Abstract
Rationale: Asthmatic airways have an increased number and size of vascular structures, which contribute to airflow obstruction and hyperresponsiveness.
Objectives: We examined whether proangiogenic mediators are elevated in bronchoalveolar lavage fluid (BALF) from subjects with asthma and if this translated to induction of angiogenesis.
Methods: Angiogenic activity in BALF from 12 healthy, nonatopic subjects and 10 atopic subjects with mild asthma was evaluated by examining tubule formation at 11 days in cocultures of human endothelial cells with dermal fibroblasts. Vascular structures were visualized by anti-CD31 labeling and quantified by image analysis. Angiogenic growth factors in BALF from healthy subjects and subjects with asthma were identified using antibody arrays and by ELISA.
Measurements and Main Results: Angiogenic activity induced by BALF from healthy subjects was not different from basal tubule formation (p > 0.05). However, induction of tubular structures by asthmatic BALF was 2.5-fold greater (p < 0.001) compared with healthy samples. Similarly, levels of proangiogenic growth factors (angiogenin, vascular endothelial growth factor [VEGF], monocyte chemotactic protein-1) were increased approximately 2.5-fold (p < 0.05) in BALF from subjects with asthma, whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. A blocking anti-VEGF antibody abolished tubule formation induced by BALF from either healthy subjects or subjects with asthma (p < 0.01). Immunodepletion of VEGF had no effect on basal tubule formation induced by healthy BALF but abrogated enhanced tubule formation by asthmatic BALF (p < 0.01).
Conclusions: BALF collected from subjects with asthma but not healthy subjects is functionally active in promoting angiogenesis in vitro. The proangiogenic capacity of BALF from subjects with asthma resides in elevated VEGF derived from asthmatic airways. This observation supports VEGF as a key factor in vascular remodeling in asthma.
The contribution of the airway microvascular bed to asthma phenotype has not been fully elucidated.
VEGF is the major component in bronchoalveolar lavage fluid from subjects with asthma promoting in vitro angiogenesis, underscoring its importance in asthma pathogenesis.
The contribution of the microvascular bed to airway remodeling in asthma is not fully elucidated. Capillary engorgement, leakage, and vasodilatation can directly increase airway wall thickness, increase airway luminal narrowing, facilitate inflammatory cell trafficking, and are believed to account for the decrease in forced expiratory flow rates seen in exercise-induced asthma (5), and after bronchial provocation challenge (6). Airway wall neovascularization, seen as increases in both the size and number of bronchial blood vessels, is a prominent feature of fatal and nonfatal asthma (7–9) that correlates with reticular basement membrane thickening (10) and airway obstruction (9, 11). Thus, subepithelial vascularity and angiogenesis appear to be important remodeling events in airway narrowing and airflow obstruction in asthma.
Angiogenesis, a complex process whereby blood vessels sprout from extant microvasculature, involves the coordination of multiple events, including degradation of the basement membrane by proteases, proliferation and migration of endothelial cells, lumen formation, basement membrane reassembling, recruitment of pericyte and/or vascular smooth muscle cells, vascular maturation, and, finally, blood flow (12). Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic factors; it stimulates endothelial cell migration and proliferation and is widely expressed in highly vascularized organs and tissues, including the lung (12, 13). Other polypeptide growth factors implicated in angiogenesis include angiogenin, platelet-derived growth factor (PDGF), and angiopoietin (Ang)-1. Angiogenin, first isolated from conditioned medium of colonic carcinoma cell cultures (14), is a potent tumor-derived angiogenic factor, but it also plays a role in several nonmalignant vasculoproliferative disorders, and, like VEGF, it induces vascular endothelial cell proliferation, migration, and tubule formation (15). Similarly, PDGF induces differentiation of mesenchymal cells toward pericytes and smooth muscle and stimulates migration of these cells to the newly formed vessels (16, 17). Ang-1 is a ligand for the endothelial cell–specific Tie2 surface receptor (18). It acts both in series with and subsequent to VEGF stimulation to promote sprouting and then maintains and stabilizes nascent vessel networks by promoting interactions between endothelial cells and surrounding support cells, including pericytes (18). Thus, VEGF and Ang-1 have been hypothesized to complement each other in angiogenic processes (18).
