miR-21 promotes keratinocyte migration and re-epithelialization during wound healing

MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-β1. Similar to the effect of TGF-β1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-β1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-β-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-β1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.     

Fibroblast state switching orchestrates dermal maturation and wound healing

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual‐tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue‐scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.     

A C-type CpG ODN accelerates wound healing via regulating fibroblasts and immune response

Abolished or delayed wound healing is a serious problem in clinical surgery, therefore, the new therapy for wound healing is needed. Synthetic oligodeoxynucleotides containing one or more CpG motifs (CpG ODN) has been reported to activate the immune system and improves skin wound healing. The aim of the present study was to evaluate the role of a new C-type CpG ODN in wound healing. We found that the CpG ODN promoted cell proliferation and collagen I production in human skin fibroblasts cells. Besides, we also investigated the effect of CpG ODN on the activation of immune cells. The macrophages and plasmacytoid dendritic cells (pDCs) were incubated with CpG ODN. CpG ODN activated macrophage and pDCs via regulating TLR9/MyD88/NF-κB pathway and TLR9/MyD88/IRF7 pathway, respectively. To further evaluate the effect of CpG ODN on wound healing in vivo a wound healing model was established in mice. The results showed that CpG ODN treatment accelerated wound healing in mice. CpG ODN increased cytokines secretion in wound skin and elevated the ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen. Our results showed that CpG ODN accelerated wound healing, which was partly due to the regulation of fibroblasts and immune response. The findings suggested that the CpG ODN might be a proper medicament for the treatment of wound healing.     

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.     

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1–7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4–6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1–6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds. © 1996 Wiley-Liss, Inc.     

Exosomes from adipose‐derived stem cells and application to skin wound healing

Skin wound healing is an intractable problem that represents an urgent clinical need. To solve this problem, a large number of studies have focused on the use of exosomes (EXOs) derived from adipose‐derived stem cells (ADSCs). This review describes the mechanisms whereby ADSCs‐EXOs regulate wound healing and their clinical application. In the wound, ADSCs‐EXOs modulate immune responses and inflammation. They also promote angiogenesis, accelerate proliferation and re‐epithelization of skin cells, and regulate collagen remodelling which inhibits scar hyperplasia. Compared with ADSCs therapeutics, ADSCs‐EXOs have highly stability and are easily stored. Additionally, they are not rejected by the immune system and have a homing effect and their dosage can be easily controlled. ADSCs‐EXOs can improve fat grafting and promote wound healing in patients with diabetes mellitus. They can also act as a carrier and combined scaffold for treatment, leading to scarless cutaneous repair. Overall, ADSCs‐EXOs have the potential to be used in the clinic to promote wound healing.     

Anti-inflammatory and wound healing potential of cashew apple juice (Anacardium occidentale L.) in mice

Cashew apple is a tropical pseudofruit consumed as juice due to its excellent nutritional and sensory properties. In spite of being well known for its important antioxidant properties, the cashew apple has not been thoroughly investigated for its therapeutic potential. Thereby, this study evaluated the antioxidant capacity, anti-inflammatory, and wound-healing activities of cashew apple juice. Juices from ripe and immature cashew apples were analyzed for antioxidant, anti-inflammatory, and wound-healing properties. Those were evaluated in murine models of xylene-induced ear edema and wound excision. Swiss mice were treated with cashew juice by gavage. Edema thickness was measured and skin lesions were analyzed by planimetry and histology. Both antioxidant content and total antioxidant activity were higher in ripe cashew apple juice (RCAJ) than in unripe cashew apple juice (UNCAJ). The UNCAJ presented the main anti-inflammatory activity by a significant inhibition of ear edema (66.5%) when compared to RCAJ (10%). Moreover, UNCAJ also showed the best result for wound contraction (86.31%) compared to RCAJ (67.54%). Despite of higher antioxidant capacity, RCAJ did not promote better anti-inflammatory, and healing responses, which may be explained by the fact that treatment increased antioxidants level leading to a redox “imbalance” turning down the inflammatory response modulation exerted by reactive oxygen species (ROS). The results suggest that UNCAJ presents a greater therapeutic activity due to a synergistic effect of its phytochemical components, which improve the immunological mechanisms as well as an optimal balance between ROS and antioxidants leading to a better wound healing process.     

