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Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

Beyeler J, Schnyder I, Katsaros C, Chiquet M - PLoS ONE (2014)

Bottom Line: Wound closure rate showed highly significant differences between fibroblast strains.Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group.Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

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Size of focal adhesions differs between the two CLP migratory groups.Immunofluorescence staining against vinculin, and rhodamine-phalloidin staining for F-actin of migrating cells 24 h after scratch wounds were applied. Two randomly chosen strains from the “fast” (TL and XB) and the “intermediate” (LP and ML) group, respectively, were included in the analysis. (A) The box plots indicate the area in µm2 covered by individual vinculin positive focal adhesions at the front end of cells (n[cells per strain] = 12–15). The whiskers indicate the maximum and minimum area per focal adhesion in a strain (***p<0.001). (B) Representative pictures from two strains depict the front end with the lamellipodium of individual cells migrating into the wound. Typically, migrating cells of the “fast” CLP group form smaller focal adhesions in comparison to cells of the “intermediate” group. Scale bar, 10 µm.
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pone-0111752-g002: Size of focal adhesions differs between the two CLP migratory groups.Immunofluorescence staining against vinculin, and rhodamine-phalloidin staining for F-actin of migrating cells 24 h after scratch wounds were applied. Two randomly chosen strains from the “fast” (TL and XB) and the “intermediate” (LP and ML) group, respectively, were included in the analysis. (A) The box plots indicate the area in µm2 covered by individual vinculin positive focal adhesions at the front end of cells (n[cells per strain] = 12–15). The whiskers indicate the maximum and minimum area per focal adhesion in a strain (***p<0.001). (B) Representative pictures from two strains depict the front end with the lamellipodium of individual cells migrating into the wound. Typically, migrating cells of the “fast” CLP group form smaller focal adhesions in comparison to cells of the “intermediate” group. Scale bar, 10 µm.

Mentions: Immunofluorescence staining for vinculin and F-actin was conducted on wounded cultures to examine whether focal adhesions of migrating fibroblasts at the wound edge differed between “fast” and “intermediate” CLP strains. For this experiment, two representative strains of each CLP group were randomly chosen, stained, and evaluated. Vinculin positive focal adhesions at and behind the front lamellipodium of individual migrating cells were measured; we quantified the area (in µm2) covered by single adhesion contacts. As shown in Fig. 2A, the average size of focal adhesions was significantly smaller in fibroblasts from “fast” migrating strains compared to strains of the “intermediate” CLP group (p<7×10−09). Images showed in addition that cells from “intermediate” strains tended to have fewer but thicker stress fibers attached to their large focal adhesions, whereas “fast” migrating fibroblasts had prominent lamellipodia, and showed thinner and more evenly distributed actin fibers connected to many small adhesion contacts (Fig. 2B; Fig. S1). As expected, these observations indicate that focal adhesion size and actin organization correlate with migratory behavior and thus with wound closure ability of the respective CLP strains in wounding assays.


Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

Beyeler J, Schnyder I, Katsaros C, Chiquet M - PLoS ONE (2014)

Size of focal adhesions differs between the two CLP migratory groups.Immunofluorescence staining against vinculin, and rhodamine-phalloidin staining for F-actin of migrating cells 24 h after scratch wounds were applied. Two randomly chosen strains from the “fast” (TL and XB) and the “intermediate” (LP and ML) group, respectively, were included in the analysis. (A) The box plots indicate the area in µm2 covered by individual vinculin positive focal adhesions at the front end of cells (n[cells per strain] = 12–15). The whiskers indicate the maximum and minimum area per focal adhesion in a strain (***p<0.001). (B) Representative pictures from two strains depict the front end with the lamellipodium of individual cells migrating into the wound. Typically, migrating cells of the “fast” CLP group form smaller focal adhesions in comparison to cells of the “intermediate” group. Scale bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4216129&req=5

pone-0111752-g002: Size of focal adhesions differs between the two CLP migratory groups.Immunofluorescence staining against vinculin, and rhodamine-phalloidin staining for F-actin of migrating cells 24 h after scratch wounds were applied. Two randomly chosen strains from the “fast” (TL and XB) and the “intermediate” (LP and ML) group, respectively, were included in the analysis. (A) The box plots indicate the area in µm2 covered by individual vinculin positive focal adhesions at the front end of cells (n[cells per strain] = 12–15). The whiskers indicate the maximum and minimum area per focal adhesion in a strain (***p<0.001). (B) Representative pictures from two strains depict the front end with the lamellipodium of individual cells migrating into the wound. Typically, migrating cells of the “fast” CLP group form smaller focal adhesions in comparison to cells of the “intermediate” group. Scale bar, 10 µm.
Mentions: Immunofluorescence staining for vinculin and F-actin was conducted on wounded cultures to examine whether focal adhesions of migrating fibroblasts at the wound edge differed between “fast” and “intermediate” CLP strains. For this experiment, two representative strains of each CLP group were randomly chosen, stained, and evaluated. Vinculin positive focal adhesions at and behind the front lamellipodium of individual migrating cells were measured; we quantified the area (in µm2) covered by single adhesion contacts. As shown in Fig. 2A, the average size of focal adhesions was significantly smaller in fibroblasts from “fast” migrating strains compared to strains of the “intermediate” CLP group (p<7×10−09). Images showed in addition that cells from “intermediate” strains tended to have fewer but thicker stress fibers attached to their large focal adhesions, whereas “fast” migrating fibroblasts had prominent lamellipodia, and showed thinner and more evenly distributed actin fibers connected to many small adhesion contacts (Fig. 2B; Fig. S1). As expected, these observations indicate that focal adhesion size and actin organization correlate with migratory behavior and thus with wound closure ability of the respective CLP strains in wounding assays.

Bottom Line: Wound closure rate showed highly significant differences between fibroblast strains.Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group.Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

Show MeSH
Related in: MedlinePlus