Although the mechanisms triggering the angiogenic switch in asthma are unknown, recent studies show an imbalance between protective antiangiogenic (endostatin) and proangiogenic factors, such as VEGF and its receptors (VEGFR1 and VEGFR2), in the airways of patients with asthma (9, 11, 19–22). Increasing evidence from animal studies supports a central role for VEGF in the pathogenesis of asthma (9, 23). Elevated levels of VEGF in airway biopsies, induced sputum, and bronchoalveolar lavage fluid (BALF) from patients with asthma correlate directly with increased total airway vascular area (9–11, 20–22) and disease severity (24), and are inversely correlated with airway caliber and airflow obstruction (9, 11). Similarly, Ang-1 and angiogenin levels are increased in asthma (9, 19, 22).
In the current study, we investigate the profile of multiple proangiogenic and antiantigenic proteins in BALF and demonstrate for the first time the proangiogenic capacity of BALF obtained from volunteers with asthma using an in vitro endothelial cell and dermal fibroblast coculture system. Furthermore, using multiple intervention strategies, we characterize the nature of the dominant proangiogenic activity present in these airway lining secretions. Some of the results of this study have been previously reported in the form of an abstract (25).
Twenty-two healthy, nonatopic volunteers and 16 glucocorticoid-naive, atopic volunteers with mild asthma were recruited. All subjects were nonsmokers and fulfilled American Thoracic Society criteria for asthma, including proven reactivity to skin allergen prick tests and a positive PC20 of less than 2 mg methacholine at screening, and were characterized using spirometry (26) and current symptom levels according to procedures approved by the research ethics committees of King's College Hospital (study no. 11–03–209) and Guy's and St. Thomas' Hospitals (study no. 05/Q0704/72). All subjects gave informed, written consent (see Table 1 for patient details). Subjects underwent bronchoscopy as described previously (26). See the online supplement for full details of patient recruitment and endobronchoscopy.
Bronchoalveolar lavage was performed by instilling 240 ml of warmed, sterile, sodium chloride into the right middle lobe, and which was immediately aspirated via the working channel of the bronchoscope. BALF was passed through a 100-μm filter and centrifuged (522 g × 7 min) to remove all cellular components. BALF was concentrated (×40) using a centrifugal filter (Amicon Ultra; Millipore Ltd, Watford, UK) with a 10-kD exclusion limit and sterilized by 0.22 μm centrifugal filtration (Amicon Ultra Free; Millipore Ltd). Aliquoted samples were stored at −80°C until use.
The proangiogenic capacity of BALF samples was investigated using an in vitro angiogenesis assay (27) comprising cocultures of human dermal fibroblasts and human umbilical vein endothelial cells (AngioKit; TCS CellWorks, Buckingham, UK). Individual BALF samples from healthy subjects or subjects with asthma were diluted in culture media supplied by the manufacturer and incubated (fourfold concentrated) in duplicate with cocultures on Day 1 and replaced on Days 4, 7, and 9. Cultures were incubated at 37°C in a humidified atmosphere with 5% CO2, and culture media alone and recombinant human (rh)VEGF-A (165 amino acid isoform; R&D Systems, Abingdon, UK) at 1.25 ng/ml served as negative and positive assay controls, respectively. After cell fixing on Day 11, vascular structures were visualized by labeling with mouse anti-CD31 according to the manufacturer's instructions (TCS CellWorks). Multiple photomicrographs (×4 objective) were taken, and angiogenesis in each field of view was quantified using image analysis (AngioSys; TCS CellWorks). The analysis software segmented the images using a gray level threshold tool to select CD31-labeled cells. Resultant binary images were skeletonized and branch points removed to determine the total length of individual tubules. Branch points were counted and the total area of CD31 labeling determined from the original binary image, permitting overall numbers of vascular junctions, tubules, and tubule length to be determined (27, 28).
