Cutaneous Tissue Repair Research Articles

LncRNA-H19 in Fibroblasts Promotes Wound Healing in Diabetes.

Cutaneous wound healing in diabetes is impaired and would develop into nonhealing ulcerations. However, the molecular mechanism underlying the wound-healing process remains largely obscure. Here, we found that cutaneous PDGFRα+ fibroblast-expressing lncRNA-H19 (lncH19) accelerates the wound-healing process via promoting dermal fibroblast proliferation and macrophage infiltration in injured skin. PDGFRα+ cell-derived lncH19, which is lower in contents in the wound-healing cutaneous tissue of patients and mice with type 2 diabetes, is required for wound healing through promoting proliferative capacity of dermis fibroblasts as well as macrophage recruitments. Mechanistically, lncH19 relieves the cell cycle arrest of fibroblasts and increases macrophage infiltration in injured tissues via inhibiting p53 activity and GDF15 releasement. Furthermore, exosomes derived from adipocyte progenitor cells efficiently restore the impaired diabetic wound healing via delivering lncH19 to injured tissue. Therefore, our study reveals a new role for lncRNA in regulating cutaneous tissue repair and provides a novel promising insight for developing clinical treatment of diabetes.

Skin cell-derived extracellular vesicles: a promising therapeutic strategy for cutaneous injury

Wound healing refers to the healing process that occurs after the skin and other tissues are separated or damaged by internal or external forces. It is a complex combination of tissue regeneration, granulation tissue hyperplasia, and scar formation, and shows the synergistic effects of these processes. After skin damage, the environment around the wound and the cells at site of the damage respond immediately, and a range of cytokines and growth factors are released. In cutaneous injury, extracellular vesicle (EV) signaling plays a vital role in the healing process via paracrine and endocrine mechanisms. EVs are natural intercellular and inter-organ communication tools that carry various bioactive substances for message exchange. Stem cells and stem cell EVs facilitate tissue repair, showing promising potential in regenerative medicine. Nevertheless, EVs derived from specific skin tissue cells, such as epidermal cells, fibroblasts, vascular endothelial cells and inflammatory cells, also play important roles in cutaneous tissue repair. Here, we describe the characteristics of wound healing, concentrating on the production and functions of EVs derived from specific skin cells, and provide new ideas for wound therapy using EVs.

Environmentally Relevant Levels of Depleted Uranium Impacts Dermal Fibroblast Proliferation, Viability, Metabolic Activity, and Scratch Closure.

Uranium (U) is a heavy metal used in military and industrial settings, with a large portion being mined from the Southwest region of the United States. Uranium has uses in energy and military weaponry, but the mining process has released U into soil and surface waters that may pose threats to human and environmental health. The majority of literature regarding U’s human health concern focuses on outcomes based on unintentional ingestion or inhalation, and limited data are available about its influence via cutaneous contact. Utilizing skin dermis cells, we evaluated U’s topical chemotoxicity. Employing soluble depleted uranium (DU) in the form of uranyl nitrate (UN), we hypothesized that in vitro exposure of UN will have cytotoxic effects on primary dermal fibroblasts by affecting cell viability and metabolic activity and, further, may delay wound healing aspects via altering cell proliferation and migration. Using environmentally relevant levels of U found in water (0.1 M to 100 M [UN]; 23.8–23,800 ppb [U]), we quantified cellular mitosis and migration through growth curves and in vitro scratch assays. Cells were exposed from 24 h to 144 h for a time-course evaluation of UN chemical toxicity. The effects of UN were observed at concentrations above and below the Environmental Protection Agency threshold for safe exposure limits. UN exposure resulted in a dose-dependent decrease in the viable cell count; however, it produced an increase in metabolism when corrected for the viable cells present. Furthermore, cellular proliferation, population doubling, and percent closure was hindered at levels ≥10 M UN. Therefore, inadvertent exposure may exacerbate pre-existing skin diseases in at-risk demographics, and additionally, it may substantially interfere in cutaneous tissue repair processes.

