): [in this window] [in a new window]   Fig 1. (A) Baseline pulmonary vascular resistance (PVR) in left pulmonary artery (LPA) and control animals and after acute LPA occlusion in the control (CON) animals. (B) Changes in PVR in response to inhaled nitric oxide (NO). (C) Changes in PVR after acetylcholine (ACH) infusion. Note paradoxical vasoconstriction in animals with increased pulmonary blood flow secondary to LPA ligation (WU = Wood units.)

View larger version (95K): [in this window] [in a new window]   Fig 2. Photomicrographs of equally distended intraacinar arteries from control (A) and left pulmonary artery ligation animals (B). The wall thickness of the two arteries are not different. The lumen of each is filled with barium/Hypaque. (x400 before 51% reduction.)

	Comment

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Celermajer and colleagues [11] evaluated endothelial function in children with congenital heart disease and increased pulmonary artery blood flow. In normal children, flow velocity increased in response to both ACH and sodium nitroprusside, demonstrating normal endothelial-dependent and -independent vasorelaxation. In children with increased pulmonary blood flow, the response to ACH was diminished while the response to sodium nitroprusside was maintained, consistent with impaired endothelial-dependent vasorelaxation. In children with established pulmonary vascular disease, the responses to both ACH and sodium nitroprusside were diminished. These findings are consistent with the findings of our study and suggest that the early response to increased pulmonary blood flow is impaired endothelial-dependent vasodilatation, which may be important in the pathogenesis of pulmonary vascular disease.

The response of vessels in different vascular beds to alterations in blood flow may be very different. Kamiya and Togawa [12] evaluated the effects of increased blood flow in systemic arteries by creating an arteriovenous fistula between the carotid artery and jugular vein in dogs. Acutely, shear stress was significantly increased. Chronically, the vessel dilated to normalize shear stress, suggesting an autoregulatory mechanism. Miller and associates [13] created femoral arteriovenous fistulas in dogs and assessed endothelial-dependent vasodilatation in isolated arterial rings. Relaxation in response to ACH was enhanced in arteries exposed to chronically increased flow. NaDaud and colleagues [14] demonstrated increased expression of NO synthase in systemic arteries exposed to increased flow. Tronc and colleagues [15] evaluated the role of NO in flow-induced remodeling of the carotid artery secondary to an arteriovenous fistula model. Blockade of NO production attenuated the adaptive increase in vessel diameter.

The response of the pulmonary vasculature to increases in blood flow appears to be very different. Fullerton and associates [16] created femoral arteriovenous shunts in mature dogs to increase pulmonary blood flow. Endothelial-dependent and -independent vasodilatation were studied in isolated pulmonary artery rings. There was progressive endothelial dysfunction with significantly decreased vasorelaxation in response to ACH. In addition, there was a decreased response to endothelial-independent vasodilatation. In the current study, there was paradoxical vasoconstriction in response to ACH, suggesting that endothelium dysfunction in response to increased flow may be more severe in immature animals. The response to inhaled NO was slightly diminished compared with controls. Paradoxical vasoconstriction to ACH has been reported in patients with primary pulmonary hypertension [17, 18]. Vasoconstriction in response to ACH may be mediated by muscarine receptors on the vascular smooth muscle.

Significant pulmonary vascular development continues after birth [19]. The effects of increased blood flow are more severe in immature animals than in mature animals. Acute occlusion of a pulmonary artery in a mature animal does not cause pulmonary hypertension in the contralateral lung but rather a fall in PVR with maintenance of cardiac output [4]. However, in immature animals occlusion of a pulmonary artery results in a significant increase in pressure in the contralateral lung. Aortopulmonary shunts result in more severe pulmonary hypertension with increased muscularization of arteries in immature swine (4 weeks of age) as compared with more mature animals [5]. The effects of LPA ligation in neonatal pigs have been characterized by Haworth and others [8, 9]. Ligation of the LPA produces only modest increases in flow to the right lung. In this model, mild pulmonary hypertension and increased muscularization are present by 16 to 20 weeks of age as compared with age-matched controls. Animals in the current study underwent hemodynamic evaluation at 12 weeks of age to allow assessment of endothelial function before the onset of smooth muscle proliferation.

The use of an intact animal model has advantages and disadvantages. The use of an intact animal model mimics the clinical situation more closely than in vitro techniques; however, all of the potential variables cannot be tightly controlled. In particular, the LPA animals did not grow as well as the control animals; therefore, there is a discrepancy in body weight and cardiac output, which results in a higher nonindexed PVR in the LPA animals. It is interesting to note that the animals that underwent sham operation were similar in weight to the controls, which did not undergo any operation. There was no evidence of respiratory distress, infection, or other compromise in the experimental animals; therefore, the poor growth is likely secondary to the experimental intervention. Compensatory growth of the right lung and pulmonary artery occurs in response to the increased flow and thus the relationship between the PVR and body weight or surface area may not be the same as in control animals. Use of indexed PVR, however, would not alter the directional change in PVR in response to either ACH or NO. Because the hemodynamic measurements were performed in an intact animal model, the findings represent the sum of changes in all vascular segments. Thus, it is not possible to determine the level of the vasculature responsible for the hemodynamic changes.

In summary, we have examined the effects of increased pulmonary blood flow on endothelial function in the developing lung in vivo. This study demonstrates that increased blood flow produces endothelial dysfunction with loss of endothelial dependent vasodilatation. These findings are consistent with previous study of the effects of increased pulmonary blood flow in mature animals and suggest that pulmonary arteries adapt differently than systemic arteries to increased blood flow. Endothelial dysfunction occurs before the onset of significant pulmonary hypertension or histologic evidence of smooth muscle dysfunction. Endothelial-derived factors may be important mediators of vessel remodeling in response to alterations of flow.

	Acknowledgments

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Supported in part by a grant from the Mary L. Smith Charitable Trust (04269-06-5) and by a grant from the Tommy Martin Memorial Fund.

	References

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