Recent investigations into asthma pathogenesis have begun to focus, not on the acute inflammatory events involved with asthmatic airway inflammation, but on aberrant repair mechanisms which appear to be present [28–30]. This work focused on vasodilator-stimulated phosphoprotein (VASP) which is predicted to be involved in epithelial repair mechanisms by mediating focal adhesion, actin filament binding and polymerization, and ultimately, epithelial cell mobility. Reports published during the course of our studies have shown that detachment of kidney epithelial cells increases protein kinase A activity and its phosphorylation of VASP , as well as a transient increase in VASP expression . Although there was some increase in total VASP (46 KD + 50 KD forms) in asthmatics compared to normals on day 2 in the recovery from injury protocol, the difference was not significant, and probably does not contribute substantially to cell adherence and motility. We hypothesized that asthmatics would demonstrate increased VASP phosphorylation compared to nonasthmatic, normal subjects to permit actin remodeling and cell migration for repair of inflammatory injury. Instead, we observed a potential defect as shown by decreased VASP phosphorylation in asthmatic epithelial cells prior to and following allergen-induced injury compared to normal subjects. Increased VASP phosphorylation in response to segmental antigen challenge was modest in both asthmatic and nonasthmatic normal epithelial cells, but overall a profound decrease in the VASP phosphorylation was observed in asthmatic epithelial cells at all time points examined. The reduced VASP phosphorylation in epithelial cells of asthmatics was confirmed by expanded examination of an additional group of subjects, both asthmatic and normal (Table 4). It has been previously shown that blocking of phosphorylation at MENA Ser 236, which corresponds in molecular structure to VASP Ser 157, or depletion of ENA/VASP results in increased cell spreading and reduced functional control of cell motility in a number of model systems [3–5, 11]. More directly, 10 μM PGE1 converts 60% of the 46 KD VASP to 50 KD VASP and completely inhibits platelet aggregation as a measure of cell-cell adhesion . Thus, even partial conversion of VASP to its phosphorylated form has significant impact. This suggests one potential mechanism for aberrant epithelial repair in asthmatics: defective or diminished VASP phosphorylation may indicate abnormal epithelial motility. Confirming defective epithelial cell motility in asthmatics in vivo will be challenging, but is a necessary next step in this work.
While segmental antigen challenge did not significantly increase VASP phosphorylation in the recovery from injury protocol "a", there was a trend toward a significant increase, which was confirmed in asthmatics enrolled in the regular β-agonist inhalation protocol "b". In addition, leukocytes in bronchoalveolar lavage fluid from asthmatics co-cultured with autologous epithelium did significantly increase VASP phosphorylation, unlike the leukocytes from nonasthmatic normal subjects, suggesting a soluble signal from the BAL leukocytes to epithelial cells in asthmatics. What the signal(s) may be remains to be determined, but other work from our laboratory suggests certain growth factors such as TGFβ, could be involved.
Because VASP becomes phosphorylated by cAMP-dependent protein kinase, we also hypothesized that both in vivo and in vitro exposure of epithelial cells to a β-agonist, which increases cyclic AMP levels, should increase VASP phosphorylation. As predicted, regularly inhaled albuterol increased VASP phosphorylation in vivo and apparently altered epithelial cell adhesion, producing significantly greater numbers of epithelial cells shed into bronchoalveolar lavage fluid from unchallenged lung segments exposed only to the β-agonist, without any allergen-induced inflammation. The β-agonist effect on VASP phosphorylation was short-lived and the ratio of 50/46 KD VASP returned to baseline within 12 hr of the last albuterol inhalation in vivo. Whether the increased epithelial cell detachment induced by albuterol inhalation returns to baseline as quickly has not been determined. β-agonist use may therefore produce conflicting effects on asthmatic epithelium. β-agonists inhibit keratinocyte migration by β2-adrenergic receptor activation of the serine/threonine phosphatase PP2A , the principal phosphatase which dephosphorylates VASP , and at the same time activate cAMP-dependent protein kinase phosphorylation of VASP [6–9]. Thus, β-agonists may promote epithelial repair by enhancing both phosphorylation and dephosphorylation of VASP in actin filament restructuring, but may also promote epithelial damage by increasing the detachment of epithelial cells from the airway. These results also caution against considering as "baseline" or "control," samples obtained from asthmatics receiving β-agonist therapy without an appropriate washout interval.
Genetic variation and altered function of the β2-adrenergic receptor could potentially contribute to the differences in VASP phosphorylation observed between asthmatics and nonasthmatic normal subjects . However, examination of 50/46 KD VASP ratio grouped according to β2-adrenergic receptor haplotype for our study subjects did not reveal any specific haplotype bias, either homozygous or heterozygous, affecting VASP phosphorylation. Although the numbers of subjects in any one haplotype classification were limited, the largest 2/4 haplotype group, nonasthmatic normal subjects showed a significantly increased ratio compared to asthmatic subjects. It is possible that other characteristics of the β2-adrenergic receptor gene (for example, stability of message due to variation in the 3' untranslated region, ), variation in other components in the signaling cascade such as cAMP dependent protein kinase A [6–9], or activation of protein kinase C , genetic variation in VASP itself, or differences in the activity of protein phosphatases  between asthmatics and normals may contribute to the observed disparity in VASP phosphorylation here between asthmatic and normal subjects. Work is currently addressing these areas of inquiry.