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Published ahead of print on June 12, 2008, doi:10.1164/rccm.200707-1046OC
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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 460-468, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200707-1046OC

Original Article

Induction of Angiogenesis by Airway Smooth Muscle From Patients with Asthma

David E. Simcock1, Varsha Kanabar1,*, Graham W. Clarke1,*, Katharina Mahn1, Charlotta Karner1, Brian J. O'Connor1, Tak H. Lee1 and Stuart J. Hirst1

1 Division of Asthma, Allergy, and Lung Biology, King's College London, Medical Research Council & Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom

Correspondence and requests for reprints should be addressed to Stuart J. Hirst, Ph.D., Department of Physiology, Monash University, Building 13F, Clayton Campus, Melbourne Victoria 3800, Australia. E-mail: stuart.hirst{at}med.monash.edu.au


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Airway remodeling in asthma involves accumulation of airway smooth muscle (ASM) and increased vascularity due to angiogenesis. Bronchial blood vessels and ASM are found in close proximity, and ASM releases multiple proinflammatory mediators, including vascular endothelial growth factor (VEGF).

Objectives: We examined whether release of proangiogenic mediators is increased in ASM from subjects with asthma and whether this is translated to induction of angiogenesis.

Methods: Biopsy-derived ASM cells were cultured from 12 subjects with mild asthma, 8 with moderate asthma, and 9 healthy control subjects. Angiogenesis induced by cell-conditioned medium (CM) from ASM was evaluated in a tubule formation assay. Anti-CD31–labeled tubules were quantified by image analysis. Angiogenic factors in CM were quantified by antibody arrays and by enzyme-linked immunosorbent assay.

Measurements and Main Results: Induction of angiogenesis by CM from unstimulated ASM was increased in subjects with mild asthma (twofold) and moderate asthma (threefold), compared with healthy CM (P < 0.001). Levels of angiogenic factors (VEGF, angiopoietin [Ang]-1, angiogenin) were similarly elevated in CM from subjects with asthma compared with that from healthy subjects (P < 0.05), whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. VEGF, Ang-1, and angiogenin in combination increased vascularity (twofold, P < 0.01) in cultured intact biopsies. Selective VEGF immunodepletion abolished enhanced tubule formation by CM from asthmatic ASM (P < 0.01), but CM depletion of Ang-1 or angiogenin had no effect.

Conclusions: ASM cultured from subjects with mild or moderate asthma, but not from healthy control subjects, promotes angiogenesis in vitro. This proangiogenic capacity resides in elevated VEGF release and suggests that ASM regulates airway neovascularization in asthma.

Key Words: airway smooth muscle • airway wall vascular remodeling • angiogenesis • asthma • vascular endothelial growth factor



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on this Subject
The cell types regulating airway wall angiogenesis in asthma are unknown.

What This Study Adds to the Field
We demonstrate that airway smooth muscle cells from subjects with mild or moderate asthma can initiate and sustain angiogenesis in vitro and that release of vascular endothelial growth factor accounts for this activity.

 
Asthma symptoms of airways hyperresponsiveness (AHR), variable airflow obstruction, and reversibility are associated with intense persistent airway inflammation and structural remodeling. Striking features of remodeled airways include accumulation of airway smooth muscle (ASM) and subepithelial neovascularization (1–3). Vascular engorgement, vasodilatation, and microvascular leakage can directly increase airway wall thickness, increase airway luminal narrowing, and facilitate inflammatory cell trafficking into tissues, and may sustain the baseline metabolic demands of the increased ASM content in asthma, leading to increased force generation (2, 4–9). Furthermore, airway wall neovascularization, seen as increases in the number and size of bronchial blood vessels and angiogenic sprouts (3, 10, 11), is a prominent feature of fatal and nonfatal asthma (1, 8, 12) that correlates with subepithelial basement membrane thickening (13), airway obstruction, and the degree of AHR (8, 14).

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 (15). 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 (15, 16). Other polypeptide growth factors implicated in angiogenesis include angiogenin and angiopoietin (Ang)-1. Angiogenin, first isolated from conditioned medium of colonic carcinoma cell cultures (17), is a potent tumor-derived angiogenic factor but also plays a role in several nonmalignant vasculoproliferative disorders, and like VEGF it induces vascular endothelial cell proliferation, migration, and tubule formation (18). Ang-1 is a ligand for the endothelial cell–specific Tie2 surface receptor (19). 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 (19). Thus, VEGF and Ang-1 have been hypothesized to complement each other in angiogenic processes (19).

