Mechanical Injury Incudes Changes Of Stemness And Radiation Response In Fibroblasts And Its Implication For Tissue Regeneration And Repair

BackgroundWound healing is a complex biological process involving repair cells, factors, and extracellular matrix coordinatedly taking part. Skin, the largest and also esily injured organ of the body, plays important roles in resisting external injury, regulating body tempreture, maintaining body fluid balance. Thus it is of great significance to study the cautanous wound healing. Repair cells are the key factor for cautanous wound healing. Following wound, resident skin cells are activated to experience of proliferation, migration, differentiation, apoptosis, when interrupted, it would lead to abnormality of healing. Wound microenvironment, the majour factor to influence biological behavior of repair cells, contains the external wound microenvironment and the internal wound microenvironment. It is one of the key points of wound healing to study the influence of wound microenvironment upon repair cells biological behavior. Previous studies have demonstrated that fibroblasts, the majory cell type in the skin, were activated to proliferate, migrate into the wound cavity and secrete amount of excellular matrix following wounding. In addition, they also could be induced into myfibroblasts to help for wound closure. Recent studies found that fibroblasts exhibited great heterogenicity that different subpopulations showed considerable functional diversity. For example, following injury, the initial wave of wound repair is mediated by the lower lineage and upper fibroblast lineages are recruited only during re-epithelialization. In addition, the high plasticity of fibroblasts is now paid much more attenition. Fibroblasts are the most widely used initial cell type for induced pluripotent stem cells and direct lineage reprogramming including cardiomyocytes, neurons, neural progenitor cells, islet cells, hematopoietic stem cells. However,it is still not clear about the relationship between wound microenvironment and plasticity especially stemness and radiation response of fibroblasts and the inner mechanism.Methods1. The biologic property changes of fibroblasts upon mechanical injuryWe adiministrated wound scratch model to minic wound, and detected the phonotype change, proliferation with Ki67, expression of the stem cell realted markers, the colony forming ability, the transcriptional state change with RNA Sequencing.2. The biologic property changes of fibroblasts in wound microenvironment.We isolated the granulation tissue cells following wound in mice, and tested the stem cells related properties with CCK-8 for proliferation, colony forming ability for self-renew, adipogenic and osteogenic differentiation for multipotentiality. Further, we transplanted granulation tissue cells into radiation combined wound injury mice, and deteted their therapeutical effects on wound healing. In addition, we tested the roles of miR-21/ROS pathway in the stem cell related properties of granulation tissue cells and neonatal dermal cells with miR-21 antagomir and ROS scavenger.3. Mechanical injury affects radiation response of fibroblastsUsing in vitro and in vivo radiation combined wound injury model, we tested the outcome of different order of occurrence of radiation and mechanical injury by detecting the colony forming ability and expression of γH2AX. We futher measured the radiation response changes of fibroblasts following mechanical injury with comet assay, apoptosis assay with PI/Annexin V, cell cycle assay, micronuclei assay. In addition, we tested the PI3K/AKT signaling pathway activation follow mechanical injury with Western blot and Immunofluorescence. In turn, we tested the roles of β-catenin in stemness of fibroblasts and Nrf2 in antioxidant ability and DNA repair of fibroblasts with administrating siRNA respectivelyResults1. Biological property changes of skin fibroblasts induced by mechanical injury.1.1 Using a scratch wound model, we found fibroblasts were induced to be a small round cell phnotype, and activated to proliferate about 36 hours following mechanical injury. The activation of fibroblasts was not affected by wound size. The expression of Sox2 and vimentin and conlony forming ability were increased following mechanical injury,other tested markers were not changed significantly.1.2 The transcriptional state changes sifnificantly following mechanical injury. The focal adhesion, TGF-β pathway, PI3K/AKT pathway, pluripotency of stem cells pathway et al were changed significantly. Genes of focal adhesion, cell cycle, migration and DNA repair were also up-regulated.2. Stemness related properties changes of fibroblasts in wound microenvironment.2.1 We successfully isolated fibroblast in the wound tissue(granulation tissue cells), and found they showed significantly up-regulating proliferation, colony forming ability and