Expression Of Lefty In Wound Healing And Scarring

Background:Hypertrophic scarring, as an aberrant form of the normal processes of wound healing, is of priority to be resolved in medical researches on burns, trauma and rehabilitation medicine. However, fetal skin, especially those in the first and second trimesters of development, undergoes rapid healing with little or no scarring, which is called as"healing of fetal scarless wound". Convincing evidence showed that many kinds of cytokines and their receptors are involved in wound healing, which constitute a complicated networking system and result in the outcome of wound healing. But the underlying mechanisms remain unclear.Among the cytokines involved, tansforming growth factor beta (TGF-β) is the hottest. It is reported that TGF-β, especially TGF-β1 and TGF-β2, can, on one side, promote the synthesis of collagen, and on the other side, restrain the degradation of collagen. It plays a critical role in scarring and healing of scarless fetal wounds. Thus, it is regarded as the most important factor in wound healing.Lefty, a novel inhibitor of TGF-β, can inhibit the function and signal transmission of TGF-β, influence the proliferation and differentiation of fibroblast, restrain the synthesis of collagen and promote degradation of the collagen. These findings remind us that lefty may inhibit the scarring and play a certain role in the healing of fetal scarless wounds. In order to investigate the role of lefty in the scarring, we detected the expression of lefty in adult normal skin (NS), human fetal skin (FS), hypertrophic scar (HS), and in the process of scarring and healing of fetal scarless wound.Objective:In order to explore the function of lefty in wound healing and the role it plays in the healing of fetal scarless wound, we studied the expression pattern of lefty in NS, FS, HS, and in the process of scarring and healing of fetal scarless wound in improved animal models by using immunofluorescence (IMF) staining and reverse transcription polymerase chain reaction (RT-PCR). Methods:A total of 30 nude mice were equally and randomly divided into 3 groups. The first group was the improved animal model group of hypertrophic scar (IHS group). In this group, the pieces of full-thickness human skin grafts were placed into the subcutaneous pockets on the back of nude (athymic) mice. After the grafts were survived, burn injury was inflicted to them by a heated copper pillar. The second group was the animal model group of human fetal scarless wound healing (FSW group). The specimens of human fetal skin tissue were implanted into the subcutaneous pockets of the back of nude mice. Heated copper pillar was used to burn the grafts after they survived. The third group is the animal model group of analog of hypertrophic scar (AHS group). The mice underwent the transplantation of full-thickness human skin grafts onto the back of nude mice. Nothing was done after that. The statuses of local grafts were observed and histological examination of the grafts was performed after transplantation.The grafts were collected before burn and 1, 2, 3, 7 and 14 days, 1, 3 and 6 months after burn in IHS group and FSW group, and 3 months after transplantation in AHS group. Skin tissue samples from NS (10 samples), FS (8 samples) and HS (15 samples) were also collected to serve as control. Morphological and histological observation, IMF staining, RT-PCR were used to detect the transmutation of the grafts in the animal models, the appearance of the fibroblasts, the expression location of lefty in NS, FS, HS specimens and in the grafts of animal models. The expressions of lefty at protein and mRNA levels were calculated with aid of image analysis system. SPSS 13.0 was employed for statistical analysis. The results of IMF staining were statistically analyzed by Chi-squared. One way ANOVA (single factor analysis of variance) was employed for the comparison in the semi-quantitative expression of lefty in RT-PCR analysis. P <0.05 was considered as statistical difference.Results:1. Establishment of animal modelHypertrophic scar was formed in IHS group with 9 mice surviving out of 10. Before burn, the histological appearance of survived grafts was similar to that of NS control. In 1 month after burn injury, there were obvious persistent scars observed in the 8 mice. The histological features of these tissues were similar to those in HS control. The scars reached their peaks in 2 months after burn and were still higher than the surface of surrounding skin in 6 months after burn.Healing of fetal scarless wounds was observed in FSW group with all 10 mice surviving. There was no obvious difference in the morphology between these grafts and FS control. No noted hypertrophic scar was seen even 6 months after burn injury. There were 8 out of 10 nude mice surviving in AHS group, but only 6 mice developed analogical hypertrophic scar after skin graft rejection. Histological observation displayed abundant collagen deposition and inflammatory infiltration in these scars, which was similar to those in HS