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 in the airways of asthmatics (8, 11, 14, 20–22). Increasing evidence from animal studies supports a central role for VEGF in the pathogenesis of asthma (8, 23). Elevated levels of VEGF in airway biopsies, induced sputum and in bronchoalveolar lavage fluid (BALF) from asthmatics correlate directly with increased total airway vascular area (8, 11, 13, 14, 21, 22), disease severity (24) and are inversely correlated to airway calibre and airflow obstruction (8, 14). Similarly, Ang-1 and angiogenin levels are increased in asthma (8, 11, 20).

ASM cells express an array of extracellular matrix components and cell surface receptor molecules involved in lymphocyte adhesion, and are a source of multiple proinflammatory cytokines, chemokines, and polypeptide growth factors including VEGF (25, 26). Furthermore, there is accumulation of ASM in asthma and the bronchial microvasculature is in close proximity to ASM bundles (3, 12, 27) as the distance between the myobundles and the reticular basement membrane is reduced with increasing asthma severity (28). This supports the possibility that ASM participates directly in the pathogenesis of airway wall vascular remodeling to perpetuate asthmatic airway inflammation and AHR (1–4).

In the current study, we investigate the profile of proangiogenic and antiangiogenic proteins expressed by ASM and demonstrate for the first time the proangiogenic capacity of ASM cells cultured from patients with asthma using an in vitro endothelial cell and dermal fibroblast coculture system. Furthermore, we characterize the nature of the dominant proangiogenic activity secreted by ASM from subjects with asthma. Some of the results of this study have been previously reported in the form of an abstract (29, 30).


   METHODS
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 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Culture of Human Airway Smooth Muscle Cells
ASM cells from 9 healthy subjects, 12 glucocorticoid-naïve subjects with atopic mild asthma, and 8 subjects with atopic moderate asthma were obtained in accordance with procedures approved by the Research Ethics Committees of King's College Hospital (study #11-03-209) and Guy's and St. Thomas' Hospitals (study #05/Q0704/72) by deep endobronchial biopsy. (See Table 1 and online supplement for patient details, recruitment, and endobronchial biopsy.) ASM bundles were visualized using a dissecting microscope and dissected free of surrounding tissue using fine needles. Cells were grown by explant culture from ASM bundle fragments in 12.5 cm2 flasks using methods described previously (31). The presence of ASM bundles in endobronchial biopsies was confirmed histologically (supplemental information in Reference 31). Fluorescent immunocytochemistry routinely confirmed that near-confluent, fetal bovine serum (FBS)-deprived ASM cells (passage 2) stained (> 95%) for smooth muscle–specific {alpha}-actin, desmin, and calponin (supplemental information in Reference 31). Cell passages 3–8 were used in all experiments.


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  		TABLE 1. DETAILS OF ASTHMATIC AND HEALTHY SUBJECTS

 
Cell Stimulation and Immunodepletion
Cells were seeded in 75 cm2 flasks (10,000 cells/cm2) in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and left overnight at 37°C. After attachment, cells were growth-arrested for 72 hours with 8 ml FBS-free RPMI-1640 and either left unstimulated or were stimulated for 72 hours in RPMI-1640 containing 10 ng/ml recombinant human (rh) transforming growth factor (TGF)-β1 or 10 ng/ml interleukin (IL)-13 (R&D Systems, Abingdon, UK). Cell-free, cell-conditioned medium (CM) was collected and stored at –80°C before detection of proangiogenic activity in the in vitro angiogenesis assay or release of angiogenic growth factors either by enzyme-linked immunosorbent assay (ELISA) or antibody array. Manual counts were performed after collection of CM to confirm that cell numbers were unchanged after stimulation and to allow cytokine levels to be expressed in picograms per milliliter per million cells to correct for small variations in release between cell lines or reported differences in growth rates between ASM cells from healthy subjects and patients with asthma (32).

To examine if specific proangiogenic mediators were released by human ASM to support tubule formation, CM samples underwent selective immunodepletion of VEGF, angiopoietin (Ang-1), or angiogenin. Aliquots of CM were treated overnight at 4°C with monoclonal antibodies to either VEGF (2 µg/ml, clone 26503; R&D Systems) or Ang-1 (2 µg/ml, clone 171718; R&D Systems), or a goat polyclonal antibody against angiogenin (2 µg/ml; Calbiochem, Darmstadt, Germany), followed by precipitation using protein-A agarose beads (Upstate, Lake Placid, NY). Control CM samples were treated with an IgG2b isotype-matched antibody or goat IgG (R&D Systems).

