INTRODUCTION

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Cystic fibrosis (CF) is the most common life-shortening genetic disorder among white individuals, with an estimated frequency of 1:3,400 live births (1). Although a variety of therapeutic advances have allowed improved quality and length of life for those with CF, the disorder continues to be characterized by chronic pulmonary infection, inflammation, progressive bronchiectasis, and shortened life expectancy. CF pulmonary disease is characterized by robust neutrophilic inflammation that occurs early in life, prior to the onset of significant clinical manifestations (2, 3). This neutrophilic inflammation is believed to contribute significantly to progressive tissue changes in the CF lung. We have recently described increased angiogenesis in CF tissues and elevations in serum vascular endothelial growth factor (VEGF) in random serum samples from patients with CF (4). Because angiogenesis and chronic inflammation appear to be codependent in a variety of disorders (5), we hypothesize that angiogenesis contributes to progressive tissue changes in CF and in other disorders of pulmonary inflammation.

Angiogenesis, the formation of capillaries from existing blood vessels, is an important pathologic mechanism in progression of many disorders, including malignancies, rheumatoid arthritis, proliferative retinopathies, and cutaneous diseases (6). Angiogenesis is dependent on a balance between angiogenic and angiostatic mediators; inadequate angiogenesis is associated with fetal demise (7), whereas excessive tissue angiogenesis accelerates a number of pathologic processes (5). Several clinical characteristics of CF suggest the presence of excessive angiogenesis in this disorder. For example, 2.6% of patients with CF develop highly vascularized nasal polyps requiring surgical resection (8). Hemoptysis and pulmonary hemorrhage commonly occur, and digital clubbing and osteoarthropathy, both associated with neocapillarization, are frequent complications. There is also an increased incidence of digestive tract tumors in CF (9). Abnormal tissue angiogenesis may contribute to these processes.

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis and vascular permeability. VEGF is a heparin-binding factor that acts specifically on endothelial cells via membrane-spanning tyrosine kinase receptors; many cell types, including neutrophils and macrophages, secrete it (10). VEGF is induced by hypoxemia and tissue inflammation. Because CF lung disease is characterized by profound neutrophilic inflammation, we postulated that subjects with CF would have elevated serum VEGF levels, and that VEGF elevation would be greater in subjects with more severe pulmonary function abnormalities. To test this hypothesis, we measured VEGF levels in subjects with CF during periods of clinical stability. Because other diseases characterized by airway inflammation might also show elevations in serum VEGF, we studied subjects with other inflammatory lung disorders. To assess the contribution of airway infection to elevations in serum VEGF, we studied patients with CF and acute pulmonary exacerbation of CF lung disease before and after intravenous administration of antibiotics, anticipating a reduction in serum VEGF related to decreased neutrophilic inflammation after treatment.

	METHODS

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Serum VEGF levels were measured in subjects with CF over a range of age and lung disease severity. The diagnosis of CF was confirmed by characteristic clinical findings and sweat chloride concentrations > 60 mmol/L (11). The pulmonary disease group consisted of subjects with other acute and chronic pulmonary diseases with an inflammatory component such as asthma and bronchopulmonary dysplasia. Subjects were recruited when clinically stable during routine clinical encounters. Spirometry was performed on subjects who could perform reproducible spirometric maneuvers (12) using a SensorMedics Vmax 22 Spirometer (SensorMedics Inc., Yorba Linda, CA). Values were expressed as a percent of predicted for height, weight, race, and age (13, 14). Oxyhemoglobin saturation (Sp_O2) was measured by pulse oximetry (Nellcor N-100; Nellcor, Pleasanton, CA). A review of each subject's medication regimen and clinical history was undertaken to assess use of therapies that could modify VEGF levels, or complications that may be related to increased tissue angiogenesis. These included use of inhaled or oral steroids, which might alter tissue inflammation; presence or absence of Pseudomonas aeruginosa in respiratory tract culture, which may increase pulmonary inflammation; and nasal polyposis, which could be promoted via increased neocapillarization.

To assess the effect of antibiotic therapy on VEGF levels in CF, subjects were recruited at the time of a pulmonary exacerbation requiring intravenous antibiotic therapy. A pulmonary exacerbation was defined by an increase in cough and sputum production accompanied by a decline in FEV₁ of at least 10% from baseline and/or other clinical symptoms such as fever, weight loss, and fatigue (15). Serum VEGF was measured prior to or within 24 h of initiation, and again upon completion, of intravenous antibiotic therapy. Specific antibiotics used, and the duration of therapy, were based upon subject sputum culture and sensitivity results and severity of exacerbation, and were at the discretion of the treating physician; criteria for discontinuing antibiotic therapy included improvement to baseline FEV₁ (defined as best FEV₁ during the previous 12 mo) and improvement of cough, sputum production, and systemic symptoms. Subjects received a minimum of 10 d of intravenous antibiotic therapy.

