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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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Distinct CLP migratory groups persist under low serum conditions.The graphs show the RWC in scratch wound assays at 24 h in 0.3% FCS/DMEM (*p<0.05, **p<0.01, ***p<0.001). (A) Despite a general decrease of the RWC, “fast” and “intermediate” CLP groups could still be distinguished under low-serum conditions. (B, C) Effect of exogenous TGF-α diluted at 5 ng/ml (T5) and 20 ng/ml (T20), as well as of 5 µM Lapatinib (Lapa) on both CLP subgroups under low serum conditions in comparison to controls (Ctrl).
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pone-0111752-g005: Distinct CLP migratory groups persist under low serum conditions.The graphs show the RWC in scratch wound assays at 24 h in 0.3% FCS/DMEM (*p<0.05, **p<0.01, ***p<0.001). (A) Despite a general decrease of the RWC, “fast” and “intermediate” CLP groups could still be distinguished under low-serum conditions. (B, C) Effect of exogenous TGF-α diluted at 5 ng/ml (T5) and 20 ng/ml (T20), as well as of 5 µM Lapatinib (Lapa) on both CLP subgroups under low serum conditions in comparison to controls (Ctrl).

Mentions: All experiments with TGF-α and inhibitors described so far were done in standard medium containing 10% serum, which simulates conditions in a wound in vivo. Since serum contains other growth factors (such as platelet-derived PDGF-BB) that might independently or synergistically stimulate fibroblast migration in scratch wound assays, it was important to test whether effects of TGF-α were persistent in low-serum conditions. Scratch wound assays were conducted with CLP strains in 0.3% FCS/DMEM culture media. Also in low serum, the difference in the speed of wound closure between “fast” and “intermediate” CLP groups was significant (p<4×10−05): The RWC of the “fast” group was ∼1.3-fold higher than the one of the “intermediate” group (Fig. 5A). Next we tested the wound closure ability after addition of either TGF-α or Lapatinib to both groups. The “intermediate” group was rescued by 5 ng/ml TGF-α in terms of the RWC (∼1.3-fold, p<0.02; Fig. 5C). A significant but rather small increase was measured for the “fast” group (∼1.1-fold, p<0.02; Fig. 5B). The higher concentration of TGF-α caused an increase in both groups (“fast” ∼1.5-fold, p<2×10−03, “intermediate” ∼1.2-fold, p<0.04; Fig. 5B, C). When lapatinib was preincubated with cultures at 5 µM in low-serum media, the RWC of both the “fast” and the “intermediate” group was significantly decreased (p<0.01) (Fig. 5B, C). Thus, the effects of TGF-α or 5 µM Lapatinib seen under high-serum conditions could in essence been reproduced in medium containing only 0.3% FCS, although the RWCs were generally lower.


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)

Distinct CLP migratory groups persist under low serum conditions.The graphs show the RWC in scratch wound assays at 24 h in 0.3% FCS/DMEM (*p<0.05, **p<0.01, ***p<0.001). (A) Despite a general decrease of the RWC, “fast” and “intermediate” CLP groups could still be distinguished under low-serum conditions. (B, C) Effect of exogenous TGF-α diluted at 5 ng/ml (T5) and 20 ng/ml (T20), as well as of 5 µM Lapatinib (Lapa) on both CLP subgroups under low serum conditions in comparison to controls (Ctrl).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111752-g005: Distinct CLP migratory groups persist under low serum conditions.The graphs show the RWC in scratch wound assays at 24 h in 0.3% FCS/DMEM (*p<0.05, **p<0.01, ***p<0.001). (A) Despite a general decrease of the RWC, “fast” and “intermediate” CLP groups could still be distinguished under low-serum conditions. (B, C) Effect of exogenous TGF-α diluted at 5 ng/ml (T5) and 20 ng/ml (T20), as well as of 5 µM Lapatinib (Lapa) on both CLP subgroups under low serum conditions in comparison to controls (Ctrl).
Mentions: All experiments with TGF-α and inhibitors described so far were done in standard medium containing 10% serum, which simulates conditions in a wound in vivo. Since serum contains other growth factors (such as platelet-derived PDGF-BB) that might independently or synergistically stimulate fibroblast migration in scratch wound assays, it was important to test whether effects of TGF-α were persistent in low-serum conditions. Scratch wound assays were conducted with CLP strains in 0.3% FCS/DMEM culture media. Also in low serum, the difference in the speed of wound closure between “fast” and “intermediate” CLP groups was significant (p<4×10−05): The RWC of the “fast” group was ∼1.3-fold higher than the one of the “intermediate” group (Fig. 5A). Next we tested the wound closure ability after addition of either TGF-α or Lapatinib to both groups. The “intermediate” group was rescued by 5 ng/ml TGF-α in terms of the RWC (∼1.3-fold, p<0.02; Fig. 5C). A significant but rather small increase was measured for the “fast” group (∼1.1-fold, p<0.02; Fig. 5B). The higher concentration of TGF-α caused an increase in both groups (“fast” ∼1.5-fold, p<2×10−03, “intermediate” ∼1.2-fold, p<0.04; Fig. 5B, C). When lapatinib was preincubated with cultures at 5 µM in low-serum media, the RWC of both the “fast” and the “intermediate” group was significantly decreased (p<0.01) (Fig. 5B, C). Thus, the effects of TGF-α or 5 µM Lapatinib seen under high-serum conditions could in essence been reproduced in medium containing only 0.3% FCS, although the RWCs were generally lower.

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