MicroRNA 在皮肤创伤愈合中的作用

皮肤创伤愈合过程是一个复杂而连续的过程,这一过程需要多种细胞、多种因子的参与,涉及细胞增殖、细胞分化、细胞运动、细胞黏附等多个细胞生物学过程。 MicroRNA（ miRNA）是一类高度保守的非编码RNA,它通过靶向结合信使RNA（ mRNA）并使其降解或抑制其翻译,实现转录后基因表达调控。 miRNA作为基因表达的重要调控分子,几乎参与了机体所有的生理和病理过程。除了在皮肤发育中发挥重要的作用,还参与多种皮肤病、皮肤癌和皮肤创伤愈合过程的调节。主要总结了miRNA调控皮肤创伤愈合的研究进展。     

Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice

Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM.     

Anti‐aging pharmacology in cutaneous wound healing: effects of metformin,resveratrol, and rapamycin by local application

Cutaneous wounds are among the most common soft tissue injuries and are particularly hard to heal in aging. Caloric restriction (CR) is well documented to extend longevity; pharmacologically, profound rejuvenative effects of CR mimetics have been uncovered, especially metformin (MET), resveratrol (RSV), and rapamycin (RAPA). However, locally applied impacts and functional differences of these agents on wound healing remain to be established. Here, we discovered that chronic topical administration of MET and RSV, but not RAPA, accelerated wound healing with improved epidermis, hair follicles, and collagen deposition in young rodents, and MET exerted more profound effects. Furthermore, locally applied MET and RSV improved vascularization of the wound beds, which were attributed to stimulation of adenosine monophosphate‐activated protein kinase (AMPK) pathway, the key mediator of wound healing. Notably, in aged skin, AMPK pathway was inhibited, correlated with impaired vasculature and reduced healing ability. As therapeutic approaches, local treatments of MET and RSV prevented age‐related AMPK suppression and angiogenic inhibition in wound beds. Moreover, in aged rats, rejuvenative effects of topically applied MET and RSV on cell viability of wound beds were confirmed, of which MET showed more prominent anti‐aging effects. We further verified that only MET promoted wound healing and cutaneous integrity in aged skin. These findings clarified differential effects of CR‐based anti‐aging pharmacology in wound healing, identified critical angiogenic and rejuvenative mechanisms through AMPK pathway in both young and aged skin, and unraveled chronic local application of MET as the optimal and promising regenerative agent in treating cutaneous wound defects.     

Fibromodulin reduces scar size and increases scar tensile strength in normal and excessive‐mechanical‐loading porcine cutaneous wounds

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive‐mechanical‐loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule—fibromodulin (FMOD) protein—can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive‐mechanical‐loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD‐based technologies hold high translational potential and applicability to human patients suffering from scarring—especially hypertrophic scarring.     

Nestin‐expressing interfollicular blood vessel network contributes to skin transplant survival and wound healing

Using nestin‐driven green fluorescent protein (ND‐GFP) transgenic mice, we previously demonstrated an inter‐hair‐follicle blood vessel network that expresses ND‐GFP and appears to originate from ND‐GFP expressing hair‐follicle stem cells. We report here that angiogenesis of transplanted skin or healing wounds originates from this ND‐GFP‐expressing microvasculature network. ND‐GFP‐expressing blood vessels were visualized growing from the ND‐GFP‐expressing hair‐follicle stem cell area and re‐establishing the dermal microvasculature network after skin transplantation or wound healing. When the ND‐GFP stem cell area from the vibrissa (whisker) from ND‐GFP mice was transplanted to transgenic mice ubiquitously expressing RFP, we observed chimeric ND‐GFP‐RFP blood vessels, suggesting the joining of inter‐follicular blood vessel networks from the transplant and host. These observations suggest that the inter‐hair‐follicle blood‐vessel network contributes to skin transplant survival and wound healing. J. Cell. Biochem. 110: 80–86, 2010. © 2010 Wiley‐Liss, Inc.     

TGF-β receptor expression on human keratinocytes: A 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with type I and type II receptors

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-β has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-β receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-β receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-β1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-β receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-β1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-β receptors on keratinocytes, supporting the heterotetrameric model of TGF-β signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-β by markedly downregulating all four TGF-β binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with the TGF-β signalling receptors suggests that this GPI-anchored protein may modify TGF-β signalling in human keratinocytes. J. Cell. Biochem. 70:573–586, 1998. © 1998 Wiley-Liss, Inc.     

