RESUMO
TNF-α induces loss of barrier integrity of the corneal endothelium through mechanisms involving the activation of p38 MAP kinase. This study has investigated the role of matrix metalloproteinase-9 (MMP-9), known to be activated by mechanisms downstream of p38 MAP kinase, on the breakdown of the barrier integrity. Experiments were performed with primary cultures of bovine corneal endothelium. Changes in the trans-endothelial electrical resistance (TER), a measure of barrier integrity, were measured by electric cell-substrate impedance sensing. The integrity of the apical junctional assembly was imaged by immunolocalization of ZO-1. MMP-9 activity in the conditioned medium of cells treated with TNF-α was visualized by gelatin zymography. Transcriptional activation of MMP-9 was assessed by real-time RT-PCR. Exposure to TNF-α led to significant disruption of ZO-1 and also caused a continuous decline in TER for more than 20 h. These effects were opposed by cycloheximide (protein synthesis inhibitor), GM-6001 (broad spectrum inhibitor of MMPs), minocycline (MMP-2 and MMP-9 inhibitor), and MMP-9 inhibitor I (selective MMP-9 inhibitor). Cycloheximide, GM-6001, and MMP-9 inhibitor I also attenuated the increase in permeability to FITC-dextran (10 kDa). In addition, TNF-α led to an increased MMP-9 activity in the conditioned medium as well as a nearly 20-fold increase in mRNA for MMP-9 but not for MMP-2. The functional activity and increase in mRNA levels of MMP-9 were blocked by SB-203580 (selective p38 MAP kinase inhibitor) and cycloheximide. In conclusion, transcriptional and translational activation of MMP-9, downstream of p38 MAP kinase signaling, is involved in the (TNF-α)-induced loss of corneal endothelial barrier integrity.
Assuntos
Endotélio Corneano/efeitos dos fármacos , Metaloproteinase 9 da Matriz/fisiologia , Junções Íntimas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Bovinos , Células Cultivadas , Cicloeximida/farmacologia , Dextranos/metabolismo , Impedância Elétrica , Endotélio Corneano/enzimologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Regulação da Expressão Gênica/fisiologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Junções Íntimas/enzimologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Endothelial monocyte-activating polypeptide II (EMAP II) and interferon-inducible protein (IP)-10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke-exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke-induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II-induced and IP-10-induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II-induced and IP-10-induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II-induced and IP-10-induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II-induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema.
Assuntos
Apoptose , Células Endoteliais/metabolismo , Nicotiana/química , Extratos Vegetais/farmacologia , Receptores CXCR3/metabolismo , Fumaça , Regulação para Cima/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocina CXCL10/farmacologia , Meios de Cultura Livres de Soro , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Feminino , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , Proteínas de Neoplasias/farmacologia , Proteínas de Ligação a RNA/farmacologia , Receptores CXCR3/genética , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Chronic kidney disease pathogenesis involves both tubular and vascular injuries. Despite abundant investigations to identify the risk factors, the involvement of chronic endothelial dysfunction in developing nephropathies is insufficiently explored. Previously, soluble thrombomodulin (sTM), a cofactor in the activation of protein C, has been shown to protect endothelial function in models of acute kidney injury. In this study, the role for sTM in treating chronic kidney disease was explored by employing a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice. Analysis of kidneys from these mice after 3 mo showed no apparent phenotype, whereas 6-mo-old mice demonstrated infiltration of CD45-positive leukocytes accompanied by upregulated gene expression of inflammatory chemokines, markers of kidney injury, and albuminuria. Intervention with murine sTM with biweekly subcutaneous injections during this window of disease development between months 3 and 6 prevented the development of kidney pathology. To better understand the mechanisms of these findings, we determined whether sTM could also prevent chronic endothelial cell activation in vitro. Indeed, treatment with sTM normalized increased chemokines, adhesion molecule expression, and reduced transmigration of monocytes in continuously activated TNF-expressing endothelial cells. Our results suggest that vascular inflammation associated with vulnerable endothelium can contribute to loss in renal function as suggested by the tie2-TNF mice, a unique model for studying the role of vascular activation and inflammation in chronic kidney disease. Furthermore, the ability to restore the endothelial balance by exogenous administration of sTM via downregulation of specific adhesion molecules and chemokines suggests a potential for therapeutic intervention in kidney disease associated with chronic inflammation.
