RESUMEN
Aim: Pulsed current (PC) electric field (EF) devices promote healing in chronic wounds but the underpinning mechanisms are largely unknown. The gap between clinical evidence and mechanistic understanding limits device uptake in clinics. Materials & methods: Migration, proliferation and gene/protein expression profiles were investigated in the presence/absence of PCEF, in skin: keratinocytes (NHK); dermal fibroblasts (HDF); dermal microvascular endothelial cells (HDMEC) and macrophages (THP-1). Results: While PCEF had little effect on migration or proliferation, it significantly altered the expression of 31 genes and the secretion of 7 pro-angiogenic and pro-regenerative growth factors using ELISAs. Conclusion: PCEF significantly altered skin cell genomes/proteomes which provides some evidence of how PCEF devices promote healing of chronic wounds.
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Conductividad Eléctrica , Regulación de la Expresión Génica , Fenotipo , Proteoma/metabolismo , Piel/metabolismo , Cicatrización de Heridas , Movimiento Celular , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Piel/citologíaRESUMEN
Repair to damaged tissue requires directional cell migration to heal the wound. Immediately upon wounding an electrical guidance cue is created with the cathode of the electric field (EF) located at the center of the wound. Previous research has demonstrated directional migration of keratinocytes toward the cathode when an EF of physiological strength (100-150 mV/mm) is applied in vitro, but the "sensor" by which keratinocytes sense the EF remains elusive. Here we use a customized chamber design to facilitate the application of a direct current (DC) EF of physiological strength (100 mV/mm) to keratinocytes whilst pharmacologically modulating the activation of both connexin hemichannels and purinergic receptors to determine their role in EF-mediated directional keratinocyte migration, galvanotaxis. In addition, keratinocytes were exposed to DiSCAC2 (3) dye to visualize membrane potential changes within the cell upon exposure to the applied DC EF. Here we unveil ATP-medicated mechanisms that underpin the initiation of keratinocyte galvanotaxis. The application of a DC EF of 100 mV/mm releases ATP via hemichannels activating a subset of purinergic P2 Y receptors, locally, to initiate the directional migration of keratinocytes toward the cathode in vitro, the center of the wound in vivo. The delineation of the mechanisms underpinning galvanotaxis extends our understanding of this endogenous cue and will facilitate the optimization and wider use of EF devices for chronic wound treatment. J. Cell. Physiol. 230: 181-191, 2016. © 2015 Wiley Periodicals, Inc.
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Adenosina Trifosfato/metabolismo , Queratinocitos/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Transducción de Señal , Piel/citología , Cicatrización de Heridas/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Humanos , Transducción de Señal/fisiologíaRESUMEN
Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.
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Estimulación Eléctrica/métodos , Neovascularización Fisiológica/fisiología , Piel/fisiopatología , Cicatrización de Heridas/fisiología , Adulto , Femenino , Voluntarios Sanos , Humanos , Técnicas In Vitro , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.
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Movimiento Celular/fisiología , Colágeno/química , Neutrófilos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Movimiento Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Geles/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-8/farmacología , Masculino , Neutrófilos/citología , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
During wound healing, excessive inflammation, angiogenesis, and differentiated human dermal fibroblast (HDF ) function contribute to scarring, whereas hyperpigmentation negatively affects scar quality. Over 100 million patients heal with a scar every year. To investigate the role of the beta 2 adrenergic receptor (ß2AR) in wound scarring, the ability of beta 2 adrenergic receptor agonist (ß2ARag) to alter HDF differentiation and function, wound inflammation, angiogenesis, and wound scarring was explored in HDFs, zebrafish, chick chorioallantoic membrane assay (CAM), and a porcine skin wound model, respectively. Here we identify a ß2AR-mediated mechanism for scar reduction. ß2ARag significantly reduced HDF differentiation, via multiple cAMP and/or fibroblast growth factor 2 or basic FGF (FGF2)-dependent mechanisms, in the presence of transforming growth factor betaß1, reduced contractile function, and inhibited mRNA expression of a number of profibrotic markers. ß2ARag also reduced inflammation and angiogenesis in zebrafish and CAMs in vivo, respectively. In Red Duroc pig full-thickness wounds, ß2ARag reduced both scar area and hyperpigmentation by almost 50% and significantly improved scar quality. Indeed, mechanisms delineated in vitro and in other in vivo models were evident in the ß2ARag-treated porcine scars in vivo. Both macrophage infiltration and angiogenesis were initially decreased, whereas DF function was impaired in the ß2ARag-treated porcine wound bed. These data collectively reveal the potential of ß2ARag to improve skin scarring.