In Vitro Angiogenesis Assay
The proangiogenic capacity of CM samples was investigated using an in vitro angiogenesis assay (33) comprising cocultures of human dermal fibroblasts and human umbilical vein endothelial cells (AngioKit; TCS CellWorks, Buckingham, UK). Individual CM samples from ASM cells grown from healthy control subjects or from subjects with asthma were diluted twofold in culture media supplied by the manufacturer and incubated 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 rhVEGF-A (165 amino acid isoform; R&D Systems) at 1 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 instructions (TCS CellWorks). Multiple photomicrographs (x4 objective) were taken, and angiogenesis in each field of view (FOV) was quantified using image analysis (AngioSys; TCS CellWorks) as described previously (33, 34).

Culture of Endobronchial Biopsies and Immunohistochemistry
Whole endobronchial biopsies from healthy subjects were cultured in FBS-free DMEM alone or in the presence of VEGF-A, Ang-1, and angiogenin in combination (all at 50 ng/ml rh-protein; R&D Systems). After 72 hours, vascular structures in frozen tissue sections were labeled using a mouse anti-CD31 antibody. Sections were then counterstained with hematoxylin. Percentage vascular area (vascular area divided by total area of section) and number of vessels per square millimeter were obtained from digitized images with a three-chip color camera Zeiss Vision KS400 system (Carl Zeiss, Göttingen, Germany). Further details are provided in the online supplement.

Angiogenesis Protein Array and ELISA
RayBio Human Angiogenesis Antibody Arrays (RayBiotech, Norcross, GA) were employed to assay multiple proangiogenic factors in CM from ASM growth from four healthy subjects, four subjects with mild asthma and four with moderate asthma (34). Twenty different angiogenic growth factors were evaluated according to the manufacturer instructions: angiogenin, epidermal growth factor (EGF), epithelial neutrophil-activating peptide (ENA)-78, fibroblast growth factor (FGF)-2, growth-related oncogene (GRO), interferon {gamma} (IFN-{gamma}), insulin-like growth factor (IGF)-1, IL-6, IL-8, leptin, monocyte chemotactic protein (MCP)-1, PDGF-BB, placenta growth factor (PIGF), RANTES (regulated on activation, normal T cell expressed and secreted), TGF-β1, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, thrombopoietin (thrombo), 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 CM were also determined in duplicate by specific sandwich ELISA and expressed as pg/ml/million cells to correct for small variations in either the volume of culture media or cell number used for generation of CM, as described previously (34, 35). 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).

Statistical Analysis
All data are expressed as mean ± SEM of observations obtained from n subjects with or without asthma. Data were compared using one- or two-way ANOVA as appropriate with a Bonferroni post hoc test for multiple comparisons (SigmaStat 3.5; Systat Software Inc, San Jose, CA). A P value of < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive Proangiogenic Capacity of ASM from Subjects with Asthma
To assess whether soluble factors from ASM could promote angiogenesis constitutively, CM from unstimulated ASM cells, obtained from healthy subjects or subjects with mild or moderate asthma, was added to cocultures of endothelial cells and human dermal fibroblasts for an 11-day period (Figure 1). Endothelial-derived capillary-like structures were formed either spontaneously or in response to a positive assay control, 1 ng/ml rhVEGF-A (33, 34). The assay was validated by demonstrating a reproducible and linear concentration–response relationship between rhVEGF-A and vascular indices (33, 34; see Figure E1 in the online supplement). CM was diluted (1:2) to produce vascular changes that fell within the linear range of the rhVEGF-A concentration–response relationship.


Figure 1
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  		Figure 1. Airway smooth muscle cells from subjects with asthma constitutively promote in vitro angiogenesis. Representative light photomicrographs (x4 original magnification) show induction of primitive vascular tubule structures after 11 days in culture with (A) cell conditioned media (CM) from a healthy donor, (B) CM from a subject with mild asthma (Mild A), and (C) CM from a subject with moderate asthma (Mod A). In graphs, vascular changes were quantified using image analysis (see METHODS) and presented as (D) number of vascular intervessel junctions (measure of branching), (E) number of vascular tubules, and (F) length of tubules formed per field of view (FOV). Culture in 1 ng/ml recombinant human (rh) vascular endothelial growth factor (VEGF)-A (right-hatched bars) or basal media alone (left-hatched bars) served as assay positive and negative controls, respectively. Data are mean ± SEM of duplicate values from CM samples from 6 healthy subjects, 11 subjects with mild asthma, and 6 subjects with moderate asthma (*P < 0.05, **P < 0.01, ***P < 0.001).