Assuntos
Albuminúria/prevenção & controle , Inflamação/tratamento farmacológico , Falência Renal Crônica/metabolismo , Trombomodulina/uso terapêutico , Albuminúria/tratamento farmacológico , Albuminúria/genética , Albuminúria/metabolismo , Animais , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica/fisiologia , Inflamação/genética , Inflamação/metabolismo , Falência Renal Crônica/genética , Camundongos , Camundongos Transgênicos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor TIE-2 , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Recent clinical trials indicate that human adipose-derived stromal cells (hASCs) have beneficial effects on antiaging and wound healing. This study examined the morphologic changes in photodamaged human organotypic skin culture after treatment of autologous hASCs. METHODS: Abdominal skin flaps were obtained from 8 white females who underwent abdominoplasties with liposuction. The adipose layer was removed and used for hASC isolation. Sections of skin were removed and cultured in serum-free medium. To induce photodamage, some of the skin pieces were irradiated with maximum subcytotoxic doses of UVB (1600 J/m(2)) and UVA (250 J/m(2)). The effects of hASC on skin segments were evaluated by coculture as a feeder layer or by injecting intradermally. Portions of the skin samples were removed for analysis on days 3, 5, 7, and 9 of culture and analyzed histologically for morphology, viability, and proliferation status. RESULTS: Epidermal necrosis of irradiated skin was significantly reduced by the presence of hASCs. Increased parakeratosis was observed at early time points, and apoptosis in epidermis was markedly decreased by hASCs. Differences were observed in epidermal differentiation but not basal cell proliferation. Similar results were obtained by both methods of hASC treatment to the skin. CONCLUSIONS: Exposure of UV-irradiated skin to hASC attenuated cell senescence and promoted repair from photodamage in an organotypic skin culture. These results suggest that hASC treatment may have a useful therapeutic effect for salvaging photodamaged skin.
Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/efeitos da radiação , Envelhecimento da Pele/patologia , Pele/efeitos da radiação , Células Estromais/citologia , Células Estromais/efeitos da radiação , Adulto , Análise de Variância , Apoptose , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura Livres de Soro , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Raios UltravioletaRESUMO
Chronic inflammation is tightly linked to diseases associated with endothelial dysfunction including aberrant angiogenesis. To better understand the endothelial role in pro-inflammatory angiogenesis, we analyzed signaling pathways in continuously activated endothelial cells, which were either chronically exposed to soluble TNF or the reactive oxygen species (ROS) generating H2O2, or express active transmembrane TNF. Testing in an in vitro capillary sprout formation assay, continuous endothelial activation increased angiogenesis dependent on activation of p38 MAP kinase, NADPH oxidase, and matrix metalloproteinases (MMP). p38 MAP kinase- and MMP-9-dependent angiogenesis in our assay system may be part of a positive feed forward autocrine loop because continuously activated endothelial cells displayed up-regulated ROS production and subsequent endothelial TNF expression. The pro-angiogenic role of the p38 MAP kinase in continuously activated endothelial cells was in stark contrast to the anti-angiogenic activity of the p38 MAP kinase in unstimulated control endothelial cells. In vivo, using an experimental prostate tumor, pharmacological inhibition of p38 MAP kinase demonstrated a significant reduction in tumor growth and in vessel density, suggesting a pro-angiogenic role of the p38 MAP kinase in pathological angiogenesis in vivo. In conclusion, our results suggest that continuous activation of endothelial cells can cause a switch of the p38 MAP kinase from anti-angiogenic to pro-angiogenic activities in conditions which link oxidative stress and autocrine TNF production.
Assuntos
Inflamação/complicações , Inflamação/enzimologia , Neovascularização Patológica/complicações , Neovascularização Patológica/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Modelos Biológicos , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/patologia , Ratos , Solubilidade/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
RATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.