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Cicatriz/fisiopatología , Receptores Adrenérgicos beta 2/metabolismo , Cicatrización de Heridas/fisiología , Proteínas de Pez Cebra/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Membrana Corioalantoides/metabolismo , Dermis/citología , Dermis/lesiones , Modelos Animales de Enfermedad , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Adhesiones Focales/patología , Humanos , Neovascularización Fisiológica/fisiología , Sus scrofa , Pez CebraRESUMEN
Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (ß-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning ß-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. ß-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated ß-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that ß-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible ß-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, ß-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. ß-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, ß-AR agonists could be promising anti-angiogenic modulators in skin.
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AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Neovascularización Patológica/metabolismo , Receptores Adrenérgicos beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular , Células Cultivadas , Células Endoteliales/citología , Humanos , Ratones , Piel/citología , Piel/metabolismoRESUMEN
Both human ether-à-go-go-related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2-3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1-2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrate-independent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers.
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Transformación Celular Neoplásica/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Actinas/metabolismo , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Fibroblastos/efectos de los fármacos , Adhesiones Focales/metabolismo , Humanos , Ratones , Ratones Desnudos , Células 3T3 NIH , Fibras de Estrés/metabolismo , TransfecciónRESUMEN
Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re-epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane-bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage-gated channel model.
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Movimiento Celular/fisiología , Fenómenos Electromagnéticos , Queratinocitos/fisiología , Cicatrización de Heridas/fisiología , Animales , Fenómenos Biomecánicos , Cricetinae , Electricidad , Glicocálix/metabolismo , Humanos , Recién Nacido , Masculino , Modelos Biológicos , Transducción de SeñalRESUMEN
Skin wound healing is a complex process requiring the coordinated, temporal orchestration of numerous cell types and biological processes to regenerate damaged tissue. Previous work has demonstrated that a functional ß-adrenergic receptor autocrine/paracrine network exists in skin, but the role of ß2-adrenergic receptor (ß2AR) in wound healing is unknown. A range of in vitro (single-cell migration, immunoblotting, ELISA, enzyme immunoassay), ex vivo (rat aortic ring assay), and in vivo (chick chorioallantoic membrane assay, zebrafish, murine wild-type, and ß2AR knockout excisional skin wound models) models were used to demonstrate that blockade or loss of ß2AR gene deletion promoted wound repair, a finding that is, to our knowledge, previously unreported. Compared with vehicle-only controls, ß2AR antagonism increased angiogenesis, dermal fibroblast function, and re-epithelialization, but had no effect on wound inflammation in vivo. Skin wounds in ß2AR knockout mice contracted and re-epithelialized faster in the first few days of wound repair in vivo. ß2AR antagonism enhanced cell motility through distinct intracellular signalling mechanisms and increased vascular endothelial growth factor secretion from keratinocytes. ß2AR antagonism promoted wound repair processes in the early stages of wound repair, revealing a possible new avenue for therapeutic intervention.
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Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Eliminación de Gen , Receptores Adrenérgicos beta 2/genética , Piel/patología , Animales , Aorta/patología , Embrión de Pollo , Ensayo de Inmunoadsorción Enzimática/métodos , Fibroblastos/metabolismo , Inflamación , Queratinocitos/citología , Ratones , Neovascularización Patológica , Ratas , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Cicatrización de Heridas , Pez CebraRESUMEN
Cytoskeletal proteins of the tensin family couple integrins to the actin cytoskeleton. They are found in both focal adhesions and the fibrillar adhesions formed between cells and the fibronectin matrix. There are four tensin genes which encode three large (approximately 200 kDa) tensin isoforms (tensin 1, 2, 3) and one short isoform (cten). However, the subcellular localization and function of the individual isoforms is poorly understood. Using human foreskin fibroblasts (HFFs), and imaging on both fixed and live cells, we show that GFP-tensin 2 is enriched in dynamic focal adhesions at the leading edge of the cell, whereas GFP-tensin 3 translocates rearward, and is enriched in fibrillar adhesions. To investigate the possible role of tensins in cell-matrix remodeling, we used siRNAs to knockdown each tensin isoform. We discovered that tensin 2 knockdown significantly reduced the ability of HFFs to contract 3D collagen gels, whilst no effect on fibronectin fibrillogenesis was observed. This inhibition of collagen gel contraction was associated with a substantial reduction in Rho activity, and it was reversed by depletion of DLC1, a RhoGAP that binds to tensin in focal adhesions. These findings suggest that focal adhesion-localized tensin 2 negatively regulates DLC1 to permit Rho-mediated actomyosin contraction and remodeling of collagen fibers.