 
Culture with CM from ASM grown from healthy subjects induced multiple proangiogenic parameters (Figure 1A), including branching (estimated by counting vascular junctions), number of vascular tubules, and vascular tubule length, but these did not differ from the basal vascular changes found with medium alone (P > 0.05, n = 6, Figures 1D–1F). However, induction of tubular structures by CM from subjects with mild asthma was 1.9-fold greater (at least P < 0.05, n = 11) compared with ASM from healthy control subjects (n = 6) (Figure 1B), and was increased by 2.9-fold with CM from ASM cultured from subjects with moderate asthma (P < 0.001, n = 6, Figure 1C).

Regulation of Asthmatic ASM Proangiogenic Capacity by IL-13 and TGF-β1
To investigate up-regulation of ASM proangiogenic capacity by factors known to be elevated in inflammation and remodeling in asthma, ASM cells grown from healthy subjects or patients with mild or moderate asthma were treated with 10 ng/ml TGF-β1 or 10 ng/ml IL-13, and CM subsequently examined in the angiogenesis assay. TGF-β1 potentiated both indices of vascular branching (number of junctions and tubules) compared with unstimulated ASM cells (Figures 2A and 2B), but had no effect on tubule length (Figure 2C). This effect of TGF-β1 on vascular branching was restricted to ASM cells cultured either from patients with mild (P < 0.01, n = 11) or moderate (P < 0.01, n = 6) asthma. Up-regulation of vascular indices by TGF-β1 treatment of ASM from healthy subjects was not different from untreated ASM (P > 0.05, n = 6). Treatment with IL-13 of ASM cells from healthy subjects or from subjects with asthma did not induce angiogenesis above unstimulated levels (P > 0.05, n = 5–9, Figure 2). Furthermore, neither IL-13 or TGF-β1 when added directly to the angiogenesis assay increased spontaneous tubule formation (P > 0.05).


Figure 2
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  		Figure 2. CM from TGF-β1– but not IL-13–stimuated airway smooth muscle from subjects with mild (shaded bars) or moderate (solid bars) asthma enhances in vitro angiogenesis. Angiogenesis by CM from healthy subjects (open bars) is also shown. Vascular changes were quantified using image analysis (see METHODS) and presented as (A) number of vascular intervessel junctions (measure of branching), (B) number of vascular tubules, and (C) length of tubules formed per field of view (FOV). Culture in 1 ng/ml rhVEGF-A (right-hatched open bars) or basal media alone (left-hatched open bars) served as assay positive and negative controls, respectively. Neither TGF-β1 (right-hatched shaded bars) or IL-13 (left-hatched shaded bars) added directly to the assay induced tubular formation. Note: control data (Unstim) are re-shown from Figure 1. Data are mean ± SEM of duplicate values from CM samples from 6 healthy subjects, 11 subjects with mild asthma, and 6 subjects with moderate asthma (**P < 0.01).

 
Multiple Proangiogenic Proteins Produced by ASM from Subjects with Asthma
RayBio Human Angiogenesis Antibody Arrays were used to investigate the nature and compare expression of soluble proangiogenic factors in CM from unstimulated ASM cells from healthy subjects with CM from subjects with mild or moderate asthma (n = 4 per group). The array identified higher levels of five factors in CM from ASM cultured from subjects with either mild or moderate asthma: (1) VEGF (max 2.5-fold, P < 0.05), (2) angiogenin (max 4.4-fold, P < 0.01), (3) EGF (max 3.9-fold, P < 0.05), (4) IGF-1 (max 1.8-fold, P < 0.05), and (5) IFN-{gamma} (max 2.5-fold, P < 0.05) (Figure 3). There were no differences in expression of all other proangiogenic factors detected in CM samples from ASM cells obtained from healthy subjects or subjects with mild or moderate asthma (Figure E2).


Figure 3
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  		Figure 3. RayBio Human Angiogenesis Antibody Array detection of proteins constitutively released by airway smooth muscle. CM from (A) a healthy donor, (B) a subject with mild asthma, and (C) a subject with moderate asthma. Pos = positive control; Neg = negative control; BLK = blank; Angiogen = angiogenin; EGF = epidermal growth factor; ENA-78 = epithelial neutrophil-activating peptide-78; FGF-2 = fibroblast growth factor-2; GRO = growth-related oncogene; IFN-{gamma} = interferon {gamma}; IGF-1 = insulin-like growth factor-1; IL = interleukin; MCP-1 = monocyte chemotactic protein; PlGF = placenta growth factor; PDGF-BB = platelet-derived growth factor B chain homodimer; RANTES = regulated on activation, normal T cell expressed and secreted; TGF-β1 = transforming growth factor-β1; TIMP = tissue inhibitor of metalloproteinase; Thrombo = thrombopoietin. (D) Examples of detected levels of proangiogenic factors in CM from cells cultured from healthy subjects or those with mild asthma (Mild A) and moderate asthma (Mod A) are shown for VEGF (open bars), angiogenin (hatched bars), EGF (shaded bars), IGF-1 (solid bars) and IFN-{gamma} (cross-hatched bars). (E) Map for antibody array. Protein levels were quantified against an internal control on the array and shown as mean ± SEM of duplicate values from independent experiments in CM samples obtained from four different subjects in each group. *P < 0.05, **P < 0.01 compared with CM from healthy subjects.

