RESUMO
Dysfunctional inflammatory pathways are associated with an increased risk of cancer, including colorectal cancer. We have previously identified and enriched for a self-renewing, colon cancer stem cell (CCSC) subpopulation in primary sporadic colorectal cancers (CRC) and a related subpopulation in ulcerative colitis (UC) patients defined by the stem cell marker, aldehyde dehydrogenase (ALDH). Subsequent work demonstrated that CCSC-initiated tumors are dependent on the inflammatory chemokine, CXCL8, a known inducer of tumor proliferation, angiogenesis and invasion. Here, we use RNA interference to target CXCL8 and its receptor, CXCR1, to establish the existence of a functional signaling pathway promoting tumor growth initiated by sporadic and colitis CCSCs. Knocking down either CXCL8 or CXCR1 had a dramatic effect on inhibiting both in vitro proliferation and angiogenesis. Likewise, tumorigenicity was significantly inhibited due to reduced levels of proliferation and angiogenesis. Decreased expression of cycle cell regulators cyclins D1 and B1 along with increased p21 levels suggested that the reduction in tumor growth is due to dysregulation of cell cycle progression. Therapeutically targeting the CXCL8-CXCR1 signaling pathway has the potential to block sustained tumorigenesis by inhibiting both CCSC- and pCCSC-induced proliferation and angiogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/etiologia , Neoplasias do Colo/metabolismo , Inflamação/metabolismo , Interleucina-8/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores de Interleucina-8A/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Colite/complicações , Colite/genética , Colite/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Dosagem de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imunofenotipagem , Inflamação/complicações , Inflamação/genética , Interleucina-8/genética , Camundongos , Modelos Biológicos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Receptores de Interleucina-8A/genéticaRESUMO
Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.
Assuntos
Antineoplásicos/farmacologia , Apolipoproteína A-I/metabolismo , Cardiotônicos/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Apolipoproteína A-I/deficiência , Apolipoproteína A-I/farmacologia , Apolipoproteína A-II/farmacologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Humanos , Imunidade/efeitos dos fármacos , Imunocompetência/efeitos dos fármacos , Lipoproteínas HDL/metabolismo , Lisofosfolipídeos/sangue , Lisofosfolipídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Indução de Remissão , Análise de Sobrevida , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Skin cells produce soluble factors which influence keratinocyte proliferation, angiogenesis, nerve innervation and immunocyte response. OBJECTIVE: To test the hypothesis that epidermal-dermal interactions influence neural outgrowth, vascular survival, immunocyte recruitment and keratinocyte proliferation. METHODS: We genetically manipulated the epidermis to express excess vascular endothelial growth factor (VEGF) and/or angiopoietin-1 (Ang1) and then examined the epidermal and dermal phenotypes. We compared these findings with those occurring following overexpression of the Ang1 receptor Tie2 in endothelial cells or keratinocytes. RESULTS: Keratinocyte-overexpression of Ang1 resulted in increased epidermal thickness compared to control littermates. Keratinocyte-specific overexpression of Ang1 or VEGF increased dermal angiogenesis compared to control animals and combined Ang1-VEGF lead to further increases. Cutaneous leukocyte examination revealed increases in CD4(+) T cell infiltration in mice with keratinocyte-specific overexpression of Ang1, VEGF and Ang1-VEGF combined; in contrast only keratinocyte-specific Ang1 overexpression increased cutaneous F4/80(+) macrophage numbers. Interestingly, combined keratinocyte-derived Ang1-VEGF overexpression reduced significantly the number of F4/80(+) and Cd11c(+) cells compared to mice overexpressing epidermal Ang1 alone. Endothelial cell-specific Tie2 overexpression increased dermal angiogenesis but failed to influence the epidermal and immune cell phenotypes. Keratinocyte-specific Tie2 expressing mice had the highest levels of CD4(+), CD8(+) and CD11c(+) cell numbers and acanthosis compared to all animals. Finally, increases in the number of cutaneous nerves were found in all transgenic mice compared to littermate controls. CONCLUSION: These findings demonstrate that change to one system (vascular or epidermal) results in change to other cutaneous systems and suggest that individual molecules can exert effects on multiple systems.