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Adhesión Celular , Fibroblastos/citología , Proteínas Activadoras de GTPasa/genética , Proteínas de Microfilamentos/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Supresoras de Tumor/genética , Actomiosina/metabolismo , Células Cultivadas , Colágeno/metabolismo , Citoesqueleto/metabolismo , Adhesiones Focales/química , Geles , Humanos , Proteínas de Microfilamentos/análisis , Movimiento , Monoéster Fosfórico Hidrolasas/análisis , ARN Interferente Pequeño/farmacología , Tensinas , Regulación hacia Arriba/genéticaRESUMEN
Conformationally constrained mimetics of the laminin cell-adhesion site, YIGSR, are described. The site is the natural antagonist of the integrin-associated laminin receptor 1 (LAMR1) known to mediate metastatic tumor adhesion. The attachment of selected metastatic cell lines toward the constrained antagonists has been assessed. Observed differential responses prompted by folding preferences of the mimetics revealed stronger attachment activities for turnlike structures. The results permit the conformational design of antimetastatic disintegrins.
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Antineoplásicos/química , Desintegrinas/química , Laminina/química , Oligopéptidos/química , Péptidos Cíclicos/química , Animales , Antineoplásicos/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Línea Celular Tumoral , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Células HeLa , Humanos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Conformación Proteica , Receptores de Laminina/antagonistas & inhibidores , Proteínas RibosómicasRESUMEN
BACKGROUND: Stress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the beta2-adrenergic receptor (beta2AR), another study objective was to determine whether beta2AR antagonists could block epinephrine effects on healing and improve wound repair. METHODS AND FINDINGS: Migratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the beta2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver beta2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%-95% in humans, p < 0.001, and by 36%, 95% CI 24%-49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%-36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by beta2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the beta2AR, and is reproduced by incubation of keratinocytes with other beta2AR-specific agonists. Activation of the beta2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein +/- standard error of the mean: unburned control, 0.6 +/- 0.36; immediately postburn, 9.6 +/- 1.58; 2 h postburn, 3.1 +/- 1.08; 24 h post-burn, 6.7 +/- 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with betaAR antagonists results in a significant increase (44%, 95% CI 27%-61%, p < 0.00000001) in the rate of burn wound re-epithelialization. CONCLUSIONS: This work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte beta2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific beta2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.
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Antagonistas Adrenérgicos beta/farmacología , Epinefrina/antagonistas & inhibidores , Epinefrina/metabolismo , Piel/metabolismo , Estrés Fisiológico , Cicatrización de Heridas/fisiología , Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Humanos , Inmunohistoquímica , Queratinocitos/citología , Queratinocitos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Piel/citología , Piel/lesionesRESUMEN
Adrenergic receptors and their downstream effector molecules are expressed in all cell types in the skin, and it is only recently that functionality of the catecholamine agonist activated signaling in the cutaneous repair process has been revealed. In addition to responding to systemic elevations in catecholamines (as in stress situations) or to pharmacologically administered adrenergic agonists, epidermal keratinocytes themselves can synthesize catecholamine ligands. They also respond to these systemic or self-generated agonists via receptor mediated signaling, resulting in altered migration, and changes in wound re-epithelialization. Endothelial cells, inflammatory cells, dermal fibroblasts, and mesenchymal stem cells, all cells that contribute to the wound repair process, express multiple subtypes of adrenergic receptors and exhibit responses that can be either contribute or impair healing-and occasionally, depending on the species and assay conditions, results can be conflicting. There is still much to be uncovered regarding how this self-contained autocrine and paracrine signaling system contributes to cutaneous wound repair.
