The effect of EGF application in gel form on histamine content of experimentally induced wound in mice

Summary. The factors participating to the wound healing are complex and still obscure. Among these factors, epidermal growth factor (EGF) and histamine by increasing reepithelization and reparation tissue strength via enhancing collagen deposition to the wound site have a beneficial effect. This study was performed to investigate the effect of EGF dosage forms on the histamine content of the experimentally induced wound and some wound healing criters in the mice.Histological investigation of reepithelization, wound tensile strength for healing and collagen maturation, and histamine levels were assessed in the present study. Thirty two mice were divided into control, and EGF treated groups. Controls included three subgroups; untreated (n=5), 0.9% NaCl applied (n=5), and gel applied (n=5). Experimental groups were treated with two forms of EGF; EGF, solution form in 0.9% NaCl (n=5) and the gel form in 0.2% w/w in carbopol 940 (n=7). The discrepancy between these forms were evaluated. This evaluation was done by the application of two forms of EGF for 15 days on experimentally induced wound healing.Gel form of EGF by sustained release from bioadhesive polymer is found to be more effective than the soluble form, on the healing of the wound, by acceleration of reepithelization and increment of wound tensile strength. The tensile strength of the wound indicates the rate of repair and collagen maturation. It has been observed that when physiological saline and carbopol 940 exposed to incision without EGF causes a significant increase in tissue histamine content.According to the results of the present investigation; the histamine content is found to be decreased by EGF gel dosage form treatment, therefore preventing abnormal collagen formation has a beneficial effect on wound healing.     

Overexpressed transglutaminase 5 triggers cell death

Summary. Transglutaminases are a class of nine different proteins involved in many biological phenomena such as differentiation, tissue repair, endocytosis. Transglutaminase 5 was originally cloned from skin keratinocytes, and a partial biochemical characterization showed its involvment in skin differentiation. Here we demonstrate that transglutaminase 5 is able to induce cell death when intracellularly overexpressed. Transfected cells show enzymatic activity, as demonstrated by fluoresceincadaverine staining. Transfected cells died due to the formation of hypodiploid DNA content, indicating the induction of cell death under these pharmacological conditions. We also show that the primary sequence of transglutaminase 5 contains GTP binding domains which are similar to those in transglutaminase 2. This raises the possibility that transglutaminase 5 is regulated by GTP in a similar fashion to transglutaminase 2.     

Gene therapy for tissue regeneration

Tissue repair and regeneration are the normal biological responses of many different tissues in the body to injury. During the healing process, profound changes occur in cell composition and extracellular matrix (ECM) formation. Fibroblasts and equivalent reparative cells migrate to the wounded area and subsequently proliferate. These cells and reparative cells from the surrounding tissue are responsible for the rapid repair which results in tissue regeneration. Growth factors, one of which is transforming growth factor-beta (TGF-beta), stimulate fibroblasts and smooth muscle cells to proliferate and synthesize ECM proteins. This process of early repair provides a rapid way to restore new tissue and mechanical integrity. This early tissue repair process is normally followed by involution, which requires the production and activation of proteases, tissue maturation and remodeling, reorganization and finally regeneration. Alternately, failure to replace the critical components of the ECM, including elastin and basement membrane, results in abnormal regeneration of the epithelial cell layer. Although remodeling should occur during healing, provisional repair may be followed by excessive synthesis and deposition of collagen, which results in irreversible fibrosis and scarring. This excessive fibrosis which occurs in aberrant healing is at least in part mediated by persistent TGF-beta. Because of the central role of collagen in the wound healing process, the pharmacological control of collagen synthesis has been of paramount importance as a possible way to abrogate aberrant healing and prevent irreversible fibrosis. Fibrosis is an abnormal response to tissue injury.     

