Emerging role of immune cells as drivers of pulmonary fibrosis.

The pathogenesis of pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and other forms of interstitial lung disease, involves a complex interplay of various factors including host genetics, environmental pollutants, infection, aberrant repair and dysregulated immune responses. Highly variable clinical outcomes of some ILDs, in particular IPF, have made it difficult to identify the precise mechanisms involved in disease pathogenesis and thus the development of a specific cure or treatment to halt and reverse the decline in patient health. With the advent of in-depth molecular diagnostics, it is becoming evident that the pathogenesis of IPF is unlikely to be the same for all patients and therefore will likely require different treatment approaches. Chronic inflammation is a cardinal feature of IPF and is driven by both innate and adaptive immune responses. Inflammatory cells and activated fibroblasts secrete various pro-inflammatory cytokines and chemokines that perpetuate the inflammatory response and contribute to the recruitment and activation of more immune cells and fibroblasts. The balance between pro-inflammatory and regulatory immune cell subsets, as well as the interactions between immune cell types and resident cells within the lung microenvironment, ultimately determines the extent of fibrosis and the potential for resolution. This review examines the role of the innate and adaptive immune responses in pulmonary fibrosis, with an emphasis on IPF. The role of different immune cell types is discussed as well as novel anti-inflammatory and immunotherapy approaches currently in clinical trial or in preclinical development.

Using x-ray micro computed tomography to quantify intracochlear fibrosis after cochlear implantation in a Guinea pig model.

Cochlear implants (CIs) allow individuals with profound hearing loss to understand speech and perceive sounds. However, not all patients obtain the full benefits that CIs can provide and the cause of this disparity is not fully understood. One possible factor for the variability in outcomes after cochlear implantation, is the development of fibrotic scar tissue around the implanted electrode. It has been hypothesised that limiting the extent of fibrosis after implantation may improve overall CI function, and longevity of the device. Currently, histology is often used to quantify the extent of intracochlear tissue growth after implantation however this method is labour intensive, time-consuming, often involves significant user bias, and causes physical distortion of the fibrosis. Therefore, this study aimed to evaluate x-ray micro computed tomography (µCT) as a method to measure the amount and distribution of fibrosis in a guinea pig model of cochlear implantation. Adult guinea pigs were implanted with an inactive electrode, and cochleae harvested eight weeks later (n = 7) and analysed using µCT, to quantify the extent of tissue reaction, followed by histological analysis to confirm that the tissue was indeed fibrotic. Cochleae harvested from an additional six animals following implantation were analysed by µCT, before and after contrast staining with osmium tetroxide (OsO4), to enhance the visualisation of soft tissues within the cochlea, including the tissue reaction. Independent analysis by two observers showed that the quantification method was robust and provided additional information on the distribution of the response within the cochlea. Histological analysis revealed that µCT visualised dense collagenous material and new bone formation but did not capture loose, areolar fibrotic tissue. Treatment with OsO4 significantly enhanced the visible tissue reaction detected using µCT. Overall, µCT is an alternative and reliable method that can be used to quantify the extent of the CI-induced intracochlear tissue response and will be a useful tool for the in vivo assessment of novel anti-fibrotic treatments.

Using RNA-seq to identify suitable housekeeping genes for hypoxia studies in human adipose-derived stem cells.

BACKGROUND: Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative[Formula: see text], RefFinder, and the Livak method. RESULTS: Gene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions. CONCLUSIONS: We have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.

Plasma cell but not CD20-mediated B-cell depletion protects from bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.

Keloid fibroblasts have elevated and dysfunctional mechanotransduction signaling that is independent of TGF-ß.

BACKGROUND: Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. OBJECTIVES: Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. METHODS: We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-ß (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. RESULTS: KFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-ß. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-ß. TGF-ß1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. CONCLUSIONS: These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.

A cGAS-dependent response links DNA damage and senescence in alveolar epithelial cells: a potential drug target in IPF.

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5-7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.

A Senescence Bystander Effect in Human Lung Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in "naïve" non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of "senescence-induced senescence" can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.

The epigenetics of keloids.