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Receptores Adrenérgicos beta 2/fisiología , Cicatrización de Heridas/fisiología , Animales , Lesiones de la Cornea , Epitelio/fisiología , Fibroblastos/fisiología , Humanos , Inflamación/fisiopatología , Células Madre Mesenquimatosas/fisiología , Neovascularización Fisiológica , Piel/irrigación sanguíneaRESUMEN
PURPOSE: Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting RESULTS: With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing. METHODS: Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). CONCLUSIONS: The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair.
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Epitelio Corneal/fisiología , Receptores Adrenérgicos beta 2/fisiología , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Movimiento Celular , Epinefrina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Immunoblotting , Isoproterenol/farmacología , Masculino , Ratones , Ratones Noqueados , Fosforilación , Timolol/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Wound healing is a complex and well-orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of beta2-adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. Beta-adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A-mediated mechanism and decrease both electric field-directed migration and corneal wound healing. Conversely, beta-adrenergic receptor antagonists accelerate CEC migration, enhance electric field-mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (beta-adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro-motogenic effect of the beta-adrenergic antagonist is blockade of the beta2-adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs.
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Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Células Epiteliales/efectos de los fármacos , Epitelio Corneal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Adulto , Animales , Bovinos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Electrodos , Epinefrina/biosíntesis , Células Epiteliales/citología , Células Epiteliales/enzimología , Epitelio Corneal/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2RESUMEN
Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (beta)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), beta4 integrin-null human keratinocytes (beta4-), or those in which beta4 integrin was reexpressed (beta4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of beta4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding-defective beta4 (beta4+AD) or beta4 with a truncated cytoplasmic tail (beta4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the beta4 integrin ligand-binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Conductividad Eléctrica , Factor de Crecimiento Epidérmico/farmacología , Integrina beta4/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Células Cultivadas , Citoplasma/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Humanos , Integrina beta4/química , Queratinocitos/efectos de los fármacos , Laminina/metabolismo , Modelos Biológicos , Transporte de Proteínas/efectos de los fármacos , Seudópodos/efectos de los fármacosRESUMEN
The skin is our primary defense against noxious environmental agents. Upon injury, keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, essential for wound repair and restoration of barrier integrity. Keratinocytes express a high level of beta2-adrenergic receptors (beta2-ARs) that appear to play a role in cutaneous homeostasis as aberrations in either keratinocyte beta2-AR function or density are associated with various skin diseases. Here we report the novel finding that beta-AR antagonists promote wound re-epithelialization in a "chronic" human skin wound-healing model. beta-AR antagonists increase ERK phosphorylation, the rate of keratinocyte migration, electric field-directed migration, and ultimately accelerate human skin wound re-epithelialization. We demonstrate that keratinocytes express two key enzymes required for catecholamine (beta-AR agonist) synthesis, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase, both localized within keratinocyte cytoplasmic vesicles. Finally, we confirm the synthesis of epinephrine by measuring the endogenously synthesized catecholamine in keratinocyte extracts. Previously, we have demonstrated that beta-AR agonists delay wound re-epithelialization. Here we report that the mechanism for the beta-AR antagonist-mediated augmentation of wound repair is due to beta2-AR blockade, preventing the binding of endogenously synthesized epinephrine. Our work describes an endogenous beta-AR mediator network in the skin that can temporally regulate skin wound repair. Further investigation of this network will improve our understanding of both the skin repair process and the multiple modes of action of one of the most frequently prescribed class of drugs, hopefully resulting in a new treatment for chronic wounds.
Asunto(s)
Antagonistas Adrenérgicos beta/química , Epidermis/patología , Receptores Adrenérgicos beta/fisiología , Cicatrización de Heridas , Antagonistas Adrenérgicos beta/metabolismo , Animales , Proliferación Celular , Citoesqueleto/metabolismo , Epidermis/metabolismo , Epinefrina/farmacología , Humanos , Immunoblotting , Queratinocitos/citología , Queratinocitos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Dermal fibroblasts are required for skin wound repair; they migrate into the wound bed, proliferate, synthesize extracellular matrix components and contract the wound. Although fibroblasts express beta2-adrenergic receptors (beta2-AR) and cutaneous keratinocytes can synthesize beta-AR agonists (catecholamines), the functional significance of this hormonal mediator network in the skin has not been addressed. Emerging studies from our laboratory demonstrate that beta2-AR activation modulates keratinocyte migration, essential for wound re-epithelialization. Here we describe an investigation of the effects of beta2-AR activation on the dermal component of wound healing. We examined beta2-AR-mediated regulation of biological processes in dermal fibroblasts that are critical for wound repair: migration, proliferation, contractile ability and cytoskeletal conformation. We provide evidence for the activation of at least two divergent beta2-AR-mediated signaling pathways in dermal fibroblasts, a Src-dependent pro-migratory pathway, transduced through the epidermal growth factor receptor and extracellular signal-regulated kinase, and a PKA-dependent pro-proliferative pathway. beta2-AR activation attenuates collagen gel contraction and alters the actin cytoskeleton and focal adhesion distribution through PKA-dependent mechanisms. Our work uncovers a previously unrecognized role for the adrenergic hormonal mediator network in the cutaneous wound repair process. Exploiting these divergent beta2-AR agonist responses in cutaneous cells may generate novel therapeutic approaches for the control of wound healing.