control. The AHSs achieved their peaks in 4 months after transplantation and were seen still higher than the surface of surrounding skin 8 months after transplantation.2. Expression of lefty in NS, FS and HS controlsIMF staining indicated that lefty was located in the cytoplasma of fibroblast, in an irregular distribution. The lefty-positive cell rates in HS, NS and FS were 15.38%, 67.92% and 84.48% respectively. The expression was significantly lower in HS than in NS and FS (P<0.01), and was higher in FS than in NS (P<0.05).RT-PCR showed that lefty mRNA was detected in NS, FS and HS controls, with semi-quantitative results of 0.4961±0.0473, 1.2037±0.1261 and 1.6013±0.1283 respectively. The mRNA expression in HS was lower than that in NS and FS (P<0.05) and was higher in FS than in FS (P<0.05).3. Expression of lefty in the process of wound healingIMF staining showed that lefty was distributed irregularly in the cytoplasma of fibroblast. In IHS group, the positive cell percentages were 66.67%, 67.92%%, 70.91%, 66.04%, 60.78%, 51.02%, 42.22%, 19.05% and 17.07% respectively for the following time points, before burn, 1, 2, 3, 7 and 14 days, 1, 3 and 6 months after burn injury. There was statistically difference among these time points (P<0.05). The lefty expression in the beginning was similar to that in NS control (P>0.05), then began to decline from 1 month after burn (P<0.05), reached the expression amount as HS control in 3 months after burn (compared with that of HS control, P>0.05), and maintained a low level since then. In FSW group, the positive cell rates were 79.37%, 80.65%, 82.54%, 79.69%, 80.33%, 79.66%, 78.95%, 78.69% and 76.67% respectively for above-mentioned time points. No significant difference was seen among these groups (P>0.05). Lefty expressed at a high level in the process of wound healing in comparison to that in FS control, and had no obvious change before and after burn injury (P>0.05).RT-PCR analysis indicated that there was positive expression of lefty in the process of wound healing in both animal models of hypertrophic scar and healing of human fetal scarless wound. The semi-quantitative results of IHS group in the time points before burns, 1, 2, 3, 7 and 14 days, 1, 3 and 6 months after burns were 1.0316±0.0680, 1.0510±0.0838, 1.0960±0.0764, 1.0237±0.1070, 0.9410±0.0660, 0.7907±0.0807, 0.6553±0.0465, 0.2949±0.0351 and 0.2652±0.0511 respectively. Significant difference was observed among these time points (P<0.05). The expression in the beginning was similar to that in NS control ( P>0.05), then began to reduce in 7 days after burns (P<0.05), reached the expression amount as HS control in 3 months after burns (0.2949±0.0351, P>0.05), and maintained a low level after then. In HFS group, the results were 1.1058±0.0539, 1.1234±0.0545, 1.1504±0.0633, 1.1095±0.0474, 1.1199±0.0775, 1.1097±0.0494, 1.1020±0.0962, 1.0971±0.0678 and 1.0696±0.0792 respectively at above-mentioned time points. There was no statistical difference among these time points (P>0.05). Lefty expressed at a high level in the process of wound healing, in comparison to that in FS control, and had no significant change before and after burn (P>0.05).Conclusions:1. The survived grafts in IHS group are similar to that in the NS control. The similarity, repeatability, certainty, and survival rate of the animal and grafts in our established animal model of HS were superior to those models ever reported. This model develops obvious persistent scars and is an ideal animal to observe the whole process of hypertrophic scarring.2. The expression is lower in HS samples than in NS and FS samples, and its expression in FS is higher than that in FS. The results suggest that the low expression of lefty might be one of the reasons of hypertrophic scarring, and its high expression be related to the healing of fetal scarless wound.3. The wounds in IHS group produce obvious persistent HS. The expression of lefty in this group is similar to that in NS at the beginning; then began to decline in 7 days after burns at mRNA level, 1 month after burns at protein level; approached the expression in HS 3 months after burns, and maintained a low expression after then. The wounds in HFS group demonstrate as scarless wound healing. The expressions of lefty at all the time points in FSW group are similar to that in FS control, maintaining at a high level in the process of wound healing. The results are in accordance with the morphological observation, and indicate that the low expression of lefty might be one of the reasons of hypertrophic scarring and its high expression might be related to the healing of fetal scarless wound.In this study, we observed the expression pattern of lefty in NS, FS, HS, and during the process of scarring and healing of fetal scarless wound with aid of improved animal models. Our results suggest that lefty might inhibit hypertrophic scarring and its high expression might be related to the healing of fetal scarless wound. More research needs to be carried out to investigate the potential value of lefty in scarring and wound healing.