全身放射损伤对皮肤伤口成纤维细胞参与组织修复能力的影响

合并全身放射损伤的创伤（放创复合伤）是一种重要而有代表性的难愈性创伤,其难愈机制尚未完全阐明,成纤维细胞是最为重要的组织修复细胞,其辐射敏感性较低,为了明确放创复合伤时合并的放射损伤是否对伤口成纤维细胞有直接损伤作用,以及这些损伤作用对创伤愈合的影响,实验检测了分离,培养的放创复合伤和单纯创伤大鼠皮肤伤口成纤维细胞的增殖,凋亡及其他反映其参与组织修复能力的指标变化,结果发现,在去除全身因素和局部因素,特别是创伤局部细胞因子和细胞外基质对成纤维细胞的反馈作用后,放创复合伤组伤口成纤维细胞增殖力,贴壁力和粘附力均显著弱于单纯创伤组,而成纤维细胞的凋亡率则显著增加,这些细胞表明,全身放射损伤对伤口成纤维细胞有直接损伤作用,使其参与组织修复能力显著受抑,这是合并全身放射损伤时创伤难愈的重要原因。     

RGTA<Superscript>®</Superscript> or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients

The importance of extracellular matrix (ECM) integrity in maintaining normal tissue function is highlighted by numerous pathologies and situations of acute and chronic injury associated with dysregulation or destruction of ECM components. Heparan sulfate (HS) is a key component of the ECM, where it fulfils important functions associated with tissue homeostasis. Its degradation following tissue injury disrupts this delicate equilibrium and may impair the wound healing process. ReGeneraTing Agents (RGTA^®s) are polysaccharides specifically designed to replace degraded HS in injured tissues. The unique properties of RGTA^® (resistance to degradation, binding and protection of ECM structural and signaling proteins, like HS) permit the reconstruction of the ECM, restoring both structural and biochemical functions to this essential substrate, and facilitating the processes of tissue repair and regeneration. Here, we review 25 years of research surrounding this HS mimic, supporting the mode of action, pre-clinical studies and therapeutic efficacy of RGTA^® in the clinic, and discuss the potential of RGTA^® in new branches of regenerative medicine.     

The absence of oestrogen receptor beta disturbs collagen I type deposition during Achilles tendon healing by regulating the IRF5‐CCL3 axis

Achilles tendon healing (ATH) remains an unanswered question in the field of sports medicine because it does not produce tissue with homology to the previously uninjured tissue. Oestrogen receptor β (ERβ) is involved in the injury and repair processes of tendons. Our previous study confirmed that ERβ plays a role in the early stage of ATH by affecting adipogenesis, but its role in extracellular matrix (ECM) remodelling is unknown. We established a 4‐week Achilles tendon repair model to investigate the mechanism through which ERβ affects ATH at the very beginning of ECM remodelling phase. In vitro studies were performed using tendon‐derived stem cells (TDSCs) due to their promising role in tendon healing. Behavioural and biomechanical tests revealed that ERβ‐deficient mice exhibit weaker mobility and inferior biomechanical properties, and immunofluorescence staining and qRT‐PCR showed that these mice exhibited an erroneous ECM composition, as mainly characterized by decreased collagen type I (Col I) deposition. The changes in gene expression profiles between ERβ‐knockout and WT mice at 1 week were analysed by RNA sequencing to identify factors affecting Col I deposition. The results highlighted the IRF5‐CCL3 axis, and this finding was verified with CCL3‐treated TDSCs. These findings revealed that ERβ regulates Col I deposition during ATH via the IRF5‐CCL3 axis.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling

The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.     

The interplay of fibroblasts,the extracellular matrix,and inflammation in scar formation

Various forms of fibrosis, comprising tissue thickening and scarring, are involved in 40% of deaths across the world. Since the discovery of scarless functional healing in fetuses prior to a certain stage of development, scientists have attempted to replicate scarless wound healing in adults with little success. While the extracellular matrix (ECM), fibroblasts, and inflammatory mediators have been historically investigated as separate branches of biology, it has become increasingly necessary to consider them as parts of a complex and tightly regulated system that becomes dysregulated in fibrosis. With this new paradigm, revisiting fetal scarless wound healing provides a unique opportunity to better understand how this highly regulated system operates mechanistically. In the following review, we navigate the four stages of wound healing (hemostasis, inflammation, repair, and remodeling) against the backdrop of adult versus fetal wound healing, while also exploring the relationships between the ECM, effector cells, and signaling molecules. We conclude by singling out recent findings that offer promising leads to alter the dynamics between the ECM, fibroblasts, and inflammation to promote scarless healing. One factor that promises to be significant is fibroblast heterogeneity and how certain fibroblast subpopulations might be predisposed to scarless healing. Altogether, reconsidering fetal wound healing by examining the interplay of the various factors contributing to fibrosis provides new research directions that will hopefully help us better understand and address fibroproliferative diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis.     