In some experiments, a function blocking anti-human VEGF (2 μg/ml, clone 26503; R&D Systems) and the nonselective VEGF receptor antagonist suramin (100 μM; Sigma, Poole, UK) were preincubated with concentrated BALF and compared with samples either in the presence of an IgG2b isotype-matched antibody (R&D Systems) or without suramin, respectively. The effect of selective VEGF immunodepletion was also compared after overnight treatment of BALF samples with anti-VEGF (2 μg/ml; R&D Systems) and precipitation using sterile protein-A agarose beads (Upstate, Lake Placid, NY).
RayBio Human Angiogenesis Antibody Arrays (RayBiotech, Norcross, GA) were used to assay multiple proangiogenic factors in BALF from five healthy subjects and five subjects with asthma. Twenty different angiogenic growth factors were evaluated according to the manufacturer's instructions: angiogenin, epidermal growth factor, epithelial neutrophil–activating peptide-78, fibroblast growth factor (FGF)-2, growth-related oncogene, IFN-γ, insulin-like growth factor-1, IL-6, IL-8, leptin, monocyte chemotactic protein (MCP)-1, platelet-derived growth factor B-chain homodimer (PDGF-BB), placenta growth factor (PlGF), RANTES (regulated on activation, normal T-cell expressed and secreted), transforming growth factor-β1, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, thrombopoietin, VEGF, and VEGF-D. Semiquantitative growth factor levels were visualized by enhanced chemiluminescence comparison (Amersham-Pharmacia, Amersham, UK) and quantified (ImageQuant; Molecular Dynamics, Sunnyvale, CA) on autoradiographs that depicted spots within a linear range of exposure. The variation from membrane to membrane between duplicate positive control spots ranged from 0 to 8%. Protein levels were quantified against internal controls in the array and compared with other samples as fold increases. Levels of angiogenin, VEGF, Ang-1/Ang-2, and endostatin in BALF were also determined in duplicate by specific sandwich ELISA as described previously (29). Minimum detection limits were 78.1 pg/ml (angiogenin), 15.6 pg/ml (VEGF), 62.5 pg/ml (Ang-1), 46.9 pg/ml (Ang-2), and 0.31 pg/ml (endostatin).
Except for ELISA, all data are expressed as mean ± SEM of observations obtained from BALF samples from subjects with or without asthma. For ELISA, data are presented as median and range and statistical differences were analyzed by Mann-Whitney U test (SigmaStat 3.5; Systat Software, Inc., San Jose, CA). Remaining data were compared using Student's unpaired t test or one- or two-way analysis of variance as appropriate, with a Bonferroni post hoc test for multiple comparisons (SigmaStat). Relationships were analyzed by Spearman rank correlation coefficient (rs). A probability value of less than 0.05 was considered significant.
To assess whether soluble factors in airway secretions could promote angiogenesis, BALF from healthy subjects or subjects with asthma was added to cocultures of endothelial cells and human dermal fibroblasts for an 11-day period (Figures 1A and 1B). These cocultures form endothelial cell–derived capillary-like structures (27) either spontaneously or in response to a positive assay control, 1.25 ng/ml rhVEGF-A. The assay was validated by demonstrating a reproducible and linear concentration–response relationship between rhVEGF-A and vascular indices (rs > 0.85, not shown). BALF was concentrated to produce vascular changes that fell within the linear range of the rhVEGF-A concentration–response relationship.
Figure 1. Bronchoalveolar lavage fluid (BALF) from subjects with asthma promotes increased in vitro angiogenesis compared with healthy control subjects. Representative light photomicrographs (×4 original magnification) show induction of primitive vascular tubule structures after 11 days in culture with (A) BALF from a healthy donor and (B) BALF from a subject with mild asthma. In graphs, vascular changes were quantified using image analysis (see Methods) and presented as (C) number of vascular intervessel junctions (measure of branching), (D) number of vascular tubules, and (E) length of tubules formed per field of view (FOV). Culture in 1.25 ng/ml rhVEGF-A (hatched bars) or basal media alone (open bars) served as assay positive and negative controls, respectively. Data are mean ± SEM of duplicate values from BALF samples from 12 healthy subjects or 10 subjects with asthma. ***p < 0.001 compared with BALF from healthy subjects.
