Faculty Bibliography

Surgical correction of unilateral coronal synostosis offers a unique opportunity to examine the molecular differences between an abnormal and a normal cranial suture. We isolated and identified a cDNA fragment whose expression was up-regulated in the premature fusing and fused coronal sutures, as compared with normal coronal sutures. The nucleotide sequence of the full-length cDNA of this gene, human NELL-1, has approximately 61% homology with the chicken Nel gene. Both chicken Nel and human NELL-1 are comprised of six epidermal growth factor-like repeats. The human NELL-1 messages were localized primarily in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins, and within the condensing mesenchymal cells of newly formed bone in sites of premature sutural fusion. Human multiorgan tissue mRNA blot showed that NELL-1 was specifically expressed in fetal brain but not in fetal kidney, liver, or lung. We also showed that Nell-1 was expressed in rat calvarial osteoprogenitor cells and was largely absent in rat tibiae and fibroblast cell cultures. In conclusion, our data suggest that the NELL-1 gene is preferentially expressed in cranial intramembranous bone and neural tissue (both of neural crest cell origin) and is up-regulated during unilateral premature closure of the coronal suture. The precise role of this gene is unknown

The purpose of this study was to determine if aberrant apoptosis plays a role in pathologic wound healing as manifested by hypertrophic scarring and keloid formation. Apoptosis has recently been found to participate in the transition between granulation tissue and the development of definitive scar. The question that remains to be answered is what stimuli initiate apoptosis during wound healing. Hitherto, regulatory factors and pathways involved have been largely undefined. We investigated heterogeneity among fibroblasts derived from normal skin and keloid scar, by examining apoptotic profiles and pathways for these cells. Quantitative analysis of apoptotic cells using an Annexin-V-FITC binding assay showed that normal skin fibroblast cultures were found to have a two-fold higher percentage of apoptotic cells than did keloid fibroblast cultures. To study apoptotic pathways and related death-associated genes, a ribonuclease protection assay was performed for fibroblasts exposed to anti-Fas antibody and tumor necrosis factor-alpha to activate the Fas/TNF receptor apoptotic pathway. Compared with normal skin fibroblasts, keloid fibroblasts exhibited decreased expression of apoptosis-associated genes

We have used DD-PCR (differential display-polymerase chain reaction) to identify new genes that are over- or underexpressed during wound repair. DD-PCR performed on excisional wounds identified the expression of rat c49a. Cloning and sequence analysis of the rat c49a gene revealed high homology to a novel human melanoma differentiation associated gene, mda-7. The human mda-7gene isolated from melanoma cell lines, has been linked with human melanoma differentiation, and growth suppression. Moreover, transfection of human mda-7 constructs into human tumor cells suppresses the growth and colony formation of tumor cells from diverse origins. To confirm and relatively quantitate expression of rat c49a gene during repair, specific primer, reduced cycle RT-PCR (reverse transcription-PCR) was performed. RT-PCR showed an approximately 9 to 12-fold elevation of rat c49a mRNA at 12 h to 5 days above nonwounded controls that gradually decreased to approximately 1.5 to 3-fold by day 14. Cloning and sequence analysis of the entire 1200 base pair c49a gene product showed 78% nucleotide homology to human mda-7. Immunohistochemistry studies localized rat C49A expression primarily to fibroblast-like cells at the wound edge and base. The marked up-regulation of rat c49a transcripts during the inflammatory and early granulation tissue phases of wound repair where cellular processes such as re-epithelialization, angiogenesis, and fibroplasia predominate--suggest that c49a is associated with proliferation of fibroblasts in wound healing