Improved repair of dermal wounds in mice lacking microRNA‐155

Wound healing is a well-regulated but complex process that involves haemostasis, inflammation, proliferation and maturation. Recent reports suggest that microRNAs (miRs) play important roles in dermal wound healing. In fact, miR deregulation has been linked with impaired wound repair. miR-155 has been shown to be induced by inflammatory mediators and plays a central regulatory role in immune responses. We have investigated the potential role of miR-155 in wound healing. By creating punch wounds in the skin of mice, we found an increased expression of miR-155 in wound tissue when compared with healthy skin. Interestingly, analysis of wounds of mice lacking the expression of miR-155 (miR-155^−/−) revealed an increased wound closure when compared with wild-type animals. Also, the accelerated wound closing correlated with elevated numbers of macrophages in wounded tissue. Gene expression analysis of wounds tissue and macrophages isolated from miR-155^−/− mice that were treated with interleukin-4 demonstrated an increased expression of miR-155 targets (BCL6, RhoA and SHIP1) as well as, the finding in inflammatory zone-1 (FIZZ1) gene, when compared with WT mice. Moreover, the up-regulated levels of FIZZ1 in the wound tissue of miR-155^−/− mice correlated with an increased deposition of type-1 collagens, a phenomenon known to be beneficial in wound closure. Our data indicate that the absence of miR-155 has beneficial effects in the wound healing process.     

Skin Cornification Proteins Provide Global Link between ROS Detoxification and Cell Migration during Wound Healing

Wound healing is a complex dynamic process characterised by a uniform flow of events in nearly all types of tissue damage, from a small skin scratch to myocardial infarction. Reactive oxygen species (ROS) are essential during the healing process at multiple stages, ranging from the initial signal that instigates the immune response, to the triggering of intracellular redox-dependent signalling pathways and the defence against invading bacteria. Excessive ROS in the wound milieu nevertheless impedes new tissue formation. Here we identify small proline-rich (SPRR) proteins as essential players in this latter process, as they directly link ROS detoxification with cell migration. A literature-based meta-analysis revealed their up-regulation in various forms of tissue injury, ranging from heart infarction and commensal-induced gut responses to nerve regeneration and burn injury. Apparently, SPRR proteins have a far more widespread role in wound healing and tissue remodelling than their established function in skin cornification. It is inferred that SPRR proteins provide injured tissue with an efficient, finely tuneable antioxidant barrier specifically adapted to the tissue involved and the damage inflicted. Their recognition as novel cell protective proteins combining ROS detoxification with cell migration will provide new venues to study and manage tissue repair and wound healing at a molecular level.     

Skin regeneration in adult axolotls: a blueprint for scar-free healing in vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.     

Matrix metalloproteinases and epidermal wound repair

Epidermal wound healing is a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. Among the many proteins that are essential for the restoration of tissue integrity is the metalloproteinase (MMP) family. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation and cell–cell and cell–matrix signalling. MMPs are secreted by different cell types such as keratinocytes, fibroblasts and inflammatory cells at different stages and locations during wound healing, thereby regulating this process in a very coordinated and controlled way. In this article, we review the role of MMPs and their inhibitors (TIMPs), as well as the disintegrin and metalloproteinase with the thrombospondin motifs (ADAMs) family, in epithelial wound repair.     

The role of oxidised regenerated cellulose/collagen in wound repair: effects in vitro on fibroblast biology and in vivo in a model of compromised healing

Irrespective of underlying chronic wound pathology, delayed wound healing is normally characterised by impaired new tissue formation at the site of injury. It is thought that this impairment reflects both a reduced capacity to synthesize new tissue and the antagonistic activities of high levels of proteinases within the chronic wound environment. Historically, wound dressings have largely been passive devices that offer the wound interim barrier function and establish a moist healing environment. A new generation of devices, designed to interact with the wound and promote new tissue formation, is currently being developed and tested. This study considers one such device, oxidised regenerated cellulose (ORC) /collagen, in terms of its ability to promote fibroblast migration and proliferation in vitro and to accelerate wound repair in the diabetic mouse, a model of delayed wound healing. ORC/collagen was found to promote both human dermal fibroblasts proliferation and cell migration. In vivo studies considered the closure and histological characteristics of diabetic wounds treated with ORC/collagen compared to those of wounds given standard treatment on both diabetic and non-diabetic mice. ORC/collagen was found to significantly accelerate diabetic wound closure and result in a measurable improvement in the histological appearance of wound tissues. As the diabetic mouse is a recognised model of impaired healing, which may share some characteristics of human chronic wounds, the results of this in vivo study, taken together with those relating the positive effects of ORC/collagen in vitro, may predict the beneficial use of this device in the clinical setting.     

Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis

The biological roles of hyaluronan (HA) fragments in angiogenesis acceleration have been investigated recently. Studies have confirmed that oligosaccharides of HA (o-HA) are capable of stimulating neovascularization in vitro and promoting blood flow or angiogenesis in animal models. However, few laboratories have studied the function of o-HA as an exogenous treatment in injured tissue repair in vivo. It is thought that o-HA may lose its activities when used topically in vivo due to its small size, which may be absorbed quickly by the surrounding tissues. In this study, we prepared a special slow-releasing gel that contains a mixture of defined size of o-HA and studied the healing effects of o-HA by topical application to an acute wound model. We report that o-HA complex promotes the repair of tissue injury of a murine excisional dermal wound. The therapy by o-HA was compared with high molecular weight HA (HMW-HA) and the known angiogenesis stimulator, VEGF. At days 6 to 8 after treatment, significant differences were seen in wound closure rates between o-HA and control or HMW-HA groups, in which o-HA showed an increased wound recovery. Histological analysis revealed that increased neo-blood and lymph vessels were formed in wounded tissues treated by o-HA. In addition, treatments of wounds with o-HA resulted in more granulation production, collagen deposition, and fibroblast proliferation. Analysis of gene expression by real-time RT-PCR demonstrated a significant up-regulation of some cytokines or adhesion molecules in o-HA-treated wounds, which corresponds with the increased granulation tissue in these wounds. Our findings suggested that o-HA therapy may be useful in acute wound repair.     

Neutrophil signaling during myocardial infarction wound repair

Neutrophils are key effector cells of the innate immune system, serving as a first line of defense in the response to injury and playing essential roles in the wound healing process. Following myocardial infarction (MI), neutrophils infiltrate into the infarct region to propagate inflammation and begin the initial phase of cardiac wound repair. Pro-inflammatory neutrophils release proteases to degrade extracellular matrix (ECM), a necessary step for the removal of necrotic myocytes as a prelude for scar formation. Neutrophils transition their phenotype over time to regulate MI inflammation resolution and stabilize scar formation. Neutrophils contribute to the evolution from inflammation to resolution and scar formation by serving anti-inflammatory and repair functions. As anti-inflammatory cells, neutrophils contribute ECM proteins during scar formation, in particular fibronectin, galectin-3, and vimentin. The diverse and polarizing functions that contribute to MI wound repair make this innate immune cell a viable target to improve MI outcomes. Thus, understanding the signaling involved in neutrophil physiology in the context of MI may help to identify novel therapeutic targets.     

SPARC-thrombospondin-2-double-null mice exhibit enhanced cutaneous wound healing and increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges.

Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell-extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.     

Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts

Proteolytic degradation of extracellular matrix (ECM) components during tissue remodeling plays a pivotal role in normal and pathological processes including wound healing, inflammation, tumor invasion, and metastasis. Proteolytic enzymes in tumors may activate or release growth factors from the ECM or act directly on the ECM itself, thereby facilitating angiogenesis or tumor cell migration. Fibroblast activation protein (FAP) is a cell surface antigen of reactive tumor stromal fibroblasts found in epithelial cancers and in granulation tissue during wound healing. It is absent from most normal adult human tissues. FAP is conserved throughout chordate evolution, with homologues in mouse and Xenopus laevis, whose expression correlates with tissue remodeling events. Using recombinant and purified natural FAP, we show that FAP has both dipeptidyl peptidase activity and a collagenolytic activity capable of degrading gelatin and type I collagen; by sequence, FAP belongs to the serine protease family rather than the matrix metalloprotease family. Mutation of the putative catalytic serine residue of FAP to alanine abolishes both enzymatic activities. Consistent with its in vivo expression pattern determined by immunohistochemistry, FAP enzyme activity was detected by an immunocapture assay in human cancerous tissues but not in matched normal tissues. This study demonstrates that FAP is present as an active cell surface-bound collagenase in epithelial tumor stroma and opens up investigation into physiological substrates of its novel, tumor-associated dipeptidyl peptidase activity.