Assuntos
Angiopoietina-1/fisiologia , Células Epiteliais/metabolismo , Queratinócitos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Pele , Fator A de Crescimento do Endotélio Vascular/fisiologia , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos , Células Epiteliais/imunologia , Queratinócitos/imunologia , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Receptor TIE-2 , Pele/irrigação sanguínea , Pele/imunologia , Pele/inervação , Pele/patologia , Fenômenos Fisiológicos da Pele/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Recent studies on the pathogenesis of diabetic retinopathy have focused on correcting adverse biochemical alterations, but there have been fewer efforts to enhance prosurvival pathways. Bcl-2 is the archetypal member of a group of antiapoptotic proteins. In this study, we investigated the ability of overexpressing Bcl-2 in vascular endothelium to protect against early stages of diabetic retinopathy. Transgenic mice overexpressing Bcl-2 regulated by the pre-proendothelin promoter were generated, resulting in increased endothelial Bcl-2. Diabetes was induced with streptozotocin, and mice were sacrificed at 2 months of study to measure superoxide generation, leukostasis, and immunohistochemistry, and at 7 months to assess retinal histopathology. Diabetes of 2 months duration caused a significant decrease in expression of Bcl-2 in retina, upregulation of Bax in whole retina and isolated retinal microvessels, and increased generation of retinal superoxide and leukostasis. Seven months of diabetes caused a significant increase in the number of degenerate (acellular) capillaries in diabetic animals. Furthermore, overexpression of Bcl-2 in the vascular endothelium inhibited the diabetes-induced degeneration of retinal capillaries and aberrant superoxide generation, but had no effect on Bax expression or leukostasis. Therefore, overexpression of Bcl-2 in endothelial cells inhibits the capillary degeneration that is characteristic of the early stages of diabetic retinopathy, and this effect seems likely to involve inhibition of oxidative stress.
Assuntos
Capilares/metabolismo , Retinopatia Diabética/metabolismo , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose , Endotélio Vascular/embriologia , Imuno-Histoquímica/métodos , Leucostasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcirculação , Estresse Oxidativo , Superóxidos/metabolismoRESUMO
Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4(+) T cells, infiltrating epidermal CD8(+) T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-gamma, tumor necrosis factor-alpha, and interleukins 1alpha, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, beta-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis.
Assuntos
Modelos Animais de Doenças , Células Endoteliais/metabolismo , Queratinócitos/metabolismo , Psoríase/genética , Receptor TIE-2/genética , Animais , Western Blotting , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Psoríase/metabolismo , Psoríase/patologia , Receptor TIE-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/irrigação sanguínea , Pele/metabolismo , Pele/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
OBJECTIVE: Complement mediated injury of the neuromuscular junction is considered a primary disease mechanism in human myasthenia gravis and animal models of experimentally acquired myasthenia gravis (EAMG). We utilized active and passive models of EAMG to investigate the efficacy of a novel C5 complement inhibitor rEV576, recombinantly produced protein derived from tick saliva, in moderating disease severity. METHODS: Standardized disease severity assessment, serum complement hemolytic activity, serum cytotoxicity, acetylcholine receptor (AChR) antibody concentration, IgG subclassification, and C9 deposition at the neuromuscular junction were used to assess the effect of complement inhibition on EAMG induced by administration of AChR antibody or immunization with purified AChR. RESULTS: Administration of rEV576 in passive transfer EAMG limited disease severity as evidenced by 100% survival rate and a low disease severity score. In active EAMG, rats with severe and mild EAMG were protected from worsening of disease and had limited weight loss. Serum complement activity (CH(50)) in severe and mild EAMG was reduced to undetectable levels during treatment, and C9 deposition at the neuromuscular junction was reduced. Treatment with rEV576 resulted in reduction of toxicity of serum from severe and mild EAMG rats. Levels of total AChR IgG, and IgG(2a) antibodies were similar, but unexpectedly, the concentration of complement fixing IgG(1) antibodies was lower in a group of rEV576-treated animals, suggesting an effect of rEV576 on cellular immunity. INTERPRETATION: Inhibition of complement significantly reduced weakness in two models of EAMG. C5 inhibition could prove to be of significant therapeutic value in human myasthenia gravis.