Differential expression of cyclooxygenase-1 and cyclooxygenase-2 in the cornea during wound healing

Cyclooxygenases (COXs) are the key enzymes in the production of prostaglandins (PGs) and exist in two isoforms. Isoform 1 (COX-1) is constitutively expressed in most tissues, whereas cyclooxygenase-2 (COX-2) is rapidly induced by a variety of different stimuli. In this study, we have quantitatively analyzed mRNA expression of COX-1 and COX-2 and protein distribution during corneal reparative processes after wound. Total RNA was isolated from cornea samples of New Zealand rabbits that had been subjected to corneal wound by mechanical brush scraping. Quantification of RT-PCR results was made by using a DNA mimic approach. The localization and expression of the enzymes was studied by immunocytochemistry and Western blotting. In normal corneas COX-1 is expressed throughout the cornea in the whole tissue, while COX-2 is strongly expressed in stromal keratocytes. Following injury, COX-2 levels drastically increase and, at least in the epithelium, COX-2 becomes the predominant isoform of cyclooxygenases at an early stage of healing. Moreover, in the epithelium COX-2 is expressed predominantly by those cells close to the wound. These cells become migratory and move toward the injured area. In contrast, COX-1 levels remain unaffected in all corneal tissues. The system returns to the pre-injury state in about 24h. Thus, the expression of COX-2 in the corneal epithelium during wound repair is tightly regulated both temporally and spatially.     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

Scarless skin wound healing in FOXN1 deficient (nude) mice is associated with distinctive matrix metalloproteinase expression

Similar to mammalian fetuses FOXN1 deficient (nude) mice are able to restore the structure and integrity of injured skin in a scarless healing process by mechanisms independent of the genetic background. Matrix metalloproteinases (MMPs) are required for regular skin wound healing and the distinctive pattern of their expression has been implicated to promote scarless healing. In this study, we analyzed the temporal and spatial expression patterns of these molecules during the incisional skin wounds in adult nude mice. Macroscopic and histological analyses of skin wounds revealed an accelerated wound healing process, minimal granulation tissue formation and markedly diminished scarring in nude mice. Quantitative RT-PCR (Mmp-2, -3, -8, -9, -10, -12, -13, -14 and Timp-1, -2, -3), Western blots (MMP-13) and gelatin zymography (MMP-9) revealed that MMP-9 and MMP-13 showed a unique, bimodal pattern of up-regulation during the early and late phases of wound healing in nude mice. Immunohistochemically MMP-9 and MMP-13 were generally detected in epidermis during the early phase and in dermis during the late (remodeling) phase. Consistent with these in vivo observations, dermal fibroblasts cultured from nude mice expressed higher levels of types I and III collagen, MMP-9 and MMP-13 mRNA levels and higher MMP enzyme activity than wild type controls. Collectively, these finding suggest that the bimodal pattern of MMP-9 and MMP-13 expression during skin repair process in nude mice could be a major component of their ability for scarless healing.     

Skin wound healing in different aged Xenopus laevis

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8‐ and 15‐month‐old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re‐epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti‐inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti‐tumor necrosis factor (TNF)‐α, iNOS, transforming growth factor (TGF)‐β1, and matrix metalloproteinase (MMP)‐9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti‐α‐SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar‐like pattern. The quantitative PCR analysis demonstrated an up‐regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar‐like tissue formation. J. Morphol. 274:956–964, 2013. © 2013 Wiley Periodicals, Inc.     

A novel therapeutic phosphate-based glass improves full-thickness wound healing in a rat model

Wound healing is a highly dynamic process and innovative therapeutic approaches are currently developed to address challenges of providing optimal wound care. In this study, phosphate-based glasses in the (CuO)_x·(KPO₃)_79.5-x·(ZnO)₂₀·(Ag₂O)_0.5 system (CuKPO₃ZnAg), with different CuO/ KPO₃ ratios were prepared by melt-quenching technique. Constant Cu concentrations were released from the samples during immersion in Simulated Body Fluid (SBF), while Zn concentrations were slightly decreased over time. Glass surface phosphatation leading to formation of Zn crystalline salts was revealed through spectroscopic techniques. This finding was supported by SEM images that illustrated new compound formation. Subsequent cytotoxicity evaluation on HaCaT Keratinocytes using the indirect MTT cell viability assay revealed a CuO concentration-dependent cytotoxicity profile and excellent biocompatibility at low CuO concentrations, in all CuKPO₃ZnAg glasses. Furthermore, the (CuO)₅·(KPO₃)_74.5·(ZnO)₂₀·(Ag₂O)_0.5 sample (5CuKPO₃ZnAg)_, demonstrated superior antibacterial potency against S. aureus (ATCC 25923) strain compared to amoxicillin and ciprofloxacin. In vivo full-thickness wound healing evaluation showed a significantly higher regenerative effect of the 5CuKPO₃ZnAg sample, in terms of angiogenesis, collagen synthesis and re-epithelialization compared to non-treated wounds. These findings advance our understanding of the therapeutic perspectives of phosphate-based glasses, showing promising potential for wound-healing applications.     

