Systematic profiling of early regulators during tissue regeneration using zebrafish model.

Great progresses have been made in comprehension of tissue regeneration process. However, one of the central questions in regeneration research remains to be deciphered is what factors initiate regenerative process. In present study, we focused on systematic profiling of early regulators in tissue regeneration via high-throughput screening on zebrafish caudal fin model. Firstly, 53 GO-annotated regeneration-related genes, which were specifically activated upon fin amputation, were identified according to the transcriptomic analysis. Moreover, qRT-PCR analysis of a couple of randomly selected genes from the aforementioned gene list validated our sequencing results. These studies confirmed the reliability of transcriptome sequencing analysis. Fibroblast growth factor 20a (fgf20a) is a key initial factor in the regeneration of zebrafish. Through a gene expression correlation analysis, we discovered a collection of 70 genes correlating with fgf20a, whose expression increased promptly at 2 days post amputation (dpa) and went down to the basal level until the completion of fin regeneration. In addition, two genes, socs3b and nppc, were chosen to investigate their functions during the fin regeneration. Inhibition of either of those genes significantly delayed the regenerative process. Taken together, we provided a simple and effective time-saving strategy that may serve as a tool for identifying early regulators in regeneration and identified 71 genes as early regulators of fin regeneration.

The effect of hyperbaric oxygen therapy on gene expression: microarray analysis on wound healing.

Background: Hyperbaric oxygen (HBO2) therapy can have a positive effect on wound healing, angiogenesis and blood flow. No prior study has described the effects of HBO2 therapy and gene expression of this process. The goal of our research was to show the effects of HBO2 and its impact at the molecular level on angiogenesis, proliferation, differentiation, oxidative stress, inflammation, and extracellular matrix formation. Live animal subjects were used for simulating the process of wound healing under standard conditions and under the influence of HBO2. Methods: Two experimental groups were created using injured rabbits (N=24), one group (N=12) treated with hyperbaric therapy twice a day and one (N=12) with standard wound care management. Wounds were surgical, uninfected, and in healthy animal test subjects. We compared the whole genomic analysis of the transcriptome with the use of microarray technology at three intervals during treatment. Results: The induction of the wounds in rabbit skin increased expression of hundreds of genes in both treatment groups. The numbers of elevated and decreased genes gradually reduced as the wound healed. Gene expression analysis showed elevated expression of several genes associated with inflammation in both groups of injured animals. Genes connected to the process of angiogenesis, proliferation, differentiation, oxidative stress and extracellular matrix formation were without statistically significant changes. Conclusion: The evidence did not support that HBO2 had any significant effect on gene expression during wound healing. Additionally, there was no evidence to support that there were changes in gene expression in either treatment group.

Expression of Metalloproteinases and Type I and III Collagens during Healing of Excisional Skin Wound on the Abdomen and Back in Rats.

The study was carried out using a novel rat model developed in our laboratory, namely16 mm diameter circular excisional wounds were generated on the abdomen which resulted in minimal scarring. Restoration of the skin integrity was completed by day 60 after the wounding surgery. By this time, regenerates on the abdomen were stronger than on the back (at, respectively, 58 and 17.4 % of the tensile strength of the intact skin at corresponding location) and the ratio of type I and type III collagens in regenerates on the abdomen reached the level of intact skin at the same location. On days 3 to 14, the ratio of Mmp9/Timp1 expression levels on the abdomen was higher than on the back. On days 20 and 30, the Mmp9/Timp1 ratio on the abdomen was identical to the level of intact skin, whereas the increased MMPs expression levels on the back were maintained until day 30. It has been shown for the first time that according to functional and molecular characteristics, wound healing on the abdomen of an adult rat is more similar to complete regeneration than scarring repair of the back skin.

Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway.

Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.

Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress-induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress-induced mitochondrial dysfunction and stimulate bone marrow-derived EPC (BM-EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM-EPCs and prevented tert-butyl hydroperoxide (TBHP)-induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM-EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury-related wounds.

Tumor potential in rat wounds after short- and long-term administration of platelet-rich plasma.

