Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation

Abstract

We have examined the role of mechanical tension in myofibroblast differentiation using two in vivo rat models. In the first model, granulation tissue was subjected to an increase in mechanical tension by splinting a full-thickness wound with a plastic frame. Myofibroblast features, such as stress fiber formation, expression of ED-A fibronectin and alpha-smooth muscle actin (alpha-SMA) appeared earlier in splinted than in unsplinted wounds. Myofibroblast marker expression decreased in control wounds starting at 10 days after wounding as expected, but persisted in splinted wounds. In the second model, granuloma pouches were induced by subcutaneous croton oil injection; pouches were either left intact or released from tension by evacuation of the exudate at 14 days. The expression of myofibroblast markers was reduced after tension release in the following sequence: F-actin (2 days), alpha-SMA (3 days), and ED-A fibronectin (5 days); cell density was not affected. In both models, isometric contraction of tissue strips was measured after stimulation with smooth muscle agonists. Contractility correlated always with the level of alpha-SMA expression, being high when granulation tissue had been subjected to tension and low when it had been relaxed. Our results support the assumption that mechanical tension is crucial for myofibroblast modulation and for the maintenance of their contractile activity.

Figures

Figure 1.

Isometric contraction of wound granulation tissue. Maximum contraction of tissue strips taken from 3- to 12-day-old wound granulation tissue and normal dermal tissue (0 days) was quantified after stimulation with ET-1 (A) and AT-II (B). Tissue from splinted wounds (continuous line) exhibits significantly higher contractility already at early stages of development (6 days) and maintains high levels of contractility up to 10 days after wounding compared to unsplinted granulation tissue (dashed line). Normal dermal tissue shows no significant contractility (0 days). Bars indicate SD of mean values. Filled squares indicate P ≤ 0.01 for comparison between unsplinted and splinted tissue of the same age.

Figure 2.

Mechanical tension enhances expression of F-actin and ED-A FN in wound granulation tissue. A: Sections of 3-day-old (Aa, Ae), 6-day-old (Ab, Af), 9-day-old (Ac, Ag), and 12-day-old (Ad, Ah) granulation tissue were double-stained for F-actin (green) and ED-A FN (red) and examined by confocal laser-scanning microscopy. Tissues from unsplinted wounds (Aa–Ad) are compared to those from splinted wounds (Ae–Ah). B: F-actin expression in unsplinted (dashed line) and splinted granulation tissue (continuous line) from 3 days to 12 days assessed by image analysis of immunostained tissue sections (normal dermis = 0 days). In 3-day-old unsplinted granulation tissue (Aa) fibroblastic cells exhibit low levels of de novo-appearing F-actin and ED-A FN whereas fibroblasts in 3-day splinted tissue (Ae) already show important expression and co-localization of the two proteins (yellow); co-localization is also seen in vascular smooth muscle cells (Aa, arrowhead). Expression of both proteins gradually increases and with partial co-localization at 6 days to 12 days after wounding. At any wound age fibroblastic cells of splinted wound granulation tissue exhibit higher expression of ED-A FN and F-actin compared to control (B). Scale bar, 50 μm.

Figure 3.

Wound splinting promotes early expression of α-SMA in granulation tissue. Sections of 3-day-old (A, E), 6-day-old (B, F), 9-day-old (C, G), and 12-day-old (D, H) granulation tissue were double-stained for α-SMA (red) and desmin (green). Protein expression in tissue from unsplinted wounds (A–D) is compared to that in splinted wound tissue (E–H). α-SMA is constitutively expressed by smooth muscle cells, co-localizing with desmin in vessels (A, arrowheads). Fibroblastic cells in unsplinted wound tissue express α-SMA at 9 days after wounding (C) and exhibit decreased expression levels after 12 days (D). Mechanical tension results in expression of α-SMA already at 6 days (E) that gradually increases up to 12 days (H). Scale bar, 50 μm.

Figure 4.