Amniotic fluid-derived multipotent stromal cells drive diabetic wound healing through modulation of macrophages

BackgroundCutaneous wounds in patients with diabetes exhibit impaired healing due to physiological impediments and conventional care options are severely limited. Multipotent stromal cells (MSCs) have been touted as a powerful new therapy for diabetic tissue repair owing to their trophic activity and low immunogenicity. However, variations in sources and access are limiting factors for broader adaptation and study of MSC-based therapies. Amniotic fluid presents a relatively unexplored source of MSCs and one with wide availability. Here, we investigate the potential of amniotic fluid-derived multipotent stromal cells (AFMSCs) to restore molecular integrity to diabetic wounds, amend pathology and promote wound healing.MethodWe obtained third trimester amniotic fluid from term cesarean delivery and isolated and expanded MSCs in vitro. We then generated 10 mm wounds in Leprdb/db diabetic mouse skin, and splinted them open to allow for humanized wound modeling. Immediately after wounding, we applied AFMSCs topically to the sites of injuries on diabetic mice, while media application only, defined as vehicle, served as controls. Post-treatment, we compared healing time and molecular and cellular events of AFMSC-treated, vehicle-treated, untreated diabetic, and non-diabetic wounds. A priori statistical analyses measures determined significance of the data.ResultAverage time to wound closure was approximately 19 days in AFMSC-treated diabetic wounds. This was significantly lower than the vehicle-treated diabetic wounds, which required on average 27.5 days to heal (p < 0.01), and most similar to time of closure in wild type untreated wounds (an average of around 18 days). In addition, AFMSC treatment induced changes in the profiles of macrophage polarizing cytokines, resulting in a change in macrophage composition in the diabetic wound bed. We found no evidence of AFMSC engraftment or biotherapy induced immune response.ConclusionTreatment of diabetic wounds using amniotic fluid-derived MSCs encourages cutaneous tissue repair through affecting inflammatory cell behavior in the wound site. Since vehicle-treated diabetic wounds did not demonstrate accelerated healing, we determined that AFMSCs were therapeutic through their paracrine activities. Future studies should be aimed towards validating our observations through further examination of the paracrine potential of AFMSCs. In addition, investigations concerning safety and efficacy of this therapy in clinical trials should be pursued.

Recombinant Myxoma Virus-Derived Immune Modulator M-T7 Accelerates Cutaneous Wound Healing and Improves Tissue Remodeling.

Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or “fine tune” the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.

Physical plasma therapy accelerates wound re-epithelialisation and enhances extracellular matrix formation in cutaneous skin grafts.

Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full-thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full-thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal-epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD-dependent TGF-β1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival

Proteases modulate critical processes in cutaneous tissue repair to orchestrate inflammation, cell proliferation and tissue remodeling. However, the functional consequences and implications in healing impairments of most cleavage events are not understood. Using iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) we had characterized proteolytic signatures in a porcine wound healing model and identified two neo-N termini derived from proteolytic cleavage of the focal adhesion protein and mechanotransducer zyxin. Here, we assign these proteolytic events to the activity of either caspase-1 or serine protease HtrA1 and analyze the biological relevance of the resultant zyxin truncations. By cellular expression of full-length and truncated zyxin proteins, we demonstrate nuclear translocation of a C-terminal zyxin fragment that could also be generated in vitro by HtrA1 cleavage and provide evidence for its anti-apoptotic activities, potentially by regulating the expression of modulators of cell proliferation, protein synthesis and genome stability. Targeted degradomics correlated endogenous generation of the same zyxin fragment with increased cell density in human primary dermal fibroblasts. Hence, this newly identified HtrA1-zyxin protease signaling axis might present a novel mechanism to transiently enhance cell survival in environments of increased cell density like in wound granulation tissue.

Biomimetic and estrogenic fibers promote tissue repair in mice and human skin via estrogen receptor β

The dynamic changes in estrogen levels throughout aging and during the menstrual cycle influence wound healing. Elevated estrogen levels during the pre-ovulation phase accelerate tissue repair, whereas reduced estrogen levels in post-menopausal women lead to slow healing. Although previous reports have shown that estrogen may potentiate healing by triggering the estrogen receptor (ER)-β signaling pathway, its binding to ER-α has been associated with severe collateral effects and has therefore limited its use as a therapeutic agent. To this end, soy phytoestrogens, which preferentially bind to the ER-β, are currently being explored as a safer therapeutic alternative to estrogen. However, the development and evaluation of phytoestrogen-based materials as local ER-β modulators remains largely unexplored. Here, we engineered biomimetic and estrogenic nanofiber wound dressings built from soy protein isolate (SPI) and hyaluronic acid (HA) using immersion rotary jet spinning. These engineered scaffolds were shown to successfully recapitulate the native dermal architecture, while delivering an ER-β-triggering phytoestrogen (genistein). When tested in ovariectomized mouse and ex vivo human skin tissues, HA/SPI scaffolds outperformed controls (no treatment or HA only scaffolds) towards promoting cutaneous tissue repair. These improved healing outcomes were prevented when the ER-β pathway was genetically or chemically inhibited. Our findings suggest that estrogenic fibrous scaffolds facilitate skin repair by ER-β activation.

