Alterations in vasodilator-stimulated phosphoprotein (VASP) phosphorylation: associations with asthmatic phenotype, airway inflammation and beta2-agonist use

Abstract

Background: Vasodilator-stimulated phosphoprotein (VASP) mediates focal adhesion, actin filament binding and polymerization in a variety of cells, thereby inhibiting cell movement. Phosphorylation of VASP via cAMP and cGMP dependent protein kinases releases this "brake" on cell motility. Thus, phosphorylation of VASP may be necessary for epithelial cell repair of damage from allergen-induced inflammation. Two hypotheses were examined: (1) injury from segmental allergen challenge increases VASP phosphorylation in airway epithelium in asthmatic but not nonasthmatic normal subjects, (2) regular in vivo beta2-agonist use increases VASP phosphorylation in asthmatic epithelium, altering cell adhesion.

Methods: Bronchial epithelium was obtained from asthmatic and non-asthmatic normal subjects before and after segmental allergen challenge, and after regularly inhaled albuterol, in three separate protocols. VASP phosphorylation was examined in Western blots of epithelial samples. DNA was obtained for beta2-adrenergic receptor haplotype determination.

Results: Although VASP phosphorylation increased, it was not significantly greater after allergen challenge in asthmatics or normals. However, VASP phosphorylation in epithelium of nonasthmatic normal subjects was double that observed in asthmatic subjects, both at baseline and after challenge. Regularly inhaled albuterol significantly increased VASP phosphorylation in asthmatic subjects in both unchallenged and antigen challenged lung segment epithelium. There was also a significant increase in epithelial cells in the bronchoalveolar lavage of the unchallenged lung segment after regular inhalation of albuterol but not of placebo. The haplotypes of the beta2-adrenergic receptor did not appear to associate with increased or decreased phosphorylation of VASP.

Conclusion: Decreased VASP phosphorylation was observed in epithelial cells of asthmatics compared to nonasthmatic normals, despite response to beta-agonist. The decreased phosphorylation does not appear to be associated with a particular beta2-adrenergic receptor haplotype. The observed decrease in VASP phosphorylation suggests greater inhibition of actin reorganization which is necessary for altering attachment and migration required during epithelial repair.

Figure 1

Western blot of bronchial epithelial cells demonstrating 50 and 46 KD isoforms of VASP. Representative western blot of bronchial epithelial cells from an asthmatic subject developed with anti-VASP antibody to show the banding pattern for the 46 KD and phosphorylated 50 KD isoforms of VASP. Bronchial epithelial cells received either no treatment for 2 or 24 hr or treatment with 10^-7 albuterol for 2 or 24 hr. The treatment with albuterol increased the band density for the 50 KD VASP isoform relative to the 46 KD isoform, greater at 2 hr than after 24 hr chronic albuterol treatment.

Figure 2

VASP phosphorylation in epithelial cells of asthmatic and normal control subjects following allergen challenge. Ratio of 50/46 KD VASP in epithelial cells of asthmatic (squares) and nonasthmatic normal subjects (circles) from before (Day 1) and after segmental allergen challenge (Days 2, 9 and 16). Epithelial cells were cultured for 24 hr with (dashed lines) or without (solid lines) BAL cell co-culture. Segmental allergen challenge increased the phosphorylation of VASP in asthmatic and normal epithelial cells but did not reach significance (p = 0.08). BAL cell co-culture exerted a significant effect on VASP phosphorylation in asthmatic (p = 0.022) but not normal epithelial cells. Nevertheless, asthmatic epithelial cells had significantly less VASP phosphorylation (lower 50/46 KD VASP ratio) at all timepoints compared to nonasthmatic normal epithelial cells (p < 0.001 without BAL cells; p = 0.006 with BAL cells).

Figure 3

VASP phosphorylation in epithelial cells of asthmatics after regular albuterol or placebo inhalation. Ratio of 50/46 KD VASP in asthmatic epithelial cells without BAL cell co-culture from subjects either on regular albuterol inhalation (A) or on regular placebo inhalation (B). Epithelial cells were obtained from unchallenged lung segments (Days 1, and 18, broken lines), from Ag challenged lung segments (Days 2, and 19, solid lines) and from resampled lung segments on Day 25 (unchallenged Day 1 segment, and challenged Day 2 segment). Two weeks of regular β₂-agonist inhalation, but not placebo inhalation, resulted in significantly increased phosphorylation of VASP in both unchallenged and challenged lung segments' epithelium (p = 0.009). Overall, VASP phosphorylation, i.e. a larger 50/46 KD VASP ratio, was greater in epithelium from challenged than from unchallenged lung segments in the regular β₂-agonist inhalation protocol (p = 0.017), but not in the placebo inhalation protocol (B panel).

Figure 4

Numbers of epithelial cells in control segment BAL fluid from asthmatics before and after regular β-agonist or placebo inhalation. Epithelial cell numbers in unchallenged lung segment BAL fluid prior to any inhalation (A, day 1) and in unchallenged lung segment BAL fluid after two weeks regular inhalation of albuterol or placebo (B, day 18). The numbers of cells stained with Alcian Blue for mucous (gray shaded bars), nonspecific esterase for ciliated (hatched bars), other columnar cells (open bars) and the total of these three (solid bars) per ml were increased after two weeks of regularly inhaled albuterol compared to placebo (p < 0.001 for both Alcian blue and total groups). There was also a significant effect for day in Alcian blue stained cells (day 1 versus day 18, p = 0.014) and a significant group × day interaction in Alcian blue and total cell groups for albuterol inhalation (p = 0.004 and p = 0.014, respectively).