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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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Different expression levels of TGFA, PDGFC, EGFR and PDGFRB in the three migratory groups.Expression levels were determined for the various mRNAs by qRT-PCR and normalized relative to GAPDH. Results are represented by boxplots; whiskers indicate the maximum and the minimum values in each group (*p<0.05; **p<0.01). (A) TGFA was significantly up-regulated in the “fast” migratory group. The difference between the “intermediate” and “slow” migratory groups was also significant. (B) PDGFC was down-regulated in the “fast” migratory group compared to “slow” and “intermediate” migratory groups. The difference between the “slow” and “intermediate” migratory group was not significant. Expressions levels of the respective receptors EGFR and PDGFRB: (C) The mRNA of EGFR showed roughly the same tendency, but was not significant. (D) PDGFRB was significantly down-regulated in “fast” migratory group compared to the “intermediate” migratory group. Differences in expression level of the same mRNAs were persistent when comparing the “fast” with the “intermediate” migratory group within the CLP cohort: (E) TGFA; (F) PDGFC; (G) EGFR, and (H) PDGFRB.
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pone-0111752-g003: Different expression levels of TGFA, PDGFC, EGFR and PDGFRB in the three migratory groups.Expression levels were determined for the various mRNAs by qRT-PCR and normalized relative to GAPDH. Results are represented by boxplots; whiskers indicate the maximum and the minimum values in each group (*p<0.05; **p<0.01). (A) TGFA was significantly up-regulated in the “fast” migratory group. The difference between the “intermediate” and “slow” migratory groups was also significant. (B) PDGFC was down-regulated in the “fast” migratory group compared to “slow” and “intermediate” migratory groups. The difference between the “slow” and “intermediate” migratory group was not significant. Expressions levels of the respective receptors EGFR and PDGFRB: (C) The mRNA of EGFR showed roughly the same tendency, but was not significant. (D) PDGFRB was significantly down-regulated in “fast” migratory group compared to the “intermediate” migratory group. Differences in expression level of the same mRNAs were persistent when comparing the “fast” with the “intermediate” migratory group within the CLP cohort: (E) TGFA; (F) PDGFC; (G) EGFR, and (H) PDGFRB.

Mentions: We next asked whether and which genes known to be involved in both facial morphogenesis and regeneration might be responsible for the differences observed between fibroblast strains in their speed of wound closure in vitro. Therefore, fibroblasts from the second passage of individual strains were grown to near confluency and their RNA was isolated. The mRNA expression of TGFB1, TGFB3, BMP7, FGF12, EGF, TGFA, PDGFC, TGFBR2, FGFR1, EGFR, PDGFRB, MET, JAG1, TNC, TNW, FN, COL1, COL3, MMP2, MMP9, VCL, ACTA2, ADH1C, IRF6, RUNX2, SOX9 was measured by qRT-PCR, normalized against GAPDH, and statistically evaluated by Kruskal-Wallis followed by a pairwise Wilcoxon rank sum test for multiple comparisons. When comparing cell strains derived from the entire cohort (CLP, Fsk, and Phim subject groups), we did not find significant differences in expression level for most of these genes (not shown). However, our results indicated that the mean rank of TGFA mRNA expression was significantly higher (>2-fold; Fig. 3A) and of PDGFC lower (>3-fold; Fig. 3B) in the “fast” migratory group compared to the “intermediate” and “slow” groups. Interestingly, genes coding for respective receptors of these growth factors showed the same tendency, although differences were only significant for PDGFRB (>2-fold, Fig. 3D), not for TGF-α receptor EGFR (Fig. 3C).


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)

Different expression levels of TGFA, PDGFC, EGFR and PDGFRB in the three migratory groups.Expression levels were determined for the various mRNAs by qRT-PCR and normalized relative to GAPDH. Results are represented by boxplots; whiskers indicate the maximum and the minimum values in each group (*p<0.05; **p<0.01). (A) TGFA was significantly up-regulated in the “fast” migratory group. The difference between the “intermediate” and “slow” migratory groups was also significant. (B) PDGFC was down-regulated in the “fast” migratory group compared to “slow” and “intermediate” migratory groups. The difference between the “slow” and “intermediate” migratory group was not significant. Expressions levels of the respective receptors EGFR and PDGFRB: (C) The mRNA of EGFR showed roughly the same tendency, but was not significant. (D) PDGFRB was significantly down-regulated in “fast” migratory group compared to the “intermediate” migratory group. Differences in expression level of the same mRNAs were persistent when comparing the “fast” with the “intermediate” migratory group within the CLP cohort: (E) TGFA; (F) PDGFC; (G) EGFR, and (H) PDGFRB.
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pone-0111752-g003: Different expression levels of TGFA, PDGFC, EGFR and PDGFRB in the three migratory groups.Expression levels were determined for the various mRNAs by qRT-PCR and normalized relative to GAPDH. Results are represented by boxplots; whiskers indicate the maximum and the minimum values in each group (*p<0.05; **p<0.01). (A) TGFA was significantly up-regulated in the “fast” migratory group. The difference between the “intermediate” and “slow” migratory groups was also significant. (B) PDGFC was down-regulated in the “fast” migratory group compared to “slow” and “intermediate” migratory groups. The difference between the “slow” and “intermediate” migratory group was not significant. Expressions levels of the respective receptors EGFR and PDGFRB: (C) The mRNA of EGFR showed roughly the same tendency, but was not significant. (D) PDGFRB was significantly down-regulated in “fast” migratory group compared to the “intermediate” migratory group. Differences in expression level of the same mRNAs were persistent when comparing the “fast” with the “intermediate” migratory group within the CLP cohort: (E) TGFA; (F) PDGFC; (G) EGFR, and (H) PDGFRB.
Mentions: We next asked whether and which genes known to be involved in both facial morphogenesis and regeneration might be responsible for the differences observed between fibroblast strains in their speed of wound closure in vitro. Therefore, fibroblasts from the second passage of individual strains were grown to near confluency and their RNA was isolated. The mRNA expression of TGFB1, TGFB3, BMP7, FGF12, EGF, TGFA, PDGFC, TGFBR2, FGFR1, EGFR, PDGFRB, MET, JAG1, TNC, TNW, FN, COL1, COL3, MMP2, MMP9, VCL, ACTA2, ADH1C, IRF6, RUNX2, SOX9 was measured by qRT-PCR, normalized against GAPDH, and statistically evaluated by Kruskal-Wallis followed by a pairwise Wilcoxon rank sum test for multiple comparisons. When comparing cell strains derived from the entire cohort (CLP, Fsk, and Phim subject groups), we did not find significant differences in expression level for most of these genes (not shown). However, our results indicated that the mean rank of TGFA mRNA expression was significantly higher (>2-fold; Fig. 3A) and of PDGFC lower (>3-fold; Fig. 3B) in the “fast” migratory group compared to the “intermediate” and “slow” groups. Interestingly, genes coding for respective receptors of these growth factors showed the same tendency, although differences were only significant for PDGFRB (>2-fold, Fig. 3D), not for TGF-α receptor EGFR (Fig. 3C).

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