Alpha3beta1 integrin-controlled Smad7 regulates reepithelialization during wound healing in mice.
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ABSTRACT:
SUBMITTER: Reynolds LE
PROVIDER: S-EPMC2215730 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Diabetic patients are frequently afflicted with impaired wound healing where linear progression of molecular and cellular events compromised. Despite of meaningful progress in diabetic treatment, management of diabetic chronic wounds is still challenging. Jamun (Syzygium cumini) honey may be a promising candidate for diabetic wound healing and need to explore in detail. So present study was designed to evaluate the efficacy of Jamun honey (JH) for diabetic wound healing in in vitro wound (primary fibroblasts) model and in in vivo of diabetic mice (Streptozotocin induced) model. The fibroblast cell model was studied for migratory behaviour and myofibrolasts infiltration under honey interventions via scratch/migration assay, immuno-cytochemistry and western blot. We applied FDA approved Manuka honey (MH) as positive control and JH as test honey to evaluate wound re-epithelialization, sub-epithelial connective tissue modification and angiogenesis via histo-pathological and immuno-histochemical analysis. JH (0.1% v/v) dilution has notably improved wound closure, migration with concomitant α-SMA expressions in vitro. Topical application of JH in diabetic mice model showed significant (*p ≤ 0.05) wound closure, reepithelialization, collagen deposition (I/III) and balanced the myofibroblasts formation. It also modulated vital angiogenic markers (viz HIF-1α, VEGF, VEGF R-II) significantly (*p ≤ 0.05). All these observations depicted that JH promotes sequential stages of wound healing in diabetic mice model. The results of the present study established Jamun honey as good as Manuka honey considering wound closure, re-epithelialization, collagen deposition and pro-angiogenic potential.
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Project description:Monocytes (Mos)/macrophages (Mϕs) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/Mϕ heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/Mϕ heterogeneity and associated state transitions during the course of excisional skin wound healing in mice using single-cell RNA sequencing. The live CD45+CD11b+Ly6G- cells were isolated from skin wounds of C57BL/6 mice on days 3, 6, and 10 postinjury and captured using the 10x Genomics Chromium platform. A total of 2813 high-quality cells were embedded into a uniform manifold approximation and projection space, and eight clusters of distinctive cell populations were identified. Cluster dissimilarity and differentially expressed gene analysis categorized those clusters into three groups: early-stage/proinflammatory, late-stage/prohealing, and Ag-presenting phenotypes. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/Mϕ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/Mϕs inferred from our single-cell RNA sequencing dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity and adoptive transfer experiments indicated state transitions between early- and late-state Mos/Mϕs as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/Mϕs, preferentially induced the accumulation of proinflammatory Ly6C+F4/80lo/- Mos/Mϕs in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/Mϕ phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.
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Project description:The IKK? is known to regulate transcription factor NF-?B activation leading to inflammatory responses. Recent gene knockout studies have shown that IKK? can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKK? has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12(rtTA/rtTAt)/tet-O-Cre/Ikk?(F/F) (Ikk?(?CE/?CE)) mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikk?(F/F) mice. Doxycycline-induced IKK? ablation occurred in corneal epithelial cells of triple transgenic Ikk?(?CE/?CE) mice, but loss of IKK? did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKK? significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi) also showed that IKK? was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKK? was required for optimal activation of NF-?B and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKK?-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKK? in the control of epithelial cell motility and wound healing.
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Project description:Previous studies focused on the effects of sympathetic denervation with 6-hydroxydopamine (6-OHDA) on nondiabetic wounds, but the effects of 6-OHDA on diabetic wounds have not been previously reported. In this study, treated mice received intraperitoneal 6-OHDA, and control mice received intraperitoneal injections of normal saline. Full-thickness wounds were established on the backs of mice. The wounds were sectioned (four mice per group) for analysis at 2, 5, 7, 10, 14, 17, and 21 days after injury. The wound areas in the control group were larger than those in the treatment group. Histological scores for epidermal and dermal regeneration were reduced in the 6-OHDA-treated group on day 21. The mast cells (MCs) in each field decreased after sympathectomy on days 17 and 21. The expression levels of norepinephrine, epidermal growth factor (EGF), interleukin-1 beta, NG2 proteoglycan, and desmin in the treatment group were less than those in the control group. In conclusion, 6-OHDA delays reepithelialization during wound healing in diabetic mice by decreasing EGF, but increases wound contraction by reducing IL-1β levels and the number of MCs. Besides, 6-OHDA led to reduced pericyte proliferation in diabetic wounds, which might explain the vascular dysfunction after sympathetic nerve loss in diabetic wounds.
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Project description:BackgroundMatricellular proteins, including periostin, are important for tissue regeneration.Methods and findingsPresently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient ?/? mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin ?2. Periostin was associated with laminin ?2, and this association enhanced the proteolytic cleavage of the laminin ?2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin?/? mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin?/? mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-?B.ConclusionThese results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing.
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