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J Thorac Cardiovasc Surg 2001;122:518-523
© 2001 The American Association for Thoracic Surgery

Surgery for Congenital Heart Disease (CHD)

Angiogenic proteins in the lungs of children after cavopulmonary anastomosis

From The Division of Cardiac Surgery,^a Children's Hospital and Regional Medical Center and the Department of Surgery, the University of Washington School of Medicine, the Division of Cardiology,^b Children's Hospital and Regional Medical Center and the Department of Pediatrics, the University of Washington School of Medicine, and the Department of Pathology,^c Children's Hospital and Regional Medical Center, Seattle, Wash.

This work was supported by a grant from the Howard Hughes Medical Institute.

Received for publication Sept 12, 2000. Revisions requested Dec 15, 2000; revisions received Feb 15, 2001. Accepted for publication Feb 28, 2001. Address for reprints: Brian W. Duncan, MD, Cleveland Clinic Children's Hospital, Division of Pediatric and Congenital Heart Surgery, 9500 Euclid Ave/M-41, Cleveland, OH 44195 (E-mail: duncanb{at}cff.org)

Abstract

Objective: Pulmonary arteriovenous malformations may cause progressive cyanosis after cavopulmonary anastomosis and may develop as a result of abnormal angiogenesis. We used immunohistochemistry to determine whether angiogenic proteins are increased in the lungs of children after cavopulmonary anastomosis.
Methods: Lung specimens were obtained from 13 children after cavopulmonary anastomosis and from 6 control subjects. Specimens were stained with antibodies against vascular endothelial growth factor and its receptor (flk-1/KDR), basic fibroblast growth factor, -smooth muscle actin, CD31, collagen IV, fibronectin, and proliferating cell nuclear antigen. Staining was graded on a scale of 0 to 3. Vessels positive for proliferating cell nuclear antigen were counted in 10 fields per specimen, and the results were averaged.
Results: After cavopulmonary anastomosis, patients demonstrated increased staining for vascular endothelial growth factor (P = .03) and its receptor (P = .03) and decreased staining for CD31 (P = .004). Proliferating cell nuclear antigen staining in patients was equivalent to that for control subjects (P = .9).
Conclusions: Lung biopsy specimens from children after cavopulmonary anastomosis demonstrate increased expression of vascular endothelial growth factor and its receptor. These data confirm earlier findings that blood vessels forming after cavopulmonary anastomosis may have reduced intercellular junctions (decreased CD31 staining). Despite the increased numbers of pulmonary vessels that are present in these patients, these vessels are not highly proliferative (proliferating cell nuclear antigen staining equivalent to that of control subjects). These results suggest that vascular endothelial growth factor may be a mediator of angiogenesis in the lungs of children after cavopulmonary anastomosis; however, other factors, such as vascular dilation and remodeling, may also be important.

Pulmonary arteriovenous malformations (PAVMs) are a frequent cause of progressive cyanosis in children after cavopulmonary anastomosis. The development of PAVMs may represent a form of abnormal angiogenesis that is under hepatic control. ¹ We have previously reported a detailed histologic description of PAVMs in 2 children after cavopulmonary anastomosis. ² Peripheral lung biopsy specimens from these children demonstrated numerous dilated vessels, suggesting that PAVMs are angiogenically active lesions. In addition, we have shown that children demonstrate increased numbers of pulmonary blood vessels early after cavopulmonary anastomosis, even without clinical evidence of PAVMs. ³ This suggests that after cavopulmonary anastomosis, there is a constant angiogenic stimulus that may ultimately lead to the development of clinically apparent PAVMs.

Angiogenesis involves the interaction of many proteins, such as growth factors (vascular endothelial growth factor [VEGF] and basic fibroblast growth factor [bFGF]), extracellular matrix proteins, and tissue proteases. We assessed the expression of several proteins known to be important in angiogenesis in lung biopsy specimens from children after cavopulmonary anastomosis by using immunohistochemistry to determine whether these proteins are upregulated.

