Modulation of the inflammatory response by increasing fetal wound size or interleukin-10 overexpression determines wound phenotype and scar formation.

Wound size impacts the threshold between scarless regeneration and reparative healing in the fetus with increased inflammation showed in fetal scar formation. We hypothesized that increased fetal wound size increases pro-inflammatory and fibrotic genes with resultant inflammation and fibroplasia and that transition to scar formation could be reversed by overexpression of interleukin-10 (IL-10). To test this hypothesis, 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep. A subset of 8-mm wounds were injected with a lentiviral vector containing the IL-10 transgene (n = 4) or vehicle (n = 4). Wounds were harvested at 3 or 30 days for histology, immunohistochemistry, analysis of gene expression by microarray, and validation with real-time polymerase chain reaction. In contrast to the scarless 2-mm wounds, 8-mm wounds showed scar formation with a differential gene expression profile, increased inflammatory cytokines, decreased CD45+ cells, and subsequent inflammation. Lentiviral-mediated overexpression of the IL-10 gene resulted in conversion to a regenerative phenotype with decreased inflammatory cytokines and regeneration of dermal architecture. In conclusion, increased fetal wounds size leads to a unique gene expression profile that promotes inflammation and leads to scar formation and furthermore, these results show the significance of attenuated inflammation and IL-10 in the transition from fibroplasia to fetal regenerative healing.

The alarmin HMGB-1 influences healing outcomes in fetal skin wounds.

In mice, cutaneous wounds generated early in development (embryonic day 15, E15) heal scarlessly, while wounds generated late in gestation (embryonic day 18, E18) heal with scar formation. Even though both types of wounds are generated in the same sterile uterine environment, scarless fetal wounds heal without inflammation, but a strong inflammatory response is observed in scar-forming fetal wounds. We hypothesized that altered release of alarmins, endogenous molecules that trigger inflammation in response to damage, may be responsible for the age-related changes in inflammation and healing outcomes in fetal skin. The purpose of this study was to determine whether the alarmin high-mobility group box-1 (HMGB-1) is involved in fetal wound repair. Immunohistochemical analysis showed that in unwounded skin, E18 keratinocytes expressed higher levels of HMGB-1 compared with E15 keratinocytes. After injury, HMGB-1 was released to a greater extent from keratinocytes at the margin of scar-forming E18 wounds, compared with scarless E15 wounds. Furthermore, instead of healing scarlessly, E15 wounds healed with scars when treated with HMGB-1. HMGB-1-injected wounds also had more fibroblasts, blood vessels, and macrophages compared with control wounds. Together, these data suggest that extracellular HMGB-1 levels influence the quality of healing in cutaneous wounds.

SOX11 and SOX4 drive the reactivation of an embryonic gene program during murine wound repair.

Tissue injury induces changes in cellular identity, but the underlying molecular mechanisms remain obscure. Here, we show that upon damage in a mouse model, epidermal cells at the wound edge convert to an embryonic-like state, altering particularly the cytoskeletal/extracellular matrix (ECM) components and differentiation program. We show that SOX11 and its closest relative SOX4 dictate embryonic epidermal state, regulating genes involved in epidermal development as well as cytoskeletal/ECM organization. Correspondingly, postnatal induction of SOX11 represses epidermal terminal differentiation while deficiency of Sox11 and Sox4 accelerates differentiation and dramatically impairs cell motility and re-epithelialization. Amongst the embryonic genes reactivated at the wound edge, we identify fascin actin-bundling protein 1 (FSCN1) as a critical direct target of SOX11 and SOX4 regulating cell migration. Our study identifies the reactivated embryonic gene program during wound repair and demonstrates that SOX11 and SOX4 play a central role in this process.

Expression of TGF-beta and its receptors in murine fetal and adult dermal wounds.