Assuntos
Complemento C5/antagonistas & inibidores , Proteínas Inativadoras do Complemento/uso terapêutico , Miastenia Gravis Autoimune Experimental/prevenção & controle , Animais , Anticorpos/efeitos adversos , Proteínas de Artrópodes , Linhagem Celular Tumoral , Complemento C9/metabolismo , Proteínas Inativadoras do Complemento/imunologia , Proteínas Inativadoras do Complemento/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Proteínas de Insetos/imunologia , Proteínas de Insetos/uso terapêutico , Força Muscular/efeitos dos fármacos , Miastenia Gravis Autoimune Experimental/etiologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/imunologia , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Proteínas e Peptídeos Salivares/sangue , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/uso terapêutico , Índice de Gravidade de Doença , Fatores de Tempo , Redução de Peso/efeitos dos fármacosRESUMO
PURPOSE: Amblyopia is the most common cause of visual impairment in children. Early detection of amblyopia and subsequent intervention are vital in preventing visual loss. Understanding the molecular pathogenesis of amblyopia would greatly facilitate development of therapeutic interventions. An animal model of amblyopia induced by monocular vision deprivation has been extensively studied in terms of anatomic and physiologic alterations that affect visual pathways. However, the molecular events underlying these changes are poorly understood. This study aimed to characterize changes of gene expression profiles in the lateral geniculate nucleus (LGN) associated with amblyopia induced by monocular visual deprivation. METHODS: Monocular vision deprivation was generated by either opaque dark contact lens or tarsorrhaphy of newborn rhesus monkeys. LGN was harvested at two or four months following induction of vision deprivation. Laser capture microdissection was used to obtain individual LGN layers for total RNA isolation. Linear T7-based in vitro RNA amplification was used to obtain sufficient RNA to conduct DNA microarray studies. The resulting Affymetrix GeneChip Expression data were analyzed using Affymetrix GeneChip Operating Software. Real-time quantitative polymerase chain reaction and in situ hybridization were used to further analyze expression of selected genes. RESULTS: Using 52,699 microarray probe sets from a Rhesus array, we identified 116 transcripts differentially expressed between deprived and nondeprived parvocellular layers: 45 genes were downregulated and 71 genes were upregulated in deprived parvocellular layers. We also observed substantial changes in deprived magnocellular laminae: 74 transcripts exhibited altered expression, 42 genes were downregulated, and 32 genes were upregulated. The genes identified in this study are involved in many diverse processes, including binding (calcium ion binding, nucleic acid binding, and nucleotide binding), catalytic activity, and signal transducer activity. CONCLUSIONS: There were significant differences in gene expression profiles between deprived and nondeprived parvocellular layers and magnocellular laminae of LGN. These alterations in gene expression may play a critical role in the molecular pathogenesis of amblyopia. The genes identified in this study may provide potential targets for therapeutic intervention of this disease.