Keloid scarring is a fibroproliferative disorder of the skin with unknown pathophysiology, characterised by fibrotic tissue that extends beyond the boundaries of the original wound. Therapeutic options are few and commonly ineffective, with keloids very commonly recurring even after surgery and adjunct treatments. Epigenetics, defined as alterations to the DNA not involving the base-pair sequence, is a key regulator of cell functions, and aberrant epigenetic modifications have been found to contribute to many pathologies. Multiple studies have examined many different epigenetic modifications in keloids, including DNA methylation, histone modification, microRNAs and long non-coding RNAs. These studies have established that epigenetic dysregulation exists in keloid scars, and successful future treatment of keloids may involve reverting these aberrant modifications back to those found in normal skin. Here we summarise the clinical and experimental studies available on the epigenetics of keloids, discuss the major open questions and future perspectives on the treatment of this disease.

Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5'-aza-2'-deoxycytidine' treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

The contribution of animal models to understanding the role of the immune system in human idiopathic pulmonary fibrosis.

Pulmonary fibrosis occurs in a heterogeneous group of lung disorders and is characterised by an excessive deposition of extracellular matrix proteins within the pulmonary interstitium, leading to impaired gas transfer and a loss of lung function. In the past 10 years, there has been a dramatic increase in our understanding of the immune system and how it contributes to fibrogenic processes within the lung. This review will compare some of the models used to investigate the pathogenesis and treatment of pulmonary fibrosis, in particular those used to study immune cell pathogenicity in idiopathic pulmonary fibrosis, highlighting their advantages and disadvantages in dissecting human disease.

The extracellular matrix and mechanotransduction in pulmonary fibrosis.

Pulmonary fibrosis is characterised by excessive scarring in the lung which leads to compromised lung function, serious breathing problems and in some diseases, death. It includes several lung disorders with idiopathic pulmonary fibrosis (IPF) the most common and most severe. Pulmonary fibrosis is considered to be perpetuated by aberrant wound healing which leads to fibroblast accumulation, differentiation and activation, and deposition of excessive amounts of extracellular matrix (ECM) components, in particular, collagen. Recent studies have identified the importance of changes in the composition and structure of lung ECM during the development of pulmonary fibrosis and the interaction between ECM and lung cells. There is strong evidence that increased matrix stiffness induces changes in cell function including proliferation, migration, differentiation and activation. Understanding how changes in the ECM microenvironment influence cell behaviour during fibrogenesis, and the mechanisms regulating these changes, will provide insight for developing new treatments.

Identification of Differentially Methylated CpG Sites in Fibroblasts from Keloid Scars.

As a part of an abnormal healing process of dermal injuries and irritation, keloid scars arise on the skin as benign fibroproliferative tumors. Although the etiology of keloid scarring remains unsettled, considerable recent evidence suggested that keloidogenesis may be driven by epigenetic changes, particularly, DNA methylation. Therefore, genome-wide scanning of methylated cytosine-phosphoguanine (CpG) sites in extracted DNA from 12 keloid scar fibroblasts (KF) and 12 control skin fibroblasts (CF) (six normal skin fibroblasts and six normotrophic fibroblasts) was conducted using the Illumina Human Methylation 450K BeadChip in two replicates for each sample. Comparing KF and CF used a Linear Models for Microarray Data (Limma) model revealed 100,000 differentially methylated (DM) CpG sites, 20,695 of which were found to be hypomethylated and 79,305 were hypermethylated. The top DM CpG sites were associated with TNKS2, FAM45B, LOC723972, GAS7, RHBDD2 and CAMKK1. Subsequently, the most functionally enriched genes with the top 100 DM CpG sites were significantly (p ≤ 0.05) associated with SH2 domain binding, regulation of transcription, DNA-templated, nucleus, positive regulation of protein targeting to mitochondrion, nucleoplasm, Swr1 complex, histone exchange, and cellular response to organic substance. In addition, NLK, CAMKK1, LPAR2, CASP1, and NHS showed to be the most common regulators in the signaling network analysis. Taken together, these findings shed light on the methylation status of keloids that could be implicated in the underlying mechanism of keloid scars formation and remission.

Mesothelial cells regulate immune responses in health and disease: role for immunotherapy in malignant mesothelioma.