Assuntos
Alvéolos Pulmonares/efeitos dos fármacos , Enfisema Pulmonar/tratamento farmacológico , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Ceramidas/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Cloridrato de Fingolimode , Indóis/farmacologia , Lisofosfolipídeos/biossíntese , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase , Propilenoglicóis/farmacologia , Alvéolos Pulmonares/fisiopatologia , Enfisema Pulmonar/fisiopatologia , Pirróis/farmacologia , Receptores de Lisoesfingolipídeo/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Esfingosina/farmacologiaRESUMO
Senescence of endothelial cells increases with systemic aging and is thought to contribute to the development of atherosclerosis. Cell therapy with highly proliferative endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote endothelial regeneration, but little is known about their senescence and their vulnerability to inflammatory stressors. We therefore studied the senescence of proliferative human EPCs and investigated the effects of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on their senescence. Human EPCs had a significantly lower rate of senescence at baseline, compared with that of mature endothelial cells. However, EPCs up-regulated the expression of the senescence-associated cell cycle arrest protein p16(INK4a) and markedly increased measured senescence levels when exposed to chronic TNF-alpha treatment. Analysis of telomere length showed that the increases in senescence were not related to changes in telomere length. Inhibition of the p38 mitogen-activated protein kinase pathway blocked the induction of p16(INK4a) and cellular senescence. In conclusion, highly proliferative EPCs have a low rate of intrinsic senescence but are vulnerable to premature senescence induction by chronic proinflammatory stimulation. These findings will lead to a better understanding of physiological endothelial regeneration as well as to targeted therapies with the aim of promoting endothelial regeneration through endothelial progenitor cells.
Assuntos
Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Endoteliais/fisiologia , Células-Tronco/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Sangue Fetal/citologia , Humanos , Imidazóis/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Adipose stromal cells (ASC) are multipotential mesenchymal progenitor cells that are readily induced to undergo adipogenic differentiation, and we have recently demonstrated them to have functional and phenotypic overlap with pericytes lining microvessels in adipose tissues. In this study we addressed the hypothesis that modulation of ASC fate within this perivascular niche can occur via interaction with endothelial cells (EC), which serve to modulate the adipogenic potential of ASC. To this end, we investigated contact as well as paracrine effects of EC on ASC adipogenesis, in two-dimensional coculture and via conditioned medium and analyzed mutual gene expression changes by real-time reverse transcription polymerase chain reaction (PCR). A significant decrease in adipogenic differentiation was observed in ASC when they were cocultured with EC but not control fibroblasts. This endothelial cell-specific effect was accompanied by increased expression of factors involved in Wnt signaling, most prominently Wnt1, Wnt4, and Wnt10a, which are well-known inhibitors of adipogenesis. Suppression of Wnt1 but not Wnt 10a or scrambled control short interfering RNA in cocultures partially reversed the endothelial cell effect, thus increasing adipogenic differentiation, suggesting a plausible role of Wnt1 ligand in modulation of adipogenesis by the vasculature. Furthermore, addition of recombinant Wnt ligand or the Wnt signaling agonist inhibited adipogenic differentiation of ASC in the absence of EC. In conclusion, these data define the relationship in adipose tissue between ASC and EC in the perivascular niche, in which the latter act to repress adipogenesis, thereby stabilizing vasculature. It is tempting to speculate that abnormal endothelial function may be associated with pathologic derepression of adipogenesis. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Células Endoteliais/citologia , Comunicação Parácrina , Transdução de Sinais , Células Estromais/citologia , Proteínas Wnt/metabolismo , Adipogenia , Adulto , Adesão Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Inativação Gênica , Humanos , Células Estromais/metabolismo , Regulação para Cima , Proteínas Wnt/genética , Proteína Wnt1/genéticaRESUMO
Although the roles of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in pathologic neovascularization have been well characterized in certain tissues, their particular functions and expression patterns in choroidal neovascularization (CNV) have not been clearly established. After localized laser trauma to Bruch's membrane to induce CNV development, the temporal changes in mRNA and protein expression of these 3 cytokines were documented and compared histologically to areas of immunofluorescence, the proliferation of endothelial cells, neovascular development, and temporal changes in vascular permeability. Changes in mRNA and protein levels of bFGF and HGF occurred quickly and reached peak expression within hours. This activity corresponded in time to intense and localized immunofluorescence for these cytokines within the choriocapillaris within laser lesion sites. During this same initial time period, mRNA upregulation of VEGF occurred, primarily within the neural retina and this expression corresponded to intense immunolabeling of Müller cells immediately adjacent to the lesion sites. By 3 days after lasering, increased VEGF(164) protein expression was measurable, whereas early neovascular development histologically corresponded to HGF and bFGF mRNA expansion into the developing choroidal neovascular membrane (CNVM). At 7 days, CNV expansion, maturation, and increased vascular permeability corresponded to peak VEGF mRNA and protein expression and to immunofluorescence of the CNVM. Differences also occurred in the expression of precursor and activated isoforms of these cytokines in the retinal pigment epithelium/choroid as compared to those in the retina. These molecular and immunocytochemical results suggest that bFGF and HGF may be important as initial regulators neovascularization in this CNV model; whereas VEGF may be important during later phases of angiogenesis and neovascular hyperpermeability.
