Exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice

The purpose of this study was to determine the effect of exercise on wound healing and inflammation in young (3 mo) and old (18 mo) female BALB/cByJ mice. Mice were assigned to either exercise or sedentary control (control) groups. The exercise group mice were run on a motorized treadmill at a moderate intensity 30 min/day for 8 days. All mice were given four full-thickness dermal wounds, and the rate of wound closure was assessed daily for 10 days. Four months later, the aged mice were rerandomized to treatment, wounded again in different locations, and wounds were harvested at 1, 3, or 5 days postwounding. Wound tissue was analyzed for IL-1beta, IL-6, keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), and TNF-alpha protein. Myeloperoxidase (MPO) activity and F4/80 mRNA were assessed as an indirect measure of neutrophil and macrophage content, respectively. There was a trend (P = 0.10) for exercise to reduce wound size in young mice, and exercise significantly (P < 0.05) decreased wound size in old mice. TNF-alpha, KC, and MCP-1 were significantly (P < 0.05) lower in wounds from exercised old mice compared with control. No group differences were found for wound IL-1beta or IL-6, MPO activity, or F4/80 mRNA. Our data suggest that exercise accelerates the wound healing process in old mice. This improved healing response in the old mice may be the result of an exercise-induced anti-inflammatory response in the wound.     

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT–loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72 h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT–loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p < 0.01) and IL-1β (p < 0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p < 0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.     

Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis.     

Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing

The phenotype and fate of fetal microchimeric cells transfered into the maternal circulation during pregnancy are not well described. Since progenitors from distal sites mobilize during wound healing, we analyzed the recruitment and plasticity of fetal progenitors into maternal wounds. Wounds were generated on normal and bleomycin-induced fibrotic skin of parous or pregnant wild-type females with fluorescent GFP(+) fetuses. Analyses were performed on skin and blood specimens through PCR, immunohistochemistry, and flow cytometry. Controls consisted of parous and pregnant females without wounds and virgin females with wounds. Fetal cells were detected in all skin specimens of parous mice as long as healing was not achieved. During early stages of wound healing, fetal cells expressed mainly leukocyte markers, while in later phases endothelial markers prevailed. Fetally derived vessels connected to maternal circulation were also found, demonstrating the transfer of fetal endothelial progenitor cells. Wounding mobilized fetal CD34(+)ckit(-) cells into the blood during pregnancy. Most of this population was CD11b(-)VEGFR2(-). Another part was CD11b(+) with a fraction expressing VEGFR2. VEGFa-spiked Matrigel plugs partially mimicked this fetal progenitor recruitment and mobilization into the blood. In summary, fetal cells that mobilize in response to wounding are mainly progenitor cells and participate in angiogenesis and inflammation.     

Syndecans in wound healing, inflammation and vascular biology

Syndecans are heparan sulphate proteoglycans consisting of a type I transmembrane core protein modified by heparan sulphate and sometimes chondroitin sulphate chains. They are major proteoglycans of many organs including the vasculature, along with glypicans and matrix proteoglycans. Heparan sulphate chains have potential to interact with a wide array of ligands, including many growth factors, cytokines, chemokines and extracellular matrix molecules relevant to growth regulation in vascular repair, hypoxia, angiogenesis and immune cell function. This is consistent with the phenotypes of syndecan knock-out mice, which while viable and fertile, show deficits in tissue repair. Furthermore, there are potentially important changes in syndecan distribution and function described in a variety of human vascular diseases. The purpose of this review is to describe syndecan structure and function, consider the role of syndecan core proteins in transmembrane signalling and also their roles as co-receptors with other major classes of cell surface molecules. Current debates include potential redundancy between syndecan family members, the significance of multiple heparan sulphate interactions, regulation of the cytoskeleton and cell behaviour and the switch between promoter and inhibitor of important cell functions, resulting from protease-mediated shedding of syndecan ectodomains.     

Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior

The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET^+/?), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface‐resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET^+/? mouse establishes an activated state of existing surface NET proteins. The NET^+/? mice exhibit increased anxiety in the open field and light–dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET^+/? mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET^+/? mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders .     