Assuntos
Ambliopia/genética , Perfilação da Expressão Gênica , Corpos Geniculados/metabolismo , Corpos Geniculados/patologia , Lasers , Microdissecção , Visão Monocular/genética , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Regulação para Baixo/genética , Hibridização In Situ , Macaca , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genéticaRESUMO
It has been previously reported that aspirin inhibited the development of diabetic retinopathy in diabetic animals, raising the possibility that anti-inflammatory drugs may have beneficial effects on diabetic retinopathy. To further explore this, we compared effects of oral consumption of three different salicylate-based drugs (aspirin, sodium salicylate, and sulfasalazine) on the development of early stages of diabetic retinopathy in rats. These three drugs differ in their ability to inhibit cyclooxygenase but share an ability to inhibit nuclear factor-kappaB (NF-kappaB). Diabetes of 9-10 months duration significantly increased the number of TUNEL (transferase-mediated dUTP nick-end labeling)-positive capillary cells and acellular (degenerate) capillaries in the retinal vasculature, and all three salicylate-based drugs inhibited this cell death and formation of acellular capillaries without altering the severity of hyperglycemia. In short-term diabetes (2-4 months), all three salicylates inhibited the diabetes-induced loss of neuronal cells from the ganglion cell layer. Oral aspirin (as a representative of the salicylate family) inhibited diabetes-induced increase in NF-kappaB DNA-binding affinity in electrophoretic mobility shift assay and transcription factor array in nuclear extract isolated from whole retina. All three salicylates inhibited the diabetes-induced translocation of p50 (a subunit of NF-kappaB) into nuclei of retinal vascular endothelial cells of the isolated retinal vasculature, as well as of p50 and p65 into nuclei of cells in the ganglion cell layer and inner nuclear layer on whole-retinal sections. Sulfasalazine (also as a representative of the salicylates) inhibited the diabetes-induced upregulation of several inflammatory gene products, which are regulated by NF-kappaB, including vascular cell adhesion molecule, intracellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 in whole-retinal lysate. Salicylates, in doses administrated in our experiments, inhibited NF-kappaB and perhaps other transcription factors in the retina, were well tolerated, and offered new tools to investigate and inhibit the development of diabetic retinopathy.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Retinopatia Diabética/tratamento farmacológico , NF-kappa B/efeitos dos fármacos , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Salicilatos/uso terapêutico , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Aspirina/uso terapêutico , Morte Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/prevenção & controle , Inflamação/patologia , Masculino , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Retina/metabolismo , Retina/patologia , Salicilatos/farmacologia , Salicilato de Sódio/farmacologia , Salicilato de Sódio/uso terapêutico , Sulfassalazina/farmacologia , Sulfassalazina/uso terapêutico , Fator de Transcrição RelA/efeitos dos fármacosRESUMO
PURPOSE: Retinal neurons and vasculature interact with each other under normal conditions, and occlusion of the retinal vasculature can result in damage to retinal neurons. Whether damage to the neural retina will damage the vasculature, however, is less clear. This study was conducted to explore the relationship between vascular and nonvascular cells of the retina. The response of the retinal vasculature to an injury (ischemia and reperfusion; I/R) that is known to cause neuronal degeneration was studied. METHODS: I/R injury to the retinas was induced in Lewis rats and C57BL/6J mice by elevating intraocular pressure (IOP), and reperfusion was established immediately afterward. Some rats were pretreated with aminoguanidine (AMG, 50 mg/Kg BW in drinking water) before the procedure. Poly(ADP-ribose) polymerase (PARP) activity and expression of inducible nitric oxide synthase (iNOS), and cycloxygenase-2 (COX-2) were measured by Western blot analysis, and levels of TNF-alpha and intercellular adhesion molecule (ICAM)-1 mRNA were measured by qPCR at 2 and 7 days after the procedure. Also at 2 and 7 days after the I/R injury, apoptosis of retinal neural cells (demonstrated by TUNEL assay), density of cells in the ganglion cell layer, and thickness of retinas were quantitated, and the number of TUNEL-positive capillary cells and degenerated capillaries were assessed. Retinal neurodegeneration and capillary degeneration were also examined in C57BL/6J mice 2, 5, 8, and 14 days after I/R injury. RESULTS: As expected, loss of cells in the retinal ganglion cell layer was apparent 2 days after I/R injury in the rat and mouse models. In contrast, the retinal vasculature had essentially no pathology at this time in either model. Surprisingly, the number of degenerated capillaries increased greatly by 7 to 8 days after the injury. Administration of aminoguanidine significantly inhibited the I/R-induced capillary degeneration as well as neurodegeneration in the rat model. Retinal I/R caused increased PARP activity (detected by poly(ADP-ribosy)lated proteins), as well as upregulation of iNOS, COX-2, TNF-alpha, and ICAM-1 levels in rats, consistent with an inflammatory process. CONCLUSIONS: Capillary degeneration is an unrecognized component of acutely elevated IOP and develops only after neurodegeneration is severe. Thus, this finding raises the possibility that damage to the neural retina contributes to capillary degeneration. Aminoguanidine, a nonspecific inhibitor of iNOS, inhibited I/R-induced degeneration of both neuronal and vascular cells of the retina. The model of retinal ischemia and reperfusion will be a useful tool for investigating the relationship between neuronal damage and vascular damage in glaucoma and other diseases such as diabetic retinopathy.