The transforming growth factor-betas (TGF-betas) are of major importance in wound healing and have been implicated in the scar-less wound repair observed in fetuses. Few studies have characterised the role of TGF-beta in fetal wound repair and to date no studies have characterised the expression of its receptors within non-scarring fetal wounds. We have localised the TGF-beta isoforms beta1, beta2 and beta3 and its two receptors, TGF-betaRI and TGF-betaRII in both adult and fetal dermal murine wounds. We observed low level immunofluorescence of TGF-beta1 and TGF-beta2 in fetal wounds and although TGF-beta3 staining was observed in the epidermis of fetal skin, there was no upregulation in response to injury. By contrast, all three isoforms were strongly expressed in adult wounds. Similar to its ligands, TGF-beta receptor expression was increased post-wounding in the adult wounds. However, in contrast, no mRNA or protein for either of the TGF-beta receptors was observed in response to wounding in the fetal dermis although there was both mRNA and protein expression of both the receptors localised within the fetal alimentary tract, one of the few fetal organs which does scar post-injury. The differences that we observed in the expression of TGF-beta and its receptors in adult and fetal wounds could be important in the absence of scar formation that is observed in the fetus.

Differential use of Erk1/2 and transforming growth factor beta pathways by mid- and late-gestational murine fibroblasts.

BACKGROUND: Previously, we demonstrated the rapid closure of mid-gestational excisional murine wounds at 32 hours. In this study, we theorized that mid-gestational wounds would be completely regenerated, whereas late-gestational wounds would heal with scar formation at 48 hours. Furthermore, we theorized that mid- and late-gestational fibroblasts differentially use the transforming growth factor beta and mitogen-activated protein kinase pathways. METHODS: Three-millimeter excisional cutaneous wounds were made on murine mid- (embryonic day 15 [E15]) and late-gestational (E18) fetuses and harvested at 48 hours for histology. Percent wound closure was calculated. E15 and E18 fibroblasts were cultured overnight for in vitro scratch wound assay in the presence of the activin receptor-like kinase 4-5-7, Erk1/2, and p38 inhibitors. RESULTS: E15 wounds healed in a regenerative manner, whereas E18 wounds exhibited scar formation. In vitro scratch closure was similar in the E15 and E18 groups at 8 hours; yet, it increased in E15 compared with E18 groups with activin receptor-like kinase 4-5-7 and Erk1/2 inhibitors. p38 inhibition resulted in reduced scratch closure in both groups. CONCLUSION: The scarless mid-gestational excisional wounds compared with the scar-forming late-gestational wounds provides a model to study scar formation. This study also suggests that variable transforming growth factor beta and Erk1/2 signaling may influence differences in wound closure between mid- and late-gestational wounds.

Fetal wound healing using a genetically modified murine model: the contribution of P-selectin.

PURPOSE: During early gestation, fetal wounds heal with paucity of inflammation and absent scar formation. P-selectin is an adhesion molecule that is important for leukocyte recruitment to injury sites. We used a murine fetal wound healing model to study the specific contribution of P-selectin to scarless wound repair. METHODS: Linear excisional wounds were created on the dorsa of E15.5 and E17.5 gestation fetuses in wild-type and P-selectin (-/-) mice (term = 19 days). Wounds were harvested at various time-points after wounding and analyzed using histology and immunohistochemistry. RESULTS: The E15.5 wounds in both wild-type and P-selectin (-/-) fetuses healed scarlessly and with minimal inflammation, whereas E17.5 wounds healed with fibrosis and inflammation. However, the scars of the P-selectin (-/-) wounds appeared slightly different than wild-type. There were significantly more inflammatory cells in E17.5 wild-type wounds 6 hours after injury (P < .001), but the difference was no longer significant by 24 hours. Finally, reepithelialization was slower in the E15.5 knockout wounds compared to their wild-type counterparts. CONCLUSIONS: Absence of P-selectin delays inflammatory cell recruitment and reepithelialization of fetal wounds; however, scar formation still occurs in late gestation wounds. The contribution of specific molecules to fetal wound healing can be elucidated using murine knockout or transgenic models.

Early-gestation fetal scarless wounds have less lysyl oxidase expression.