Healing activity of hydrogel containing nanoemulsified β-caryophyllene

β-caryophyllene is a sesquiterpene present in the oil of many plant species, such as Copaifera sp., which has been shown to possesses potent anti-inflammatory action; however, its healing activity remains under study. The objectives of the present study were to produce a nanoemulsion containing β-caryophyllene followed by a hydrogel containing nanoemulsified β-caryophyllene, to evaluate the permeation profile in vitro, and to assess the in vivo healing activity, which is so far unexplored in the literature for pure β-caryophyllene and in pharmaceutical formulation. The nanoemulsion was obtained through high-pressure homogenization and the hydrogel by direct dispersion with hydroxyethylcellulose. Both formulations were characterized according to droplet size, polydispersity index, volume-weighted mean diameters, particle distribution, droplets diameters tracking, zeta potential, viscosity and bioadhesion behavior. β-caryophyllene content was determined by gas chromatography coupled with mass spectrometry (GC/MS). Both formulations presented a nanometric droplet size, negative zeta potential, high β-caryophyllene content, and were stable for 60 days. In agreement with the viscosity results, the hydrogel containing the β-caryophyllene nanoemulsion showed superior bioadhesiveness than the nanoemulsion. The skin permeation study in Franz cells demonstrated that isolated β-caryophyllene was unable to cross the stratum corneum and that its nanoemulsification promoted its permeation. On the other hand, in the simulated deeply wounded skin (dermis), no significant differences were observed between the formulations and isolated β-caryophyllene with respect to the amount of marker retention in the dermis, suggesting saturation of this skin layer. For the study of healing activity, the dorsal wound model was performed with an evaluation of the lesion size, anti-inflammatory markers, and antioxidant activity. The initial closure of the wound was achieved sooner in the group treated with the hydrogel containing the β-caryophyllene nanoemulsion, indicating its anti-inflammatory effect. The histological analysis indicated that on day 12 day of the lesion, the hydrogel presented similar results to those of the positive control group (Dersani® oil), proving effectiveness in cutaneous tissue repair.

Porous Biomimetic Hyaluronic Acid and Extracellular Matrix Protein Nanofiber Scaffolds for Accelerated Cutaneous Tissue Repair.

Recent reports suggest the utility of extracellular matrix (ECM) molecules as raw components in scaffolding of engineered materials. However, rapid and tunable manufacturing of ECM molecules into fibrous structures remains poorly developed. Here we report on an immersion rotary jet-spinning (iRJS) method to show high-throughput manufacturing (up to ∼1 g/min) of hyaluronic acid (HA) and other ECM fiber scaffolds using different spinning conditions and postprocessing modifications. This system allowed control over a variety of scaffold material properties, which enabled the fabrication of highly porous (70-95%) and water-absorbent (swelling ratio ∼2000-6000%) HA scaffolds with soft-tissue mimetic mechanical properties (∼0.5-1.5 kPa). Tuning these scaffolds' properties enabled the identification of porosity (∼95%) as a key facilitator for rapid and in-depth cellular ingress in vitro. We then demonstrated that porous HA scaffolds accelerated granulation tissue formation, neovascularization, and reepithelialization in vivo, altogether potentiating faster wound closure and tissue repair. Collectively, this scalable and versatile manufacturing approach enabled the fabrication of tunable ECM-mimetic nanofiber scaffolds that may provide an ideal first building block for the design of all-in-one healing materials.

Alfalfa Nanofibers for Dermal Wound Healing.

Engineering bioscaffolds for improved cutaneous tissue regeneration remains a healthcare challenge because of the increasing number of patients suffering from acute and chronic wounds. To help address this problem, we propose to utilize alfalfa, an ancient medicinal plant that contains antibacterial/oxygenating chlorophylls and bioactive phytoestrogens, as a building block for regenerative wound dressings. Alfalfa carries genistein, which is a major phytoestrogen known to accelerate skin repair. The scaffolds presented herein were built from composite alfalfa and polycaprolactone (PCL) nanofibers with hydrophilic surface and mechanical stiffness that recapitulate the physiological microenvironments of skin. This composite scaffold was engineered to have aligned nanofibrous architecture to accelerate directional cell migration. As a result, alfalfa-based composite nanofibers were found to enhance the cellular proliferation of dermal fibroblasts and epidermal keratinocytes in vitro. Finally, these nanofibers exhibited reproducible regenerative functionality by promoting re-epithelialization and granulation tissue formation in both mouse and human skin, without requiring additional proteins, growth factors, or cells. Overall, these findings demonstrate the potential of alfalfa-based nanofibers as a regenerative platform toward accelerating cutaneous tissue repair.