Transcriptional space-time mapping identifies concerted immune and stromal cell patterns and gene programs in wound healing and cancer

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Cellular and physiological upregulation of inducible nitric oxide synthase,arginase, and inducible cyclooxygenase in wound healing

Wound repair is regulated by overlapping cellular, physiological and biochemical events. Prostaglandins and nitric oxide have been a focus for inflammation research particularly since the discovery of their inducible isoforms nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Study of the cellular expression of iNOS and COX-2 and arginase which competes with iNOS for its substrate, in an in vivo model of wound healing could reveal important roles for these enzymes in the physiological progression of wound repair. Adult male rats received full thickness dermal wounds which were harvested at different times. Protein levels and activities of the enzymes were assessed by western blot and biochemical assays respectively. The cellular distribution and the colocalization were assessed by immunostaining. The protein levels and activities of iNOS, arginase, and COX-2 increased only during the inflammatory phase of wound. Immunocytochemistry showed that the three enzymes were coexpressed and the main cellular source was inflammatory cells mainly macrophages. iNOS was induced at the wound site and was the earliest to increase significantly (p < 0.05) for only up to 3 days postwounding. However, arginase and COX-2 significant ( p < 0.05) upregulation started at a later time points and continued for up to 14 days postwounding. Therefore iNOS, compared with arginase and COX-2, showed a temporal difference in expression during wound healing which could be explained by their products being required at different stages of the healing process. The coordinated expression of the three enzymes at different time points could account for the physiological progression of the healing process.     

The lactate conundrum in wound healing: Clinical and experimental findings indicate the requirement for a rapid point-of-care diagnostic

The increasing prevalence of chronic wounds has significant financial implications for nations with advanced healthcare provision. Although the diseases that predispose to hard‐to‐heal wounds are recognized, their etiology is less well understood, partly because practitioners in wound management lack specialized diagnostic support. Prognostic indicators for healing may be inherent to wound biochemistry but remain invisible under routine clinical investigation; lactate is an example of this. In this study, lactate concentration in exudate obtained from 20 patients undergoing wound management in hospital was variable but in some cases approached or exceeded 20 mM. In vitro viability studies indicated that fibroblasts and endothelial cells tolerated low levels of lactate (1–10 mM), but cell viability was severely compromised by high lactate concentrations (=20 mM). Scratched monolayer experiments revealed that cell migration was affected earlier than viability in response to increasing lactate dose, and this was shown by immunocytochemistry to be associated with cytoskeletal disruption. A prototype enzyme‐based colorimetric assay for lactate generating a color change that was rapid in the context of clinical practise, and capable of functioning within a gel vehicle, was developed with point‐of‐care dipstick applications in mind. A randomized single‐blinded trial involving 30 volunteers and using a color chart to calibrate the assay demonstrated that lactate concentration could be reliably estimated with 5 mM precision; this suggesting that “physiological” and “pathological” lactate concentration could be distinguished. The present data suggest that a dipstick‐type colorimetric assay could comprise a viable diagnostic tool for identifying patients at‐risk from high‐wound lactate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 917–924, 2012     

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1beta (IL-1beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alpha/beta (CINC-2alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process.     

Differential cytokine activity and morphology during wound healing in the neonatal and adult rat skin

Wound-healing mechanisms change during transition from prenatal to postnatal stage. Cytokines are known to play a key role in this process. The current study investigated the differential cytokine activity and healing morphology during healing of incisional skin wounds in rats of the ages neonatal (p0), 3 days old (p3) and adult, after six different healing times (2 hrs to 30 days). All seven tested cytokines (Transforming Growth Factor (TGF) alpha, TGFbeta1, -beta2 and -beta3, IGF 1, Platelet Derived Growth Factor A (PDGF A), basic Fibroblast Growth Factor (bFGF) exhibited higher expression in the adult wounds than at the ages p0 and p3. Expression typically peaked between 12 hrs and 3 days post-wounding, and was not detectable any more at days 10 and 30. The neonate specimen showed more rapid re-epithelialization, far less inflammation and scarring, and larger restitution of original tissue architecture than their adult counterparts, resembling a prenatal healing pattern. The results may encourage the use of neonatal rat skin as a wound-healing model for further studies, instead of the more complicated prenatal animal models. Secondly, the data may recommend inhibition of PDGF A, basic FGF or TGF-beta1 as therapeutic targets in efforts to optimize wound healing in the adult organism.     

Energy metabolism in the granulation tissue of diabetic rats during cutaneous wound healing

The skin cells chiefly depend on carbohydrate metabolism for their energy requirement during cutaneous wound healing. Since the glucose metabolism is greatly hampered in diabetes and this might affect wound repair process. This prompted us to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the wound tissues of normal and streptozotocin-induced diabetic rats following excision-type of cutaneous injury. The activities of key regulatory enzymes namely hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the granulation tissues of normal and diabetic rats at different time points (2, 7, 14 and 21 days) of postwounding. Interestingly, a significant alteration in all these enzyme activities was observed in diabetic rats. The activity of PFK was increased but HK, LDH and CS showed a decreased activity in the wound tissue of diabetics as compared to normal rats. However G6PD exhibited an elevated activity only at early stage of healing in diabetic rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the wound tissue of diabetic rats may affect the energy availability for cellular activity needed for repair process and this may perhaps be one of the factor responsible for impaired healing in these subjects. (Mol Cell Biochem 270: 71–77, 2005)     

Sorting of murine vascular smooth muscle cells during wound healing in the chicken chorioallantoic membrane