 
To confirm findings with the arrays, levels of VEGF, angiogenin, and Ang-1 in CM were assessed by ELISA and were found to be increased in unstimulated ASM from subjects with mild or moderate asthma by 3.1-fold (n = 8–11), 2.3-fold (P < 0.05, n = 5–10), and 2.3-fold (P < 0.05, n = 8–10), respectively, compared with CM from unstimulated ASM from healthy subjects (Figure 4A). In keeping with previous reports (36, 37), treatment with TGF-β1 increased the release of VEGF from ASM cells grown from healthy subjects or patients with asthma. Levels of VEGF in CM from ASM cells grown from patients with either mild or moderate asthma were increased by 1.9-fold (P < 0.05, n = 8–10) compared with ASM from healthy subjects (Figure 4B). In the same CM samples, no effect of TGF-β1 was found on basal levels of either Ang-1 or angiogenin from healthy or asthmatic ASM cells (not shown). Consistent with an earlier report (38), IL-13 was a weak stimulus for release of VEGF from ASM (Figure 4B) and was without effect on basal levels of Ang-1 or angiogenin released by either healthy or asthmatic ASM (not shown).


Figure 4
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  		Figure 4. Imbalance of (A) proangiogenic (vascular endothelial growth factor [VEGF], angiopoietin [ANG], angiopoietin-1 [Ang-1]) versus (C) antiangiogenic (endostatin, [Endo], angiopoietin-2 [Ang-2]) factors elevated in cell conditioned (CM) media from airway smooth muscle from healthy subjects (open bars), subjects with mild asthma (gray bars), and subjects with moderate asthma (solid bars), detected by enzyme-linked immunosorbent assay (ELISA). (B) Effects of stimulation of cells with IL-13 or TGF-β1. Mean cell counts were 867,416 ± 540, 935,436 ± 592, and 989,200 ± 438 for subjects who were healthy or who had mild or moderate asthma, respectively. Data are mean ± SEM of duplicate values from independent experiments in CM samples obtained from 5 (healthy) or 10 (mild and moderate asthma) different subjects. *P < 0.05, **P < 0.01, #P < 0.05 compared with unstimulated in B.

 
Constitutive levels of the antiangiogenic proteins endostatin and Ang-2 did not vary between healthy and asthmatic ASM (P > 0.05, Figure 4C), and were not regulated by treatment with either TGF-β1 or IL-13 (not shown).

Induction of Vascularity in Intact Endobronchial Biopsies
To investigate if VEGF, angiogenin, and Ang-1, which were each found to be elevated in CM from ASM from subjects with asthma (Figures 4A and E2), could induce tissue neovascularisation, intact endobronchial biopsies from healthy subjects were treated for 72 hours with the combination of these proangiogenic factors (all at 50 ng/ml). This resulted in a 1.9-fold increase in subepithelial vascular area (P < 0.01) and a 1.8-fold increase in the number of microvessels (P < 0.01), identified by CD31-positve labeling (Figure E3).

Characterization of Proangiogenic Activity from Asthmatic ASM
Based on the profile of proangiogenic factors released by ASM from individuals 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 agreement with findings presented in Figure 1, CM from ASM cultured from subjects with asthma induced more vascular tubules than CM from healthy ASM (P < 0.05, n = 4) (Figures 5 and E4 in the online supplement). Addition of an anti-VEGF–neutralizing antibody to the coculture abrogated the formation of vascular tubules induced by the rhVEGF-A positive control and by CM from healthy or asthmatic ASM (at least P < 0.05, n = 4) (Figure E4).