[More] [Minimize]Culture with concentrated BALF (fourfold at final dilution) samples from healthy subjects induced multiple proangiogenic parameters, including branching (estimated by counting vascular junctions), number of vascular structures, and vascular tubule length, but these did not differ from the basal vascular changes found with medium alone (p > 0.05, n = 12). However, induction of tubular structures by BALF from subjects with mild asthma was at least twofold greater (p < 0.001, n = 10) compared with BALF from healthy control subjects (Figure 1).
RayBio Human Angiogenesis Antibody Arrays (RayBiotech) were used to investigate the nature and compare the expression of soluble proangiogenic factors present in BALF collected from five healthy subjects and five subjects with asthma (Figure 2). The array identified higher levels of five factors in BALF from subjects with asthma: (1) VEGF (2.1-fold, p < 0.05), (2) angiogenin (1.9-fold, p < 0.05) (Figure 2C), (3) MCP-1 (1.9-fold, p < 0.01), (4) TIMP-1 (1.7-fold, p < 0.05), and (5) TIMP-2 (2.0-fold, p < 0.05). All other proangiogenic factors, including FGF-2 (0.8-fold, p > 0.05) and VEGF-D (1.0-fold, p > 0.05) (Figure 2C) did not differ in BALF samples from healthy subjects and subjects with asthma (Figure E1 in the online supplement).
Figure 2. RayBio Human Angiogenesis Antibody Array (RayBiotech) detection (with corresponding map below A and B) of proteins present in BALF from (A) a healthy and (B) an asthmatic donor. BLK = blank; EGF = epidermal growth factor; ENA-78 = epithelial neutrophil–activating peptide-78; FGF-2 = fibroblast growth factor-2; GRO = growth-related oncogene; IGF = insulin-like growth factor; MCP-1 = monocyte chemotactic protein (MCP)-1; PDGF-BB = platelet-derived growth factor B-chain homodimer; PlGF = placenta growth factor; RANTES = regulated on activation, normal T-cell expressed and secreted; thrombo = thrombopoetin; TGFβ1 = transforming growth factor-β1; TIMP = tissue inhibitor of metalloproteinase. (C) Examples of detected levels of proangiogenic factors in BALF are shown for FGF-2 (open bars), VEGF (gray bars), VEGF-D (black bars), and angiogenin (hatched bars). Protein levels were quantified against an internal control on the array and shown as mean ± SEM of duplicate values from independent experiments in BALF samples obtained from five different subjects in each group. *p < 0.05 compared with BALF from healthy subjects.
[More] [Minimize]To confirm findings with the arrays, levels of VEGF and angiogenin were assessed by ELISA and found to be increased in BALF from subjects with mild asthma by 1.6-fold (p < 0.05, n = 16) and 9.9-fold (p < 0.05, n = 8), respectively, compared with BALF from healthy subjects (Figures 3A and 3B). In the same BALF samples, levels of the proangiogenic molecule Ang-1 were unchanged and at the assay detection limit (p > 0.05, n = 4) (Figure 3C). Likewise, the antiangiogenic proteins endostatin and Ang-2 were not different (p > 0.05, n = 8–9; Figures 3D and 3E), but Ang-2 levels were at the minimal detection limit.
Figure 3. Imbalance in proangiogenic (VEGF, angiogenin, Ang-1; A, B, and C, respectively) versus antiangiogenic (endostatin, Ang-2 D and E, respectively) factors elevated in BALF collected from subjects with asthma compared with healthy subjects, detected by ELISA. Data are median and range of duplicate values from independent experiments in BALF samples obtained from 4 (Ang-1), 8–11 (Ang-2), 8–10 (angiogenin), 9–14 (endostatin), or 14–16 (VEGF) different subjects in each group. *p < 0.05 compared with BALF from healthy subjects.
