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1.
Science ; 364(6441): 653-658, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31097661

RESUMO

Unlike mammals, Xenopus laevis tadpoles have a high regenerative potential. To characterize this regenerative response, we performed single-cell RNA sequencing after tail amputation. By comparing naturally occurring regeneration-competent and -incompetent tadpoles, we identified a previously unrecognized cell type, which we term the regeneration-organizing cell (ROC). ROCs are present in the epidermis during normal tail development and specifically relocalize to the amputation plane of regeneration-competent tadpoles, forming the wound epidermis. Genetic ablation or manual removal of ROCs blocks regeneration, whereas transplantation of ROC-containing grafts induces ectopic outgrowths in early embryos. Transcriptional profiling revealed that ROCs secrete ligands associated with key regenerative pathways, signaling to progenitors to reconstitute lost tissue. These findings reveal the cellular mechanism through which ROCs form the wound epidermis and ensure successful regeneration.


Assuntos
Epiderme/fisiologia , Reepitelização/fisiologia , Cauda/fisiologia , Xenopus laevis/fisiologia , Animais , Células Epidérmicas/fisiologia , Reepitelização/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Proteínas de Xenopus/genética , Xenopus laevis/genética
2.
Dev Biol ; 445(2): 271-279, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30476483

RESUMO

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.


Assuntos
Caveolina 1/fisiologia , Cicatrização/fisiologia , Animais , Queimaduras/genética , Queimaduras/patologia , Queimaduras/fisiopatologia , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Epidérmicas/patologia , Células Epidérmicas/fisiologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/genética , Ratos , Reepitelização/genética , Reepitelização/fisiologia , Células-Tronco/patologia , Células-Tronco/fisiologia , Regulação para Cima , Cicatrização/genética
3.
Trends Cardiovasc Med ; 29(3): 131-137, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30143275

RESUMO

Diabetic wound healing is an incompletely understood pathophysiological state. It comprises a range of potentially devastating and common complications of diabetes mellitus (DM) leading to intractable infections, lower extremity amputations, and associated cardiovascular morbidity and mortality. MicroRNAs (miRNAs) have emerged as important regulators in various physiological processes in health and disease through their ability to fine-tune cellular responses. Herein, we summarize the versatile roles of miRNAs implicated in diabetic wound healing in key stages including inflammation, angiogenesis, re-epithelialization, and remodeling. Furthermore, we highlight current evidence through which miRNAs exert control of gene expression and signaling pathways in the reparative response that may provide opportunities for therapeutic intervention for this potentially devastating disease state.


Assuntos
Complicações do Diabetes/genética , MicroRNAs/genética , Cicatrização/genética , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Complicações do Diabetes/terapia , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Reepitelização/genética , Transdução de Sinais
4.
Science ; 362(6417)2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30467144

RESUMO

During tissue repair, myofibroblasts produce extracellular matrix (ECM) molecules for tissue resilience and strength. Altered ECM deposition can lead to tissue dysfunction and disease. Identification of distinct myofibroblast subsets is necessary to develop treatments for these disorders. We analyzed profibrotic cells during mouse skin wound healing, fibrosis, and aging and identified distinct subpopulations of myofibroblasts, including adipocyte precursors (APs). Multiple mouse models and transplantation assays demonstrate that proliferation of APs but not other myofibroblasts is activated by CD301b-expressing macrophages through insulin-like growth factor 1 and platelet-derived growth factor C. With age, wound bed APs and differential gene expression between myofibroblast subsets are reduced. Our findings identify multiple fibrotic cell populations and suggest that the environment dictates functional myofibroblast heterogeneity, which is driven by fibroblast-immune interactions after wounding.


Assuntos
Macrófagos/fisiologia , Miofibroblastos/fisiologia , Reepitelização/fisiologia , Pele/lesões , Cicatrização/fisiologia , Adipócitos/fisiologia , Animais , Proliferação de Células , Matriz Extracelular/metabolismo , Fibrose , Integrina beta1/genética , Queloide/patologia , Lectinas Tipo C/análise , Lectinas Tipo C/metabolismo , Linfocinas/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Derivado de Plaquetas/metabolismo , Reepitelização/genética , Pele/imunologia , Pele/patologia , Envelhecimento da Pele/fisiologia , Transcriptoma , Cicatrização/genética
5.
Sci Rep ; 8(1): 13140, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30177722