Matrix composition in 3-D collagenous bioscaffolds modulates the survival and angiogenic phenotype of human chronic wound dermal fibroblasts

There is a substantial need for new strategies to stimulate cutaneous tissue repair in the treatment of chronic wounds. To address this challenge, our team is developing modular biomaterials termed “bead foams”, comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive three-dimensional (3-D) network. In the current study, bead foams were fabricated from human decellularized adipose tissue (DAT) or commercially-sourced bovine tendon collagen (COL) to explore the effects of ECM composition on human wound edge dermal fibroblasts (weDF) sourced from chronic wound tissues. The DAT and COL bead foams were shown to be structurally similar, but compositionally distinct, containing different levels of glycosaminoglycan content and collagen types IV, V, and VI. In vitro testing under conditions simulating stresses within the chronic wound microenvironment indicated that weDF survival and angiogenic marker expression were significantly enhanced in the DAT bead foams as compared to the COL bead foams. These findings were corroborated through in vivo assessment in a subcutaneous athymic mouse model. Taken together, the results demonstrate that weDF survival and paracrine function can be modulated by the matrix source applied in the design of ECM-derived scaffolds and that the DAT bead foams hold promise as cell-instructive biological wound dressings. Statement of SignificanceBiological wound dressings derived from the extracellular matrix (ECM) can be designed to promote the establishment of a more permissive microenvironment for healing in the treatment of chronic wounds. In the current work, we developed modular biomaterials comprised of fused networks of porous ECM-derived beads fabricated from human decellularized adipose tissue (DAT) or commercially-available bovine collagen. The bioscaffolds were designed to be structurally similar to provide a platform for investigating the effects of ECM composition on human dermal fibroblasts isolated from chronic wounds. Testing in in vitro and in vivo models demonstrated that cell survival and pro-angiogenic function were enhanced in the adipose-derived bioscaffolds, which contained higher levels of glycosaminoglycans and collagen types IV, V, and VI. Our findings support that the complex matrix composition within DAT can induce a more pro-regenerative cellular response for applications in wound healing.

Adipose tissue-derived extracellular fraction characterization: biological and clinical considerations in regenerative medicine

BackgroundAdipose tissue-derived stem cells are considered to be a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using adipose tissue or purified stem cells, intercellular molecule exchange by the adipose tissue complex, a vast array of bioactive secretory factors, demonstrated beneficial effects by reducing tissue damage and stimulation of endogenous repair. However, for therapeutic purposes, the use of secretome derivatives, such as full conditioned media or purified exosomes generated in vitro, may present considerable disadvantages for cell manufacturing, storage, product safety, and their potential as a ready-to-go therapeutic product.MethodsIn this study, the effect of a liquid fraction of lipoaspirates isolated intraoperatively from 28 healthy donors was evaluated for their protective effect against oxidative stress and senescence, proliferation, and migration in vitro on normal human melanocytes, keratinocytes, and fibroblasts. Immunoenzymatic quantification of several growth factors and important signal molecules was used to define the biological profile of physiological adipose tissue secretome.ResultsAdipose tissue extracellular fraction (AT-Ex), isolated from lipoaspirate, exhibited significant potential for skin repair. AT-Ex augmented dermal and epidermal cell proliferation in a dose-dependent manner without promoting cancer cell growth. Moreover, migration of dermal fibroblasts, an important phenomenon implicated in endogenous repair, was enhanced by AT-Ex treatment. AT-Ex has a positive impact on oxidative stress damage when cells are exposed to extrinsic hostile factors and prevent a fibroblast senescence phenotype including paracrine functions associated with skin aging.ConclusionsCollectively, our findings propose natural systems carrying the physiological balance of in-vivo produced secretome that could improve cutaneous wound healing and tissue repair. This approach, representing an innovative perspective and therapeutic strategy in regenerative medicine, could also be combined with autologous stem cell grafts to treat chronic nonhealing wounds, stable vitiligo, severe burns, and post-oncological scarring.