Platelet-rich plasma (PRP) has been recognized as an effective strategy for tissue regeneration, how-ever, the safety of PRP in wound healing in terms of tumorigenicity has not yet been addressed. Therefore, the aim of this study was to examine the impact of PRP administration on the expression of the inflammatory marker, tenascin-C (TnC) and the myofibroblast markers, α-smooth muscle actin (α-SMA) and vimentin. The immune suppressive response was examined by determining the level of forkhead box protein 3 (Foxp3). PRP was administered for both long-term (two times weekly for four weeks) and short-term (for the fourth week only) post-wounding. Collagen I (col1) and lysyl oxidase (LOX) were used to indicate complete healing, after which any increase in the myofibroblast or in the inflammatory markers would suggest tumor potential. Collagen III (col3), a marker for granulation tissue, was used to remark non-healing. Quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR) and Western blot showed that after long-term administration of PRP, the expression of TnC, α-SMA and vimentin was barely detected, while being markedly expressed in the wounded non-treated group and in the short-term administration group. Moreover, the active expression of α-SMA in the two groups was associated positively with the expression of col3 and negatively with the expression of col1. The low expression of Foxp3 after short-term administration relative to the control group indicated active immunity against tumor development. In conclusion, these findings indicate that PRP can be safely used in short- and long-term administration without tumorigenesis concern.

Human gingiva transcriptome during wound healing.

OBJECTIVES: To investigate the gene expression profile of human gingiva following surgical wounding. METHODS: Ten volunteers had one side of the palate wounded. Five days later, biopsies were harvested from both wounded (healing gingiva) and contra-lateral site (normal gingiva). Tissue samples were processed for gene expression (RNA-Seq, real-time PCR) and immunohistochemistry. Gene set enrichment/pathway analysis was also performed. RESULTS: Seven hundred genes were significantly differentially expressed in healing gingiva. Among genes with >twofold change (FC) in expression, 399 genes were up-regulated and 88 down-regulated, several not previously reported expressed in gingiva. Most increased in expression (≥30-FC) were MMP1, CCL18, SPP1, MUC21, CTHRC1, MMP10, and SERPINE1; most decreased (≥7-FC) were COCH, SIAH3, MT4, IGFL3, KY, and SYT16. Real-time PCR confirmed significantly changed mRNA levels for selective genes tested. Gene set enrichment analysis revealed several significantly enriched biological pathways. Immunohistochemistry confirmed protein expression of MUC21, CTHRC1, CTGF, and SYT16 in normal and healing gingiva. CONCLUSIONS: This first comprehensive analysis of the human gingival transcriptome during surgical wound healing offers novel insights into the participating molecular and biological mechanisms. The present results could serve as basis for future investigations into gingival wound healing following surgical, traumatic, or other type of injury.

Topical Application of Virus-Derived Immunomodulating Proteins and Peptides to Promote Wound Healing in Mouse Models.

Immune modulators play critical roles in the progression of wounds to normal or conversely delayed healing, through the regulation of normal tissue regrowth, scarring, inflammation, and growth factor expression. Many immune modulator recombinants are under active preclinical study or in clinical trial to promote improved acute or chronic wound healing and to reduce scarring. Viruses have evolved highly efficient immune modulators for the evasion of host-defensive immune responses that target and kill invasive viruses. Recent studies have proven that some of these virus-derived immune modulators can be used to promote wound healing with significantly improved speed and reduced scarring in rodent models. Mouse full-thickness excisional wound model is one of the most commonly used animal models used to study wound healing for its similarity to humans in the healing phases and associated cellular and molecular mechanisms. This chapter introduces this mouse dermal wound healing model in detail for application in studying viral immune modulators as new treatments to promote wound healing. Details of hydrogel, protein construction, and topical application methods for these therapeutic proteins are provided in this chapter.

Knockout of E2F1 enhances the polarization of M2 phenotype macrophages to accelerate the wound healing process.

Wound healing is a complex process, which is classically divided into inflammation, proliferation, and remodeling phases. Macrophages play a key role in wound healing, however, whether E2F1 mediates the M1/M2 polarization during the wound healing process is not known. Skin wounds were surgically induced in E2F1-/- mice and their WT littermates. At day 2 and day 7 post-surgery, the wounded skin tissues including 2 to 3 mm normal skin were obtained. The wounded skin tissues were used for the analyses of immunofluorescence staining (CD68, iNOS, CD206), western blotting (CD68, iNOS, CD206, PPAR-γ) and Co-immunoprecipitation (E2F1-PPAR-γ interactions). E2F1-/- mice exhibited faster wound healing process. At day 2, the M2 macrophages were remarkably increased in the E2F1-/- mice. Surprisingly, in the border zone of the wound, E2F1-/- mice had also more M2 macrophages and fewer M1 macrophages at day 7 post-surgery, suggesting a certain degree of polarization amongst the M1 and M2 phenotypes. Co-IP revealed that E2F1 indeed interacted with PPAR-γ, meanwhile western blotting and RT-PCR showed higher expression of PPAR-γ in the E2F1-/- mice as compared to that in the WT mice. Therefore, the findings suggest that wound healing process could be accelerated with enhanced M2 polarization through increased PPAR-γ expression in E2F1 knockout mice.

