Nod-Like Receptor Protein-3 Inflammasome Plays an Important Role during Early Stages of Wound Healing

The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.     

Effects of Topically Applied Vitamin D during Corneal Wound Healing

Vitamin D is an important regulator of immune function and largely acts to dampen chronic inflammatory events in a variety of tissues. There is also accumulating evidence that vitamin D acts to enhance initial inflammation, beneficial during both infection and wound healing, and then promotes resolution and prevention of chronic, damaging inflammation. The current study examines the effect of topical vitamin D in a mouse of model of corneal epithelial wound healing, where acute inflammation is necessary for efficient wound closure. At 12 and 18 hours post-wounding, vitamin D treatment significantly delayed wound closure by ~17% and increased infiltration of neutrophils into the central cornea. Basal epithelial cell division, corneal nerve density, and levels of VEGF, TGFβ, IL-1β, and TNFα were unchanged. However, vitamin D increased the production of the anti-microbial peptide CRAMP 12 hours after wounding. These data suggest a possible role for vitamin D in modulating corneal wound healing and have important implications for therapeutic use of vitamin D at the ocular surface.     

Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β₃-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.     

Effect of Mixed Transplantation of Autologous and Allogeneic Microskin Grafts on Wound Healing in a Rat Model of Acute Skin Defect

The treatment of extensive thermal injuries with insufficient autologous skin remains a great challenge to burn surgeons. In this study, we investigated the influence of the ratio of autologous and allogeneic tissue in mixed microskin grafts on wound healing in order to develop an effective method for using limited donor skin to cover a large open wound. Four different mixtures were tested: autologous microskin at an area expansion ratio of 10∶1 with allogeneic microskin at an area expansion ratio of 10∶1 or 10∶3 and autologous microskin at an expansion ratio of 20∶1 with allogeneic microskin at an expansion ratio of 20∶3 or 20∶6. Wound healing, wound contraction, and integrin β1 expression were measured. Mixed microskin grafting facilitated wound healing substantially. The mixture of autologous microskin at an expansion ratio of 10∶1 with the same amount of allogeneic microskin achieved the most satisfactory wound healing among the 4 tested mixtures. Histological examination revealed the presence of obviously thickened epidermis and ectopic integrin β1 expression. Keratinocytes expressing integrin β1 were scattered in the suprabasal layer. Higher levels of integrin β1 expression were associated with faster wound healing, implying that ectopic expression of integrin β1 in keratinocytes may play a pivotal role in wound healing. In conclusion, this study proves that this new skin grafting technique may improve wound healing.     

Accelerated Wound Closure In Vitro by Fibroblasts from a Subgroup of Cleft Lip/Palate Patients: Role of Transforming Growth Factor-α

In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely “fast”, “intermediate”, and “slow”. Most cleft lip/palate fibroblasts were distributed between the “fast” (5 strains) and the “intermediate” group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the “fast” group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the “intermediate” migratory group to the level of the “fast”, but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of “fast” cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.     

Identification of the Critical Therapeutic Entity in Secreted Hsp90α That Promotes Wound Healing in Newly Re-Standardized Healthy and Diabetic Pig Models

Chronic and non-healing skin wounds represent a significant clinical, economic and social problem worldwide. Currently, there are few effective treatments. Lack of well-defined animal models to investigate wound healing mechanisms and furthermore to identify new and more effective therapeutic agents still remains a major challenge. Pig skin wound healing is close to humans. However, standardized pig wound healing models with demonstrated validity for testing new wound healing candidates are unavailable. Here we report a systematic evaluation and establishment of both acute and diabetic wound healing models in pigs, including wound-creating pattern for drug treatment versus control, measurements of diabetic parameters and the time for detecting delayed wound healing. We find that treatment and control wounds should be on the opposite and corresponding sides of a pig. We demonstrate a strong correlation between duration of diabetic conditions and the length of delay in wound closure. Using these new models, we narrow down the minimum therapeutic entity of secreted Hsp90α to a 27-amino acid peptide, called fragment-8 (F-8). In addition, results of histochemistry and immunohistochemistry analyses reveal more organized epidermis and dermis in Hsp90α-healed wounds than the control. Finally, Hsp90α uses a similar signaling mechanism to promote migration of isolated pig and human keratinocytes and dermal fibroblasts. This is the first report that shows standardized pig models for acute and diabetic wound healing studies and proves its usefulness with both an approved drug and a new therapeutic agent.     