Written informed consent was obtained from all participants, or from parents or legal guardians of minors; written assent was obtained from children 12 to 17 yr of age. The protocols were approved by the Institutional Review Board of the Children's Memorial Institute for Education and Research.

VEGF Assay

Blood was obtained by venipuncture and immediately centrifuged. Serum specimens were processed immediately or stored at 20° C. VEGF was measured using an Enzyme Immuno Assay (R&D Systems, Minneapolis, MN). A quantitative sandwich enzyme immunoassay was performed using VEGF-specific antibody bound to microtiter plates. Standards and serum samples were added to the wells and unbound material washed off. An enzyme-linked polyclonal antibody, specific to VEGF, was added, and unbound antibody was removed by washing the plate. Tetramethylbenzidine and hydrogen peroxide was added to the wells, developed, and read on a microplate spectrophotometer at 450-nm wavelength.

Statistical Methods

Student's t test was used to compare means of normally distributed data; the Mann-Whitney U test was used to compare means of data that were not normally distributed. Spearman's correlation was performed to assess correlation between VEGF and continuous variables. Stepwise forward multiple logistic regression was performed to assess correlation with categorical variables. A p value of < 0.05 was considered significant. Results are expressed as mean ± standard deviation.

	RESULTS

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VEGF Levels in Subjects with CF and Other Pulmonary Disorders

Thirty-eight subjects with CF 2 mo to 25 yr of age and 25 subjects 2 to 59 yr of age with other pulmonary disorders were recruited. Diagnoses in patients with other disorders included asthma (n = 16), bronchopulmonary dysplasia (n = 2), tracheoesophageal fistula with chronic bronchitis (n = 2), infectious pleural effusion (n = 3), scleroderma, and bronchiectasis (one each). Patient characteristics are summarized in Table 1. There was no significant difference in mean FEV₁ (p = 0.84) or Sp_O2 (p = 0.09) between groups.

The mean VEGF level was elevated in subjects with CF and in those with other lung disorders compared with published normal values (16, 17). Although there was a wide range of VEGF levels in both subject groups, levels were significantly higher in subjects with CF: 403 ± 280 versus 255 ± 169 pg/ml, p = 0.02. When patients receiving oral steroids were excluded from analysis, VEGF was not significantly changed: 389 ± 251 in patients with CF (n = 34) and 255 ± 184 in control subjects (n = 17). VEGF showed a significant negative correlation with FEV₁ in subjects with CF: r = 0.51, p = 0.007 (Figure 1). In a forward stepwise conditional logistic regression model, patients with CF and VEGF  300 pg/ml were compared with those with VEGF < 300 pg/ml. When FEV₁ was entered in the model, there was no association between higher VEGF levels and age (p = 0.4), Sp_O2 (p = 0.2), female sex (p = 0.6), use of oral (p = 0.6) or inhaled (p = 0.7) corticosteroids, or colonization with P. aeruginosa (p = 0.7). There was a trend towards correlation with nasal polyposis (p = 0.1). VEGF was not correlated with FEV₁, age, or Sp_O2 in control patients.

View larger version (10K): [in this window] [in a new window]   Figure 1.   VEGF versus FEV1 in cystic fibrosis. Each point represents a single subject with CF.

Effect of Pulmonary Exacerbation and Intravenous Antibiotic Therapy on VEGF Levels in CF

Ten subjects with CF (7 F) 9 to 23 yr of age (mean, 17.2 yr) were enrolled at the time of a pulmonary exacerbation. All patients had P. aeruginosa isolated from sputum culture; nine had mucoid strains of P. aeruginosa, and three had additional sputum isolates (S. aureus, S. maltophilia, and A. xylosisidans). Patients received an average of 14.7 d of intravenous antibiotic therapy (range, 10 to 25 d). The mean FEV₁ (n = 9) was 42 ± 18% predicted at the time of enrollment and 51 ± 22% predicted at completion of antibiotic therapy (p = 0.01, paired t test); two subjects with severe pulmonary disease had no significant improvement in FEV₁, but they had a significant improvement in respiratory symptoms and Sp_O2. Serum VEGF levels were elevated at the time of exacerbation and decreased significantly after intravenous antibiotic therapy: 537 ± 220 versus 259 ± 176 pg/ml, p = 0.001. A decrease in VEGF was noted in the two subjects who did not show improvement in FEV₁. Individual patient data are shown in Figure 2.