Uterine Scarring Leads to Adverse Pregnant Consequences by Impairing the Endometrium Response to Steroids.

Uterine surgical scarring is an increasing risk factor for adverse pregnant consequences that threaten fetal-maternal health. The detailed molecular features of scar implantation remain largely unknown. We aim to study the pathologic features of uterine surgical scarring and the mechanisms of compromised pregnancy outcomes of scar implantation. We generated a mouse model of uterine surgical scarring with a uterine incision penetrating the myometrium to endometrium to examine the pathologic changes and transcriptome profiles of uterine scarring at various postsurgery (PS) time points, as well as features of the feto-maternal interface during scar implantation. We found that uterine surgical scar recovery was consistently poor at PS3 until PS90, as shown by a reduced number of endometrial glands, inhibition of myometrial smooth muscle cell growth but excessive collagen fiber deposition, and massive leukocyte infiltration. Transcriptome annotation indicated significant chronic inflammation at the scarring site. At the peri-implantation and postimplantation stages, abnormal expression of various steroid-responsive genes at the scarring site was in parallel with lumen epithelial cell hyperplasia, inappropriate luminal closure, and disorientation of the implanted embryo, restricted stromal cell proliferation, and defective decidualization. High embryonic lethality (around 70%) before E10.5 was observed, and the small amount of survival embryos at E10.5 exhibited restricted growth and aberrant placenta defects including overinvasion of trophoblast cells into the decidua and insufficient fetal blood vessel branching in the labyrinth. The findings indicate that chronic inflammation and compromised responses to steroids in uterine scar tissues are the pivotal molecular basis for adverse pregnancy consequences of scar implantation.

Controlled release of lawsone from polycaprolactone/gelatin electrospun nano fibers for skin tissue regeneration.

In this study, we aimed to evaluate the role of electrospun nanofibers containing lawsone (2-hydroxy-1,4-naphthoquinone) in wound healing. Different concentrations of lawsone (0.5%, 1%, 1.5%) were electrospun in polycaprolactone-gelatin (PCL/Gel) polymers in core-shell architecture. Nanofibers were characterized using scanning electron microscopy (SEM), Transmission electron microscopy (TEM). Coaxial electrospinning of PCL/Gel/lawsone (PCL/Gel/Law) scaffolds prolonged the release of lawsone over a period of 20 days. In vitro bioactivity of fibers on human gingiva fibroblast cells (HGF) was evaluated by MTT assay. The PCL/Gel/Law 1% increased cell attachment and proliferation significantly. Additionally, the in vitro gene expression of transforming growth factor ß (TGF-B1), collagen (COL1) and epidermal growth factor (EGF) was monitored using RT-qPCR technique, which there was significant increase in TGF-B1 and COL1 gene expression in PCL/Gel/Law 0.5% and 1% mats. In vivo wound healing activity of lawsone loaded scaffolds in rat wound model revealed that the PCL/Gel/Law 1% had the highest impact on healing by increasing re-epithelialization of the wound after 14 days. It can be inferred that lawsone 1% incorporation in the core of PCL/Gel electrospun nanofibers has excellent characteristics and can be used as wound dressing patch in medicine.

Role of Trpv1 and Trpv4 in surgical incision-induced tissue swelling and Fos-like immunoreactivity in the central nervous system of mice.

Pain management remains a major concern regarding the treatment of postoperative patients. Transient receptor potential (TRP) channels are considered to be new therapeutic targets for pain control. We investigated whether the genes Trpv1 and Trpv4 are involved in hind paw swelling caused after surgical incision in mice or in incision-induced Fos-like immunoreactivity (Fos-LI) levels in the central nervous system. Mice were divided into four groups: wild-type (WT) control, WT incision, Trpv1 knockout (Trpv1-/-) incision, and Trpv4 knockout (Trpv4-/-) incision. Mice were anesthetized, and only those in the incision, and not control, groups received a surgical incision to their right plantar hind paw. Changes in paw diameter and in Fos-LI levels in the dorsal horn of the spinal cord, paraventricular nucleus of the hypothalamus (PVN), paraventricular nucleus of the thalamus, and central amygdala were evaluated 2 h after the incision. There was no significant difference in the paw diameter among groups. In contrast, in laminae I-II of the dorsal horn of the spinal cord and PVN, Fos-LI was significantly higher in all incision groups than in the WT control group. A significant increase in Fos-positive cells was also observed in the dorsal horn laminae III-IV in Trpv1-/- and Trpv4-/- incision groups compared with the WT incision group. Our results indicate that surgical incision activates the PVN and that Trpv1 and Trpv4 might be involved in neuronal activity in the dorsal horn laminae III-IV after surgical incision.