Assuntos
Traumatismo por Reperfusão/complicações , Degeneração Retiniana/etiologia , Vasos Retinianos/patologia , Animais , Apoptose , Western Blotting , Capilares , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Guanidinas/administração & dosagem , Marcação In Situ das Extremidades Cortadas , Molécula 1 de Adesão Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Degeneração Retiniana/metabolismo , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/patologia , Vasos Retinianos/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12(-/-) mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage(1).
Assuntos
Oxirredutases do Álcool/metabolismo , Degeneração Retiniana/enzimologia , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/genética , Animais , Sequência de Bases , Primers do DNA/genética , Eletrorretinografia , Feminino , Humanos , Luz/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/prevenção & controle , Retinoides/metabolismoRESUMO
To address potential regulatory roles of TGF-beta1 in muscle inflammation and fibrosis associated with dystrophin deficiency, we performed quantitative RT-PCR and in situ hybridization to characterize the temporal and spatial mRNA expression patterns of TGF-beta1 and other TGF-beta subfamily members, TGF-beta2 and TGF-beta3, as well as their receptors, in quadriceps and diaphragm muscles of mdx mice. TGF-beta1 mRNA was markedly upregulated in the endomysial inflammatory cells and regenerating fibers of mdx quadriceps and diaphragm, with the mRNA levels correlated with the degree of endomysial inflammation. Upregulation of TGF-beta2, beta3, and their receptors was also appreciated but to a much lesser degree. While high levels of TGF-beta1 mRNA remained in the aging mdx quadriceps but not the diaphragm, progressive fibrosis only occurred in the diaphragm. Our data support a regulatory role for TGF-beta1 in muscle inflammation in mdx mice. It also suggests different susceptibility of quadriceps and diaphragm muscles to fibrosis induced by TGF-beta1 signaling pathway.
Assuntos
Expressão Gênica/fisiologia , Camundongos Endogâmicos mdx/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Imunofluorescência/métodos , Hibridização In Situ/métodos , Masculino , Camundongos , Camundongos Endogâmicos mdx/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Crescimento Transformador beta/genéticaRESUMO
Hyperglycemia induces apoptotic cell death in a variety of cell types in diabetes, and the mechanism remains unclear. We report here that culture of rat retinal glial Müller cells in 25 mM glucose for 72 h significantly inactivated Akt and induced apoptosis. Likewise, hyperglycemia caused a significant dephosphorylation of Akt at serine-473 in Müller cells in the retina of streptozotocin-induced diabetic rats. Inactivation of Akt was associated with dephosphorylation of BAD, increased cytochrome c release, and activation of caspase-3 and caspase-9. Upregulation of Akt activity by overexpression of constitutively active Akt inhibited elevated glucose-induced apoptosis, whereas downregulation of Akt activity by overexpression of dominant negative Akt exacerbated elevated glucose-induced apoptosis, as assessed by caspase activity and nucleic acid staining. These data suggest that apoptosis induced by chronically elevated glucose is at least in part mediated by downregulation of Akt survival pathway in cultured Müller cells. It has been reported that antiapoptotic effect of Akt requires glucose in growth factor withdrawal-induced apoptosis. Our data suggest that although acutely elevated glucose may be beneficial to the cell survival, chronically elevated glucose can cause apoptosis via downregulation of Akt survival signaling.
