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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 14, 2002 as doi:10.1096/fj.01-0496fje. |
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,23,
,2,**
,
,4
* Department of Surgery, University Children’s Hospital of Zürich;
Institute of Physiology and
Institute of Veterinary Physiology, University of Zürich;
Institute of Physiology, and
Department of Anesthesiology, Medical University of Lübeck, D-23538 Lübeck, Germany;
Center of Experimental Rheumatology;
|| Institute of Laboratory Animal Science and
** Department of Obstetrics and Gynecology, University Hospital of Zürich, CH-8096 Zürich, Switzerland; and
Department of Biology, University of California, San Diego, California 92093, USA
3Correspondence: Department of Surgery, University Children’s Hospital, Steinwiesstrasse 75, CH-8032 Zürich, Switzerland. E-mail: ascheid@physiol.unizh.ch and mmeuli{at}kispi.unizh.ch
SPECIFIC AIMS
Understanding environmental molecular regulation in scarless early fetal wound healing may reveal scar-limiting therapeutical strategies for the prevention of postnatal scarring wound repair. We wanted to know whether fetal physiological hypoxia leads to constitutive expression of hypoxia-inducible factor 1
(HIF-1) in fetal skin during development and fetal wound healing. In an in vivo sheep model, we investigated three gestational ages correlating with the three types of wound healing observed: scarless wound healing at gestational day (GD) 60, beginning scar formation at GD 100 (‘transitional wounds’), and adult-like scar formation at GD 120 (term=145 days). To elucidate the functional significance of HIF-1 expression, we analyzed its effects on migratory capabilities of mouse embryonic fibroblasts (MEFs) and their expression of transforming growth factor (TGF) -ß1 and -ß3 mRNA in vitro.
PRINCIPAL FINDINGS
Overlapping expression patterns of HIF-1 and TGF-ß3 in vivo
Immunohistochemistry for TGF-ß3 and HIF-1 was performed in the fetal skin and wound tissues. HIF-1 and TGF-ß3 expression patterns correlated: the higher nuclear HIF-1 expression, the higher cytoplasmic/perinuclear overlapping TGF-ß3 staining. In fetal skin wounds, HIF-1 expression is absent in nonscarring wounds (GD 60); strong, prolonged, and fibroblast-specific in transitional wounds (GD 100); and moderate, temporary, and predominantly inflammatory cell-specific in scarring wounds (GD 120). These results suggest that HIF-1-dependent regulation of TGF-ß3 is pathophysiologically relevant to the wound repair process in vivo. Figure 1
exemplifies findings in a 2 day wound at GD 100.
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HIF-1-dependent mRNA regulation of TGF-ß family members in vitro
Hypoxia-dependent regulation of TGF-ß family members has been reported that could underlie HIF-1-dependent regulation. TGF-ß family members are known to be growth factor stimulated, and fetal fibroblasts find themselves under growth factor-deprived conditions when compared with adult fibroblasts. We therefore analyzed mRNA expression levels of TGF-ß family members in HIF-1+/+ and HIF-1-/- MEFs by RNA blotting. As shown in Fig. 2
a, HIF-1+/+ but not HIF-1-/- MEFs expressed HIF-1 mRNA, which was not dependent on oxygen or serum concentrations. VEGF mRNA expression patterns were investigated to confirm hypoxic stimulation of the MEFs. No consistent effects of serum concentrations on VEGF mRNA expression levels were observed (Fig. 2a
). TGF-ß1 mRNA expression was serum inducible but not hypoxia inducible in HIF-1+/+ and HIF-1-/- MEFs (Fig. 2a
). In contrast, TGF-ß3 mRNA expression was hypoxia but not serum inducible, and basal TGF-ß3 levels were nearly undetectable in HIF-1-/- MEFs, demonstrating that TGF-ß3 might be a HIF-1 target gene (Fig. 2a
).
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Influence of HIF-1 expression on fibroblast migration
An important aspect of wound healing involves the capacity of fibroblasts to migrate toward the wound edge. We were interested in whether HIF-1 expression in MEFs influences their migratory capabilities and so measured the migratory rates of HIF-1+/+ and HIF-1-/- MEFs.
In one assay, we tested the ability of both MEFs to close an in vitro ‘wound’ in a monolayer grown on plastic (Fig. 2b
). HIF-1+/+ MEFs migrated faster under 1% O2 than under 20% O2, suggesting a role for hypoxia-induced HIF-1 in cellular migration (Fig. 2b
). Consistent with this, HIF-1-/- MEFs demonstrated clearly impaired migratory capabilities vs. HIF-1+/+ MEFs (Fig. 2b
). The difference between migration capabilities of HIF-1+/+ and HIF-1-/- MEFs was most pronounced at 1% O2.
We then performed Boyden chamber experiments to measure the number of cells crossing a membrane to reach the other side, where they were stained and counted. This assay enabled us to compare the chemotactic potential of HIF-1+/+ and HIF-1-/- MEF-conditioned medium. HIF-1+/+ MEFs showed significantly enhanced migration against HIF-1+/+-conditioned medium compared with their migration against HIF-1-/--conditioned or unconditioned medium (Fig. 2c
). HIF-1+/+ MEF migration against HIF-1-/--conditioned medium did not differ significantly from their migration against unconditioned medium (Fig. 2c
). The migratory capabilities of HIF-1-/- MEFs against HIF-1+/+-conditioned medium, HIF-1-/--conditioned medium, and unconditioned medium showed no significant differences (Fig. 2c
). HIF-1+/+ MEF migration against HIF-1+/+-conditioned medium was significantly enhanced vs. HIF-1-/- MEF migration against (the same) HIF-1+/+-conditioned medium (Fig. 2c
).