MicroRNA-132 promotes fibroblast migration via regulating RAS p21 protein activator 1 in skin wound healing.

MicroRNA (miR)-132 has been identified as a top up-regulated miRNA during skin wound healing and its inhibition impairs wound repair. In a human in vivo surgical wound model, we showed that miR-132 was induced in epidermal as well as in dermal wound-edge compartments during healing. Moreover, in a panel of cells isolated from human skin wounds, miR-132 was found highly expressed in human dermal fibroblasts (HDFs). In HDFs, miR-132 expression was upregulated by TGF-ß1. By overexpression or inhibition of miR-132, we showed that miR-132 promoted HDF migration. Mechanistically, global transcriptome analysis revealed that RAS signaling pathway was regulated by miR-132 in HDFs. We found that RAS p21 protein activator 1 (RASA1), a known target of miR-132, was downregulated in HDFs upon miR-132 overexpression. Silencing of RASA1 phenocopied the pro-migratory effect of miR-132. Collectively, our study reveals an important role for miR-132 in HDFs during wound healing and indicates a therapeutic potential of miR-132 in hard-to-heal skin wounds.

Ozone oil promotes wound healing by increasing the migration of fibroblasts via PI3K/Akt/mTOR signaling pathway.

Skin injury affects millions of people via the uncontrolled inflammation and infection. Many cellular components including fibroblasts and signaling pathways such as transforming growth factor-ß (TGF-ß) were activated to facilitate the wound healing to repair injured tissues. C57BL/6 female mice were divided into control and ozone oil treated groups. Excisional wounds were made on the dorsal skin and the fibroblasts were isolated from granulation tissues. The skin injured mouse model revealed that ozone oil could significantly decrease the wound area and accelerate wound healing compared with control group. QPCR and Western blotting assays showed that ozone oil up-regulated collagen I, α-SMA, and TGF-ß1 mRNA and protein levels in fibroblasts. Wound healing assay demonstrated that ozone oil could increase the migration of fibroblasts. Western blotting assay demonstrated that ozone oil increased the epithelial-mesenchymal transition (EMT) process in fibroblasts via up-regulating fibronectin, vimentin, N-cadherin, MMP-2, MMP-9, insulin-like growth factor binding protein (IGFBP)-3, IGFBP5, and IGFBP6, and decreasing epithelial protein E-cadherin and cellular senescence marker p16 expression. Mechanistically, Western blotting assay revealed that ozone oil increased the phosphorylation of PI3K, Akt, and mTOR to regulate the EMT process, while inhibition of PI3K reversed this effect of ozone oil. At last, the results from Cytometric Bead Array (CBA) demonstrated ozone oil significantly decreased the inflammation in fibroblasts. Our results demonstrated that ozone oil facilitated the wound healing via increasing fibroblast migration and EMT process via PI3K/Akt/mTOR signaling pathway in vivo and in vitro The cellular and molecular mechanisms we found here may provide new therapeutic targets for the treatment of skin injury.

Targeted killing of myofibroblasts by biosurfactant di-rhamnolipid suggests a therapy against scar formation.

Pathological myofibroblasts are often involved in skin scarring via generating contractile force and over-expressing collagen fibers, but no compound has been found to inhibit the myofibroblasts without showing severe toxicity to surrounding physiological cells. Here we report that di-rhamnolipid, a biosurfactant secreted by Pseudomonas aeruginosa, showed potent effects on scar therapy via a unique mechanism of targeted killing the myofibroblasts. In cell culture, the fibroblasts-derived myofibroblasts were more sensitive to di-rhamnolipid toxicity than fibroblasts at a concentration-dependent manner, and could be completely inhibited of their specific functions including α-SMA expression and collagen secretion/contraction. The anti-fibrotic function of di-rhamnolipid was further verified in rabbit ear hypertrophic scar models by presenting the significant reduction of scar elevation index, type I collagen fibers and α-SMA expression. In this regard, di-rhamnolipid treatment could be suggested as a therapy against skin scarring.