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.     

Swatting flies: modelling wound healing and inflammation in Drosophila

Aberrant wound healing can lead to a variety of human pathologies, from non-healing chronic wounds that can become dangerously infected, to exuberant fibrotic healing in which repair is accompanied by excessive inflammation. To guide therapeutic intervention, we need a better understanding of the fundamental mechanisms driving tissue repair; this will require complementary wound-healing studies in several model organisms. Drosophila has been used to model genetic aspects of numerous human pathologies, and is being used increasingly to gain insight into the molecular and genetic aspects of tissue repair and inflammation, which have classically been modelled in mice or cultured cells. This review discusses the advantages and disadvantages of Drosophila as a wound-healing model, as well as some exciting new research opportunities that will be enabled by its use.     

Normal development, wound healing, and adenovirus susceptibility in beta5-deficient mice

Integrins have been shown to play important roles in embryonic development, wound healing, metastasis, and other biological processes. alphavbeta5 is a receptor for RGD-containing extracellular matrix proteins that has been suggested to be important in cutaneous wound healing and adenovirus infection. To examine the in vivo function of this receptor, we have generated mice lacking beta5 expression, using homologous recombination in embryonic stem cells. Mice homozygous for a null mutation of the beta5 subunit gene develop, grow, and reproduce normally. Keratinocytes harvested from beta5(-/-) mice demonstrate impaired migration on and adhesion to the alphavbeta5 ligand, vitronectin. However, the rate of healing of cutaneous wounds is not different in beta5(-/-) and beta5(+/+) mice. Furthermore, keratinocytes and airway epithelial cells obtained from null mice show adenovirus infection efficiency equal to that from wild-type mice. These data suggest that alphavbeta5 is not essential for normal development, reproduction, adenovirus infection, or the healing of cutaneous wounds.     

The plasminogen receptor,Plg-RKT,plays a role in inflammation and fibrinolysis during cutaneous wound healing in mice

Wound healing is a complex physiologic process that proceeds in overlapping, sequential steps. Plasminogen promotes fibrinolysis and potentiates the inflammatory response during wound healing. We have tested the hypothesis that the novel plasminogen receptor, Plg-R_KT, regulates key steps in wound healing. Standardized burn wounds were induced in mice and time dependence of wound closure was quantified. Healing in Plg-R_KT^−/− mice was significantly delayed during the proliferation phase. Expression of inflammatory cytokines was dysregulated in Plg-R_KT^−/− wound tissue. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase was observed in mice in which Plg-R_KT was specifically deleted in myeloid cells. Following wound closure, the epidermal thickness was less in Plg-R_KT^−/− wound tissue. Paradoxically, deletion of Plg-R_KT, specifically in keratinocytes, significantly accelerated the rate of healing during the proliferation phase. Mechanistically, only two genes were upregulated in Plg-R_KT^−/− compared with Plg-R_KT^+/+ wound tissue, filaggrin, and caspase 14. Both filaggrin and caspase 14 promote epidermal differentiation and decrease proliferation, consistent with more rapid wound closure and decreased epidermal thickness during the remodeling phase. Fibrin clearance was significantly impaired in Plg-R_KT^−/− wound tissue. Genetic reduction of fibrinogen levels to 50% completely abrogated the effect of Plg-R_KT deletion on the healing of burn wounds. Remarkably, the effects of Plg-R_KT deletion on cytokine expression were modulated by reducing fibrinogen levels. In summary, Plg-R_KT is a new regulator participating in different phases of cutaneous burn wound healing, which coordinately plays a role in the interrelated responses of inflammation, keratinocyte migration, and fibrinolysis.Subject terms: Extracellular matrix, Mechanisms of disease     

Marked airway eosinophilia prevents development of airway hyper-responsiveness during an allergic response in IL-5 transgenic mice