The mesothelium when first described was thought to function purely as a non-adhesive surface to facilitate intracoelomic movement of organs. However, the mesothelium is now recognized as a dynamic cellular membrane with many important functions that maintain serosal integrity and homeostasis. For example, mesothelial cells interact with and help regulate the body's inflammatory and immune system following infection, injury, or malignancy. With recent advances in our understanding of checkpoint molecules and the advent of novel immunotherapy approaches, there has been an increase in the number of studies examining mesothelial and immune cell interaction, in particular the role of these interactions in malignant mesothelioma. This review will highlight some of the recent advances in our understanding of how mesothelial cells help regulate serosal immunity and how in a malignant environment, the immune system is hijacked to stimulate tumor growth. Ways to treat mesothelioma using immunotherapy approaches will also be discussed.

The Role of IL-6 in Skin Fibrosis and Cutaneous Wound Healing.

The timely resolution of wound healing is critical for restoring the skin as a protective barrier. The switch from a proinflammatory to a reparative microenvironment must be tightly regulated. Interleukin (IL)-6 is a key modulator of the inflammatory and reparative process: it is involved in the differentiation, activation, and proliferation of leukocytes, endothelial cells, keratinocytes, and fibroblasts. This review examines the role of IL-6 in the healing of cutaneous wounds, and how dysregulation of IL-6 signaling can lead to either fibrosis or a failure to heal. The role of an IL-6/TGF-ß feedback loop is discussed in the context of fibrogenesis, while IL-6 expression and responses in advanced age, diabetes, and obesity is outlined regarding the development of chronic wounds. Current research on therapies that modulate IL-6 is explored. Here, we consider IL-6's diverse impact on cutaneous wound healing.

Senescence of IPF Lung Fibroblasts Disrupt Alveolar Epithelial Cell Proliferation and Promote Migration in Wound Healing.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease marked by excessive accumulation of lung fibroblasts (LFs) and collagen in the lung parenchyma. The mechanisms that underlie IPF pathophysiology are thought to reflect repeated alveolar epithelial injury leading to an aberrant wound repair response. Recent work has shown that IPF-LFs display increased characteristics of senescence including growth arrest and a senescence-associated secretory phenotype (SASP) suggesting that senescent LFs contribute to dysfunctional wound repair process. Here, we investigated the influence of senescent LFs on alveolar epithelial cell repair responses in a co-culture system. Alveolar epithelial cell proliferation was attenuated when in co-culture with cells or conditioned media from, senescence-induced control LFs or IPF-LFs. Cell-cycle analyses showed that a larger number of epithelial cells were arrested in G2/M phase when co-cultured with IPF-LFs, than in monoculture. Paradoxically, the presence of LFs resulted in increased A549 migration after mechanical injury. Our data suggest that senescent LFs may contribute to aberrant re-epithelialization by inhibiting proliferation in IPF.

MicroRNA Signatures in Malignant Pleural Mesothelioma Effusions.

Malignant pleural mesothelioma (MPM) is an incurable cancer of the pleura that can be difficult to diagnose. Biomarkers for an easier and/or earlier diagnosis are needed. Approximately 90% of MPM patients develop a pleural effusion (PE). PEs are ideal sources of biomarkers as the fluid would almost always require drainage for diagnostic and/or therapeutic reasons. However, differentiating MPM PE from PE caused by other diseases can be challenging. MicroRNAs are popular biomarkers given their stable expression in tissue and fluid. MicroRNAs have been analysed in PE cytology samples for the diagnosis of MPM but have not been measured in frozen/fresh PE. We hypothesise that microRNAs expressed in PE are biomarkers for MPM. TaqMan OpenArray was used to analyse over 700 microRNAs in PE cells and supernatants from 26 MPMs and 21 other PE-causing diseases. In PE cells, combining miR-143, miR-210, and miR-200c could differentiate MPM with an area under the curve (AUC) of 0.92. The three-microRNA signature could also discriminate MPM from a further 40 adenocarcinomas with an AUC of 0.9887. These results suggest that the expression of miR-143, miR-210, and miR-200c in PE cells might provide a signature for diagnosing MPM.

