Stress and matrix-responsive cytoskeletal remodeling in fibroblasts

Abstract

In areolar "loose" connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross-sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in, and dissociated from, areolar and dense connective tissue in response to 2 h of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large "sheet-like" morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch-induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues.

Figure 1. Experimental Setup

Schematic demonstrating different layers of mouse subcutaneous tissue that were dissected in this study (A). Collagen gel held between 2 rubber grips, and secured with outer metal grips (B). The grips mounted into the tissue stretching device (C).

Figure 2. Experimental measurements of cell body cross-sectional area used to quantify different cell morphologies

Examples of morphometric measurements for “dendritic” (A) and “sheetlike” (B) cells (Bar=20 µm, merged z-stack).

Figure 3. Effect of static tissue stretch for 2 hours on dense and areolar connective tissue ex vivo

Morphology of fibroblasts within dense connective tissue unstretched (A) and stretched (B) compared to areolar connective tissue unstretched (C) and stretched (D). Cell cross-sectional area (E) was significantly increased as a function of force in areolar (β=1.4, F_{1, 40}=4.4, p<.05), but not in dense connective tissue (β= −0.38, F_1,40=2.3, p=.20).

Figure 4. Determination of connective tissue and collagen gel stiffness

Dense connective tissue had a higher dynamic modulus than areolar connective tissue (A) demonstrated by both the strain sweep and frequency sweep curves (B)(n=6, t-test, p values shown for each parameter). The magnitude of the phase angle curve (δ_1%) and the exponent to the frequency sweep curve (α_|G*|) were significantly higher for the areolar connective tissue compared to the dense connective tissue. The preglycated 12 days gel had a higher dynamic modulus than the unglycated gel and preglycated 6 days gel (C) indicated by both the strain sweep and frequency sweep (D, n=6, one way ANOVA, strain sweep p values shown for each parameter, Fisher’s LSD p<0.05). Gel groups sharing a common letter were not significantly different. Error bars represent SEM.

Figure 5. Effect of static stretch for 2 hours on fibroblasts dissociated from dense and areolar connective tissue and seeded in unglycated collagen gels

Cells from dense connective tissue (A,B) and cells from areolar connective tissue (C,D) were seeded into collagen gels having a similar stiffness to areolar connective tissue. The left images represent the no stretch condition (A,C) with images on the right representing the stretch condition (B,D). Cells derived from both tissue sources responded to stretch with an increase in cell cross sectional area (Phalloidin = red, Dapi = blue, bar=50 µm, merged z-stack).

Figure 6. Morphological measurements of fibroblasts dissociated from dense and areolar connective tissue and seeded in collagen gels of varying crosslinking

Fibroblasts dissociated from dense connective tissue were seeded into unglycated collagen gels (similar modulus to areolar connective tissue) and responded to stretch with an increase in cell body cross-sectional area (A)(n=6, t-test, p = .006). Fibroblasts dissociated from areolar connective tissue were seeded into gels of different glycation durations corresponding to different stiffnesses (6 days had a similar modulus to areolar connective tissue while preglycated 12 days had a similar modulus to dense connective tissue) and cell body cross-sectional area was compared with and without static stretching of the gel. Interactions between the 6 groups demonstrated significant differences between stretch and no stretch for the unglycated and preglycated 6 days gels for cell cross-sectional area with no differences between the no stretch and stretch condition for the preglycated 12 days gels(interaction p<.001, Fisher’s LSD, p<.05). Gel groups sharing a common letter were not significantly different. Error bars represent SEM.

Figure 7. Morphological effects are dynamic

There are no significant differences in cell body cross-sectional area of fibroblasts stretched for 2 hours and then released for 10 minutes and fibroblasts that were not stretched (n=3, t-test, area p=.26). Error bars represent SEM.

Figure 8. Schematic representation of results

Fibroblast expansion in response to stretch was only observed in tissue and gels (unglycated and 6 days preglycated) with a similar modulus to areolar connective tissue regardless of fibroblast tissue of origin.