Annexin A2 Regulates β1 Integrin Internalization and Intestinal Epithelial Cell Migration

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of β1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface β1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface β1 integrin that was accompanied by decreased β1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of β1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.     

Lumican Accelerates Wound Healing by Enhancing α2β1 Integrin-Mediated Fibroblast Contractility

Lumican is a dermatan sulfate proteoglycan highly expressed in connective tissue and has the ability to regulate collagen fibril assembly. Previous studies have shown that lumican is involved in wound healing, but the precise effects of lumican on reepithelialization and wound contraction, the two pivotal aspects of skin wound healing, have not been investigated. Here we explored the roles of lumican in fibroblast contractility, a main aspect of skin wound healing, by adopting mice skin wound healing model and the corresponding in vitro cellular experiments. Our results showed that lumican can promote skin wound healing by facilitating wound fibroblast activation and contraction but not by promoting keratinocyte proliferation and migration. Silencing of integrin α2 completely abolished the pro-contractility of lumican, indicating lumican enhances fibroblast contractility via integrin α2. Our study for the first time demonstrated that lumican can affect fibroblast’s mechanical property, which is pivotal for many important pathological processes, such as wound healing, fibrosis, and tumor development, suggesting that lumican might have a potential to be used to modulate these processes.     

Extracellular Heat Shock Protein 90 Signals through Subdomain II and the NPVY Motif of LRP-1 Receptor to Akt1 and Akt2: a Circuit Essential for Promoting Skin Cell Migration In Vitro and Wound Healing In Vivo

Normal cells secrete heat shock protein 90 alpha (Hsp90α) in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90α during invasion and metastasis. The sole function of extracellular Hsp90α (eHsp90α) is to promote cell motility, a critical event for both wound healing and tumor progression. The mechanism of promotility action by eHsp90α, however, has remained elusive. A key issue is whether eHsp90α still acts as a chaperone outside the cells or is a new and bona fide signaling molecule. Here, we have provided evidence that eHsp90α utilizes a unique transmembrane signaling mechanism to promote cell motility and wound healing. First, subdomain II in the extracellular part of low-density lipoprotein receptor-related protein 1 (LRP-1) receives the eHsp90α signal. Then, the NPVY but not the NPTY motif in the cytoplasmic tail of LRP-1 connects eHsp90α signaling to serine 473 but not threonine 308 phosphorylation in Akt kinases. Individual knockdown of Akt1, Akt2, or Akt3 revealed the importance of Akt1 and Akt2 in eHsp90α-induced cell motility. Akt gene rescue experiments suggest that Akt1 and Akt2 work in concert, rather than independently, to mediate eHsp90α promotility signaling. Finally, Akt1 and Akt2 knockout mice showed impaired wound healing that cannot be corrected by topical application with the eHsp90α protein.     

Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers

Background

Stress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.

Methods and Findings

Migratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization.

Conclusions

This work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.     

Physiological ER Stress Mediates the Differentiation of Fibroblasts

Recently, accumulating reports have suggested the importance of endoplasmic reticulum (ER) stress signaling in the differentiation of several tissues and cells, including myoblasts and osteoblasts. Secretory cells are easily subjected to ER stress during maturation of their secreted proteins. Skin fibroblasts produce and release several proteins, such as collagens, matrix metalloproteinases (MMPs), the tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAGs), and the production of these proteins is increased at wound sites. Differentiation of fibroblasts into myofibroblasts is one of the key factors for wound healing and that TGF-β can induce fibroblast differentiation into myofibroblasts, which express α-smooth muscle actin. Well-differentiated myofibroblasts show increased production of collagen and TGF-β, and bring about wound healing. In this study, we examined the effects of ER stress signaling on the differentiation of fibroblasts, which is required for wound healing, using constitutively ER stress-activated primary cultured fibroblasts. The cells expressed positive α-smooth muscle actin signals without TGF-β stimulation compared with control fibroblasts. Gel-contraction assays suggested that ER stress-treated primary fibroblasts caused stronger shrinkage of collagen gels than control cells. These results suggest that ER stress signaling could accelerate the differentiation of fibroblasts to myofibroblasts at injured sites. The present findings may provide important insights for developing therapies to improve wound healing.     

