New insights in collagen turnover in orofacial cleft patients.
OBJECTIVE
We aimed to characterize the fibroblast phenotype of patients by analyzing gene and protein expression of cleft lip and/or cleft palate fibroblasts in relation to collagen turnover and extracellular matrix remodeling.


METHODS
Human palatal fibroblasts were obtained from three healthy subjects without cleft lip and/or cleft palate and from three subjects with nonsyndromic cleft lip and/or cleft palate. Collagen turnover-related gene and protein expression were analyzed by real-time polymerase chain reaction, Western and dot blots, and sodium dodecyl sulfate zymography.


RESULTS
Cleft lip and/or cleft palate fibroblasts, compared with controls, displayed a down-regulation of collagens type I and III messenger RNA (p < .0001 and p < .001, respectively) but an opposite tendency to increase protein levels. Cleft lip and/or cleft palate cells had higher lysyl hydroxylase-2b messenger RNA levels expressed in relation to collagen type I messenger RNA, down-regulated matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1, and Secreted Protein Acidic and Rich in Cysteine messenger RNA (p < .0001 and p < .01, respectively). Pro-matrix metalloproteinase-1 tended to decrease, and pro-matrix metalloproteinase-2 and -9 were down-regulated (p < .01, p < .05, respectively), as was Secreted Protein Acidic and Rich in Cysteine protein expression (p < .05).


CONCLUSIONS
Our results suggest that the cleft lip and/or cleft palate fibroblast phenotype is characterized by a tendency toward interstitial collagen deposition due to posttranslational modifications, such as decreased collagen degradation by matrix metalloproteinases and increased collagen cross-links. These findings may contribute to the knowledge of the cleft lip and/or cleft palate fibroblast phenotype and may be useful to the surgeon when considering the potential wound contraction and subsequent undesired scarring in cleft lip and/or cleft palate ocurring after the surgical closure of a cleft palate.
Gagliano
Nicoletta
N
Department of Human Morphology and Biomedical Sciences–Città Study, Extracellular Matrix Laboratory, University of Milan, Italy. nicoletta.gagliano@unimi.it
Carinci
Francesco
F
Moscheni
Claudia
C
Torri
Carlo
C
Pezzetti
Furio
F
Scapoli
Luca
L
Martinelli
Marcella
M
Gioia
Magda
M
Stabellini
Giordano
G
D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D002971:Cleft Lip / Q000378:metabolism* / Q000601:surgery; D002972:Cleft Palate / Q000378:metabolism* / Q000601:surgery; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D020782:Matrix Metalloproteinases / Q000378:metabolism; D015676:Osteonectin / Q000378:metabolism; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
D015676:Osteonectin; D012333:RNA, Messenger; C577241:SPARC protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D003094:Collagen; D020782:Matrix Metalloproteinases