Asunto(s)
Movimiento Celular/fisiología , Dermis/fisiología , Fibroblastos/fisiología , Receptores Adrenérgicos beta 2/metabolismo , Cicatrización de Heridas/fisiología , Actinas/metabolismo , Agonistas Adrenérgicos/farmacología , Agonistas de Receptores Adrenérgicos beta 2 , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dermis/citología , Fibroblastos/citología , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/fisiología , Humanos , Transducción de Señal , Cicatrización de Heridas/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, necessary for wound closure and restoration of barrier function. They solely express the beta2-adrenergic receptor (beta2-AR) subtype of beta-ARs and can also synthesize beta-AR agonists generating a hormonal mediator network in the skin. Emerging studies from our laboratory demonstrate that beta-AR agonists decrease keratinocyte migration via a protein phosphatase (PP) 2A-dependent mechanism. Here we have extended our investigations to observe the effects of beta2-AR activation on keratinocyte polarization, migration, and ERK phosphorylation at the wound edge, cytoskeletal organization, phospho-ERK intracellular localization, proliferation, human skin wound re-epithelialization, wound-induced ERK phosphorylation, and murine skin wound healing. We demonstrate that in keratinocytes, beta2-AR activation is anti-motogenic and anti-mitogenic with both mechanisms being PP2A dependent. beta2-AR activation dramatically alters the organization of the actin cytoskeleton and prevents localization of phospho-ERK to the lamellipodial edge and its colocalization with vinculin. Finally, we demonstrate a beta2-AR-mediated delay in re-epithelialization and decrease in wound-induced epidermal ERK phosphorylation in human skin wounds and a delay in re-epithelialization in murine tail-clip wounds. Our work uncovers novel keratinocyte biology and a previously unrecognized role for the adrenergic hormonal mediator network in the wound repair process.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2 , Receptores Adrenérgicos beta 2/metabolismo , Cicatrización de Heridas/fisiología , Antagonistas de Receptores Adrenérgicos beta 2 , Animales , Adhesión Celular , Proliferación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ácido Ocadaico/farmacología , Fosforilación , Transporte de Proteínas , Seudópodos/metabolismo , Piel , Vinculina/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/metabolismoRESUMEN
Dermal fibroblasts actively contribute to wound healing by migrating to the wound, synthesizing extracellular matrices, and generating mechanical forces within the wound to initiate wound contraction. Fibroblast-seeded collagen gels provide an in vitro model to study wound contraction. The authors are evaluating the role of the adrenergic signaling system in cutaneous wound repair and recently found that beta2-adrenergic receptor (beta2-AR) activation markedly decreases keratinocyte migration, an essential step in wound reepithelialization. Because the beta2-ARs are also expressed on dermal fibroblasts, a study was initiated to determine the effects of beta-adrenergic agonists on dermal fibroblast-mediated collagen gel contraction. A beta-agonist (isoproterenol) delayed gel contraction in a dose-dependent manner. A beta2-AR specific antagonist (ICI 118,551) prevented the delay, indicating that the beta2-AR alone mediated the delay. The active cyclic adenosine monophosphate (cAMP) analog also delayed collagen gel contraction, whereas an inactive cAMP analog partially prevented the delay, suggesting that the mechanism for beta-AR agonist-mediated delay was partly cAMP-dependent. Identifying and characterizing agents that modulate wound contraction improves understanding of the wound healing process and could result in novel therapeutic strategies for preventing unwanted wound contraction in burn and trauma patients.