A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G− cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G−) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.

Chitosan-Based Nanomaterials for Skin Regeneration

Chitosan (CS) is a renewable polysaccharide widely used for the preparation of biomaterials due to its special properties such as its biodegradability and biocompatibility, mucoadhesive behavior, and antibacterial and anti-inflammatory effects. These features are very important for biomedical applications, especially for tissue engineering and skin regeneration. From the clinical point of view, there is an increasing demand for the development of new materials/scaffolds with dual roles: the promotion of skin tissue repair and the simultaneous exertion of potent antimicrobial effects. Nanotechnology has been extensively employed in several pharmacological applications, including cutaneous tissue repair and antimicrobial treatments. The combination of CS and nanotechnology might create new avenues in tissue engineering. In this sense, this review presents and discusses recent advantages and challenges in the design of CS-based nanomaterials (in the form of nanofibers, composite nanoparticles, and nanogels) for cutaneous tissue regeneration. The combination of CS-based nanomaterials with other polymers, active drugs and metallic nanoparticles is also discussed from the viewpoint of designing suitable platforms for regenerative medicine and tissue engineering.

Biotechnological Management of Skin Burn Injuries: Challenges and Perspectives in Wound Healing and Sensory Recovery.

Many wound management protocols have been developed to improve wound healing after burn with the primordial aim to restore the barrier function of the skin and also provide a better esthetic outcome. Autologous skin grafts remain the gold standard in the treatment of skin burn, but this treatment has its limitation especially for patients presenting limited donor sites due to extensive burn areas. Deep burn injuries also alter the integrity of skin-sensitive innervation and have an impact on patient's quality of life by compromising perceptions of touch, temperature, and pain. Thus, patients can suffer from long-term disabilities ranging from cutaneous sensibility loss to chronic pain. The cellular mechanisms involved in skin reinnervation following injury are not elucidated yet. Depending on the depth of the burn, nerve sprouting can occur from the wound bed or the surrounding healthy tissue, but somehow this process fails to provide correct reinnervation of the wound during scarring. In addition, several clinical observations indicate that damage to the peripheral nervous system influences wound healing, resulting in delayed wound healing or chronic wounds, underlining the role of innervation and neuromediators for normal cutaneous tissue repair development. Promising tissue engineering strategies, including the use of biomaterials, skin substitutes, and stem cells, could provide novel alternative treatments in wound healing and help in improving patient's sensory recovery.

Fixing a hole: CCN2 closes chronic wounds.

Chronic wounds, especially on the feet, are a clinical feature resulting from diabetes and often result in limb amputation. Identification of strategies that promote closure of chronic wounds are essential. In a study authored by Henshaw et al. (J Diabetes Res. 2015;2015:236238), CCN2/CTGF was able to accelerated closure of wounds in diabetic rats. Moreover, in humans, the ability of wounds in diabetic patients to heal correlated with CCN2 expression. Thus CCN2 might, in the future, be used to promote healing of chronic wounds. Diabetic foot ulcers are a significant cause of morbidity and are a substantial financial burden. Approximately 25 % of diabetics have foot ulcers, and these account for up to 70 % of non traumatic lower limb amputations (Most and Sinnock 1983). About 6.5 million people in the USA suffer from chronic wounds, costing ~ $25 billion dollars (Sen et al. 2009). Developing novel methods of promoting closure of chronic diabetic wounds is therefore of paramount importance. CCN2 is a member of the CCN family of matricellular proteins that is induced by TGFβ and in response to wounding (Klaassen et al. 2015; Cicha and Goppelt-Struebe 2009). Although CCN2 is dispensible for normal cutaneous tissue repair kinetics, CCN2 is responsible for the recruitment and differentiation of progenitor cells into myofibroblasts (Liu et al. 2014; Tsang and Leask 2014). CCN2 is overexpressed in fibrotic disease including diabetes, and is believed to contribute to the kidney and eye fibrosis observed in this disorder (Mason 2009; Klaassen et al. 2015). However, the role of CCN2 in diabetic wounds is unknown. In a recent study from the Twigg laboratory (Henshaw et al. 2015), Sprague–Dawley rats were made diabetic using streptozotocin and subjected to dermal wounding. Wounds in diabetic rats closed more slowly than those in control rats. Wounds in diabetic rats treated with recombinant CCN2 showed enhanced closure; CCN2 had no effect on control wounds. Diabetic wounds showed reduced levels of type IV collagen (which assists in repithelialization) and a-smooth muscle actin and CCN2 treatment somewhat alleviated this phenotype. CCN2 treatment of diabetic mice trended higher α-SMA staining score in fibroblasts and endothelial cells compared with scores in untreated diabetic rats although this did not reach statistical significance. Macrophage number was increased in CCN2-treated wounds, but this result did not achieve statistical significance, Young’s modulus was increased by in animals that were treated with CCN2 compared to untreated control and diabetic mice. Thus, although CCN2 promoted closure of diabetic wounds, only a few parameters were affected in a statistically significant fashion. Perhaps CCN2 might work with other matricellular proteins to promote closure of diabetic wounds.