Methods

Patients
After obtaining consent, we obtained lung biopsy specimens at the time of the Fontan procedure from 13 children who had previously undergone a cavopulmonary anastomosis. Four of these children had documented PAVMs on angiography performed before the Fontan procedure. Lung biopsy specimens were obtained from the lingula or the right middle lobe through the sternotomy incision with full lung inflation and immediately fixed. Control lung specimens from 4 children without congenital heart disease or significant lung disease were obtained from archived specimens as described, ³ and 2 control specimens were obtained as lung biopsy specimens from children with acyanotic congenital heart disease at the time of operative repair (Ross procedure; aortic coarctation repair). Two archived control specimens were lung biopsy specimens from a child with an acute viral illness and a child with systemic lupus erythematosis. The other 2 archived control specimens were autopsy specimens from a child who died of acute viral myocarditis and a child who died of rabies encephalitis. All archived control specimens were obtained from patients who had a duration of illness of less than 1 week before the time the specimens were obtained. The study was approved by the Institutional Review Board of Children's Hospital and Regional Medical Center (Seattle, Wash).

Histology and immunohistochemistry
All surgical and autopsy specimens were fixed in formalin, embedded in paraffin, and processed as described. ² In brief, 5-µm sections were deparaffinized in xylene and rehydrated in graded alcohols. Primary antibodies were diluted in 1% bovine serum albumin (BSA) in phosphate-buffered saline solution (PBS) and applied to sections. Antibodies used included a monoclonal antibody against VEGF (Santa Cruz Biotechnology, Santa Cruz, Calif) diluted 1:200, a polyclonal antibody against VEGF receptor (flk-1/KDR; kindly provided by Dr Rolf Brekken, Division of Vascular Biology, Hope Heart Institute, Seattle, Wash) diluted 1:10, a monoclonal antibody against bFGF (Oncogene, Cambridge, Mass) diluted 1:250, a monoclonal antibody against fibronectin (Chemicon, Temecula, Calif) diluted 1:250, a monoclonal antibody against -smooth muscle actin diluted 1:250, a monoclonal antibody against CD31 diluted 1:50, a monoclonal antibody against collagen IV diluted 1:300, and a monoclonal antibody against proliferating cell nuclear antigen (PCNA) diluted 1:50 (all from DAKO Corporation, Carpinteria, Calif). Before the addition of primary antibody against bFGF, flk-1/KDR, fibronectin, collagen IV, and CD31, sections were predigested with 0.01% protease or trypsin for 10 minutes. Biotinylated horse anti-mouse or goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, Calif) diluted 1:500 in 1% BSA/PBS were then applied, and sections were incubated for 30 minutes. Avidin-biotin enzyme complex (Vector Laboratories) was added, and slides were incubated for 30 minutes at room temperature. The sections were developed with diaminobenzidine tetrahydrochloride (Sigma Laboratories, St Louis, Mo) and counterstained in methyl green. Appropriate positive controls were used, and negative controls consisting of sections incubated in 1% BSA/PBS in place of the primary antibody were examined to ensure that staining was specific.

Specimen grading and statistical analysis
All sections were graded by 3 investigators using a scale of 0 to 3. A score of 0 qualified as no staining, 1 as minimal staining, 2 as moderate staining, and 3 as intense staining. Staining scores were then compared between patients who had undergone cavopulmonary anastomosis and control subjects with the Fisher exact test. For sections stained with PCNA, positively stained vessels were counted in 10 high-power fields (HPFs; 250x) by 2 investigators, and these results were averaged for each patient and control subject. The average of the mean number of vessels per HPF from each individual was then determined for the following groups: asymptomatic patients after cavopulmonary anastomosis; patients with clinically evident PAVMs after cavopulmonary anastomosis; and control subjects. The averages of the means for all patients after cavopulmonary anastomosis were then compared with those of control subjects by the Student t test. The averages of the means for each of these 3 groups were also compared by 1-way analysis of variance. All statistical analyses were performed with SAS software version 6.12 (SAS Institute, Cary, NC).