BACKGROUND: Lysyl oxidase cross-links collagen and elastin. Because cross-linking likely influences collagen architecture, the authors compared lysyl oxidase expression during scarless and scarring fetal dermal wound repair. METHODS: Excisional dermal wounds were made on E17 (gestational day 16.5) and E19 (gestational day 18.5) mouse fetuses. Skin and wound RNA was collected at 8, 12, and 24 hours. Quantitative real-time polymerase chain reaction was performed for lysyl oxidase. The effect of transforming growth factor (TGF)-beta1 on lysyl oxidase expression in fetal fibroblasts was tested. Confluent primary fetal and postnatal fibroblast cultures were stimulated with TGF-beta1 for 24 hours, and lysyl oxidase expression was quantitated by performing real-time polymerase chain reaction. Lysyl oxidase expression was also quantitated in unwounded fetal skin to determine its expression profile during development. RESULTS: E17 and E19 fetal skin had approximately 2-fold greater lysyl oxidase expression than postnatal skin (p < 0.01), and fetal fibroblasts had greater baseline lysyl oxidase expression than postnatal fibroblasts. After TGF-beta1 stimulation, fetal and postnatal fibroblasts responded with increases in lysyl oxidase expression. In E17 early-gestation scarless fetal wounds, lysyl oxidase had small increases (<1.5-fold) in expression from 1 to 12 hours. In late-gestation E19 scarring fetal wounds, lysyl oxidase increased 1.8-fold at 8 hours and 2-fold at 12 hours, which was significantly greater than the changes observed in E17 scarless wounds (p < 0.01 for each). CONCLUSIONS: Lysyl oxidase has greater expression in E19 late-gestation wounds that heal with scar compared with E17 early-gestation scarless wounds. This suggests a role for lysyl oxidase in scar formation.

The extracellular matrix of the fetal wound: hyaluronic acid controls lymphocyte adhesion.

Adhesive interactions between lymphocytes and components of the extracellular matrix (ECM) within a wound environment play a crucial role in determining the inflammatory response following tissue injury. In fetal wounds the extracellular matrix is composed predominantly of hyaluronic acid. Within this environment the inflammatory reaction as a result of injury is minimal. We propose that this lack of an inflammatory cell response in the fetal wound is due to the high levels of hyaluronic acid within the ECM and the inability of lymphocytes to adhere to this matrix component. Therefore, we examined the adhesive properties of fetal lymphocytes to fibronectin, vitronectin, collagen types I, III, IV, V, and hyaluronic acid--ECM components involved in fetal and adult wound environments. Fetal lymphocytes from both spleen and thymus demonstrated significant binding capabilities to fibronectin, vitronectin, and collagen types I and III. No intrinsic binding capabilities were detected to hyaluronic acid. Adhesion was not affected by the addition of IL-1, IFN-gamma, or phorbol dibutyrate. The inability of lymphocytes to adhere to hyaluronic acid helps to explain the lack of inflammation found in fetal wounds and serves to demonstrate the importance of ECM-lymphocyte interactions in determining the inflammatory response during fetal wound healing.

Phenotypic and functional features of myofibroblasts in sheep fetal wounds.

The myofibroblast is a mesenchymal cell with functional and structural characteristics in common with fibroblasts and smooth muscle cells. These cells play a critical role in wound closure and in the pathologic sequelae of healing. It has been shown in adult humans and experimental animals that the myofibroblast expresses alpha -smooth muscle actin (ASMA) temporarily during wound contraction and more persistently during fibrocontractive diseases; however, it is unclear whether this cell makes any contribution to tissue repair in utero. Experimental work in fetal animal models has demonstrated that wound repair in fetal skin occurs by reconstitution of epidermal appendages and organized restoration of the dermal collagen network. Fetal lamb wound healing studies have shown that a transition from scarless tissue repair to healing with scar formation occurs late in gestation. In this study we examined the ontogeny of myofibroblasts in fetal lamb wounds at early through late gestation, using transmission electron microscopy (TEM) and ASMA immunohistochemistry. Dramatic differences were observed in ASMA content of early as compared to late gestation fetal wound granulation tissue: ASMA was absent in wounds made at 75 days gestation but was present in progressively greater amounts in wounds made at 100 and 120 days gestation (term = 145 days). TEM studies also demonstrated progressive development and organization of microfilament bundles. Early in development microfilament bundles were sparse and disorganized, but as gestation progressed the bundles became more prevalent and formed tightly parallel arrangements. The organization of microfilament bundles was also accompanied by fibronexus formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of growth factors in fetal wound healing.

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.