RESUMO

Woundhealing disorders characterized by impaired or delayed re-epithelialization are a serious medical problem that is painful and difficult to treat. Gelsolin (GSN), a known actin modulator, supports epithelial cell regeneration and apoptosis. The aim of this study was to estimate the potential of recombinant gelsolin (rhu-pGSN) for ocular surface regeneration to establish a novel therapy for delayed or complicated wound healing. We analyzed the influence of gelsolin on cell proliferation and wound healing in vitro, in vivo/ex vivo and by gene knockdown. Gelsolin is expressed in all tested tissues of the ocular system as shown by molecular analysis. The concentration of GSN is significantly increased in tear fluid samples of patients with dry eye disease. rhu-pGSN induces cell proliferation and faster wound healing in vitro as well as in vivo/ex vivo. TGF-ß dependent transcription of SMA is significantly decreased after GSN gene knockdown. Gelsolin is an inherent protein of the ocular system and is secreted into the tear fluid. Our results show a positive effect on corneal cell proliferation and wound healing. Furthermore, GSN regulates the synthesis of SMA in myofibroblasts, which establishes GSN as a key protein of TGF-ß dependent cell differentiation.


Assuntos
Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Síndromes do Olho Seco/genética , Gelsolina/genética , Reepitelização/genética , Actinas/genética , Actinas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Túnica Conjuntiva/patologia , Córnea/patologia , Síndromes do Olho Seco/sangue , Síndromes do Olho Seco/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Pálpebras/citologia , Pálpebras/metabolismo , Feminino , Gelsolina/sangue , Regulação da Expressão Gênica , Humanos , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Masculino , Camundongos , Miofibroblastos/citologia , Ducto Nasolacrimal/citologia , Ducto Nasolacrimal/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/genética
6.
J Invest Dermatol ; 138(12): 2653-2665, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29906410

RESUMO

Wound healing is a dynamic process involving gene-expression changes that drive re-epithelialization. Here, we describe an essential role for polyamine regulator AMD1 in driving cell migration at the wound edge. The polyamines, putrescine, spermidine, and spermine are small cationic molecules that play essential roles in many cellular processes. We demonstrate that AMD1 is rapidly upregulated following wounding in human skin biopsies. Knockdown of AMD1 with small hairpin RNAs causes a delay in cell migration that is rescued by the addition of spermine. We further show that spermine can promote cell migration in keratinocytes and in human ex vivo wounds, where it significantly increases epithelial tongue migration. Knockdown of AMD1 prevents the upregulation of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor on wounding and results in a failure in actin cytoskeletal reorganization at the wound edge. We demonstrate that keratinocytes respond to wounding by modulating polyamine regulator AMD1 in order to regulate downstream gene expression and promote cell migration. This article highlights a previously unreported role for the regulation of polyamine levels and ratios in cellular behavior and fate.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Movimento Celular/genética , Epiderme/fisiologia , Queratinócitos/fisiologia , Cicatrização , Ferimentos e Lesões/metabolismo , Citoesqueleto de Actina/metabolismo , Adenosilmetionina Descarboxilase/genética , Biópsia , Sinalização do Cálcio , Células Cultivadas , Humanos , RNA Interferente Pequeno/genética , Reepitelização/genética , Espermina/metabolismo , Regulação para Cima , Ferimentos e Lesões/genética
7.
Wound Repair Regen ; 26(1): 102-107, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29418044

RESUMO

Epithelia have the ability to repair injuries through an evolutionary conserved wound healing mechanism. Wound healing events can be classified into the transcription-independent signals involving mobilization of ionic currents and cytoskeletal rearrangements or the transcription-dependent response with activation of repair genes. The vacuolar H+ -ATPase (V-ATPase) has been implicated in the regeneration of vertebrate structures, but the underlying cellular mechanisms remain unclear. Here, we use wounding assays on the epidermis of Drosophila embryos to assess the role of the V-ATPase in the healing response. We show that a deficient V-ATPase induces a defective wound healing response by delaying re-epithelialization and preventing the ERK-dependent transcriptional activation of repair around the wound site. Our data suggests that the V-ATPase plays an evolutionary conserved role in the activation of genes necessary for the wound healing response.


