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Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells.

Robinson BK, Cortes E, Rice AJ, Sarper M, Del Río Hernández A - Biol Open (2016)

Bottom Line: We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling.Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer.We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Faculty of Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

No MeSH data available.


Related in: MedlinePlus

PSCs’ ability to remodel ECM is dependent on actomyosin machinery and activation state. (A) Bright-field images of matrix remodelled by PSCs (control, BBI, ATRA and β1 integrin inhibition). (B) Immunofluorescence of PSCs (red) and SHG imaging of collagen-I (green) for remodelled matrices. (C) Images in B represented through the BoneJ plugin to calculate fibre thickness where larger spheres fit along fibres represent greater thickness. (D) Graphs of matrix contraction, collagen alignment and collagen thickness, respectively. Each point represents a matrix for percentage contraction. Acellular, n=10; control, n=14; BBI, n=10; ATRA, n=12; β1, n=5. In the box-and-whisker plot for alignment, the central box represents values from the lower to upper quartile, the middle line represents the mean. The vertical line extends from the minimum to the maximum value. For alignment: control, n=28; BBI, n=20; ATRA, n=78; β1, n=40. Histograms of fibre thickness are plotted as mean±s.e.m. Control, n=10; BBI, n=50; ATRA, n=20; β1, n=30. ***P<0.0001 (unpaired t-test). Scale bars: 50 μm.
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BIO017632F5: PSCs’ ability to remodel ECM is dependent on actomyosin machinery and activation state. (A) Bright-field images of matrix remodelled by PSCs (control, BBI, ATRA and β1 integrin inhibition). (B) Immunofluorescence of PSCs (red) and SHG imaging of collagen-I (green) for remodelled matrices. (C) Images in B represented through the BoneJ plugin to calculate fibre thickness where larger spheres fit along fibres represent greater thickness. (D) Graphs of matrix contraction, collagen alignment and collagen thickness, respectively. Each point represents a matrix for percentage contraction. Acellular, n=10; control, n=14; BBI, n=10; ATRA, n=12; β1, n=5. In the box-and-whisker plot for alignment, the central box represents values from the lower to upper quartile, the middle line represents the mean. The vertical line extends from the minimum to the maximum value. For alignment: control, n=28; BBI, n=20; ATRA, n=78; β1, n=40. Histograms of fibre thickness are plotted as mean±s.e.m. Control, n=10; BBI, n=50; ATRA, n=20; β1, n=30. ***P<0.0001 (unpaired t-test). Scale bars: 50 μm.

Mentions: The ability of myofibroblasts to remodel the matrix relies on the ability to apply contractile forces (Calvo et al., 2013; Goetz et al., 2011). To gain more insight into the mechanisms underlying the ECM remodelling capacity by PSCs, we analysed collagen topology and structure in the presence of blebbistatin (BBI) and all-trans retinoic acid (ATRA). BBI blocks myosin II ATPase activity, and thus actomyosin contraction (Kovacs et al., 2004), and ATRA is the active metabolite of vitamin A and induces quiescence on PSCs (Froeling et al., 2011). Inhibition of actomyosin contraction in PSCs using BBI or inducing quiescence with ATRA significantly reduced the capacity of PSCs to contract the matrix (Fig. 5A,D). In both cases, the collagen alignment score and the fibre thickness significantly reduced with respect to the control condition (Fig. 5A-D; Fig. S1B). A significant decrease in the collagen intensity was also noted within the matrix embedded with PSCs treated with BBI and ATRA (Fig. S2C). We further observed a significant decrease in the spacing between collagen fibres in the matrices treated with BBI and ATRA (Fig. S2E) Taken together these results indicate that PSCs' capacity to remodel the matrix is only present in the active state and depends on their actomyosin machinery.Fig. 5.


Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells.

Robinson BK, Cortes E, Rice AJ, Sarper M, Del Río Hernández A - Biol Open (2016)

PSCs’ ability to remodel ECM is dependent on actomyosin machinery and activation state. (A) Bright-field images of matrix remodelled by PSCs (control, BBI, ATRA and β1 integrin inhibition). (B) Immunofluorescence of PSCs (red) and SHG imaging of collagen-I (green) for remodelled matrices. (C) Images in B represented through the BoneJ plugin to calculate fibre thickness where larger spheres fit along fibres represent greater thickness. (D) Graphs of matrix contraction, collagen alignment and collagen thickness, respectively. Each point represents a matrix for percentage contraction. Acellular, n=10; control, n=14; BBI, n=10; ATRA, n=12; β1, n=5. In the box-and-whisker plot for alignment, the central box represents values from the lower to upper quartile, the middle line represents the mean. The vertical line extends from the minimum to the maximum value. For alignment: control, n=28; BBI, n=20; ATRA, n=78; β1, n=40. Histograms of fibre thickness are plotted as mean±s.e.m. Control, n=10; BBI, n=50; ATRA, n=20; β1, n=30. ***P<0.0001 (unpaired t-test). Scale bars: 50 μm.
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Related In: Results  -  Collection

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BIO017632F5: PSCs’ ability to remodel ECM is dependent on actomyosin machinery and activation state. (A) Bright-field images of matrix remodelled by PSCs (control, BBI, ATRA and β1 integrin inhibition). (B) Immunofluorescence of PSCs (red) and SHG imaging of collagen-I (green) for remodelled matrices. (C) Images in B represented through the BoneJ plugin to calculate fibre thickness where larger spheres fit along fibres represent greater thickness. (D) Graphs of matrix contraction, collagen alignment and collagen thickness, respectively. Each point represents a matrix for percentage contraction. Acellular, n=10; control, n=14; BBI, n=10; ATRA, n=12; β1, n=5. In the box-and-whisker plot for alignment, the central box represents values from the lower to upper quartile, the middle line represents the mean. The vertical line extends from the minimum to the maximum value. For alignment: control, n=28; BBI, n=20; ATRA, n=78; β1, n=40. Histograms of fibre thickness are plotted as mean±s.e.m. Control, n=10; BBI, n=50; ATRA, n=20; β1, n=30. ***P<0.0001 (unpaired t-test). Scale bars: 50 μm.
Mentions: The ability of myofibroblasts to remodel the matrix relies on the ability to apply contractile forces (Calvo et al., 2013; Goetz et al., 2011). To gain more insight into the mechanisms underlying the ECM remodelling capacity by PSCs, we analysed collagen topology and structure in the presence of blebbistatin (BBI) and all-trans retinoic acid (ATRA). BBI blocks myosin II ATPase activity, and thus actomyosin contraction (Kovacs et al., 2004), and ATRA is the active metabolite of vitamin A and induces quiescence on PSCs (Froeling et al., 2011). Inhibition of actomyosin contraction in PSCs using BBI or inducing quiescence with ATRA significantly reduced the capacity of PSCs to contract the matrix (Fig. 5A,D). In both cases, the collagen alignment score and the fibre thickness significantly reduced with respect to the control condition (Fig. 5A-D; Fig. S1B). A significant decrease in the collagen intensity was also noted within the matrix embedded with PSCs treated with BBI and ATRA (Fig. S2C). We further observed a significant decrease in the spacing between collagen fibres in the matrices treated with BBI and ATRA (Fig. S2E) Taken together these results indicate that PSCs' capacity to remodel the matrix is only present in the active state and depends on their actomyosin machinery.Fig. 5.

Bottom Line: We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling.Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer.We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Faculty of Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

No MeSH data available.


Related in: MedlinePlus