A. Capetandes’s research while affiliated with Nassau University Medical Center and other places

Beta(2)-adrenergic receptor agonists have been shown to modulate airway epithelial cell and smooth muscle release of cytokines and growth factors transforming growth factor (TGF) beta, associated with remodeling, is known to up-regulate the synthesis of vascular endothelial growth factor (VEGF) and stimulate differentiation of fibroblasts to the myofibroblast phenotype. VEGF and fibronectin can promote angiogenesis and (S)-albuterol can induce VEGF secretion from normal human lung fibroblasts (NHLF). We hypothesize that (S)-albuterol could stimulate myofibroblast secretion and expression of VEGF and fibronectin in the presence of Dermatophagoides pteronyssinus extract. Cultured NHLFs were stimulated with IL-1beta, TGF-beta, D. pteronyssinus, and treated with (R)- and (S)-enantiomers of albuterol. VEGF and fibronectin and basic fibroblast growth factor (bFGF) were measured by ELISA and mRNA. VEGF secretion by fibroblasts was twofold higher with 10(-7) M of (R) relative to (S) (p < 0.05). Myofibroblast secretion of VEGF was increased twofold over fibroblasts, but there was no difference between enantiomers. (S)-albuterol at 10(-8)-10(-4) M caused an increase in VEGF mRNA that paralleled VEGF secretion relative to 10(-8)-10(-4) M. Fibronection secretion by myofibroblasts but not fibroblasts was increased by 10(-5) M of (S) relative to (R) in the presence of recombinant interleukin 1 (rhIL-1)beta and D. pteronyssinus (S)-albuterol at 10(-6) M increased bFGF. The 10(-6) M of (S)-albuterol, but not (R)-albuterol, may promote angiogenesis. Increased fibronectin or bFGF by (S)-albuterol could enhance matrix deposition and remodeling in a subset of asthmatic patients.

Airway remodeling may lead to increased secretion of TGF-beta. TGF-beta is known to activate fibroblasts and to stimulate the secretion of vascular endothelial growth factor (VEGF). Soluble and cell-associated VEGF have been identified in subjects with chronic severe asthma. Dermatophagoides sp. has been shown to contribute to the pathogenesis of asthma. The aim of this study was to determine if the Dermatophagoides sp. extract can stimulate confluent A549 (cA549) to express soluble or cell-associated VEGF and to secrete other factors that stimulate normal human lung fibroblasts (NHLFs) to secrete VEGF. Immunocytochemistry for cell-associated VEGF (pg/mL) was performed on cA549 stimulated with 300, 600, and 1000 AU/mL of dialyzed Dermatophagoides sp. extract for 24 hours with serum-free media (SFM). The subsequent conditioned media (CM) was transferred to NHLF for 24 hours (CM-NHLF). VEGF in CM, CM-NHLF, and control media (CTLM; extract without cA549) were measured by ELISA and normalized to cell density as measured by absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide at A550 nm (VEGF pg/mL:A550). Parametric data were analyzed by t-test or ANOVA at alpha = 0.05. Dermatophagoides sp. extract increased normalized VEGF secretion by cA549. Immunochemical VEGF expression by cA549 was increased qualitatively by Dermatophagoides sp. extract. The 1000 CM-NHLF showed increased normalized VEGF relative to CTLM. Dermatophagoides sp. stimulates cA549 to increase soluble and immunochemical VEGF and to secrete mediators that stimulate NHLF to increase secretion of VEGF. This suggests that inhalation of dust-mite allergen may stimulate airway cells to increase VEGF secretion, potentially contributing to edema in airway remodeling.