Figure 5
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  		Figure 5. Induction of in vitro angiogenesis by airway smooth muscle from subjects with asthma is VEGF-dependent. Representative light photomicrographs (x4 original magnification) show reduced vascular tubule formation after immunodepletion of VEGF from CM from cells cultured from a donor with mild asthma (A). CM samples were divided into two separate aliquots and treated either with an isotype IgG2b antibody control (solid bars) or anti-VEGF antibody (open bars) before addition to the angiogenesis assay (inset shows tubule formation in CM treated with an isotype-matched control antibody). After confirmation of VEGF depletion by ELISA, induction of (D–F) vascular changes was quantified using image analysis (see METHODS) and presented as (D) number of vascular tubules formed per FOV. CM samples were also depleted of Ang-1 (B, E) and angiogenin (C, F). Culture in 1 ng/ml rhVEGF-A (right-hatched bars) or basal media alone (left-hatched bars) or in VEGF-depleted media (cross-hatched bars) served as assay controls. Data are mean ± SEM of duplicate values from CM samples from four healthy subjects or four subjects with mild (Mild A) or moderate asthma (Mod A). *P < 0.05, **P < 0.01, ***P < 0.001.

 
To confirm the observed vascular changes required VEGF derived specifically from ASM rather than the cells that constituted the angiogenesis assay, CM samples from ASM were depleted of VEGF with an anti-VEGF antibody followed by protein-A immunoprecipitation before addition to the angiogenesis assay. Immunodepletion of VEGF abolished vascular tubule formation induced by either the rhVEGF-A assay positive control (P < 0.001, n = 4) or CM from unstimulated ASM grown from subjects with mild or moderate asthma (P < 0.05 to < 0.01, n = 4) (Figures 5A and 5D). This contrasted with induction of vascular tubules by CM from healthy ASM, where VEGF depletion was without effect (P > 0.05, n = 4, Figure 5D). Depletion of VEGF also abolished increases in angiogenic indices induced by CM from TGF-β1–stimulated ASM cells cultured from either healthy subjects or patients with mild or moderate asthma (not shown). Furthermore, immunodepletion of either Ang-1 (Figures 5B and 5E) or angiogenin (Figures 5C and 5F) from CM samples had no effect on angiogenesis induced by unstimulated ASM from either healthy subjects or subjects with asthma (P > 0.05, n = 4). VEGF, Ang-1, and angiogenin were readily detected by ELISA in all CM samples from healthy or asthmatic ASM treated with the IgG2b isotype control antibody, but were undetectable in the same CM samples after incubation with selective antibodies to VEGF, Ang-1, or angiogenin, confirming the depletion (not shown).


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we report that ASM cells from patients with either mild or moderate asthma induce the formation of endothelial-derived capillary-like structures in an in vitro angiogenesis assay employing cocultures of endothelial cells and human dermal fibroblasts. A key finding was that the induced angiogenesis occurred both constitutively and in response to TGF-β1 stimulation of ASM from subjects with asthma and was dependent on asthma severity, but did not occur with ASM cells cultured from healthy subjects. Antibody arrays and ELISA demonstrated an imbalance in basal release of soluble factors by ASM from patients with asthma that favored induction of angiogenesis (VEGF, Ang-1, angiogenin, EGF, IGF-1, and IFN-{gamma}) without changes in the release of antiangiogenic factors (Ang-2, endostatin, TIMP-1, TIMP-2). Furthermore, when endobronchial biopsies from healthy subjects were treated with VEGF, Ang-1, and angiogenin in combination, submucosal vascularity scores were doubled. Stimulation with TGF-β1, but not IL-13, enhanced the angiogenic activity of ASM from subjects with asthma in line with the production of VEGF. Neutralization of VEGF abrogated the proangiogenic capacity of ASM cells from subjects with asthma, demonstrating a requirement for VEGF in the angiogenesis assay. Moreover, immunodepletion of VEGF, but not depletion of Ang-1 or angiogenin, from CM abolished tubule formation induced by either unstimulated or TGF-β1–stimulated ASM from subjects with asthma, confirming that VEGF specifically derived from asthmatic ASM accounted for the observed angiogenesis. Collectively, these findings provide strong evidence that ASM can sustain active tissue neovascularization processes in the airways of individuals with asthma.

