. Isometric contraction was stimulated with various smooth muscle agonists in the following order of efficiency: ET-1 (10−7 mol/L), AT-II (10−5 mol/L), and serotonin (10−4 mol/L). Wound tissue contracted only weakly after stimulation with histamine (10−4 mol/L) and potassium (4 × 10−3 mol/L). Control tissues (0 days) did not show any significant response after stimulation with all agonists (not shown). The contraction of unsplinted and splinted granulation tissue increased from 3-day-old to 9-day-old tissue after stimulation with ET-1 (Figure 1A) ; moreover, splinted tissues contracted more strongly at 6 days (266%) and 8 days (185%) compared to unsplinted tissues. Surprisingly, splinted and unsplinted tissues exhibited comparable contraction after 9 days. At 10 days the contractile activity of unsplinted tissues decreased (67% compared to 9 days), whereas the contractile activity of splinted tissues was unchanged compared to 9 days. At 12 days unsplinted tissues showed a further decrease in contractility (71% compared to 10 days); splinted wounds also decreased their contractility compared to 10 days (78%) but maintained a higher contractility (168%) compared to unsplinted tissues. Results obtained after stimulation with AT-II (Figure 1B) and serotonin (not shown) were similar to those obtained after ET-1 stimulation.
| 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 (more ...) |
Mechanical Tension Results in an Early Expression of Myofibroblast Markers in Wound Granulation Tissue
To assess whether mechanical tension stimulates myofibroblast differentiation in vivo, we compared the expression of myofibroblast markers such as F-actin, ED-A FN, α-SMA, TGF-β-RII, TGF-β1, desmin, SMMHC, and NMMHC in the different situations. F-actin was not present in fibroblasts of normal dermis (not shown), started to be visible in 3-day-old unsplinted wounds (Figure 2Aa ) and gradually increased (Figure 2, Ab to Ad ). Splinting induced noticeable accumulation of F-actin already at 1 day (not shown) and importantly enhanced F-actin levels throughout the following days (Figure 2, Ae to Ah ). Moreover, splinting improved the alignment of F-actin along the lines of mechanical tension, ie, parallel to the wound surface. F-actin bundles were devoid of α-SMA up until day 6 in unsplinted and until day 4 in splinted tissue, as evaluated by double staining (not shown). Quantification of Alexa 488-phalloidin fluorescence intensity confirmed higher levels of F-actin in splinted compared to unsplinted granulation tissues at any wound age (Figure 2B , between 376 and 226% increase). No significant changes of marker distribution were observed in the different wound locations. Quantification of granulation tissue cellularity by means of DAPI staining demonstrated a linear increase in cell number from 3- to 12-day-old granulation tissue (120%) and no difference between unsplinted and splinted tissue. ED-A FN was detected in low levels in 3-day-old unsplinted granulation tissue (Figure 2Aa ), expression increased between day 6 and day 9 (Figure 2, Ab and Ac ), and was reduced in 12-day-old granulation tissue (Figure 2Ad ). Splinting induced ED-A FN expression starting from day 1 (not shown) and enhanced its expression compared to unsplinted granulation tissue at all stages of wound healing (Figure 2, Ae to Ah ). No differences in vascularity were observed in splinted versus unsplinted wounds. | 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 (more ...) |
α-SMA was not expressed in fibroblasts of normal dermis (not shown) and of 3-day-old granulation tissue (Figure 3, A and E) , and was not detected in fibroblasts of 6-day-old unsplinted granulation tissue (Figure 3B) . In unsplinted wounds, it was first detected after 7 days (not shown), was strongly expressed after 9 days (Figure 3C) , and expression was clearly reduced after 12 days (Figure 3D) . In contrast, splinting induced α-SMA expression by granulation tissue fibroblasts already at 4 to 6 days after wounding (Figure 3F) ; the level of α-SMA expression increased in splinted tissue to a maximum at 9 days (Figure 3; E to G ) and remained at an apparently similar level at 12 days (Figure 3H) . Three-day-old to 12-day-old splinted wounds exhibited increased expression of both TGF-β1 and TGF-β-RII; however, no clear differences were observed between splinted and unsplinted wound tissue by means of immunohistochemistry (not shown). Desmin and SMMHC were not expressed by granulation tissue fibroblasts, and expression of NMMHC did not change in any condition as tested by means of immunofluorescence and Western blotting (not shown).