CONCLUSIONS AND SIGNIFICANCE
We did not detect HIF-1 protein in fetal skin at early gestation but saw an impressive up-regulation of HIF-1 protein in fibroblasts at GD 100. Our findings agree with the immunoblot analysis of HIF-1 expression in HIF-1+/+ whole mouse embryo lysates, where HIF-1 protein was barely detectable at E8.5, then showed highly increased expression up to E18. Fetal skin tissue pO2 levels were persistently low throughout development, so that a switch in tissue pO2 levels can be eliminated as the sole driving force behind the impressive up-regulation of HIF-1 at later gestation.
In fetal skin and wound tissue, HIF-1 and TGF-ß3 expression patterns correlated: the higher nuclear HIF-1 expression, the higher cytoplasmic/perinuclear TGF-ß3 staining. A growing number of studies indicate that whether a wound heals scarlessly or by scar formation is a question of differing equilibria of antifibrotic vs. profibrotic growth factors at the wound site. Addition of TGF-ß1 to nonscarring fetal wounds induced scar formation whereas addition of neutralizing antibodies against TGF-ß1 and 2 or addition of TGF-ß3 to scarring adult wounds decreased scar formation. That we could not detect HIF-1 or TGF-ß3 staining in nonscarring GD 60 wounds clearly demonstrates that other anti-scarring, nonHIF-1 regulated genes contribute to scarless fetal wound healing. However, identification of strong, prolonged, and fibroblast-specific expression of HIF-1 and TGF-ß3 in transitional wounds with reduced scar formation at GD 100 suggests that fibroblast expression of HIF-1 in transitional wounds at GD 100 may be a key regulatory factor for up-regulation of anti-scarring TGF-ß3 in transitional wounds. HIF-1 and TGF-ß3 expression in scarring late-gestational (GD 120) wounds was far less pronounced and was limited to late inflammation. This may lead to an overruling of profibrotic (HIF-1 independent) TGF-ß1 over its anti-fibrotic (HIF-1-dependent) counterpart TGF-ß3 (Fig. 3
).
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Our studies of mRNA expression patterns of TGF-ß1 and ß3 in wild-type and HIF-1-/- MEFs after longer term exposure to (physiologically) low pO2 levels revealed anti-fibrotic TGF-ß3 as a potentially oxygen (HIF-1) -regulated growth factor. This agrees with recent findings by Caniggia et al. in the placenta demonstrating reduced TGF-ß3 expression in human villous explants cultured at 3% O2 after antisense inhibition of HIF-1. We are now identifying the functionally significant HIF-1 binding sites in the mouse TGF-ß3 promoter to analyze the involvement of HIF-1 in oxygen-regulated expression of this growth factor.
We report on the fetal fibroblast as the principal cell expressing HIF-1 in GD 100 skin and transitional wounds. Fetal fibroblasts show enhanced migratory capabilities vs. adult fibroblasts in vitro and appear at the wound edge earlier than adult fibroblasts in vivo. In vivo fetal fibroblasts at the wound edge express HIF-1 whereas their adult counterparts do not (A. Scheid et al., unpublished observations). We examined whether constitutive HIF-1 expression influences migratory capabilities of embryonic fibroblasts by performing a comparative in vitro scratch assay and a Boyden chamber assay with wild-type and HIF-1-/- MEFs. HIF-1-deficient MEFs showed impaired migratory capabilities, especially under pO2 levels similar to those during development in vivo. The fact that in the Boyden chamber assay, HIF-1+/+ MEFs show enhanced migration against HIF-1+/+-conditioned medium when compared with migration against HIF-1-/--conditioned or unconditioned medium suggests that HIF-1+/+-conditioned medium contains a secreted chemotactic agent not present in either HIF-1-/--conditioned or unconditioned medium. HIF-1 expression thus seems to modulate migration via induced secretion of a chemotactic substance. We are testing whether this substance could in fact be TGF-ß3 itself. The enhanced migratory capabilities of HIF-1+/+ MEFs vs. HIF-/- MEFs when migrating against HIF-1+/+-conditioned medium were impressive and suggest that HIF-1 has a second, direct modulatory effect on cell migration, possibly via regulation of genes controlling actin-based locomotion.
HIF-1 is not expressed in nonscarring fetal wounds but is expressed by fibroblasts in transitional fetal wounds and by inflammatory cells in late-gestational scarring wounds. Constitutive HIF-1 expression by fetal mesenchymal cells may contribute to their enhanced migratory capabilities and regulate expression of a central anti-scarring growth factor (TGF-ß3) during fetal wound healing. Its regulation of a potent anti-scarring cytokine may provide new strategies for anti-scarring manipulation of wound healing.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0496fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0496fje 
2 These two authors contributed equally to the work.
4 The contribution of the two senior authors was equivalent.
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