Assuntos
Proteínas de Drosophila/genética , Drosophila/enzimologia , Epiderme/lesões , MAP Quinases Reguladas por Sinal Extracelular/genética , Reepitelização/genética , ATPases Vacuolares Próton-Translocadoras/genética , Cicatrização/genética , Animais , Embrião não Mamífero , Epiderme/metabolismo , Modelos Animais , Reepitelização/fisiologia , Sensibilidade e Especificidade , Transdução de Sinais , Ativação Transcricional/genética , Cicatrização/fisiologia
8.
J Tissue Eng Regen Med ; 12(2): e905-e917, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28097806

RESUMO

A porous structure is critically important for wound dressing or tissue engineering scaffolds. However, the influence of the pore sizes on cell proliferation, tissue regeneration and the underlying mechanism remains unclear. In this study, silicone rubber membranes with different pore sizes were prepared using certain constituents of liquid silicone rubber precursor/liquid paraffin/hexane based on our previous studies. It was found that pore size had a significant impact on cell proliferation and wound healing. The CCK8 analysis revealed that the membrane with a certain pore size (110.47 µm, middle pore membrane, MPM) was suitable for cell proliferation compared with the membranes with other pore sizes (218.03 µm, large pore membrane, LPM; 5.27 µm, small pore membrane, SPM; non-porous membrane, NPM). Further studies demonstrated that the MPM promoted cell proliferation via activating the Wnt/ß-catenin signalling pathway. More importantly, wound healing experiments showed that 7 days post-wounding, the rate of wound healing was 89.25% with the MPM, which was significantly higher than with LPM, SPM or NPM. The in vivo data indicated that wound healing was accelerated by treatment with a silicone rubber membrane with a pore size of 110.47 µm. Our results strongly suggest that different pore structures might affect cell proliferation and wound healing and that a silicone rubber membrane with a specific pore size could potentially be used as a promising wound dressing. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Membranas Artificiais , Regeneração/efeitos dos fármacos , Elastômeros de Silicone/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Camundongos Endogâmicos BALB C , Porosidade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Reepitelização/efeitos dos fármacos , Reepitelização/genética , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , beta Catenina/metabolismo
9.
Diabetes ; 66(8): 2254-2265, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28546424

RESUMO

We have previously reported that the topical application of erythropoietin (EPO) to cutaneous wounds in rats and mice with experimentally induced diabetes accelerates their healing by stimulating angiogenesis, reepithelialization, and collagen deposition, and by suppressing the inflammatory response and apoptosis. Aquaporins (AQPs) are integral membrane proteins whose function is to regulate intracellular fluid hemostasis by enabling the transport of water and glycerol. AQP3 is the AQP that is expressed in the skin where it facilitates cell migration and proliferation and re-epithelialization during wound healing. In this report, we provide the results of an investigation that examined the contribution of AQP3 to the mechanism of EPO action on the healing of burn wounds in the skin of pigs with experimentally induced type 1 diabetes. We found that topical EPO treatment of the burns accelerated their healing through an AQP3-dependent mechanism that activates angiogenesis, triggers collagen and hyaluronic acid synthesis and the formation of the extracellular matrix (ECM), and stimulates reepithelialization by keratinocytes. We also found that incorporating fibronectin, a crucial constituent of the ECM, into the topical EPO-containing gel, can potentiate the accelerating action of EPO on the healing of the burn injury.


Assuntos
Indutores da Angiogênese/administração & dosagem , Aquaporina 3/metabolismo , Queimaduras/tratamento farmacológico , Eritropoetina/administração & dosagem , Cicatrização/efeitos dos fármacos , Cicatrização/genética , Administração Tópica , Animais , Queimaduras/genética , Colágeno/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Matriz Extracelular/genética , Fibronectinas/administração & dosagem , Ácido Hialurônico/biossíntese , Queratinócitos/metabolismo , Neovascularização Fisiológica , Reepitelização/genética , Pele/metabolismo , Suínos
10.
Endocrinology ; 158(6): 1929-1938, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28368538