Results

The demographics for the 13 children who had undergone cavopulmonary anastomosis are summarized inTable 1. The median age for patients after cavopulmonary anastomosis was 4.1 years (range, 1.8-12.0 years), and the median age was 4.6 years (range, 0.8-15.0 years) for control subjects. Four of the 13 patients had documented PAVMs on angiography performed before the Fontan procedure; 3 of these patients were significantly hypoxemic as well. The other 9 patients had no angiographic or clinical evidence of PAVMs after cavopulmonary anastomosis. The control specimens showed only minor abnormalities on microscopic examination: 3 specimens were completely normal; 2 showed mild focal inflammation; and 1 showed mild focal fibrosis. Eight of the patients who underwent cavopulmonary anastomosis and 4 of the control subjects have had their pulmonary microvessel densities compared and reported, as determined by von Willebrand factor staining. ³

View larger version (14K): [in this window] [in a new window]   Fig. 1. Staining grade for VEGF, VEGF receptor (flk-1/KDR), CD31, bFGF, -smooth muscle actin, collagen IV, and fibronectin. Filled squares represent patients with clinically evident PAVMs and filled circles represent patients without clinically evident PAVMs after cavopulmonary anastomosis. Open circles represent control patients. Staining was graded on a scale of 0 (no staining) to 3 (intense staining).

View larger version (124K): [in this window] [in a new window]   Fig. 2. Representative staining for VEGF (A, D), VEGF receptor (flk-1/KDR) (B, E), and CD31 (C, F) in a patient after cavopulmonary anastomosis (A-C) and a control subject (D-F). (Arrowheads demonstrate blood vessels.)

Discussion

PAVMs may develop in the lungs of children after palliative cardiac operations, most often after a cavopulmonary anastomosis. These abnormal vascular lesions result in right-to-left shunting, which causes significant morbidity because of the development of progressive cyanosis. PAVMs developing after cavopulmonary anastomosis may represent a form of abnormal angiogenesis, although little is known about the mechanism of their formation or the specific factors involved. We have previously reported a detailed histologic analysis of these lesions in 2 of the patients included in the present study. ² Lung biopsy specimens from these patients with documented PAVMs after cavopulmonary anastomosis demonstrated large numbers of clustered microvessels (chains) and large, dilated, thin-walled vessels (lakes). These vessels demonstrated discontinuity of the basement membrane and endothelium by means of electron microscopy. Although providing the first detailed microscopic analysis of these lesions, this initial study did not provide much evidence as to their likely cause.

The present study provides the first report of the possible involvement of specific angiogenic factors in the cause of PAVMs that develop after cavopulmonary anastomosis. VEGF and bFGF are 2 potent mediators of angiogenesis that have been shown to be increased in tumors from numerous tissue types. ^4-6 In addition, VEGF and bFGF have been shown to be upregulated in nonneoplastic conditions associated with angiogenesis, such as proliferative retinopathy and psoriasis. ⁷ In the present study, after cavopulmonary anastomosis, patients demonstrated increased staining for VEGF but not bFGF. This pattern was fairly consistent, with all but one patient demonstrating increased VEGF expression after cavopulmonary anastomosis. We also demonstrated increased staining for the VEGF receptor flk-1/KDR in our patients. The VEGF receptor flk-1/KDR is a tyrosine kinase receptor that has been linked to the mitogenic response of endothelial cells to VEGF. ⁸ This receptor is downregulated in normal differentiated adult endothelium, but its expression is increased in angiogenic states. ⁹ The finding of increased flk-1/KDR expression in the lungs of children who have undergone cavopulmonary anastomosis also supports an increased angiogenic state in these patients, which may be mediated by VEGF.

The pattern of development of PAVMs in children after cavopulmonary anastomosis suggests that synthetic or metabolic functions of the liver may play a causative role. PAVMs form in the ipsilateral lung after a classic Glenn shunt ¹⁰ and bilaterally after a bidirectional Glenn shunt or Kawashima procedure. ¹¹ In each of these cases, PAVMs develop in the portion of the pulmonary circulation that is devoid of hepatic venous effluent. Performing a Fontan procedure reconstitutes hepatic venous drainage to the lungs, which results in regression of PAVMs. ¹² Further evidence for the role of the liver in this phenomenon comes from the observation that a similar clinical picture is seen in some patients with liver failure who have progressive cyanosis from intrapulmonary shunting. This phenomenon, termed the hepatopulmonary syndrome, closely resembles the angiographic and histologic picture of PAVMs occurring after cavopulmonary anastomosis and resolves after liver transplantation. ^13,14