[More] [Minimize]Based on the profile of proangiogenic factor up-regulation in BALF from subjects with asthma, the dependency of VEGF for the induction of vascular tubules was investigated in the coculture angiogenesis assay of endothelial cells and human dermal fibroblasts. In keeping with findings presented in Figure 1, BALF samples from subjects with mild asthma increased the number of vascular structures (p < 0.05 – 0.01, n = 4) compared with BALF from healthy donors (Figures 4C and 4D). Addition of anti-VEGF neutralizing antibody during the tubule growth period abrogated vascular structure formation induced by the rhVEGF-A–positive control and by BALF (Figure 4). Where present, tubules were both shorter and poorly branched (at least p < 0.05, n = 4). Inclusion of the VEGFR1 and VEGFR2 antagonist suramin (100 μM) also abolished (p < 0.05 – < 0.001, n = 4) tubule formation induced by either rhVEGF-A or BALF (Figure E2).
Figure 4. BALF-induced in vitro angiogenesis is VEGF dependent. Representative light photomicrographs (×4 original magnification) show induction of primitive vascular tubule structures by (A) BALF from a donor with mild asthma and (B) abrogation by treatment with a VEGF-neutralizing antibody. (C–E) Vascular changes induced by BALF in the presence of an isotype IgG2b antibody control (black bars) or presence of anti-VEGF antibody (gray bars) were quantified using image analysis (see Methods) and presented as (C) number of vascular intervessel junctions (measure of branching), (D) number of vascular tubules, (E) length of tubules formed per field of view (FOV). Culture in 1.25 ng/ml rhVEGF-A (hatched bars) or basal media alone (open bars) or in the presence of anti-VEGF (cross-hatched bars) served as assay controls. Data are mean ± SEM of duplicate values from BALF samples from four healthy subjects or four subjects with asthma. *p < 0.05, **p < 0.01, ***p < 0.001 compared with BALF from healthy subjects.
[More] [Minimize]To confirm that the observed vascular changes required VEGF derived from airway secretions rather than cells in the angiogenesis assay, BALF samples were treated with an anti-VEGF antibody and bound VEGF was depleted by protein-A immunoprecipitation. Depletion of VEGF prevented induction of vascular structures after 11 days, induced by either the rhVEGF-A assay positive control (p < 0.001, n = 3) or by BALF from subjects with asthma (p < 0.05 – < 0.01, n = 3). This contrasted with the baseline formation of vascular structures by BALF from healthy subjects in whom VEGF depletion was without effect (p > 0.05, n = 3; Figure 5). VEGF was readily detected by ELISA in all BALF samples treated with the IgG2b isotype control antibody but was undetectable in BALF samples incubated with anti-VEGF, confirming the depletion (not shown).
Figure 5. VEGF in BALF from subjects with asthma is required for in vitro angiogenesis. Representative light photomicrographs (×4 original magnification) show induction of primitive vascular tubule structures by (A) BALF from a donor with mild asthma and (B) abrogation by immunodepletion of VEGF. (C–E) BALF samples were divided into two separate aliquots and treated either with an isotype IgG2b antibody control (black bars) or anti-VEGF antibody (gray bars). After confirmation of VEGF depletion by ELISA, induction of vascular changes were quantified using image analysis (see Methods) and presented as (C) number of vascular intervessel junctions (measure of branching), (D) number of vascular tubules, (E) length of tubules formed per field of view (FOV). Culture in 1.25 ng/ml rhVEGF-A (hatched bars) or basal media alone (open bars) or in VEGF-depleted media (cross-hatched bars) served as assay controls. Data are mean ± SEM of duplicate values from BALF samples from three healthy subjects or three subjects with asthma. *p < 0.05, **p < 0.01, ***p < 0.001 compared with BALF from healthy subjects.
[More] [Minimize]In the present study, we report that airway lining fluid from subjects with mild asthma but not healthy subjects promotes the formation of endothelial cell–derived capillary-like structures in an in vitro angiogenesis assay using cocultures of endothelial cells and human dermal fibroblasts. Antibody arrays compared expression of soluble proangiogenic factors elevated in BALF collected from subjects with asthma and identified five factors (VEGF, angiogenin, MCP-1, TIMP-1, and TIMP-2). Strategies that neutralized the activity of VEGF either by targeting VEGF or its receptors abrogated the proangiogenic capacity of BALF from subjects with asthma, demonstrating a requirement for VEGF in the assay system. Moreover, immunodepletion of VEGF from BALF also abolished the proangiogenic capacity of BALF collected from subjects with asthma, confirming that the proangiogenic capacity of BALF from these subjects resided predominantly in VEGF derived from asthmatic airways. These multiple indices of proangiogenic responses provide strong evidence for active tissue vascularization processes occurring in the airways of subjects with asthma and support a growing consensus that VEGF is an active participant of the microvascular remodeling process and asthma phenotype.