Rationale Granulocyte-macrophage colony-stimulating factor (GM-CSF) is overproduced by human keratinocytes (HKs) in chronic lesions of atopic dermatitis (AD) (Boguniewicz M. JACI 2006;117 Suppl:475-80). Increased concentration of GM-CSF could explain persistent inflammation with activation of dendritic cells. Additionally, Staphylococcus aureus exacerbates chronic lesions in AD by secreting superantigens, stimulating marked activation of T cells, macrophages, and eosinophils. Methods Confluent monolayers of HKs (ATCC CRL 2309) were stimulated with a cytomix composed of 100 mg/mL staphylococcal enterotoxin B and 10 U/mL rhIL-1β as determined by dose-response studies. HKs were stimulated with cytomix ± 10⁻⁶ to 10⁻¹⁰ M pimecrolimus (dosing range determined by dose-response studies) for 24 hours in serum-free media supplemented with insulin-transferrin-selenium (SFM) using standard cell culture conditions. The conditioned media was assayed for GM-CSF secretion by ELISA at the end of 24 hours of incubation. The HKs were analyzed for cell viability and proliferation using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] incorporation assay. The GM-CSF data (pg/mL) were normalized to the MTT absorption data (A550 nm). Parametric data are expressed below as the fold change mean ± 2 SD of normalized GM-CSF secretion by HKs incubated with cytomix plus drug relative to control (cytomix without drug) and analyzed by the two-tailed t-test (a = 0.05). Power for all studies was > 0.6. Results HKs showed > 95% viability by the MTT incorporation test in the presence of SFM. HK viability decreased to 50 to 70% in the presence of cytomix. The cytomix stimulated normalized GM-CSF secretion over SFM by 2.0 ± 0.9-fold. 10⁻⁶ to 10⁻¹⁰ M pimecrolimus did not improve the cell viability relative to cytomix; 10⁻⁶ and 10⁻⁷ M pimecrolimus in the presence of cytomix decreased normalized GM-CSF secretion by 2.0 ± 0.4 and 4.2 ± 3.0-fold, respectively, relative to cytomix alone (p < .05); 10⁻⁸ to 10⁻¹⁰ M pimecrolimus showed no statistical differences in the percent change in normalized GM-CSF secretion relative to cytomix alone. Conclusion 10⁻⁶ and 10⁻⁷ M pimecrolimus inhibited cytomix-induced increased normalized GM-CSF secretion by HKs in SFM. Funding: Novartis Pharmaceuticals Corporation.

Chronic severe persistent asthma is associated with damaged epithelial cells with discontinuous tight junctions that contribute to dysregulated fibroblast and endothelial cell (mesenchymal) growth. Dermatophagoides species-derived proteases have been shown to cause damage to epithelial cell tight junctions. To determine whether Dermatophagoides species can stimulate confluent A549 (cA549), a cell type with discontinuous tight junctions that approximate differentiated type II cells, to undergo altered growth and secrete putative soluble factors that affect the growth of human lung fibroblasts and microvascular endothelial cells. Dialyzed Dermatophagoides pteronyssinus or Dermatophagoides farinae extracts (0, 300, 600, and 1000 AU/mL) were cultured with and without cA549 in serum-free media for 24 hours. After changes in cA549 growth were recorded, conditioned media from extracts with cA549 (CM) and without cA549 (control media [CTLM]) were transferred to fibroblasts and endothelial cells for 24 hours. Fibroblast and endothelial cell growth responses to CM and CTLM were observed and measured. All conditions showed greater than 95% cell viability. Confluent A549 showed dose-dependent growth changes characterized by increased aggregation when incubated with 300, 600, and 1000 AU/mL of D pteronyssinus in serum-free media relative to control. The CM, but not the CTLM, induced dose-dependent aggregation by fibroblasts and endothelial cells. Fibroblasts also showed decreased adhesion when incubated with CM. Dermatophagoides farinae-treated cA549 showed similar but weaker results. The use of serum, boiled CM, or boiled extract inhibited these findings. Dialyzed Dermatophagoides species extracts altered cA549 growth and stimulated the secretion of factors that dysregulate mesenchymal cell growth in vitro.