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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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Effect of TGF-α, anti-TGF-α, and EGFR/ERBB2-inhibitor on wound closure by “fast” versus “intermediate” CLP strains.Boxplots depict the RWC in scratch wound assays at 24 h of “fast” (A, C, E) and “intermediate” (B, D, F) CLP strains, in either the absence (control; Ctrl) or the presence of the following agents diluted in 10% FCS/D-MEM: TGF-α at 5 ng/ml (T5) or 20 ng/ml (T20); TGF-α neutralizing antibody at 0.5 µg/ml (AntiT); Lapatinib at 5 µM (Lapa); TGF-α plus anti-TGF-α (T5+AntiT); or TGF-α plus Lapatinib (T5+Lapa; T20+Lapa) (*p<0.05, **p<0.01, ***p<0.001). The micrographs show representative examples of scratch wound assays at 0 and 24 h in the absence or presence of the drugs indicated at the bottom. Area of scale bar corresponds to 10% RWC or 0.32mm2.
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pone-0111752-g004: Effect of TGF-α, anti-TGF-α, and EGFR/ERBB2-inhibitor on wound closure by “fast” versus “intermediate” CLP strains.Boxplots depict the RWC in scratch wound assays at 24 h of “fast” (A, C, E) and “intermediate” (B, D, F) CLP strains, in either the absence (control; Ctrl) or the presence of the following agents diluted in 10% FCS/D-MEM: TGF-α at 5 ng/ml (T5) or 20 ng/ml (T20); TGF-α neutralizing antibody at 0.5 µg/ml (AntiT); Lapatinib at 5 µM (Lapa); TGF-α plus anti-TGF-α (T5+AntiT); or TGF-α plus Lapatinib (T5+Lapa; T20+Lapa) (*p<0.05, **p<0.01, ***p<0.001). The micrographs show representative examples of scratch wound assays at 0 and 24 h in the absence or presence of the drugs indicated at the bottom. Area of scale bar corresponds to 10% RWC or 0.32mm2.

Mentions: In a first attempt to find out whether the observed differences in TGF-α expression levels between “fast” and “intermediate” CLP groups might be causally linked to the speed of wound closure by individual fibroblast strains, we tested the effect of adding exogenous growth factor to the cultures during the assay. Three hours before wounds were inflicted to fibroblast monolayers, TGF-α was added to the standard culture media at either 5 ng/ml or 20 ng/ml. The addition of 5 ng/ml TGF-α significantly increased the RWC (∼1.5-fold; p<7×10−05) in the “intermediate” CLP group, whereas it had no effect in the “fast” group (Fig. 4A, B). The higher concentration of TGF-α (20 ng/ml) caused an increase in both groups, but less in the “fast” (∼1.2-fold, p<7×10−03) than in the “intermediate” (∼1.4-fold, p<1×10−05) (Fig. 4A, B).


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)

Effect of TGF-α, anti-TGF-α, and EGFR/ERBB2-inhibitor on wound closure by “fast” versus “intermediate” CLP strains.Boxplots depict the RWC in scratch wound assays at 24 h of “fast” (A, C, E) and “intermediate” (B, D, F) CLP strains, in either the absence (control; Ctrl) or the presence of the following agents diluted in 10% FCS/D-MEM: TGF-α at 5 ng/ml (T5) or 20 ng/ml (T20); TGF-α neutralizing antibody at 0.5 µg/ml (AntiT); Lapatinib at 5 µM (Lapa); TGF-α plus anti-TGF-α (T5+AntiT); or TGF-α plus Lapatinib (T5+Lapa; T20+Lapa) (*p<0.05, **p<0.01, ***p<0.001). The micrographs show representative examples of scratch wound assays at 0 and 24 h in the absence or presence of the drugs indicated at the bottom. Area of scale bar corresponds to 10% RWC or 0.32mm2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111752-g004: Effect of TGF-α, anti-TGF-α, and EGFR/ERBB2-inhibitor on wound closure by “fast” versus “intermediate” CLP strains.Boxplots depict the RWC in scratch wound assays at 24 h of “fast” (A, C, E) and “intermediate” (B, D, F) CLP strains, in either the absence (control; Ctrl) or the presence of the following agents diluted in 10% FCS/D-MEM: TGF-α at 5 ng/ml (T5) or 20 ng/ml (T20); TGF-α neutralizing antibody at 0.5 µg/ml (AntiT); Lapatinib at 5 µM (Lapa); TGF-α plus anti-TGF-α (T5+AntiT); or TGF-α plus Lapatinib (T5+Lapa; T20+Lapa) (*p<0.05, **p<0.01, ***p<0.001). The micrographs show representative examples of scratch wound assays at 0 and 24 h in the absence or presence of the drugs indicated at the bottom. Area of scale bar corresponds to 10% RWC or 0.32mm2.
Mentions: In a first attempt to find out whether the observed differences in TGF-α expression levels between “fast” and “intermediate” CLP groups might be causally linked to the speed of wound closure by individual fibroblast strains, we tested the effect of adding exogenous growth factor to the cultures during the assay. Three hours before wounds were inflicted to fibroblast monolayers, TGF-α was added to the standard culture media at either 5 ng/ml or 20 ng/ml. The addition of 5 ng/ml TGF-α significantly increased the RWC (∼1.5-fold; p<7×10−05) in the “intermediate” CLP group, whereas it had no effect in the “fast” group (Fig. 4A, B). The higher concentration of TGF-α (20 ng/ml) caused an increase in both groups, but less in the “fast” (∼1.2-fold, p<7×10−03) than in the “intermediate” (∼1.4-fold, p<1×10−05) (Fig. 4A, B).

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