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In Vitro Modeling of Fibroblast-Derived Matrix Deposition, Stiffness, and Remodeling
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A4361 - In Vitro Modeling of Fibroblast-Derived Matrix Deposition, Stiffness, and Remodeling
Author Block: A. Haak, D. Sicard, A. Diaz Espinosa, D. J. Tschumperlin; Physiology and Biomed Eng, Mayo Clinic, Rochester, MN, United States.
Rationale: Pulmonary fibrosis is characterized by progressive and ultimately fatal end-stage lung scarring associated with uncontrolled deposition, stiffening and complex remodeling of fibrous connective tissue proteins. Activated resident fibroblasts represent the pathological driver of these events and are often the cell-of-choice for modeling fibrosis in vitro: however, assays which can directly measure deposition of extracellular matrix proteins and quantitatively analyze the stiffness and remodeling of cell-derived matrices are lacking.
Methods: We developed two assays to measure matrix deposition, stiffness, and remodeling. The first is a multiplex high-throughput immuno-extracellular matrix (iECM) assay in which pulmonary fibroblasts are plated at confluence and allowed to deposit matrix into a 96-well tissue culture plate over 3 days to identify cell culture conditions and test reagents that enhance or suppress deposition of collagen type I and fibronectin. We translated these results to a larger scale 10-day matrix stiffness and remodeling assay. For this assay lung fibroblasts are cultured for 10 days in varying conditions and the stiffness (Young’s modulus) of the cells and their cell-derived matrices are analyzed by atomic force microscopy (AFM) microindentation followed by RNA isolation.
Results: In the initial iECM assays we observed enhanced collagen type I and fibronectin deposition in the presence of TGF-beta, ascorbic acid, and copper, consistent with known roles for these components in fibroblast activation, ECM deposition and collagen crosslinking. In the cell-derived matrix stiffness assay, AFM measurements detected a ~2X fold increase in stiffness with TGF-beta (~10kPa vs. ~20kPa Young’s modulus) after 6-10 days in culture. Following stiffness measurements, we isolated RNA and measured enhanced expression of extracellular matrix genes (COL1A1 and FN1), matrix crosslinking genes (LOX, LOXL1, 2, 3, and 4) and reduced expression of genes associated with extracellular matrix proteolysis and degradation (MMP14, CTSK, PLAT, and PLAU).
Conclusions: The development of these assays enhances the ability to model the major pathological role of fibroblasts in pulmonary fibrosis and represents an expanded experimental toolkit for determining the efficacy and mechanistic effects of potential antifibrotic therapeutics.
Funding: NIH HL092961, and The American Lung Association
Author Block: A. Haak, D. Sicard, A. Diaz Espinosa, D. J. Tschumperlin; Physiology and Biomed Eng, Mayo Clinic, Rochester, MN, United States.
Rationale: Pulmonary fibrosis is characterized by progressive and ultimately fatal end-stage lung scarring associated with uncontrolled deposition, stiffening and complex remodeling of fibrous connective tissue proteins. Activated resident fibroblasts represent the pathological driver of these events and are often the cell-of-choice for modeling fibrosis in vitro: however, assays which can directly measure deposition of extracellular matrix proteins and quantitatively analyze the stiffness and remodeling of cell-derived matrices are lacking.
Methods: We developed two assays to measure matrix deposition, stiffness, and remodeling. The first is a multiplex high-throughput immuno-extracellular matrix (iECM) assay in which pulmonary fibroblasts are plated at confluence and allowed to deposit matrix into a 96-well tissue culture plate over 3 days to identify cell culture conditions and test reagents that enhance or suppress deposition of collagen type I and fibronectin. We translated these results to a larger scale 10-day matrix stiffness and remodeling assay. For this assay lung fibroblasts are cultured for 10 days in varying conditions and the stiffness (Young’s modulus) of the cells and their cell-derived matrices are analyzed by atomic force microscopy (AFM) microindentation followed by RNA isolation.
Results: In the initial iECM assays we observed enhanced collagen type I and fibronectin deposition in the presence of TGF-beta, ascorbic acid, and copper, consistent with known roles for these components in fibroblast activation, ECM deposition and collagen crosslinking. In the cell-derived matrix stiffness assay, AFM measurements detected a ~2X fold increase in stiffness with TGF-beta (~10kPa vs. ~20kPa Young’s modulus) after 6-10 days in culture. Following stiffness measurements, we isolated RNA and measured enhanced expression of extracellular matrix genes (COL1A1 and FN1), matrix crosslinking genes (LOX, LOXL1, 2, 3, and 4) and reduced expression of genes associated with extracellular matrix proteolysis and degradation (MMP14, CTSK, PLAT, and PLAU).
Conclusions: The development of these assays enhances the ability to model the major pathological role of fibroblasts in pulmonary fibrosis and represents an expanded experimental toolkit for determining the efficacy and mechanistic effects of potential antifibrotic therapeutics.
Funding: NIH HL092961, and The American Lung Association
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