RESUMO

When the skin is injured, keratinocytes proliferate, migrate, and differentiate to regenerate the epidermis. We recently showed that ablation of the vitamin D receptor (Vdr) in keratinocytes delays wound re-epithelialization in mice also fed a low-calcium diet, implicating a cooperative role of Vdr and calcium signaling in this process. In this study, we examined the role of vitamin D and calcium signaling in wound healing by deleting their receptors, Vdr and the calcium-sensing receptor (Casr). Gene expression profiling of neonatal epidermis lacking both Vdr and Casr [Vdr and Casr double knockout (DKO)] specifically in keratinocytes revealed that DKO affects a number of pathways relevant to wound healing, including Vdr, ß-catenin, and adherens junction (AJ) signaling. In adult skin, DKO caused a significant delay in wound closure and re-epithelialization, whereas myofibroblast numbers and matrix deposition were unaffected. The injury-induced proliferation of epidermal keratinocytes was blunted in both epidermis and hair follicles, and expression of ß-catenin target genes was reduced in the DKO. Expression of E-cadherin and desmoglein 1 was reduced in the shortened leading edges of the epithelial tongues re-epithelializing the wounds, consistent with the decreased migration rate of DKO keratinocytes in vitro. These results demonstrate that Vdr and Casr are required for ß-catenin-regulated cell proliferation and AJ formation essential for re-epithelialization after wounding. We conclude that vitamin D and calcium signaling in keratinocytes are required for a normal regenerative response of the skin to wounding.


Assuntos
Reepitelização/genética , Receptores de Calcitriol/genética , Receptores Acoplados a Proteínas-G/genética , Cicatrização/genética , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Queratinócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Detecção de Cálcio , Pele/metabolismo , Pele/fisiopatologia , Fatores de Tempo , beta Catenina/metabolismo
11.
Cytotherapy ; 19(4): 543-554, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28209525

RESUMO

BACKGROUND: Although human stromal vascular fraction (SVF) has been regarded as an attractive stem cell source, its therapeutic mechanism in wound healing has not been fully elucidated. AIMS: In this study, we investigated the molecular characteristics and therapeutic property of SVF for wound healing. METHODS: Microarray data showed that SVF cells are enriched with a higher level of wound healing or epithelium development-related genes and micro RNA. RESULTS: Quantitative polymerase chain reaction (PCR) and reverse transcriptase PCR results revealed that the epithelialization growth factor, epidermal growth factor (EGF), chemokines, stromal cell-derived factor (SDF-1 or CXCL12), neutrophil-activating protein-2 (NAP-2 or CXCL7), chemokine receptors (CXCR1, CCR2 and CCR3) and wound healing genes were up-regulated in SVF compared with those in adipose-derived mesenchymal stem cells (ASCs). An in vitro scratch wound closure experiment demonstrated that co-culture with SVF substantially accelerated the wound closure of fibroblasts. Wounds in nude mice were created by skin excisions followed by injections of SVF with Pluronic hydrogel. SVF implantation highly accelerated wound closure and increased cellularity and re-epithelialization. In addition, the transplanted SVF exhibited high engraftment rates in the wound area, suggesting direct benefits for cutaneous closure. CONCLUSIONS: Taken together, these data suggest that SVF possesses high therapeutic capability for wound healing via the secretion of epithelialization and chemotactic growth factors and enhanced engraftment properties.


Assuntos
Vasos Sanguíneos/citologia , Quimiotaxia , Reepitelização/fisiologia , Células Estromais/fisiologia , Células Estromais/transplante , Animais , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Reepitelização/genética , Células Estromais/citologia , Regulação para Cima
12.
Exp Dermatol ; 26(2): 116-123, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27513293

RESUMO

Wound healing is a complex process which involves proliferation and migration of keratinocyte for closure of epidermal injuries. A member of microRNA family, let-7b, has been expressed in mammalian skin, but its exact role in keratinocyte migration is still not in knowledge. Here, we showed that let-7b regulates keratinocyte migration by targeting the insulin-like growth factor IGF2BP2. Overexpression of let-7b led to reduced HaCaT cell migration, while knockdown of let-7b resulted in enhanced migration. Furthermore, let-7b was decreased during wound healing in wild-type mice, which led us to construct the transgenic mice with overexpression of let-7b in skin. The re-epithelialization of epidermis of let-7b transgenic mice was reduced during wound healing. Using bioinformatics prediction software and a reporter gene assay, we found that IGF2BP2 was a target of let-7b, which contributes to keratinocyte migration. Introduction of an expression vector of IGF2BP2 also rescued let-7b-induced migration deficiency, which confirms that IGF2BP2 is an important target for let-7b regulation. Our findings suggest that let-7b significantly delayed the re-epithelialization possibly due to reduction of keratinocyte migration and restraints IGF2BP2 during skin wound healing.


