RESUMEN
Growing evidence supports the concept that polymorphonuclear neutrophils (PMN) are critically involved in inflammation-mediated angiogenesis which is important for wound healing and repair. We employed an oligonucleotide microarray technique to gain further insight into the molecular mechanisms underlying the proangiogenic potential of human PMN. In addition to 18 known angiogenesis-relevant genes, we detected the expression of 10 novel genes, namely midkine, erb-B2, ets-1, transforming growth factor receptor-beta2 and -beta3, thrombospondin, tissue inhibitor of metalloproteinase 2, ephrin A2, ephrin B2 and restin in human PMN freshly isolated from the circulation. Gene expression was confirmed by the RT-PCR technique. In vivo evidence for the role of PMN in neovascularization was provided by studying neovascularization in a skin model of wound healing using CD18-deficient mice which lack PMN infiltration to sites of lesion. In CD18-deficient animals, neovascularization was found to be significantly compromised when compared with wild-type control animals which showed profound neovascularization within the granulation tissue during the wound healing process. Thus, PMN infiltration seems to facilitate inflammation-mediated angiogenesis which may be a consequence of the broad spectrum of proangiogenic factors expressed by these cells.
Asunto(s)
Antígenos CD18/genética , Neovascularización Fisiológica/fisiología , Neutrófilos/fisiología , Piel/irrigación sanguínea , Piel/lesiones , Adulto , Animales , Ciclooxigenasa 2/genética , Humanos , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Análisis de Secuencia por Matrices de Oligonucleótidos , Activador de Plasminógeno de Tipo Uroquinasa/genética , Cicatrización de Heridas/fisiologíaRESUMEN
We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.
Asunto(s)
Antígenos CD18/genética , Diferenciación Celular/fisiología , Fibroblastos/citología , Síndrome de Deficiencia de Adhesión del Leucocito/fisiopatología , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/fisiología , Actinas/metabolismo , Animales , Western Blotting , Antígenos CD18/metabolismo , Movimiento Celular/fisiología , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Inmunohistoquímica , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/metabolismo , Neutrófilos/fisiología , Fagocitosis/fisiología , Factor de Crecimiento Transformador beta1 , Cicatrización de Heridas/genéticaRESUMEN
Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.
Asunto(s)
Heterocigoto , Nitroglicerina/farmacología , Estrés Oxidativo , Superóxido Dismutasa/fisiología , Aldehído Deshidrogenasa/metabolismo , Animales , Aorta/enzimología , Tolerancia a Medicamentos , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/enzimología , Superóxido Dismutasa/genéticaRESUMEN
To circumvent the early lethality of manganese superoxide dismutase (SOD2)-deficient mice, we have used a skin-specific strategy with introduction of loxP sites flanking exon 3 of the SOD2 gene. To our surprise, when breeding a female keratin 14 Cre transgenic mouse to a SOD2 "floxed" male mouse, due to keratin 14 promoter-driven Cre expression in the oocytes, all offspring were heterozygous for SOD2. In sharp contrast to initial publications on SOD2(+/-) mice, the herein reported mice on a mixed genetic background (C57BL/6 x 129/Ola) in their heterozygous state (SOD(+/-)) revealed distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation, and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo. Strikingly, and so far unreported, we found a substantial decrease in the activity of the cytosolic copper, zinc superoxide dismutase (SOD1) in the heart tissue of SOD2(+/-) mice, suggesting that the breakdown of mitochondrial membranes in the heart of SOD2(+/-) mice results in the enhanced release of superoxide anion radicals or derivatives thereof with subsequent inactivation of cytosolic SOD1. This model may be particularly suited to long-term studies on age-related heart failure as well as other age-related diseases and the polygenic base of tissue-specific responses to oxidative injury.
Asunto(s)
Apoptosis , Heterocigoto , Miocardio/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/fisiología , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Embrión de Mamíferos/citología , Femenino , Vectores Genéticos , Glutatión Peroxidasa/metabolismo , Inmunohistoquímica , Integrasas/metabolismo , Peroxidación de Lípido , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Genéticos , Miocardio/metabolismo , Oocitos/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN/metabolismo , ARN Mensajero/metabolismo , Recombinación Genética , Ribonucleasas/metabolismo , Transducción de Señal , Células Madre/citología , Superóxido Dismutasa/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismoRESUMEN
We recently identified the gene encoding the activin betaA chain as a novel injury-regulated gene. We showed that activin over-expression in the skin of transgenic mice enhances the speed of wound healing but also the scarring response. By contrast, inhibition of activin action by over-expression of the activin antagonist follistatin caused a severe delay in wound repair, but the quality of the healed wound was improved. In a search for activin-regulated genes in keratinocytes we identified the Mad1 transcription factor as a direct target of activin in these cells. Since Mad1 inhibits proliferation and induces differentiation of various cell types, our results suggest that activin regulates these processes in keratinocytes via induction of mad1. In addition to its role in the skin, we recently identified activin as a novel neuroprotective factor in vivo. Together with results from other laboratories, these findings suggest that activin is an important player in inflammation, repair and cytoprotection in various organs.
Asunto(s)
Activinas/fisiología , Activinas/antagonistas & inhibidores , Activinas/genética , Activinas/farmacología , Animales , Fibrosis , Folistatina/genética , Folistatina/farmacología , Humanos , Fármacos Neuroprotectores , Transfección , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) exhibits high specific activity in reducing phosphatidylcholine hydroperoxides (PCOOHs) and thus may play a central role in protecting the skin against UV irradiation-triggered detrimental long term effects like cancer formation and premature skin aging. Here we addressed the role of PHGPx in the protection against UV irradiation-induced expression of matrix metalloproteinase-1 (MMP-1). For this purpose, we created human dermal fibroblast cell lines overexpressing human PHGPx. Overexpression led to a significant increase in PHGPx activity. In contrast to a maximal 4.5-fold induction of specific MMP-1 mRNA levels in vector-transfected cells at 24 h after UVA irradiation, no MMP-1 induction occurred at any studied time point after UVA treatment of PHGPx-overexpressing fibroblasts. As interleukin-6 (IL-6) was earlier shown to mediate the UVA induction of MMP-1, we studied whether PHGPx overexpression might interfere with the NFkappaB-mediated IL-6 induction and downstream signaling. Using transient transfections of IL-6 promoter constructs containing NFkappaB binding sites, we observed a high induction of the reporter gene luciferase in vector-transfected control cells and a significantly lower induction in PHGPx-overexpressing fibroblasts following UVA irradiation. Consistently both UVA irradiation and treatment of fibroblasts with PCOOHs led to phosphorylation and nuclear translocation of the p65 subunit, whereas cells overexpressing PHGPx exhibited impaired NFkappaB activation, p65 phosphorylation, and nuclear translocation. In line with this, the PHGPx-overexpressing fibroblasts showed a reduced constitutive and UVA irradiation-induced IL-6 release. After incubating PHGPx-overexpressing cells with PCOOHs a reduced induction of IL-6 was observed. This together with the suppression of UVA irradiation-induced IL-6 release in the presence of Trolox, a chain breaker of PCOOH-initiated lipid peroxidation, indicates that UVA irradiation-induced PCOOHs and subsequent lipid peroxides initiate the NFkappaB-mediated induction of IL-6, which mediates the induction of MMP-1. Our finding is particularly relevant in light of the already available small molecule mimetics of PHGPx.