Mice lacking TGF-β3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-β3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.Members of the transforming growth factor-β (TGF-β) gene family have biological activities that control cell proliferation, migration and differentiation, regulation of extracellular matrix deposition and epithelial-mesenchymal transformation [1][2][3] . Mammals contain three highly conserved isoforms of TGF-β, termed TGF-β1, TGF-β2 and TGF-β3, which display distinctive, although at times overlapping, spatial and temporal expression patterns [4][5][6] . Previous studies suggested that TGF-β3 may play a crucial role during palatogenesis 7-9 , Meckel's cartilage formation 10 , cardiac morphogenesis 11 , mammary gland development 12 and wound healing 13 . Other tissues expressing TGF-β3 in significant levels are cartilage, bone, brain and lung [4][5][6]14 .In mammalian palatogenesis apposition of the palatal shelves, adhesion of the medial edge epithelia (MEE) and subsequent elimination of the epithelial seam lead to a seamless mesenchymal shelf separating the oral and nasal cavities 15 . In vitro organ culture studies indicate that TGF-β1 and TGF-β2 accelerate palatal shelf fusion 16,17 and that antisense oligodeoxynucleotides or neutralizing antibodies to TGF-β3, but not to TGF-β1 or TGF-β2, block the fusion process 9 . We have now created mice deficient in TGF-β3, and show that this factor has a role in palatal shelf fusion by means of an intrinsic, primary mechanism and not by effects secondary to craniofacial morphometrics. A comparison of this defect to the inflammatory disorder of TGF-β1-deficient mice [18][19][20][21] Mutation of TGF-β3 in ES cellsThe TGF-β3 gene was mutated in ES cells (Fig. 1a) by replacing exon 6, the first full exon encoding sequences of the active domain of the protein, with the neomycin-resistance gene from pMC1neo 22 . Diagnostic Southern blots of the clone I98 indicated that the locus was successfully targeted; the proper genomic regions flanking both sides of the target site remained intact (Fig. 1b). Probing with a neo-gene probe indicated that there was only one integration site (not shown). Consequently, only the TGF-β3 locus has been disrupted. RT-PCR analysis of whole 11.5- (Fig. 1c) and 15.5-day embryos (not shown) indicated no TGF-β3 expression in homozygous mutant embryos, and revealed no significant change in the expression of TGF-β1 or TGF-β2 in the absence of TGF-β3. Cleft palate in TGF-β3 null mutantsThe targeted ES cell clone I98 was used to produce chi-maeric mice, which were mated with CF-1, C57BL/6 or 129/Sv mice. Heterozygous offspring showed no apparent phenotype. Interc...
The effects of intrinsic aging on the cutaneous wound healing process are profound , and the resulting acute and chronic wound morbidity imposes a substantial burden on health services. We have investigated the effects of topical estrogen on cutaneous wound healing in healthy elderly men and women , and related these effects to the inflammatory response and local elastase levels , an enzyme known to be up-regulated in impaired wound healing states. Eighteen health status-defined females (mean age , 74.4 years) and eighteen males (mean age , 70.7 years) were randomized in a double-blind study to either active estrogen patch or identical placebo patch attached for 24 hours to the upper inner arm , through which two 4-mm punch biopsies were made. The wounds were excised at either day 7 or day 80 post-wounding. Compared to placebo , estrogen treatment increased the extent of wound healing in both males and females with a decrease in wound size at day 7 , increased collagen levels at both days 7 and 80 , and increased day 7 fibronectin levels. In addition , estrogen enhanced the strength of day 80 wounds. Estrogen treatment was associated with a decrease in wound elastase levels secondary to reduced neutrophil numbers , and decreased fibronectin degradation. In vitro studies using isolated human neutrophils indicate that one mechanism underlying the altered inflammatory response involves both a direct inhibition of neutrophil chemotaxis by estrogen and an altered expression of neutrophil adhesion molecules. These data demonstrate that delays in wound healing in the elderly can be significantly diminished by topical estrogen in both male and female subjects. (Am J Pathol 1999, 155:1137-1146) Acute and chronic wound healing morbidity in the elderly imposes a substantial burden on health services.1 Healing of an acute wound involves a reduction in wound size secondary to contraction and re-epithelialization and is accompanied by an increase in collagen deposition.
To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.
The concept that aging impairs wound healing is largely unsubstantiated, the literature being contradictory because of poor experimental design and a failure to adequately characterize animal models. This study tested the hypothesis that aging retards the rate of wound repair using standardized cutaneous incisional wounds in a well-characterized aging mouse colony. Against the background of age-related changes in normal dermal composition, marked differences in healing were observed. Immunostaining for fibronectin was decreased in the wounds of the old mice, with a delay in the inflammatory response, re-epithelialization, and the appearance of extracellular matrix components. Heparan sulfate and blood vessel staining were both unexpectedly increased in the wounds of the old animals at late time points. Despite an overall decrease in collagen I and III deposition in the wounds of old mice, the dermal organization was surprisingly similar to that of normal dermal basket-weave collagen architecture. By contrast, young animals developed abnormal, dense scars. Intriguingly, some of these age-related changes in scar quality and inflammatory cell profile are similar to those seen in fetal wound healing. The rate of healing in young animals appears to be increased at the expense of the scar quality, perhaps resulting from an altered inflammatory response.
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