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Softening Cellular Microenvironments Augments Glucocorticoid Actions
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A2931 - Softening Cellular Microenvironments Augments Glucocorticoid Actions
Author Block: A. B. Mitke1, T. Harris1, S. Langenbach1, M. Schuliga2, F. Jativa3, P. Lee3, J. Jaffar4, G. Westall4, A. Stewart1; 1Pharmacology and Therapeutics, The University of Melbourne, Melbourne, Australia, 2Hunter Medical Research Institute, Univ of Newcastle, Callaghan, Australia, 3Mechanical Engineering, The University of Melbourne, Melbourne, Australia, 4Alfred Hospital, Melbourne, Australia.
Rationale: Chronic airway diseases, which are characterized by airway remodeling and deposition of extracellular matrix generate a stiffer mechanical environment. Matrix stiffness alters cell morphology, cell cycle and responsiveness to pharmacological agents. Glucocorticoids are less effective in severe asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). In this study, the role of the mechanical microenvironment and dimensionality (2D vs 3D) on glucocorticoid responsiveness is investigated in vitro.
Methods: Human pulmonary fibroblasts and human airway smooth muscle (HASM) cells were established in three culture settings: conventional 2D (2D stiff) cultures established on uncoated 24-well plates; 2D soft cultures generated on 24-well plates coated with rat tail type I collagen solution; and, 3D cultures (spheroids) generated in low-adherence polyhydroxyethylmethacrylate (poly-HEMA) coated round-bottom 96-well plates by centrifugation at 1,000 RCF for 10 minutes. The expression of glucocorticoid inducible genes, pro-remodeling genes and mechanosensors was measured using qRT-PCR. Cytokines levels in cultures medium were measured by ELISA.
Results: In 3D fibroblast (both non-IPF and IPF) and HASM cell cultures incubated with dexamethasone for 16 hours, pyruvate dehydrogenase kinase isozyme 4 (PDK4) expression was strongly induced by comparison with responses in 2D stiff culture. Similarly, the expression of glucocorticoid-induced leucine zipper (GILZ) and MAP kinase phosphatase-1 (MKP-1) was augmented in 2D soft and 3D cultures. Dexamethasone concentration-response curves in 2D soft and 3D cultures were amplified, showing increased maxima without substantial changes in the effective concentration eliciting 50% maximal response (EC50), compared to curves generated in 2D stiff cultures. TGF-β1, which induces glucocorticoid resistance in 2D stiff cultured cells, did not affect responsiveness to dexamethasone in 2D soft or 3D cultures. Elevated expression of cyclooxygenases 2 (COX-2) in 2D soft and 3D cultures, was suppressed by dexamethasone in a concentration-dependent manner.
Conclusion: The efficacy of glucocorticoids increases in softer cellular microenvironments. Drugs that decrease stiffness of fibrotic tissue may also augment glucocorticoid activity.
Author Block: A. B. Mitke1, T. Harris1, S. Langenbach1, M. Schuliga2, F. Jativa3, P. Lee3, J. Jaffar4, G. Westall4, A. Stewart1; 1Pharmacology and Therapeutics, The University of Melbourne, Melbourne, Australia, 2Hunter Medical Research Institute, Univ of Newcastle, Callaghan, Australia, 3Mechanical Engineering, The University of Melbourne, Melbourne, Australia, 4Alfred Hospital, Melbourne, Australia.
Rationale: Chronic airway diseases, which are characterized by airway remodeling and deposition of extracellular matrix generate a stiffer mechanical environment. Matrix stiffness alters cell morphology, cell cycle and responsiveness to pharmacological agents. Glucocorticoids are less effective in severe asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). In this study, the role of the mechanical microenvironment and dimensionality (2D vs 3D) on glucocorticoid responsiveness is investigated in vitro.
Methods: Human pulmonary fibroblasts and human airway smooth muscle (HASM) cells were established in three culture settings: conventional 2D (2D stiff) cultures established on uncoated 24-well plates; 2D soft cultures generated on 24-well plates coated with rat tail type I collagen solution; and, 3D cultures (spheroids) generated in low-adherence polyhydroxyethylmethacrylate (poly-HEMA) coated round-bottom 96-well plates by centrifugation at 1,000 RCF for 10 minutes. The expression of glucocorticoid inducible genes, pro-remodeling genes and mechanosensors was measured using qRT-PCR. Cytokines levels in cultures medium were measured by ELISA.
Results: In 3D fibroblast (both non-IPF and IPF) and HASM cell cultures incubated with dexamethasone for 16 hours, pyruvate dehydrogenase kinase isozyme 4 (PDK4) expression was strongly induced by comparison with responses in 2D stiff culture. Similarly, the expression of glucocorticoid-induced leucine zipper (GILZ) and MAP kinase phosphatase-1 (MKP-1) was augmented in 2D soft and 3D cultures. Dexamethasone concentration-response curves in 2D soft and 3D cultures were amplified, showing increased maxima without substantial changes in the effective concentration eliciting 50% maximal response (EC50), compared to curves generated in 2D stiff cultures. TGF-β1, which induces glucocorticoid resistance in 2D stiff cultured cells, did not affect responsiveness to dexamethasone in 2D soft or 3D cultures. Elevated expression of cyclooxygenases 2 (COX-2) in 2D soft and 3D cultures, was suppressed by dexamethasone in a concentration-dependent manner.
Conclusion: The efficacy of glucocorticoids increases in softer cellular microenvironments. Drugs that decrease stiffness of fibrotic tissue may also augment glucocorticoid activity.
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