CCN2 is required for recruitment of Sox2-expressing cells during cutaneous tissue repair.

Connective tissue growth factor (CTGF/CCN2), a member of the CCN family of matricellular proteins is upregulated in both fibrosis as well as tissue repair. Recently, we showed that, in mice, CCN2 expression by fibroblasts was required for dermal fibrogenesis, but not for cutaneous tissue repair. Lineage tracing analysis linked the ability of CCN2 to promote fibrosis to the requirement for CCN2 to recruit cells expressing the progenitor cell marker Sox2 to fibrotic connective tissue and for differentiating these cells into myofibroblasts. Herein, we show that although loss of CCN2 expression by Sox2-expressing cells does not impair cutaneous tissue repair, CCN2 was required for recruitment of cells derived from Sox2-expressing cells to the wound area. Collectively, these results are consistent with the notion that neither CCN2 nor Sox2-expressing progenitor cells are essential for cutaneous tissue repair and that CCN2 represents a specific anti-fibrotic target.

CCN2 expression by fibroblasts is not required for cutaneous tissue repair

The CCN family of matricellular proteins, which includes CCN2 and CCN1, is believed to have a major in vivo role in controlling tissue morphogenesis and repair. In adult skin, the proadhesive matricellular protein connective tissue growth factor (CTGF/CCN2) is specifically up-regulated in fibrosis and wound healing. In mice, CCN2 is required for dermal fibrogenesis, but whether CCN2 is required for cutaneous tissue repair is unknown. To address this question, in this report we subjected adult mice bearing a fibroblast-specific deletion of CCN2 to the dermal punch model of cutaneous tissue repair. Loss of CCN2 did not appreciably affect the kinetics of tissue repair, collagen content, or the number of α-smooth muscle actin-positive cells. CCN1 (cyr61), which has in vitro effect similar to CCN2, is also induced in cutaneous tissue repair. Fibroblast-specific CCN1/CCN2 double knockout mice were also generated; loss of both CCN1 and CCN2 together did not appreciably affect cutaneous tissue repair. However, loss of CCN2 resulted in impaired recruitment of NG2-positive pericyte-like cells to the wound area. Collectively, these results indicate that neither CCN2 nor CCN1 is essential for cutaneous tissue repair; CCN2 appears to be required for recruitment of pericyte-like cells and may represent a specific antifibrotic target.

TAK1 Is Required for Dermal Wound Healing and Homeostasis

Dermal connective tissue is a supportive structure required for skin's barrier function; dysregulated dermal homeostasis results in chronic wounds and fibrotic diseases. The multifunctional cytokine transforming growth factor (TGF) β promotes connective tissue deposition, repair, and fibrosis. TGF-β acts through well-defined canonical pathways; however, the non-canonical pathways through which TGF-β selectively promotes connective tissue deposition are unclear. In dermal fibroblasts, we show that inhibition of the non-canonical TGF-β-activated kinase 1 (TAK1) selectively reduced the ability of TGF-β to induce expression of a cohort of wound healing genes, such as collagens, CCN2, TGF-β1, and IL-6. Fibroblast-specific TAK1-knockout mice showed impaired cutaneous tissue repair and decreased collagen deposition, α-smooth muscle actin and CCN2 expression, proliferating cell nuclear antigen staining, and c-Jun N-terminal kinase and p38, but not Smad3, phosphorylation. TAK1-deficient fibroblasts showed reduced cell proliferation, migration, cell attachment/spreading, and contraction of a floating collagen gel matrix. TAK1-deficient mice also showed progressively reduced skin thickness and collagen deposition. Thus, TAK1 is essential for connective tissue deposition in the dermis.