Bronchial smooth muscle cells of asthmatics promote angiogenesis through elevated secretion of CXC-chemokines (ENA-78, GRO-α, and IL-8)

Abstract

Background: Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors.

Objective: To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors.

Methods: Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay.

Results: Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC.

Conclusions: BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC.

Implications: CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.

Figure 1. Milligan’s trichrome stained sections of airway tissue from non-asthmatic (A) and asthmatic (B) patients.

Images are representative of tissues obtained from 3 non-asthmatic and 3 asthmatic patients. Nuclei and muscle: magenta, collagen: green, RBC: orange. Note epithelial hyperplasia, thickening of muscle bundles and basement membrane, and increased micro-vessel density in asthmatic airways. E = epithelium, MB = muscle bundles, BM = basement membrane, MV = micro-vessels.

Figure 2. Angiogenesis assay in vitro.

A, Images illustrating sprout outgrowth from EC-spheroids incubated with unconditioned control medium or CM from BSMC. B, Lengths of sprouts outgrowing from every spheroid were measured and the mean of the longest 10 sprouts was calculated. Experiments were performed on 3 separate occasions using control medium and CM derived from 3 different non-asthmatic (NA) and asthmatic (A) patients. Values are given as mean ± SD. *** p < 0.001.

Figure 3. Human angiogenesis antibody array.

Examples of the angiogenesis antibody array (exp. 3) comparing CM from BSMC of non-asthmatic (A) and asthmatic (B) patients. C, Antibody array map. Standard abbreviations for the detected proteins are used, Pos: positive control, Neg: negative control, IC1-IC3: internal controls 1-3. D, Quantitative analysis from each of the 4 independent experiments performed. Intensity ratios (A:NA) in a paired analysis are shown. Upregulation in any single experiment is indicated by a cross. nq = not quantifiable (out of range).

Figure 4. Chemokine release from BSMC derived from asthmatics and non-asthmatics.

Concentrations of GRO-α (A), IL-8 (B) and ENA-78 (C) in CM were collected from BSMC of non-asthmatic (NA) and asthmatic (A) patients after 24 h and 72 h were determined by ELISA.* p < 0.05 (n = 6).

Figure 5. Involvement of CXCR2 in BSMC-induced neovascularization.

A, RT-PCR of CXCR2 in HMEC-1. B, Immunofluorescence detection of CXCR2 on HMEC-1. C, D, HMEC-1 monolayers under normal EC growth conditions were cultured without or with SB 265610 for 48h (C) or 24h (D) (mean ± SD, n=3) and evaluated for proliferation (C) and viability (D). E, Effect of SB 265610 on sprout outgrowth induced by CM of BSMC from asthmatics. Values are mean ± SEM after normalization to the control condition. *** p < 0.001 (n ≥ 3).