| 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). (more ...) |
Analysis of myofibroblast marker expression in wound granulation tissue by Western blotting (Figure 4) essentially confirmed immunolocalization results. In unsplinted tissue ED-A FN expression was continuously increased from 3 days to 9 days (630%) and exhibited a reduction by 160% between 9 days and 12 days (Figure 4, A and B) . Splinting enhanced the amount of ED-A FN (Figure 4A) with the largest difference at 3 days (Figure 4B , 347%). Expression of α-SMA moderately increased from 3-day to 6-day unsplinted tissue (195%), steeply increased from 6 days to 9 days (400%), and was reduced by 145% between 9 days and 12 days (Figure 4, A and C) . Splinted tissue exhibited increased α-SMA expression levels at 6 days (Figure 4C , 420%) and showed similar α-SMA expression at 9 days compared to unsplinted granulation tissue. Splinting maintained high α-SMA expression levels at 12 days in contrast to the decreased expression in unsplinted wound tissue (Figure 4C) . TGF-β-RII expression increased from 0 to 12 days after wounding with no significant difference between splinted and unsplinted granulation tissue (Figure 4, A and D) . To relate changes in myofibroblast marker expression to enhanced tissue contractility during the evolution of unsplinted and splinted wound granulation tissue, we calculated correlation coefficients. Highest correlation was observed between the temporal course of ET-1-stimulated tissue contraction (Figure 1A) and expression of α-SMA (r 2 = 0.98). Lower correlation was calculated for TGF-β-RII (r 2 = 0.92), ED-A FN (r 2 = 0.90), and F-actin (r 2 = 0.84), no correlation for β-actin (r 2 = 0.69), NMMHC (r 2 = 0.61), desmin (r 2 = 0.57), and vimentin (r 2 = 0.55).
| 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 (more ...) |
Loss of Mechanical Tension Decreases Tissue Contractility and Expression of Myofibroblast Markers
Because increased mechanical tension seems to induce myofibroblast differentiation and increase granulation tissue contractility, we sought to investigate whether reduction of tension may have the opposite effect. First, wounds were splinted for 7 days, then released from tension for 1 or 2 days and compared to granulation of the same age but still splinted. One-day-released granulation tissue contracted similarly compared to 8-day splinted tissue (96 ± 7%). However, tension release for 2 days significantly reduced contraction compared to 9-day splinted tissue to 63 ± 3% after stimulation with AT-II and to 63 ± 2% after stimulation with ET-1. Contraction of 2-day-released granulation tissue was also lower (71 ± 4%) when compared to 9-day-unsplinted granulation tissue. Characterization of released tissue by immunostaining and Western blotting revealed no changes after 1 day, but a decrease of F-actin and α-SMA after 2 days. Expression levels of ED-A FN, TGF-β-RII, TGF-β1, NMMHC, desmin, and vimentin were not significantly changed 2 days after release (not shown).To investigate further the chronological relationship between tissue contraction and myofibroblast modulation after release of mechanical tension, we used the granuloma pouch model. 29 Tension was released in granuloma pouch tissue at 14 days by evacuating the exudate and isometric contraction was measured 1 to 7 days after release (Figure 5) . Contraction of 21-day control pouch tissue was maximally stimulated by ET-1 (10−7 mol/L) and by the following agonists in percentage of ET-1-response: AT-II (10−5 mol/L), 60%; serotonin (10−4 mol/L), 48%; potassium (4 × 10−3 mol/L), 45%; and histamine (10−4 mol/L), 6%. Results are presented for ET-1 (Figure 5A) and AT-II (Figure 5B) . Tissue contractility was higher in intact 21-day-old compared to 14-day-old pouches (Figure 5 , dashed lines), confirming previous observations. 30,38 Release of mechanical tension at 14 days decreased pouch tissue contraction beginning 2 days after release, exhibited a minimum at 3 days after release (14d+3dR), and kept this level until 7 days. Compared to intact pouches of the same age (21 days), tension release resulted in a significantly lower tissue contraction (38% for ET-1 and 46% for AT-II). As a control, we substituted the evacuated exudate with an identical volume of physiological saline for 7 days (Figure 5 , 14+7dS). Saline-injected pouch tissue contraction did not differ from intact pouches of the same age (21 days).