Assuntos
Apoptose/fisiologia , Glucose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Citocromos c/metabolismo , Neuroglia/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Retina/metabolismo , Proteína de Morte Celular Associada a bclRESUMO
Carbonic anhydrase (CA) accelerates contractile function, particularly in fast-twitch skeletal muscles. Since the extraocular muscles are considered to be amongst the fastest skeletal muscles in mammals, this study tested two hypotheses: (1) CA is expressed at higher levels in rat extraocular muscles than in extensor digitorum longus (EDL, a fast limb muscle), and (2) inhibition of CA activity increases twitch duration and force in the extraocular muscles to a greater extent than in EDL. By real-time quantitative PCR we determined that the expression of CA3 isoform, typically high in skeletal muscles, is significantly depressed in extraocular muscles. Message levels for the CA2 and CA4 isoforms were higher in the extraocular muscles, while CA5 expression was equivalent in both muscles. Strong CA activity was demonstrated by histochemistry in frozen EDL muscle sections, in particular along the sarcolemma and in capillaries. By contrast, extraocular muscle had very low sarcolemmal or cytosolic CA activity. CA inhibition with 6-ethoxyzolamide (ETZ) reversibly increased twitch duration and force in EDL muscle bundles. In the extraocular muscles, ETZ did not alter twitch kinetics. Based on these results, we reject our initial hypotheses and conclude that CA does not influence the fast contractile kinetics characteristic of the extraocular muscles.
Assuntos
Anidrases Carbônicas/biossíntese , Isoenzimas/biossíntese , Músculos Oculomotores/enzimologia , Animais , Inibidores da Anidrase Carbônica/farmacologia , Etoxzolamida/farmacologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/enzimologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Decay-accelerating factor (DAF or CD55) is expressed on colonic epithelial cells but its function in the mucosa is unknown. In humans, a proportion of DAF-deficient (Cromer INAB) patients develop inflammatory bowel disease (IBD). To evaluate how DAF deficiency may contribute to gut inflammation and thus could play a role in IBD pathogenesis, we compared the severity of dextran sulfate sodium-induced colitis in Daf1 gene-targeted and control mice. Seven days after consuming 3% dextran sulfate sodium in their drinking water, Daf1(-/-) mice suffered markedly greater weight loss (-24.7 +/- 7.5% vs -14.2% +/- 4.9%), exhibited uniformly bloody diarrhea as compared with soft stool in control mice, developed shortened colons, and had larger spleens. Histological examination of distal colons showed massively increased neutrophilic and mononuclear cell infiltration, greater epithelial cell destruction, and increased ulcerations. Cytokine production in organ cultures of colonic explants showed increased levels of IL-12 and IL-6. Fourteen days after switching back to regular water, in contrast to the Daf1(+/+) controls which showed little stool abnormality, all Daf1(-/-) mice continued to have diarrhea. Organ culture cytokine measurements at this time point, i.e., the end of the recovery phase, showed markedly increased levels of IL-10 (6-fold), IL-12 (4-fold), and IL-6 (2-fold), as well as TNF-alpha (>10-fold) compared with the controls. Our findings argue that, as shown for IL-10 in IL-10(-/-) mice and IL-2 in IL-2(-/-) mice, DAF control of complement additionally is important in regulating gut homeostasis and consequently its activity may participate in protecting against IBD.