Combination of Adrenomedullin with Its Binding Protein Accelerates Cutaneous Wound Healing

Cutaneous wound continues to cause significant morbidity and mortality in the setting of diseases such as diabetes and cardiovascular diseases. Despite advances in wound care management, there is still an unmet medical need exists for efficient therapy for cutaneous wound. Combined treatment of adrenomedullin (AM) and its binding protein-1 (AMBP-1) is protective in various disease conditions. To examine the effect of the combination treatment of AM and AMBP-1 on cutaneous wound healing, full-thickness 2.0-cm diameter circular excision wounds were surgically created on the dorsum of rats, saline (vehicle) or AM/AMBP-1 (96/320 μg kg BW) was topically applied to the wound daily and wound size measured. At days 3, 7, and 14, skin samples were collected from the wound sites. AM/AMBP-1 treated group had significantly smaller wound surface area than the vehicle group over the 14-day time course. At day 3, AM/AMBP-1 promoted neutrophil infiltration (MPO), increased cytokine levels (IL-6 and TNF-α), angiogenesis (CD31, VEGF and TGFβ-1) and cell proliferation (Ki67). By day 7 and 14, AM/AMBP-1 treatment decreased MPO, followed by a rapid resolution of inflammation characterized by a decrease in cytokines. At the matured stage, AM/AMBP-1 treatment increased the alpha smooth muscle actin expression (mature blood vessels) and Masson-Trichrome staining (collagen deposition) along the granulation area, and increased MMP-9 and decreased MMP-2 mRNA expressions. TGFβ-1 mRNA levels in AM/AMBP-1 group were 5.3 times lower than those in the vehicle group. AM/AMBP-1 accelerated wound healing by promoting angiogenesis, collagen deposition and remodeling. Treatment also shortened the days to reach plateau for wound closure. Thus, AM/AMBP-1 may be further developed as a therapeutic for cutaneous wound healing.     

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Age‐associated expression of p21and p53 during human wound healing

In mice, cellular senescence and senescence‐associated secretory phenotype (SASP) positively contribute to cutaneous wound healing. In this proof‐of‐concept study, we investigated the expressions of p16, p21, and other senescence‐associated biomarkers during human wound healing in 24 healthy subjects using a double‐biopsy experimental design. The first punch biopsy created the wound and established the baseline. The second biopsy, concentric to the first and taken several days after wounding, was used to probe for expression of biomarkers by immunohistochemistry and RNA FISH. To assess the effects of age, we recruited 12 sex‐matched younger (30.2 ± 1.3 years) and 12 sex‐matched older (75.6 ± 1.8 years) subjects. We found that p21 and p53, but not p16, were induced during healing in younger, but not older subjects. A role for Notch signaling in p21 expression was inferred from the inducible activation of HES1. Further, other SASP biomarkers such as dipeptidyl peptidase‐4 (DPP4) were significantly induced upon wounding in both younger and older groups, whereas matrix metallopeptidase 9 (MMP9) was induced only in the younger group. Senescence‐associated β‐galactosidase (SA‐β‐gal) was not detectable before or after wounding. This pilot study suggests the possibility that human cutaneous wound healing is characterized by differential expression of p21 and p53 between younger and older subjects.     

Knockout of Endothelial Cell-Derived Endothelin-1 Attenuates Skin Fibrosis but Accelerates Cutaneous Wound Healing

Endothelin (ET)-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF)-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF)-α and connective tissue growth factor (CTGF) were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.