Assuntos
Movimento Celular/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Reepitelização/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos/fisiologia , Camundongos Transgênicos , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fenômenos Fisiológicos da Pele
13.
Int J Mol Sci ; 17(12)2016 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-27898044

RESUMO

Mice and human patients with impaired vitamin D receptor (VDR) signaling have normal developmental hair growth but display aberrant post-morphogenic hair cycle progression associated with alopecia. In addition, VDR-/- mice exhibit impaired cutaneous wound healing. We undertook experiments to determine whether the stress-inducible regulator of energy homeostasis, DNA damage-inducible transcript 4 (Ddit4), is involved in these processes. By analyzing hair cycle activation in vivo, we show that VDR-/- mice at day 14 exhibit increased Ddit4 expression within follicular stress compartments. At day 29, degenerating VDR-/- follicular keratinocytes, but not bulge stem cells, continue to exhibit an increase in Ddit4 expression. At day 47, when normal follicles and epidermis are quiescent and enriched for Ddit4, VDR-/- skin lacks Ddit4 expression. In a skin wound healing assay, the re-epithelialized epidermis in wildtype (WT) but not VDR-/- animals harbor a population of Ddit4- and Krt10-positive cells. Our study suggests that VDR regulates Ddit4 expression during epidermal homeostasis and the wound healing process, while elevated Ddit4 represents an early growth-arresting stress response within VDR-/- follicles.


Assuntos
Folículo Piloso/metabolismo , Reepitelização/fisiologia , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Animais , Epiderme/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica , Reepitelização/genética , Receptores de Calcitriol/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Estresse Fisiológico , Fatores de Transcrição/genética , Cicatrização/fisiologia
14.
Stem Cell Res Ther ; 7(1): 73, 2016 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-27194135

RESUMO

BACKGROUND: Proliferation and migration of endothelial progenitor cells (EPCs) play important roles in restoring vascular injuries. ß2 adrenergic receptors (ß2ARs) are widely expressed in many tissues and have a beneficial impact on EPCs regulating neoangiogenesis. The aim of the present study was to determine the effect of overexpressing ß2ARs in infused peripheral blood (PB)-derived EPCs on the re-endothelialization in injured vessels. METHODS: Induction of endothelial injury was performed in male nude mice that were subjected to wire-mediated injury to the carotid artery. Human PB-derived EPCs were transfected with an adenovirus serotype 5 vector expressing ß2AR (Ad5/ß2AR-EPCs) and were examined 48 h later. ß2AR gene expression in EPCs was detected by real-time polymerase chain reaction and Western blot analysis. In vitro, the proliferation, migration, adhesion, and nitric oxide production of Ad5/ß2AR-EPCs were measured. Meanwhile, phosphorylated Akt and endothelial nitric oxide synthase (eNOS), which are downstream of ß2AR signaling, were also elevated. In an in vivo study, CM-DiI-labeled EPCs were injected intravenously into mice subjected to carotid injury. After 3 days, cells recruited to the injury sites were detected by fluorescent microscopy, and the re-endothelialization was assessed by Evans blue dye. RESULTS: In vitro, ß2AR overexpression augmented EPC proliferation, migration, and nitric oxide production and enhanced EPC adhesion to endothelial cell monolayers. In vivo, when cell tracking was used, the number of recruited CM-DiI-labeled EPCs was significantly higher in the injured zone in mice transfused with Ad5/ß2AR-EPCs compared with non-transfected EPCs. The degree of re-endothelialization was also higher in the mice transfused with Ad5/ß2AR-EPCs compared with non-transfected EPCs. We also found that the phosphorylation of Akt and eNOS was increased in Ad5/ß2AR-EPCs. Preincubation with ß2AR inhibitor (ICI118,551), Akt inhibitor (ly294002), or eNOS inhibitor (L-NAME) significantly attenuated the enhanced in vitro function and in vivo re-endothelialization capacity of EPCs induced by ß2AR overexpression. CONCLUSIONS: The present study demonstrates that ß2AR overexpression enhances EPC functions in vitro and enhances the vascular repair abilities of EPCs in vivo via the ß2AR/Akt/eNOS pathway. Upregulation of ß2AR gene expression through gene transfer may be a novel therapeutic target for endothelial repair.


