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First published online 27 September 2005
doi: 10.1242/jcs.02605

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Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro

¹ Department of Chemical and Biological Engineering, Northwestern University, Evanston, 633 Clark Street, Chicago, IL 60208, USA
² Laboratory of Cell Biophysics, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
³ Integrative Biosciences Institute, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland

View larger version (76K): [in a new window]   Fig. 1. Experimental set-up and alignment determination. (A) Design features of radial interstitial flow tissue culture chamber. The chamber is made of porous polyethylene and surface-modified glass materials to anchor the ECM and allow direct visualization. (B) Algorithm for image quantification of alignment and orientation. The confocal image (i) is modified (ii) to remove edge effects. (iii) A FFT transformation is performed to obtain a power spectrum from which (iv) an intensity frequency histogram is plotted and an alignment index (=(/( + ))/(/( + ))ideal) and the peak angle (peak) extracted.

View larger version (60K): [in a new window]   Fig. 2. Alignment of human dermal fibroblasts in a collagen matrix subjected to radial interstitial flow. Confocal images of cells (A) and matrix fibers (B) with their corresponding FFT analyzed intensity frequency histograms. f-actin is labelled green with the confocal reflection in red; arrow indicates flow direction. These observations were quantified by alignment index (C) and peak angle (D) for cell and matrix alignment, respectively. Unpaired t-tests were used for statistical analysis of the means; significant differences (**P<0.01) were observed in alignment index in both cells and matrix under flow conditions compared to that measured under static conditions using Mann-Whitney test. Bar, 200 µm (A); 20 µm (B).

View larger version (78K): [in a new window]   Fig. 3. Interstitial flow induces -SMA expression in fibroblasts. (A) Confocal images of cells at 2 and 5 days showing -SMA expression under mechanically constrained static and interstitial flow conditions (green, f-actin; red, -SMA; arrow indicates flow direction). (B) Significantly higher levels of -SMA expression are seen in fibroblasts undergoing interstitial flow at both time points (**P<0.01 using Dunn's test). Box plot shows 95% confidence intervals with midline showing the median. (C) The percentage of proliferating (Ki67+) cells after 2 days was higher under interstitial flow conditions than either constrained or relaxed static controls (bar represents the mean value and error bars, s.d.; **P<0.01 using Dunnett's test). Bar, 200 µm.

View larger version (59K): [in a new window]   Fig. 4. TGF-ß1 mediates flow-induced myofibroblast differentiation. (A) Confocal images showing TGF-ß1 and -SMA expression in mechanically constrained static (top) and flow conditions, without (middle) and with (bottom) TGF-ß1 neutralizing antibody (red, f-actin; green, TGF-ß1; blue, -SMA; arrow indicates flow direction). (B) TGF-ß1 expression quantification (**P<0.01 using Dunn's test) in fibroblasts under constrained static and interstitial flow conditions for 2 days. Box plot shows 95% confidence intervals with midline showing the median. (C) -SMA expression quantification in normal compared to bioneutralized flow conditions (**P<0.01 using Mann-Whitney test). (D,E) Quantification of cell density in terms of number of cells per mm3 and cell spreading in terms of projected cell area (µm2/cell) (**P<0.01 using Dunnett's test). (F) Alignment and (G) orientation of the cells and matrix fibers under each condition (*P<0.05; **P<0.01 using Dunnett's test). Bar, 200 µm.

View larger version (52K): [in a new window]   Fig. 5. Interstitial flow effects are mediated through 1ß1 integrins. (A) Confocal images of cells under normal, 1ß1- and 2ß1-neutralized flow conditions for 2 days (green, f-actin; red, -SMA; arrow indicates flow direction). (B) Bar graphs indicating -SMA expression levels for the respective normal and bioneutralized conditions (**P<0.01 using Dunn's test); box plot shows 95% confidence intervals with midline showing the median. (C,D) Effects of integrin blocking on cell spreading and density (**P<0.01 using Dunnett's test). (E,F) Effects of integrin blocking on the alignment and orientation of cell and collagen fibers under the various experimental conditions (*P<0.05; **P<0.01 using Dunnett's test). Error bars indicate s.d. (G) Contraction of free-floating gels by fibroblasts after no treatment (control) or treatment with either of the integrin-blocking antibodies as indicated; only neutralizing 2ß1 integrin prevents contraction. Bar, 200 µm.

View larger version (34K): [in a new window]   Fig. 6. Proposed mechanism of interstitial flow-driven myofibroblast differentiation and matrix remodeling. First, flow itself can impose shear stress on the cells directly and strain on the cells via stresses on the ECM fibers to which the cells attach. Either of these may trigger TGF-ß1 expression, the latter through 1ß1 integrin signaling. TGF-ß1 drives -SMA expression as the fibroblasts differentiate into myofibroblasts and align the matrix fibers.

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