Several growth factors with proven or potential roles in tissue neovascularization and remodeling have been identified in the context of various diseases (12). Here, we performed ELISA and antibody arrays to compare expression of soluble proangiogenic growth factors in BALF collected from healthy subjects and subjects with asthma. These proteomic approaches identified elevated levels of VEGF, angiogenin, MCP-1, TIMP-1, and TIMP-2 in the airway lining fluid of subjects with mild asthma. Increased levels of VEGF in BALF or induced sputum from subjects with asthma have been widely reported (11, 20, 22, 30), and Hoshino and colleagues (9) have reported a significant correlation between the number of angiogenin-positive cells and the percentage of vessel area in asthmatic airways. Recently, angiogenin was reported to be elevated in induced sputum from children with acute asthma, and together with VEGF, it correlates with disease severity (31). Here we report for the first time that angiogenin levels are increased in BALF collected from adults with mild asthma at baseline. The contribution of angiogenin to tubule formation remains to be explored, but the discrepancy between the fold increases in angiogenin measured by the arrays versus ELISA may reflect saturation of the array signal. MCP-1, a CC chemokine, is expressed by endothelial cells, monocytes, smooth muscle cells, and fibroblasts (32). Recent evidence suggests that MCP-1 contributes to angiogenesis by a direct effect on endothelial cells, which is VEGF dependent (32). For this reason, we did not test whether elevated MCP-1 levels in BALF from subjects with asthma were required for angiogenic activity but instead chose to target VEGF directly as it was also increased in BALF from subjects with asthma.
Feltis and colleagues (19, 22) demonstrated recently that Ang-1–positive vessels were increased in bronchial biopsies from subjects with asthma compared with healthy control subjects. However, no studies have reported Ang-1 levels in airway secretions including BALF. Here, we observed that, despite increases in VEGF and angiogenin in BALF from subjects with asthma when compared with healthy control subjects, levels of Ang-1 were barely detectable and were not different between the two groups. The reason for this discrepancy between elevated Ang-1 staining in airway tissues but not in BALF from subjects with asthma is unclear but suggests that Ang-1 in BALF may not represent bronchial mucosa Ang-1, perhaps because BALF mainly assesses the distal airways and alveoli. It is hypothesized that where increased VEGF levels occur without detectable changes in Ang-1, this reflects an active proangiogenic environment, perhaps prior to stabilization of nascent vascular structures (19, 33). The finding that several but not all growth factors were elevated in asthmatic BALF when evaluated by independent methods (antibody arrays and ELISA) suggests that observed changes in angiogenic growth factor levels were unlikely to have resulted from gross differences in a dilution/concentration factor, introduced as part of the BALF collection or processing. Thus, our collective findings suggest multiple proangiogenic growth factors are up-regulated in BALF from subjects with asthma and are potentially important in neovascularization of the airways.