induced adipogenic and osteogenic differentiation ability compared to normal fibroblasts. Further transplanting them into radiation combined wound injury mice could sifnificantly accelecte wound closure and decrease collagen deposition.2.2 mi R-21 was up regulated in granulation tissue cells. When antagonized by mi R-21 antagomir, granulation tissue cells showed increased proliferation and conlony formation ability but decreased migration and induced adipogenic and osteogenic differentiation, which suggested miR-21 could negatively regulated the stemness of granulation tissue cells.2.3 ROS was found to be up-regulated by miR-21 in granulation tissue cells, and administration of NAC could increase proliferation and conlony formation ability but decrease migration and induced adipogenic and osteogenic differentiation, which suggested mi R-21 could negatively regulate the stemness of granulation tissue cells via ROS pathway.2.4 We further proved that miR-21 could negatively regulate the stemness but promote migration and mult-differentiation via ROS pathway in neonatal dermal cells3. Mechanical injury affects the radiation response of fibroblats3.1 Using in vitro radition combined mechanical injury model, we found that fibroblasts in the post-wound IR group formed the most colonies, while there were fewer colonies in the pre-wound IR group than in the IR alone group. Fibroblast colonies in post-wound IR group were also more numerous than in the IR alone group after exposing to different IR doses, which suggested that mechanical injury could increase radioresistance of fibroblasts. However, these changes were not observed in tumor cells3.2 Expression of γH2AX, the olive tail moment and tail DNA percentage, micronuclei formation, and apoptosis in post-wound IR group were showed significantly less than in IR alone group fibroblasts. However, expression ofγH2AX is more in post-wound IR tumor cells.3.3 Following IR, confluent fibroblasts arrested in G1/S phase, however, scratched fibroblasts arrested in G2/M phage, and scratched fibroblasts recovered more quickly. Detecting the DNA repair related proteins following IR, the expression of RAD51 in scratched cells were much higher than confluent cells, however, the expression of NHEJ related proteins showed no difference. The expression of these proteins in tumor cells showed no difference.3.4 In vivo experiments, the population of γH2AX-positive cells and the signal intensity of γH2AX staining were much lower in post-wound IR skin fibroblasts. Granulation tissue-derived fibroblasts from the mice in post-wound IR group formed more and larger colonies than those in the pre-wound IR group.3.5 Expression of active AKT in fibroblasts were increased 2h following mechanical injury, and in turn up-regulated the expression of P-S9 GSK-3β. The down stream Nrf2 and β-catenin were found up-regulated for the first time accompanying with the inhibition of GSK-3β. Administrating PI3 K inhibitor and GSK-3βinhibitor further verified the activation of PI3K/AKT pathway following mechanical injury.3.6 β-catenin was up-regulated following mechanical injury, and in turn regulating the expression of Sox2, and finally contributed to radioresistance of fibroblasts. siRNA of β-catenin could reverse it.3.7 Nrf2 was up-regulated by activation of PI3K/AKT pathway following mechanical injury, and in turn regulating the expression of SOD1 and RAD51 to increase the antioxidant ability and DNA repair ability respectively, finally contributed to radioresistance of fibroblasts. siRNA of Nrf2 could significantly reverse it.Conclusions1.Mechanical injury could activate fibroblasts to proliferate about 36 hours following injury, and the proliferation activation was independent of wound size. Further, we found mechanical injury could enhance the stemness of fibroblasts for the first time by detecting the change of phenotype, transcriptional state, and stem cell specific markers.2.In vivo, mechanical injury could also increase the stem cell related properties of fibroblasts including proliferation, self-renew and multipotentiality. In addition, we found mi R-21 negatively regulated the stemness of fibroblasts, however, promoted migration and differentiation.3. We observed for the first time that the order of occurrence of radiation and mechanical injury affected the outcome of combined effects such that survivals of fibroblasts improved when mechanical injury occurred prior to IR exposure in vivo and in vitro. However these changes were not observed in tumor cells.4. Mechanical injury could activate the PI3K/AKT/GSK-3β pathway, and both of the downstream proteins β-catenin and Nrf2 were found up-regulated for the first time. Subsequent nuclear translocation of Nrf2 and β-catenin strengthenes the stemness, antioxidant capabilities, and DNA double-strand break repair abilities of fibroblasts respectively, ultimately contributing to increased radioresistance.