Tissue eosinophilia probably plays important roles in the pathophysiology of bronchial asthma and allergic disorders; however, this concept was challenged recently by controversial results in mouse models of bronchial asthma treated with anti-IL-5 Ab and the failure of anti-IL-5 therapy in humans. We have now used a unique model, IL-5 transgenic (TG) mice, to address a fundamental question: is airway eosinophilia beneficial or detrimental in the allergic response? After sensitization and challenge with OVA, IL-5 TG mice showed a marked airway eosinophilia. Surprisingly, these IL-5 TG mice showed lower airway reactivity to methacholine. Immunohistochemical analysis of the lungs revealed a marked peribronchial infiltration of eosinophils, but no eosinophil degranulation. In vitro, mouse eosinophils from peritoneal lavage fluids did not produce superoxide anion, but did produce an anti-inflammatory and fibrotic cytokine, TGF-beta 1. Indeed, the TGF-beta 1 levels in bronchoalveolar lavage fluid specimens from IL-5 TG mice directly correlated with airway eosinophilia (r = 0.755). Furthermore, anti-IL-5 treatment of IL-5 TG mice decreased both airway eosinophilia and TGF-beta 1 levels in bronchoalveolar lavage fluids and increased airway reactivity. Thus, in mice, marked eosinophilia prevents the development of airway hyper-reactivity during an allergic response. Overall, the roles of eosinophils in asthma and in animal models need to be addressed carefully for their potentially detrimental and beneficial effects.     

NIPSNAP1 deficient mice exhibit altered liver amino acid,lipid and nucleotide metabolism

4-Nitrophenyl phosphatase domain and non-neuronal SNAP25-like protein homolog1 (NIPSNAP1) is an evolutionarily conserved protein found in a variety of species ranging from C. elegans to human. NIPSNAP1 protein is localized in mitochondria and is highly expressed in liver, brain and kidney. The molecular and cellular roles of NIPSNAP1 are still unknown. To gain insights into the function of NIPSNAP1, we generated a mouse model with a disruption of Nipsnap1 gene and performed metabolomic analysis on their liver tissues. Liver samples from 13 to 15 month old NIPSNAP1 deficient (n = 7) and wild-type (n = 8) mice were extracted and processed for analysis using liquid/gas chromatography followed by mass spectrometry (LC/MS and GC/MS). We examined a total of 291 compounds in liver samples and found 45 compounds whose levels were significantly altered (p < 0.05, Welch’s t test) in NIPSNAP1 deficient mice compared to controls. These compounds were associated with a variety of processes, including metabolism of nucleotides, amino acids and lipids. In addition, we found a significant reduction in reduced glutathione (GSH) (0.63-fold change, p < 0.05) and elevation in cysteine–glutathione disulfide (2.77-fold change, p < 0.05). Our results suggest that NIPSNAP1 deficiency affects multiple processes in intermediate metabolism and results in oxidative stress in the liver.     

Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.     

Short- and long-term effects of IL-1 and TNF antagonists on periodontal wound healing

The present study tested the effects of local injection of IL-1 and TNF soluble receptors on a periodontal wound-healing model in nonhuman primates. In this model, periodontal lesions were developed for 16 wk, followed by open flap surgery. Starting at the time of surgery, groups of animals received localized injections of both soluble cytokine receptors or else PBS three times per week for 3, 14, or 35 days. Periodontal wound healing was analyzed for each group at the end of the treatment regimen. Fourteen days after surgery, a significant decrease was observed between the animals treated with soluble receptors and the untreated group with respect to recruitment of inflammatory cells in deep gingival connective tissue. Concurrent apoptosis of inflammatory cells in those tissues increased significantly in treated animals compared with untreated animals. All other outcome parameters of periodontal wound healing were likewise significantly improved in treated animals compared with untreated animals. In marked contrast, however, 35 days after surgery, there was a significant increase in the number of inflammatory cells that had infiltrated into deep gingival connective tissue in treated compared with untreated animals. Outcome parameters of periodontal wound healing worsened in treated animals when compared with untreated. These results indicate that proinflammatory cytokines may play different functional roles in early vs late phases of periodontal wound healing. Short-term blockade of IL-1 and TNF may facilitate periodontal wound healing, whereas prolonged blockade may have adverse effects.