Asai and colleagues (20) reported recently that there is an imbalance between VEGF and endostatin (a potent endogenous inhibitor of angiogenesis and tumor growth [34]) levels in induced sputum from subjects with asthma in whom the VEGF:endostatin ratio was increased by approximately twofold. Thus, in the present study, it was important to measure multiple growth factors as well as physiologically opposing factors. In addition, few reports have described levels of antiangiogenic factors in asthmatic airways or secretions. Here, we show a trend toward up-regulation of proangiogenic protein expression in the airway lining fluid of subjects with asthma. In keeping with the imbalance hypothesis (20), we demonstrate that levels of proangiogenic factors, such as VEGF, angiogenin, and MCP-1, were increased in BALF from subjects with asthma, whereas antiangiogenic molecules, such as endostatin and Ang-2 (disrupts blood vessel formation by competing with equal affinity with Ang-1 at the endothelial cell–specific Tie2 receptor [18]) were unchanged between healthy subjects and subjects with asthma. However, the angiogenesis arrays also identified increased expression of TIMP-1 and TIMP-2 in BALF from subjects with mild asthma. TIMP-1 and TIMP-2 inhibit endothelial cell migration in type I collagen, and TIMP-2 is a known inhibitor of angiogenesis and decreases endothelial cell proliferation in vitro (35, 36). Physiologically, it would be expected that factors that initially promote endothelial cell migration and proliferation (e.g., VEGF, angiogenin, and MCP-1) would need to be aided by factors that then limit migration (TIMP-1, TIMP-2, endostatin, and Ang-2) in favor of differentiation and capillary formation within the airway wall in asthma. Thus, it may be an oversimplification to view neovascularization in asthma as a process occurring due to a global imbalance between pro- and antiangiogenic growth factor ratios; rather, specific temporal combinations of opposing factors may be required for angiogenesis to occur.
Given the complex mixture of growth factors and their potential proangiogenic versus antiangiogenic properties in the airway lining fluid of subjects with asthma, it was important to evaluate the overall biological activity of BALF from subjects with asthma and healthy control subjects. A coculture of endothelial cells and human dermal fibroblasts was used in which BALF from subjects with asthma but not healthy subjects induced the formation of CD31-positive capillary-like structures that were at least twofold longer, more numerous, and more highly branched than those that formed either spontaneously or in response to BALF from healthy control subjects. In general, similar quantitative differences were found between the observed induction of CD31-positive vascular tubules and the fold increase in proangiogenic growth factors, detected by either antibody array or ELISA, in BALF from subjects with asthma. This was especially true with VEGF. In addition, because numerous reports describe elevated levels of VEGF in induced sputum in airway secretions from subjects with asthma (11, 20, 22, 30) as well as positive correlations with increased total airway vascular area (7–9) and asthma severity (24), we targeted VEGF as the component of BALF most likely to drive the vascular changes in the in vitro angiogenesis assay. Abrogation of tubule formation induced by BALF from either subjects with asthma or healthy control subjects by treatment with a VEGF-neutralizing antibody indicated that VEGF was required but not whether it was derived from the BALF or from cells in the in vitro angiogenesis assay. To exclude the latter, VEGF was first depleted from BALF. The observed abrogation of tubule formation in VEGF-depleted BALF from subjects with asthma but not healthy control subjects suggested that the proangiogenic capacity of BALF from subjects with asthma resided predominantly with VEGF from the asthmatic airway. This, with the increased angiogenic growth factor content of asthmatic BALF, provides strong evidence for active tissue vascularization processes occurring in the airways of subjects with mild asthma and supports a growing consensus that VEGF is an active participant of microvascular remodeling processes and asthma phenotype. However, increased VEGF expression is reported in the pathophysiology of other common respiratory disorders, such as acute lung injury, chronic obstructive pulmonary disease, obstructive sleep apnea, idiopathic pulmonary fibrosis, pulmonary hypertension, pleural disease, and lung cancer. Thus, elevated levels of VEGF in BALF from patients with asthma may reflect its wider importance as a marker of airway inflammation and remodeling (37–39).
In summary, we report that airway lining fluid from subjects with asthma but not healthy subjects promotes the formation of endothelial cell–derived capillary-like structures in an in vitro angiogenesis assay using cocultures of endothelial cells and human dermal fibroblasts. Antibody arrays and ELISA of BALF from subjects with asthma suggest an imbalance in the production of proangiogenic versus antiangiogenic growth factors. Selective immunodepletion of VEGF from BALF obtained from subjects with asthma abolished the proangiogenic capacity, confirming that the proangiogenic capacity of BALF from subjects with asthma resided predominantly in VEGF from the asthmatic airway. These findings suggest that VEGF is present in the asthmatic airway and is necessary for asthma-induced angiogenesis in vitro. This observation supports the notion that VEGF is a key factor in the pathogenesis of asthma.
The authors thank the research nursing and technical staff in the department for recruitment and screening of volunteers and for assistance with bronchoscopy and bronchoalveolar lavage collection.
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