Assuntos
Lesões das Artérias Carótidas/genética , Células Progenitoras Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Proteínas Proto-Oncogênicas c-akt/genética , Reepitelização/genética , Receptores Adrenérgicos beta 2/genética , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Adesão Celular , Movimento Celular , Proliferação de Células , Cromonas/farmacologia , Células Progenitoras Endoteliais/citologia , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Injeções Intravenosas , Masculino , Camundongos Nus , Morfolinas/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Propanolaminas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Transfecção
15.
J Invest Dermatol ; 136(5): 1031-1041, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26827763

RESUMO

Transmembrane collagen XVII is traditionally viewed as an important hemidesmosomal attachment component that promotes stable dermal-epidermal adhesion in the skin. However, its expression is highly elevated at the leading edges of cutaneous wounds or invasive carcinomas, suggesting alternative functions in cell migration. The collagenous ectodomain of collagen XVII is constitutively shed from the cell surface by a disintegrin and metalloproteinases, and this shedding is strongly induced during wound healing. Recently, we investigated the physiological relevance of collagen XVII shedding by generating knock-in mice, which exclusively express a functional non-sheddable collagen XVII mutant. Prevention of ectodomain shedding in these mice caused no spontaneous phenotype in resting skin, but accelerated re-epithelialization on skin wounding. Here, we investigated the mechanistic function of shedding during wound healing. Using the non-shedding collagen XVII mice as a model, we uncovered ectodomain shedding as a highly dynamic modulator of in vivo proliferation and motility of activated keratinocytes through tight coordination of α6ß4 integrin-laminin-332 interactions and dampening of mechanistic target of rapamycin signaling at the wound edges. Thus, our studies identify ectodomain shedding of collagen XVII as an interactive platform that translates shedding into a signal for directed cell growth and motility during skin regeneration.


Assuntos
Autoantígenos/metabolismo , Regulação da Expressão Gênica , Queratinócitos/citologia , Colágenos não Fibrilares/metabolismo , Reepitelização/genética , Serina-Treonina Quinases TOR/genética , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Distribuição Aleatória , Transdução de Sinais , Ferimentos e Lesões/genética , Ferimentos e Lesões/patologia
16.
PLoS One ; 11(1): e0146451, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26752054

RESUMO

Wound healing in a pre-existing Th2-dominated skin milieu was assessed by using an epidermal specific interleukin-4 (IL-4) transgenic (Tg) mouse model, which develops a pruritic inflammatory skin condition resembling human atopic dermatitis. Our results demonstrated that IL-4 Tg mice had delayed wound closure and re-epithelialization even though these mice exhibited higher degrees of epithelial cell proliferation. Wounds in IL-4 Tg mice also showed a marked enhancement in expression of inflammatory cytokines/chemokines, elevated infiltration of inflammatory cells including neutrophils, macrophages, CD3+ lymphocytes, and epidermal dendritic T lymphocytes. In addition, these mice exhibited a significantly higher level of angiogenesis as compared to wild type mice. Furthermore, wounds in IL-4 Tg mice presented with larger amounts of granulation tissue, but had less expression and deposition of collagen. Taken together, an inflamed skin condition induced by IL-4 has a pronounced negative influence on the healing process. Understanding more about the pathogenesis of wound healing in a Th2- dominated environment may help investigators explore new potential therapeutic strategies.


Assuntos
Dermatite Atópica/fisiopatologia , Epiderme/metabolismo , Interleucina-4/metabolismo , Animais , Citocinas/metabolismo , Dermatite Atópica/genética , Feminino , Tecido de Granulação/metabolismo , Interleucina-4/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neutrófilos/metabolismo , Reepitelização/genética , Reepitelização/fisiologia , Linfócitos T/metabolismo , Cicatrização/genética , Cicatrização/fisiologia
17.
J Cell Biochem ; 117(4): 1000-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26403263