Quantification of myofibroblast markers in granulation tissue by means of Western blot. A: Expression of β-actin, vimentin, and the myofibroblast markers TGFβ-RII, ED-A FN, and α-SMA was investigated. Extracts of 3- to 12-day-old granulation tissue from splinted wounds (sp) are compared to those of unsplinted wounds (un) and of normal dermis (derm). Expression of myofibroblast markers gradually increases in evolving granulation tissues up to 9 days after wounding. No change in expression of β-actin and vimentin is observed. B–D: Protein bands were scanned, quantified by image analysis, and normalized to vimentin. Expression of ED-A FN is significantly higher in 3 day and 6 day splinted tissue compared to the respective control (B), as observed for α-SMA expression at 6 days and 12 days (C). Expression of TGFβ-RII is similar in unsplinted and splinted wound granulation tissue at any age (D). Filled squares indicate P ≤ 0.01 for comparison between unsplinted and splinted tissue of the same age.

Figure 5.

Loss of mechanical tension decreases granuloma pouch tissue contraction. Maximum contraction of tissue strips from granuloma pouches was quantified after stimulation with ET-1 (A) and AT-II (B). Pouches were either left intact for 14 days and 21 days or were released from tension by removing exudate after 14 days for 1 to 7 days. A significant increase in tissue contraction is observed between 14-and 21-day-old intact pouches (dashed lines). Loss of tension decreases granuloma pouch tissue contractility time-dependently and starts 2 days after release (14d+2dR). Note the dramatic difference in tissue contractility between 21-day intact pouches (21 day) and 21-day-old pouches, which were released for 7 days (14d+7dR). Exchange of exudate by physiological saline shows no effect (14d+7dS), whereas injection of TGF-β-sRII significantly decreases pouch tissue contraction (14d+7dT). **, P ≤ 0.005 compared to tissue contraction mean values of 14-day intact pouches.

Figure 6.

Loss of mechanical tension reduces myofibroblast differentiation in granuloma pouch tissue. Tissue sections of intact (A, D) and tension-released granuloma pouches (B, C) of different ages were double-stained for α-SMA (red) and ED-A FN (green) or stained for F-actin (insets, red) and examined by confocal laser-scanning microscopy. α-SMA and ED-A FN show important expression by fibroblastic cells and co-localization (yellow) in tissue from 14-day (A) and 21-day (D) intact granuloma pouches, as well as in vascular smooth muscle cells (arrowheads). Loss of tension in 14-day pouches results in decreased levels of F-actin and α-SMA expression 3 days after release (B) followed by decreased expression of ED-A FN 7 days after release (C). Note that expression of both proteins in small vessels is not affected by pouch relaxation. Scale bars, 50 μm.

Figure 7.

Quantification of myofibroblast markers in granuloma pouch tissue by means of Western blot. A: Protein expression in intact granuloma pouches (14d, 21d) was compared to 14-day-old pouches, which were subsequently released from mechanical tension for 1 to 7 days. Expression of α-SMA gradually decreases starting 2 days after release (14d+2dR), whereas decrease in ED-A FN expression occurs later (14d+7dR) after release. TGF-β-RII expression shows a moderate decrease after 7 days and no change is observed for β-actin and vimentin expression. Exchange of exudate by saline has no effect (14d+7dS), whereas injection of TGF-β-sRII significantly decreases expression of all myofibroblast markers (14d+7dT). B: Protein bands obtained from 21-day intact (21d) and 7-day-tension-released (14d+7dR) pouch tissue were scanned, their density was quantified by image analysis, and obtained values were normalized to vimentin. Tissue from 7-day-released pouches exhibits significantly reduced levels of α-SMA and ED-A FN expression, moderately reduced TGF-β-RII expression and no change in β-actin compared to 21-day intact pouches. *, P ≤ 0.01; **, P ≤ 0.005.

Figure 8.

Evolution of granulation tissue contractility. The chronological evaluation of granulation tissue contractile activity distinguished three phases characterized by: 1) slow increase (red), 2) steep increase (blue), and 3) decrease (green). These phases correlate with the onset of F-actin and ED-A FN expression (phase 1), de novo expression of α-SMA (phase 2), and decrease of α-SMA expression (phase 3) as indicated by the arrows. The first two phases clearly start earlier and the third phase is delayed in splinted (continuous line) compared to unsplinted wounds (dotted line).