| 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 (more ...) |
Alexa 568-phalloidin (Figure 6 , insets) revealed an intense F-actin staining in 14-day-old granuloma pouch fibroblasts, that was clearly decreased already 2 days after release (Figure 6B) ; this decrease was more important at 7 days after release (Figure 6C , inset). ED-A FN and α-SMA were co-localized and their expression increased in 21-day-old compared to 14-day-old intact granuloma pouch tissue (Figure 6, A and D) . Three days after tension release α-SMA expression was reduced (Figure 6B) ; 7 days after tension release, ED-A FN expression was also reduced and α-SMA was hardly detectable (Figure 6C) . TGF-β1, previously shown to be expressed in granuloma pouch tissue, 39,40 and TGF-β-RII were both predominantly expressed in the external collagen-rich layer of the pouch tissue. TGF-β1 and TGF-β-RII expression patterns in 7-day-released pouches did not differ significantly from 1- to 5-day-released tissue or 21-day intact pouch tissue, but the thickness of the collagen-rich layer decreased progressively during the 7-day period of tension release to ~70% compared to 21-day intact pouches (not shown).
| 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) (more ...) |
As determined by Western blotting, α-SMA was significantly reduced 2 to 3 days after release, compared to 14-day-old intact pouch tissue, whereas decrease of ED-A FN expression started 4 days after release (Figure 7) . TGF-β-RII expression was only moderately diminished throughout the 7-day period after release and no change was observed for β-actin and vimentin expression (Figure 7A) . Compared to 21-day-old intact pouch tissue, 7-day tension release reduced α-SMA expression to 51%, ED-A FN to 47%, and TGF-β-RII to 81% (Figure 7B) . In contrast to wound granulation tissue fibroblasts, which did not express desmin and SMMHC, low expression of both proteins was observed in fibroblastic cells of 14-day and 21-day intact granuloma pouches. Tension release did not change desmin and NMMHC expression and only moderately diminished expression of SMMHC 7 days after release as evaluated by Western blotting and immunofluorescence (not shown). When removed exudate was replaced by physiological saline, no changes in protein expression were detected (Figure 7A , 14d+7dS).
| 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 (more ...) |
Injection of soluble TGF-β-RII into the granuloma pouch for 7 days decreased tissue contraction to 49% after stimulation with ET-1 (Figure 5A , 14d+7dT) and to 48% after AT-II stimulation (Figure 5B , 14d+7dT) compared to 21-day-old control pouch tissue. It reduced the expression levels of TGF-β, TGF-β-RII, ED-A FN, F-actin, and α-SMA significantly compared to 21-day-old control pouch tissue as demonstrated by Western blotting (Figure 7A) and immunofluorescence (not shown).
Discussion It is accepted that mechanical tension plays an important role in the modulation of cultured fibroblasts into myofibroblasts by favoring the formation of stress fibers and the expression of α-SMA. 27 Here, we demonstrate that mechanical tension also influences myofibroblast differentiation in vivo. Fibroblasts populating the granulation tissue of splinted wounds exhibit earlier formation of stress fibers, expression of ED-A FN and of α-SMA compared to nonsplinted wounds. Inversely, release of tension in wound granulation tissue and in granuloma pouch leads to a sequential loss of stress fibers, α-SMA, and ED-A FN. Moreover, our study indicates a correlation between the level of α-SMA expression and contractility of the tissues obtained during the different experimental situations. The acquisition of contractile activity by granulation tissue on stimulation with smooth muscle agonists is a well-accepted phenomenon and has been suggested to represent a useful indication of the retractile potential of granulation tissue. 29,30,41 Acquisition of an increased contractile activity by wound splinting 42 has been correlated to the induction of microfilament bundles in fibroblasts. 18 Our results corroborate these observations and establish a correlation between changes in tissue contractility and cytoskeletal protein expression and/or organization after mechanical load modulation. In both splinted and unsplinted wounds, the granulation tissue strip contractile activity evolves according to three phases, as depicted schematically in Figure 8 . During the first phase (red) the contractility of granulation tissue increases slowly, whereas during the second phase (blue), the increase is steep; the third phase (green) corresponds to a decrease of contractile activity. In the first phase there is an increase of F-actin and of ED-A FN expression that is clearly more important in splinted wounds. The onset of high contractile activity correlates always with the de novo expression of α-SMA, assembled into stress fibers. It is noteworthy that the first phase is shorter and the second phase is longer in splinted compared to unsplinted wounds and corresponds to the maximal level of α-SMA expression. Interestingly, the maximal level of contractile capacity is similar in both situations. Finally, a decrease in contractile activity takes place more slowly in splinted than in unsplinted wounds. The observed correlation between α-SMA expression and granulation tissue contractility is in agreement with the previously reported correlation between α-SMA expression and contraction of fibroblast-populated anchored collagen gels. 8,9 More recently we have shown that 3T3 fibroblasts exhibit a significantly higher contractile activity after transient and/or stable transfection with α-SMA cDNA compared to fibroblasts transfected with β-actin, γ-actin, or even α-cardiac actin cDNA in the absence of any change of myosin heavy chain expression. 