The patterns of development of PAVMs seen in these various conditions combined with the data presented in the present study now allow us to develop a hypothesis regarding the cause of PAVMs that develop after cavopulmonary anastomosis. An inhibitor of angiogenesis that is normally present in hepatic venous effluent may be absent in the pulmonary arterial circulation after cavopulmonary anastomosis. Alternatively, a proangiogenic substance that is normally degraded by the liver may now be present in higher concentrations in the pulmonary circulation after cavopulmonary anastomosis. The result of either of these derangements would be the activation of VEGF-mediated angiogenesis in the lungs of these children. We have partially purified a novel inhibitor of angiogenesis from hepatocyte-conditioned media that would support the presence of a hepatic derived inhibitor of angiogenesis that normally holds pulmonary vascular proliferation in check. ¹ The present study suggests that upregulation of local expression of both VEGF and VEGF receptor is increased as a result of these mechanisms. As further evidence for this hypothesis, we have recently shown that there appears to be a constant angiogenic stimulus in the lungs of children after cavopulmonary anastomosis by demonstrating that a marker of angiogenesis (microvessel density) is increased in children early after cavopulmonary anastomosis, even in the absence of clinically evident PAVMs. ³

In the present study we also sought to confirm previous preliminary immunohistochemical analysis of lung biopsy specimens from children who have undergone cavopulmonary anastomosis. The pattern of CD31 staining that we observed here is the same as that found in our earlier study. ² CD31 is an adhesion molecule that is present at endothelial intercellular junctions. ¹⁵ We have consistently found decreased staining for this marker after cavopulmonary anastomosis compared with that seen in control subjects. This correlates with the finding of basement membrane and endothelial discontinuity on electron microscopy in children after PAVM. ² Increased numbers of vessels with diminished intercellular junctions might contribute to the clinical phenomenon of capillary leak and pleural effusions that arise as a result of elevated central venous pressure after the Fontan procedure. VEGF is also known to increase vascular permeability ¹⁶ and may be linked to the decrease in CD31 expression seen in our patients.

The present study also confirms our earlier observations that despite increased numbers of vessels in these patients, these vessels do not show evidence of intense proliferation, as determined with PCNA staining. PCNA is a nuclear protein that is associated with rapidly proliferating cells. ¹⁷ Most of the specimens in the present study did not demonstrate vessels with increased PCNA staining. One explanation for this finding is that the phase of intense cellular proliferation may have occurred earlier during the development of PAVMs because PCNA is known to be expressed early in proliferating tissue and only for a limited time. ¹⁷

There are limitations to this study. Importantly, the present study does not definitively rule out alternative causes for PAVM development. For example, there is evidence that dilatation of existing embryonal vascular channels in the lungs of these children may also contribute to the development of PAVM after cavopulmonary anastomosis. ¹⁸ The presence of mechanisms related to vascular remodeling and dilatation in this condition may help to explain the minimal PCNA staining that we have observed. Another potential causative factor for which the present study fails to control is the influence of hypoxia on the expression of angiogenic proteins in the lungs of these children. Hypoxia is a potent stimulus of angiogenesis and upregulates angiogenic factors, such as VEGF. ¹⁹ Interestingly, we have shown increased VEGF levels, determined by means of enzyme-linked immunosorbent assay, in the serum of children with cyanotic congenital heart disease compared with that seen in control subjects. ²⁰ Although these results were found in the serum of cyanotic patients, it is unknown whether chronic hypoxia may result in elevations of VEGF or its receptor in the tissues.

We believe that the absence of a hepatic derived inhibitor of angiogenesis in the pulmonary circulation will prove to be a major factor leading to PAVM formation; however, this process is complex and undoubtedly involves many components. mRNA levels for VEGF and VEGF receptor should be determined for lung biopsy specimens from these children to confirm the findings from the present study on the basis of immunohistochemical changes. In addition, protein expression and mRNA levels of mediators of vascular remodeling and dilatation will need to be examined.

Finally, the effect of chronic cyanosis on the tissue expression of angiogenic proteins could be assessed in the future by including analyses of lung biopsy specimens from hypoxic patients who have not undergone a cavopulmonary anastomosis. The importance of the development of an animal model for the ultimate understanding of this condition cannot be overstated and remains an ongoing objective of our laboratory investigation.

Surgery
VEGF and its receptor appear to be increased in lung biopsy specimens from children after cavopulmonary anastomosis; however, these vessels do not appear to be highly proliferative. This is the first reported example of increased expression of any angiogenic factor in patients after cavopulmonary anastomosis. However, PAVM development that occurs in these children is undoubtedly caused by multiple processes that may include angiogenesis, recruitment of existing embryonal channels, and vascular remodeling.

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