MiR‐21‐5p/dual‐specificity phosphatase 8 signalling mediates the anti‐inflammatory effect of haem oxygenase‐1 in aged intracerebral haemorrhage rats

Intracerebral haemorrhage (ICH) is a severe neurological disorder caused by bleeding within the brain tissue. Inflammation has been implicated in ICH pathogenesis and is a potential therapeutic target for ICH. Haemin, an activator of haem oxygenase‐1 (HO‐1), rapidly increases HO‐1 protein expression and activity and has been shown to distinctly affect anti‐inflammatory functions after central nervous system (CNS) injury. However, less is known about the mechanisms that underlie the anti‐inflammatory effects of haemin in aged rats post‐ICH. Here, we performed microarray analysis to identify miRNAs that respond strongly to HO‐1 regulation in ICH rats and found that miR‐21‐5p induced the most significant change. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) analysis, we focused on dual‐specificity phosphatase 8 (DUSP8) from the predicted miR‐21‐5p targets. Luciferase reporter assays confirmed that miR‐21‐5p bound directly to DUSP8. MiR‐21‐5p upregulation in vitro downregulated DUSP8 expression. Importantly, intracerebroventricularly injecting antagomir for miR‐21‐5p (A‐miR‐21‐5p), which was used to inhibit miR‐21‐5p in aged ICH rats, significantly reduced the neurological defects, repaired cognitive impairment, alleviated blood–brain barrier (BBB) permeability, inhibited neuronal apoptosis posthaemorrhage and accelerated haematoma absorption. In addition, serum miR‐21‐5p levels were notably elevated in patients relative to healthy individuals and were correlated with National Institutes of Health Stroke Scale (NIHSS) scores and clinical outcomes. In summary, A‐miR‐21‐5p increased HO‐1 expression in cerebral haematomas, thus eliciting the DUSP8‐modulated perifocal neuroprotective effect of haemin. MiR‐21‐5p with haemin therapy may be a potential therapy post‐ICH.     

Pro‐apoptotic effect of haem oxygenase‐1 in human colorectal carcinoma cells via endoplasmic reticular stress

Several biological effects of haem oxygenase (HO)‐1, including anti‐inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO‐1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO‐1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)‐3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT‐15, LOVO and HT‐29 cells in serum‐free (SF) conditions with increased HO‐1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP‐induced apoptosis and HO‐1 protein expression in human colon cancer cells. CoPP‐induced apoptosis of colon cancer cells was prevented by the addition of the pan‐caspase inhibitor, Z‐VAD‐FMK (VAD), and the Casp‐3 inhibitor, Z‐DEVD‐FMK (DEVD). N‐Acetyl cysteine inhibited reactive oxygen species‐generated H₂O₂‐induced cell death with reduced intracellular peroxide production, but did not affect CoPP‐induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO‐1 expression by those cells, and knockdown of HO‐1 protein expression by HO‐1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO₄ or biliverdin, induced DNA ladders, and cleavage of Casp‐3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R‐like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM‐ and CoPP‐induced apoptosis. Increased GRP78 level and formation of the HO‐1/GRP78 complex were detected in CORM‐ and CoPP‐treated human CRC cells. A pro‐apoptotic role of HO‐1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.     

Changes in the levels and distribution of glycogen during regeneration of cutaneous wounds in Heteropneustes fossilis: a histochemical investigation

Incisional wounds of the same length and depth were made in the skin of catfish Heteropneustes fossilis , and the response in terms of levels of glycogen in cells during healing was studied histochemically. After injury, glycogen disappeared completely from the cells in the migrating epidermis. This disappearance is correlated with the migration of the epidermal cells toward the wound gap for epithelialization and the increased production of mucous cells at the initial stages of healing. The gradual accumulation of glycogen in the epithelial cells and the basal cells after epithelialization of the wound during the early stages of healing suggests that in these cells gluconeogenesis predominates. The gradual disappearance of glycogen from the epithelial cells 72 h after injury and from the basal cells 36 h after injury indicates the gradual resumption of their metabolic activities. The increased gradients of glycogen in club cells that are undergoing vacuolization and disintegration are discussed. No significant levels of glycogen were observed during the formation of granulation tissue and biosynthesis of the sub-cutis. The depletion of glycogen in the degenerating muscle bundles is correlated with the synthesis of lactic acid which contributes to local acidity and favours the autolysis of muscles.