7 All these data are compatible with the assumption that α-SMA expression alone plays a role in the enhancement of fibroblast contractility in vitro. Our results allow to hypothesize that also in vivo α-SMA expression plays a crucial role in the development of granulation tissue contractile capacity. In all situations, increased tension results in a greater expression of α-SMA and an increased force production, whereas decreased tension has the opposite effect. 2,21,43,44 Although in vivo and in vitro data indicate that α-SMA expression is stimulated by mechanical stress, it remains unclear how fibroblasts translate the physical signal into protein expression. In cells such as cardiomyocytes, endothelial and smooth muscle cells, mechanical stress was shown to stimulate the production and secretion of growth factors that mediate stress-induced cell responses. 45 In particular, expression of TGF-β1, the major inducer of α-SMA expression 13,14 and contractility in myofibroblasts, 8,21 is up-regulated in mechanically stressed mesangial cells. 46 Under our conditions the expression of TGF-β1 and of its receptor TGF-β-RII was gradually increased, but similarly in splinted tissue and unsplinted tissues. Nevertheless, blocking TGF-β1 by an excess of soluble receptor without changing the mechanical load reduced α-SMA expression and tissue contraction in our experimental conditions, implying a role of TGF-β1. Enhanced mechanical stress increases growth factor sensitivity 23,24,47 and loss of mechanical tension reduces the growth factor response of cultured fibroblasts. 48,49 It remains to be examined whether TGF-β1 activation changes in our experimental situations. | 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 (more ...) |
Changes in mechanical stress may directly affect the level of extracellular matrix proteins. 50-52 Recently, it was shown that during wound healing ED-A FN expression 53,54 precedes the appearance of α-SMA-positive myofibroblasts and is essential to mediate TGF-β1-induced α-SMA expression. 17 Splinting induces ED-A FN expression in granulation tissue, suggesting a possible regulatory function of this protein during stress-induced myofibroblast differentiation. Regulation may occur at the level of FN expression, 55 alternative splicing, 56,57 fibril assembly, 22 or accessibility of functional residues in the ED-A domain of FN 58 as it has been demonstrated for plasma FN. 59 Integrins of focal adhesions are potential receptors and transducers of these extracellular signals. 60 When myofibroblasts were released from mechanical stress by removing the wound splint or by evacuating the granuloma pouch, disappearance of α-SMA preceded the decrease of ED-A FN and TGF-β1 levels. Thus, ED-A FN and TGF-β1 are crucial for the induction of α-SMA expression but are not sufficient to maintain myofibroblast differentiation in the absence of a mechanical stimulus. Taking into account the early formation of F-actin during wound healing and the rapid loss of actin stress fibers after load release, we suggest that the physical integrity of stress fibers 61 provides an important prerequisite for α-SMA expression and myofibroblast contraction. Mechanical stress induces actin polymerization and stress fiber formation when applied externally 62 or by increasing intracellular tension. 10,63 The ratio between G-actin/F-actin was shown to directly influence β-actin synthesis. 64,65 However, the role of the G-actin/F-actin ratio for the expression of other actin isoforms, such as α-SMA, has not been evaluated. It is conceivable that mechanical tension induces actin expression without discriminating among isoforms whereas factors such as TGF-β and ED-A FN exert a specific stimulation of α-SMA. 17 A similar phenomenon has been shown for the stress-dependent expression of α-skeletal actin by cardiomyocytes in the presence of AT-II. 45 In conclusion our results show that mechanical tension is a prerequisite for the development and maintenance of myofibroblast differentiation and hence of granulation tissue contraction. Given the reciprocal relationship between fibroblast contractility and the mechanical state of the matrix, the modulation of extracellular and intracellular tension may help to influence wound healing and development of fibrocontractive diseases. |
Acknowledgments We thank Drs. Victor Koteliansky (Biogen Inc., Cambridge, MA) for providing the soluble TGF-β-RII, Luciano Zardi (National Institute for Cancer Research, Laboratory of Cell Biology, Genoa, Italy) for providing IST-9 antibodies, and Jo de Mey (Department of Pharmacology, University of Maastricht, Maastrich, The Netherlands) for expert advice and teaching; A. Geinoz, M. Bacchetta, P. Henchoz, and S. Coutant-Zimmerli for technical assistance; J. C. Rumbeli and E. Denkinger for photographic work; and Mrs. S. Josseron for secretarial work. |
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