RESUMO

Deposition of collagen-based extracellular matrix by fibroblasts during wound healing leads to scar formation--a typical outcome of the healing process in soft tissue wounds. The process can, however, be skewed in favor of tissue regeneration by manipulation of wound environment. Low oxygen conditions and supplementation with FGF2 provide extracellular cues that drive wound fibroblasts towards a pro-regenerative phenotype. Under these conditions, fibroblasts dramatically alter expression of many genes among which the most significantly deregulated are extracellular matrix and adhesion molecules. Here we investigate the mechanism of a collagen I binding integrin α11 (ITGA11) deregulation in response to low oxygen-mediated FGF2 effects in dermal fibroblasts. Using RT-PCR, qRT-PCR, Western blotting, and immunocytochemistry, we describe significant down-regulation of ITGA11. Decrease in ITGA11 is associated with its loss from focal adhesions. We show that loss of ITGA11 requires FGF2 induced ERK1/2 activity and in the presence of FGF2, ITGA11 expression cannot be rescued by TGFß1, a potent activator of ITGA11. Our results indicate that FGF2 may be redirecting fibroblasts towards an anti-fibrotic phenotype by overriding TGFß1 mediated ITGA11 expression.


Assuntos
Cicatriz/prevenção & controle , Fatores de Crescimento de Fibroblastos/farmacologia , Fibroblastos/efeitos dos fármacos , Cadeias alfa de Integrinas/genética , Reepitelização/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Hipóxia Celular , Cicatriz/genética , Cicatriz/metabolismo , Cicatriz/patologia , Metilação de DNA/efeitos dos fármacos , Derme/efeitos dos fármacos , Derme/lesões , Derme/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Adesões Focais/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Cadeias alfa de Integrinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oxigênio/farmacologia , Cultura Primária de Células , Reepitelização/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo
18.
Adv Exp Med Biol ; 888: 291-305, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26663189

RESUMO

microRNAs (miRNAs) are small noncoding RNA molecules which play pivotal roles in wound healing. The increased expression of certain genes and expression of some others represent a key component of the wound biology and are largely under the regulation of naturally occurring miRNAs. Understanding the dysregulated miRNAs in chronic wound biology will therefore enable the development of newer therapies. This chapter focuses on the miRNAs that can be potentially targeted for improving skin wound healing and the challenges in miRNA therapy, including considerations in miRNA target identification and delivery.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Pele/metabolismo , Cicatrização/genética , Animais , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Modelos Genéticos , Neovascularização Fisiológica/genética , Reepitelização/genética , Reepitelização/fisiologia , Pele/irrigação sanguínea , Pele/fisiopatologia , Dermatopatias/genética , Dermatopatias/fisiopatologia
19.
PLoS One ; 10(10): e0139787, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26440795

RESUMO

BACKGROUND: Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates. AIM: To examine the role of CD24 in the wound healing process. METHODS: An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA-/-) compared to wild-type (WT) mice. RESULTS: Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA+/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA-/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice. CONCLUSIONS: Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.


Assuntos
Antígeno CD24/genética , Tecido de Granulação/metabolismo , Reepitelização/genética , Cicatrização/genética , Animais , Antígeno CD24/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fatores de Tempo
20.
Exp Dermatol ; 24(10): 785-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26121181

RESUMO

The mucin-like transmembrane protein podoplanin (PDPN) is prominently represented in tumor-associated gene expression signatures of numerous types of cancer including squamous cell carcinoma, and gain-of-function and knockdown approaches in tissue culture strongly suggested an important role of PDPN in cell proliferation, migration and adhesion. PDPN is absent during epidermal homeostasis but is highly expressed in basal keratinocytes during cutaneous wound healing. Enhanced motility of immortalized keratinocytes upon ectopic PDPN overexpression argues for wound healing defects upon podoplanin deficiency in keratinocytes; however, in vivo data that unequivocally define the impact of PDPN by functional studies in a physiologically relevant system are still missing. Here, we have applied an in vivo loss-of-function approach by generating a novel transgenic mouse line with keratinocyte-specific podoplanin deficiency. Performing cutaneous full-thickness excisional wounds to examine re-epithelialization capacity, unexpectedly, no defects were observed in wound healing properties of mutant mice. Similarly, PDPN-deficient primary keratinocytes showed no impairment in migration, adhesion or proliferation. Thus, PDPN function is not rate-limiting for re-epithelialization but may be functionally compensated by an as yet unknown protein. Our data also call for in vivo functional studies on PDPN in settings of skin tumor development and progression to clarify PDPN's role in skin pathology.


Assuntos
Queratinócitos/fisiologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Reepitelização/genética , Pele/lesões , Animais , Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Camundongos , Camundongos Transgênicos
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