Several growth factors with proven or potential roles in tissue neovascularization and remodeling have been identified in the context of various diseases (15). Here, we performed ELISA and antibody arrays to compare expression of soluble proangiogenic growth factors in CM from ASM cultured from healthy subjects and from subjects with asthma. These combined proteomic approaches identified elevated levels of VEGF, angiogenin, Ang-1, EGF, IGF-1, and IFN-{gamma} in CM of subjects with asthma. Although VEGF, EGF, IGF-1, and IFN-{gamma} are known to be expressed by nonasthmatic human ASM (25, 26, 36, 37, 39), this is the first report to demonstrate release of angiogenin or Ang-1 or to show enhanced release of multiple proangiogenic mediators from ASM cultured from subjects with mild or moderately severe asthma. Increased levels of VEGF in BALF or induced sputum from individuals with asthma have been widely reported (9, 11, 14, 21, 34), and recently we have shown induction of angiogenesis by BALF collected from subjects with asthma but not healthy control subjects (34). Hoshino and colleagues (8) have reported a significant correlation between the number of angiogenin-positive cells and the percentage vessel area in asthmatic airways. Recently, angiogenin was reported to be elevated in induced sputum from children with acute asthma and, along with VEGF, it correlates with disease severity (40) and is increased in BALF from adults with mild asthma (34). Feltis and colleagues (11, 20) have demonstrated that Ang-1–positive vessels are increased in bronchial biopsies from individuals with asthma compared with those from healthy control subjects. The present finding that several but not all growth factors were elevated in CM from ASM cells cultured from patients with mild or moderate asthma suggests that observed changes in angiogenic growth factor levels were unlikely to have resulted from gross differences in a dilution/concentration factor or a cell culture artifact. We performed manual cell counts after collection of CM to confirm cell numbers were unchanged after stimulation with IL-13 or TGF-β1, and that cell numbers did not vary according to reported differences in growth rates between ASM cells from healthy subjects and those from patients with asthma (32). Thus, our collective findings suggest that release of multiple proangiogenic growth factors is increased by ASM from individuals with asthma and that this cell type is potentially important in airway neovascularization processes in asthma.

It is reported that an imbalance exists between VEGF and endostatin (a potent endogenous inhibitor of angiogenesis and tumor growth [41]) levels in induced sputum from subjects with asthma in which the VEGF:endostatin ratio was increased by approximately twofold (21). Thus, in the present study, it was important to measure multiple factors implicated in angiogenesis including physiologically opposing factors. In keeping with the imbalance hypothesis (21), we demonstrate enhanced release of the proangiogenic factors VEGF, angiogenin, and Ang-1 by ASM cells cultured from patients with mild or moderate asthma compared with ASM from healthy subjects, whereas release of antiangiogenic factors including endostatin, Ang-2 (disrupts blood vessel formation by competing with equal affinity with Ang-1 at the endothelial cell-specific Tie2 receptor [20]), TIMP-1, and TIMP-2 was unchanged between healthy subjects and subjects with asthma. Previously, in BALF from subjects with mild asthma we have detected increased expression of TIMP-1 and TIMP-2 (34). 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 (42, 43). Physiologically, it would be expected that factors that initially promote endothelial cell migration and proliferation (such as VEGF and angiogenin) 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 the 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 diversity of angiogenic factors we detected in CM from ASM cells and their potential proangiogenic versus antiangiogenic properties, it was important to evaluate net activity in a biological assay system. A coculture of endothelial cells and human dermal fibroblasts was employed in which CM from ASM cultured from individuals with asthma induced the formation of CD31-positive capillary-like structures that were at least two- to threefold longer, more numerous, and more highly branched than those that formed either spontaneously or in response to CM from ASM cultured from healthy control subjects. 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. This was especially true with VEGF, Ang-1, and angiogenin. In a second model having greater relevance to airway vascularity, the capacity of these combined proangiogenic factors to induce neovascularization was evaluated in endobronchial biopsies from healthy subjects. The combination of VEGF, Ang-1, and angiogenin induced a near doubling of the CD31 vascularity score, which is similar in magnitude to the vascularity increase reported in mild and moderate asthma identified by vessel labeling with CD31 (44) or collagen IV (7, 8, 12, 13). Future studies are required to establish which of these angiogenic factors or combinations are required for induction of vascularity ex vivo.

Previously, we and others have demonstrated release of VEGF from nonasthmatic human ASM cells in response to various stimuli, including TGF-β, IL-1β, and TNF-{alpha} (25, 26, 36, 37). A consensus of new data indicates the presence of an inherent abnormality of ASM in asthma that not only claims enhanced contractile (45) and proliferative (32) properties, but now includes enhanced secretory responses involving the release of eotaxin (31) as well as multiple proangiogenic factors. Hasaneen and colleagues (46) reported recently that constitutive VEGF production is increased by cyclic mechanical strain involving multiple intracellular pathways leading to enhanced expression of HIF (hypoxia-inducible factor)-1{alpha}, a transcription factor that initiates and directly activates transcription of the VEGF gene. The importance of HIF-1{alpha} activation and expression in the underlying proangiogenic activity of ASM from individuals with asthma remains to be evaluated.