The vascular wall is built up of a heterogeneous population of smooth muscle cells, which exhibit not only morphological distinctions but also important differences in the composition of their structural and contractile proteins. "Epithelioid" smooth muscle cells correspond to an intimal-like type and display features associated with immaturity, whereas "spindle-shaped" cells closely resemble the more typical medial smooth muscle population. We have investigated the integration of these two cell types into the vascular architecture of an in vivo wound-healing model. Stably transfected with the beta-galactosidase gene, intima- and media-like cells were injected intravenously into the chicken chorioallantoic membrane, within which superficial foci of granulation tissue had been created by thermal or chemical injury. At 24 to 72 h after injection, cells had honed in on the lesion sites and were observed in juxtaposition to the endothelial lining of the capillaries. They began to deposit laminin, thereby indicating an impending role in the formation of the vascular wall. Intima- and media-like smooth muscle cells did not differ in their capacity to associate with capillaries, and, in so doing, their biochemical lineage characteristics became indistinguishable from one another. However, intima-like cells also penetrated the adventitial and medial layers of arteries. These findings reveal vascular smooth muscle cells to possess an extraordinary degree of plasticity, being able to adapt flexibly to changes in functional demands.     

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.     

Temporal and differential proteomic profile of molecular mediators associated with chronic and acute wound healing

The underlying pathophysiology of nonhealing chronic wounds is poorly understood due to the changes occurring at the gene level and the complexity arising in their proteomic profile. Here, we elucidated the temporal and differential profile of the normal and diabetic wound-healing mediators along with their interactions and associated pathways. Skin tissues corresponding to normal and diabetic wounds were isolated at Days 0, 3, 6, and 9 representing different healing phases. Temporal gene expression was analyzed by quantitative real-time PCR. Concurrently, differential protein patterns in the wound tissues were identified by Nano LC-ESI-TOF mass spectrometry and later confirmed by Western blot analysis. Gene ontology annotation, protein-protein interaction, and protein pathway analysis were performed using DAVID, PANTHER, and STRING bioinformatics resources. Uniquely identified proteins (complement C3, amyloid beta precursor protein, and cytoplasmic linker associated protein 2) in the diabetic wound tissue implied that these proteins are involved in the pathogenesis of diabetic wound. They exhibit enhanced catalytic activity, trigger pathways linked with inflammation, and negatively regulate wound healing. However, in the normal wound tissue, axin 1, chondroitin sulfate proteoglycan 4, and sphingosine-1-phosphate receptor were identified, which are involved in proliferation, angiogenesis, and remodeling. Our findings demonstrate the correlation between elevated gene expression of tumor necrosis factor-α, interleukin (IL)-1β, and identified mediators: aryl hydrocarbon receptor nuclear translocator, 5′-aminolevulinate synthase 2, and CXC-family, that inflicted an inflammatory response by activating downstream MAPK, JAK-STAT, and NF-κB pathways. Similarly, in normal wound tissue, the upregulated IL-4 and hepatocyte growth factor levels in conjunction with the identified proteins, serine/threonine-protein kinase mTOR and peroxisome proliferator-activated receptor gamma, played a significant role in the cellular response to platelet-derived growth factor stimulus, dermal epithelialization, and cell proliferation, processes associated with the repair mechanism. Furthermore, Western blot analysis indicated elevated levels of inflammatory markers and reduced levels of proliferative and angiogenic factors in the diabetic wound.     

角质细胞生长因子2对细胞生长、移行及创伤愈合的作用

角质细胞生长因子2(KGF-2)是成纤维细胞生长因子超家族的一员,由间质细胞合成并分泌,能特异促进上皮细胞增殖、分化与迁移,对脊椎动物多种组织和器官的发育起重要调控作用．通过PCR从人的肾组织cDNA文库中克隆分离获得了KGF-2的cDNA,表明该因子在成人肾中有表达．采用大肠杆菌表达并纯化重组蛋白用于生物学功能研究的结果显示：KGF-2在体外不仅能够促进角质细胞的生长和增殖,而且对其凋亡具有抑制作用,还对细胞的移行具有影响．在动物实验中,KGF-2能促进皮肤切除产生的伤口愈合,提示该蛋白质可以作为创伤治疗或辅助用药的候选分子．     

鹿茸角柄断面伤口无疤痕愈合过程中骨形态蛋白的表达及功能分析

为研究骨形态蛋白（bone morphogenetic proteins ,BMP）在鹿茸再生早期角柄断面伤口无疤痕愈合过程中的功能,本研究通过免疫组化技术对比分析了BMP 2 和BMP 4在正常皮肤及茸皮中的表达及分布差异,同时利用添加外源性蛋白分析了BMP 4对鹿真皮成纤维细胞和毛乳头细胞的影响。结果显示：(1)原代培养的真皮成纤维细胞表达波形蛋白阳性率几乎为100%;(2) BMP 2和BMP 4强烈表达于茸皮中新生毛囊的毛基质细胞中;(3) BMP 4可促进鹿真皮成纤维细胞向脂肪细胞转分化;(4) BMP 4可促进鹿真皮毛乳头细胞成团。以此推测BMP在鹿毛囊形成及伤口愈合过程中发挥重要作用。