STAT3 Regulates the Onset of Oxidant-induced Senescence in Lung Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown cause with a median survival of only 3 years. Other investigators and we have shown that fibroblasts derived from IPF lungs display characteristics of senescent cells, and that dysregulated activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) correlates with IPF progression. The question of whether STAT3 activation is involved in fibroblast senescence remains unanswered. We hypothesized that inhibiting STAT3 activation after oxidant-induced senescence would attenuate characteristics of the senescent phenotype. We aimed to characterize a model of oxidant-induced senescence in human lung fibroblasts and to determine the effect of inhibiting STAT3 activity on the development of senescence. Exposing human lung fibroblasts to 150 µM hydrogen peroxide (H2O2) resulted in increased senescence-associated ß-galactosidase content and expression of p21 and IL-6, all of which are features of senescence. The shift into senescence was accompanied by an increase of STAT3 translocation to the nucleus and mitochondria. Additionally, Seahorse analysis provided evidence of increased mitochondrial respiration characterized by increased basal respiration, proton leak, and an associated increase in superoxide (O2-) production in senescent fibroblasts. Targeting STAT3 activity using the small-molecule inhibitor STA-21 attenuated IL-6 production, reduced p21 levels, decreased senescence-associated ß-galactosidase accumulation, and restored normal mitochondrial function. The results of this study illustrate that stress-induced senescence in lung fibroblasts involves the activation of STAT3, which can be pharmacologically modulated.

Inhibition of collagen production delays malignant mesothelioma tumor growth in a murine model.

Malignant mesothelioma is an aggressive fibrous tumor, predominantly of the pleura, with a very poor prognosis. Cell-matrix interactions are recognized important determinants of tumor growth and invasiveness but the role of the extracellular matrix in mesothelioma is unknown. Mesothelioma cells synthesize collagen as well as transforming growth factor-beta (TGF-ß), a key regulator of collagen production. This study examined the effect of inhibiting collagen production on mesothelioma cell proliferation in vitro and tumor growth in vivo. Collagen production by mesothelioma cells was inhibited by incubating cells in vitro with the proline analogue thiaproline (thiazolidine-4-carboxylic acid) or by oral administration of thiaproline in a murine tumor model. Cell cytotoxicity was measured using neutral red uptake and lactate dehydrogenase assays. Proliferation was measured by tritiated thymidine incorporation, and inflammatory cell influx, proliferation, apoptosis and angiogenesis in tumors examined by immunohistochemical labelling. Tumor size was determined by tumor weight and collagen production was measured by HPLC. Thiaproline at non-toxic doses significantly reduced basal and TGF-ß-induced collagen production by over 50% and cell proliferation by over 65%. In vivo thiaproline administration inhibited tumor growth at 10 days, decreasing the median tumor weight by 80%. The mean concentration of collagen was 50% lower in the thiaproline-treated tumors compared with the controls. There were no significant differences in vasculature or inflammatory cell infiltration but apoptosis was increased in thiaproline treated tumors at day 10. In conclusion, these observations strongly support a role for collagen in mesothelioma growth and establish the potential for inhibitors of collagen synthesis in mesothelioma treatment.

Ephrin-A2 affects wound healing and scarring in a murine model of excisional injury.

Ephrin ligand/Eph receptor signaling is important in both tissue development and homeostasis. There is increasing evidence that Ephrin/Eph signaling is important in the skin, involved in hair follicle cycling, epidermal differentiation, cutaneous innervation and skin cancer. However, there is currently limited information on the role of Ephrin/Eph signaling in cutaneous wound healing. Here we report the effects of the Ephrin-A2 and A5 ligands on wound healing. Using Ephrin-A2-/-, Ephrin-A5-/- and Ephrin-A2A5-/- transgenic mice, in vitro wound healing assays were conducted using isolated keratinocytes and fibroblasts. Ephrin-A2-/-, Ephrin-A2A5-/- and wild type mice with excisional wounds were used to analyze the impact of these ligands on wound closure, scar outcome, collagen orientation and re-innervation in vivo. The absence of the Ephrin-A2 and A5 ligands did not have any effect on dermal fibroblast proliferation or on fibroblast or keratinocyte migration. The loss of Ephrin-A2 and A5 ligands did not impact on the rate of wound closure or re-innervation after injury. However, changes in the gross morphology of the healed scar and in collagen histology of the scar dermis were observed in transgenic mice. Therefore Ephrin-A2 and A5 ligands may play an important role in final scar appearance associated with collagen deposition and structure.