View larger version (17K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 2.   Effect of intravenous antibiotic therapy on VEGF and FEV1 in subjects with cystic fibrosis. Each unique symbol represents one subject. One subject (closed circles) was unable to perform reproducible spirometric maneuvers. (Top panel ) FEV1% pred before, and FEV1% pred after intravenous antibiotic therapy. (Bottom panel ) VEGF pretreatment-VEGF level before intravenous antibiotic therapy; VEGF post-treatment-VEGF level after intravenous antibiotic therapy.

	DISCUSSION

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These data demonstrate that serum VEGF levels are elevated in CF and other pulmonary inflammatory disorders. This VEGF elevation probably reflects inflammation, a characteristic present in the variety of disorders studied; higher levels in CF may reflect the severity and neutrophil-predominance of inflammation in this disorder. Profound neutrophilic inflammation, demonstrated by increased neutrophil counts, activity of free neutrophil elastase, and increased interleukin-8, is noted in bronchoalveolar lavage fluid from infants with CF (2). Circulating neutrophils release VEGF in response to a variety of stimuli; furthermore, neutrophils infiltrating inflamed tissues contain VEGF (7). Further studies that include subjects with chronic bronchitis and bronchiectasis would further clarify the relationship between infection, inflammation, and VEGF.

Tissue hypoxia may also play a role in VEGF elevations in lung disease; VEGF mRNA is elevated in hypoxic tumor cells and in nontumor cells grown in hypoxic conditions (18). The lack of association of VEGF with Sp_O2 in this study may be related to the small number of patients who had abnormal Sp_O2, or by the insensitivity of this measurement for detecting local tissue hypoxia.

Higher VEGF levels in CF are correlated with more severe pulmonary disease as measured by FEV₁. VEGF levels decrease by 50% after antibiotic therapy for acute exacerbation of CF lung disease, approaching those of subjects with other, non-CF lung diseases. It is interesting that VEGF levels decreased even in patients with severe lung disease who did not have an improvement in FEV₁ after intravenous antibiotic therapy. These findings suggest that VEGF elevation in CF is related to airway infection and that VEGF may be a sensitive surrogate marker of airway inflammation associated with airway infection.

Because VEGF is a potent inducer of angiogenesis, patients with CF may be at risk for development of organ-system abnormalities related to excessive angiogenesis. Angiogenesis is triggered when the production of inducers increases and/or the production of inhibitors decreases. It is of note that a mouse model lacking thrombospondin-1, a cytokine with angiostatic properties, has been noted to have morphologic disturbances that overlap with those in CF. These changes include pulmonary inflammation and, in the proximal airways, increased Clara and goblet cells and excessive mucinous secretions (19). Pathologic changes are also seen in the pancreas, with relative hypoplasia of the exocrine pancreas and interstitial matrix.

Angiogenesis appears to contribute to tissue inflammation and abnormal remodeling in a variety of disorders. Many inflammatory mediators promote angiogenesis either directly or indirectly. Interleukin-8, which is elevated in bronchoalveolar lavage fluid in subjects with CF (20), is associated with endothelial cell chemotaxis and is a potent inducer of angiogenesis by psoriatic keratinocytes (21). Other inflammatory mediators, including IL-6 and IL-1, increase VEGF mRNA levels. New blood vessels maintain chronic inflammation by facilitating the diapedesis of inflammatory cells to the site of inflammation; furthermore, because endothelial cells are a significant source of proinflammatory cytokines, increasing endothelial surface area may enhance the inflammatory process (5). Attenuating angiogenesis may reduce tissue inflammation and remodeling. For example, in an animal model of rheumatoid arthritis, collagen-induced arthritis, angiostatic agents prevent the onset of and suppress active disease (22).

VEGF-mediated angiogenesis may be an important pathologic mechanism in the progression of CF respiratory tract disease, CF-related complications, and persistence of inflammation in other pulmonary disorders. Serum VEGF appears to be a sensitive marker of pulmonary infection in CF. The relationship of elevated VEGF and other proinflammatory cytokines should be further elucidated. Additional studies are needed to elucidate the mechanisms that lead to elevated VEGF in CF and in other disorders, and the significance of this finding. If excessive angiogenesis plays a significant role in promoting inflammation in the lung, angiostatic agents may provide a new strategy in therapy.