The contribution of factors secreted by ASM to the overall composition of airway lining secretions is unknown. Increased levels of VEGF are present in induced sputum in airway secretions from individuals with asthma (9, 11, 14, 21), as well as in ASM bundles in patients with chronic obstructive pulmonary disease (47), and positive correlations exist between elevated VEGF and increased total airway vascular area (1, 8, 12) and asthma severity (24). In light of the findings we report here, it will be important in future studies to determine if airway vascularity scores in asthma correlate with either ASM content or expression levels of VEGF in ASM. Hoshino and colleagues (8) have reported that VEGF is expressed by eosinophils within the bronchial mucosa of subjects with asthma. Elsewhere, VEGF levels correlate with the percentage of eosinophils and eosinophil chemotactic peptide levels in induced sputum from individuals with asthma (14). Other cell types acting as potential major sources of VEGF within the bronchial mucosa of individuals with asthma include macrophages and CD34+ cells, although epithelial cells and infiltrating T lymphocytes also express VEGF (16, 21–23, 47). We therefore targeted VEGF as the component of CM from asthmatic ASM most likely to drive the vascular changes in the in vitro angiogenesis assay. Possible discrepancies in the extent of tubule formation induced by rhVEGF and that corresponding to levels of VEGF released by ASM may reflect functional differences between rhVEGF and endogenous VEGF or the involvement of unidentified factors that synergize or otherwise support the efficacy of endogenous VEGF. Abrogation of tubule formation induced by ASM from healthy subjects or patients with asthma by treatment with a VEGF-neutralizing antibody indicated that VEGF was required, but not whether it was derived from the ASM itself or from the constituent cells of the in vitro angiogenesis assay. To exclude the latter, VEGF was depleted from CM before addition to the tubule formation assay. The observed abrogation of tubule formation in VEGF-depleted, but not Ang-1– or angiogenin-depleted, CM from asthmatic ASM suggested that the proangiogenic capacity of ASM cultured from patients with mild or moderate asthma was dependent primarily on VEGF. This, with the increased angiogenic growth factor content of CM from asthmatic ASM, provides strong evidence for active tissue neovascularisation processes occurring in the airways of individuals with mild and moderate asthma and supports the growing consensus that VEGF is an active participant of microvascular remodeling processes and asthma phenotype.

The effects of prior drug treatment on asthmatic cell function should be considered, as the majority of patients with asthma are treated with inhaled β2-agonists and/or glucocorticoids. In the present study all subjects with mild asthma were glucocorticoid-naïve, and only two of eight of the subjects with moderate asthma were regularly taking inhaled glucocorticoids (Table 1). It is unlikely that a direct effect of prior treatment would persist after ASM cells have been isolated and maintained in culture for many weeks, although treatment may have exerted a selection pressure in the airway inflammatory environment that could be translated to the cell cultures.

In summary, we report that ASM cultured from patients with mild or moderate asthma, but not from healthy subjects, promotes the formation of endothelial-derived capillary-like structures in an in vitro angiogenesis assay employing cocultures of endothelial cells and human dermal fibroblasts. Antibody arrays and ELISA of CM from asthmatic ASM suggest an imbalance in the production of proangiogenic versus anti-angiogenic growth factors. Selective immunodepletion of VEGF abolished the proangiogenic capacity of CM obtained from ASM from patients with asthma, confirming that the proangiogenic capacity was dependent on VEGF derived from ASM. These findings suggest that ASM has the capacity to initiate and sustain neovascularization processes in the asthmatic airway. Furthermore, the fact that ASM from patients with asthma were constitutively active in this respect, and bronchial vessels and ASM bundles are found in close proximity (3, 12, 27), suggests a bidirectional relationship in which ASM may directly regulate airway wall vascularity to sustain the baseline metabolic demands of the increased ASM content that characterizes even mild asthma (4, 28, 48).


   Acknowledgments
 
The authors gratefully acknowledge the invaluable contribution of Professor Chris Corrigan and the research nursing staff in the department (Amanda Chaytor, Wei-Yee James, Kheem Jones, Marianne Morgan and Cherylin Reinholtz) for recruitment and screening of volunteers and for assistance with bronchoscopy.


   FOOTNOTES
 
* These authors contributed equally to this study. Back

Supported by a King's College London Studentship (D.E.S) and Asthma UK (No. 05/022, No. 05/027, and No. 07/034).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.200707-1046OC on June 12, 2008

Conflict of Interest Statement: D.E.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. V.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.W.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.J.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.J.H. was a discussant at a scientific meeting organized and financed by GlaxoSmithKline in January 2004.

Received in original form July 16, 2007; accepted in final form June 11, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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