Assuntos
Neovascularização de Coroide/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Western Blotting/métodos , Permeabilidade Capilar , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/patologia , Neovascularização de Coroide/fisiopatologia , Progressão da Doença , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fotocoagulação a Laser , Masculino , Microscopia de Fluorescência/métodos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos BN , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Activation of the vascular endothelium with cytokines such as TNF is widely used to study the role of the vasculature in proinflammatory disease. To gain insight into mechanisms of prolonged vascular endothelial activation we compared changes in gene expression induced by continuous activation in stable tmTNF-expressing cells with changes due to acute TNF challenge in vitro. Affymetrix Genechip analysis was performed on RNA from control, acute and continuous TNF-activated endothelial cells. Only 36% of the significant changes in gene expression were convergent between the acute and continuously activated endothelial cells compared with the control. From the divergently regulated genes, for example the cytokine ENA-78 was specifically induced in chronically activated cells, while E-selectin, a cell adhesion molecule, was upregulated only in acutely activated endothelial cells. Antioxidant SOD gene induction was noted in acute activation, while a regulatory NADPH oxidase subunit was selectively upregulated in continuously activated endothelium in accordance with significant reactive oxygen species induction occurred only in these cells. Accordingly, p38 and ERK1/2, two MAP kinases downstream of reactive oxygen species, were activated in stable transmembrane-spanning precursor (tm) TNF-expressing cells and were refractory to activation with soluble TNF or VEGF. In consequence, the increased p38 MAP kinase activity contributed to increased endothelial cell migration in tmTNF-expressing cells. These data suggest that continuous activation of endothelial cells leads to specific expression and functional changes, consistent with alterations observed in dysfunctional endothelium exposed to or involved in chronic inflammation.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Animais , Movimento Celular , Citocinas/metabolismo , Selectina E/metabolismo , Humanos , Camundongos , Modelos Biológicos , NADPH Oxidases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Recruitment of endothelial progenitor cells to the sites of ischemia has recently been suggested as a mechanism of tissue repair. Here we address the hypothesis that the hypoxia-inducible full-length endothelial-monocyte-activating polypeptide II (EMAP II) provides a mechanism to recruit late outgrowth highly proliferating endothelial progenitor cells (EPCs). MATERIALS AND METHODS: We tested in a transwell migration assay EMAP II for its ability to induce migration of EPCs. Furthermore, we measured changes in cellular calcium levels in EPC to assess the ability of EMAP II to induce intracellular signaling. Finally, we employed neutralizing antibodies and binding competition studies in order to identify the receptor mediating these activities of EMAP II in EPCs. RESULTS: EMAP II elicits dose-dependent migration and intracellular calcium mobilization in EPCs. Functional blocking and binding studies with radiolabeled interferon-gamma-induced protein (IP-10) indicate that EMAP II employs the CXCR3 receptor for these activities in EPCs. Indeed, EMAP II-induced migration of EPCs can be abolished by prior treatment of cells with anti-CXCR3 antibodies or with IP-10. CONCLUSIONS: These data suggests a novel function for EMAP II and a hitherto undescribed role of the CXCR3 chemokine receptor in EPC recruitment.
Assuntos
Citocinas/farmacologia , Células Endoteliais/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Neoplasias/farmacologia , Proteínas de Ligação a RNA/farmacologia , Receptores de Quimiocinas/fisiologia , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL10 , Quimiocinas CXC/fisiologia , Relação Dose-Resposta a Droga , Humanos , Receptores CXCR3RESUMO
Eye diseases characterized by excessive angiogenesis such as wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity are major causes of blindness. Cremastranone is an antiangiogenic, naturally occurring homoisoflavanone with efficacy in retinal and choroidal neovascularization models and antiproliferative selectivity for endothelial cells over other cell types. We undertook a cell-based structure-activity relationship study to develop more potent cremastranone analogues, with improved antiproliferative selectivity for retinal endothelial cells. Phenylalanyl-incorporated homoisoflavonoids showed improved activity and remarkable selectivity for retinal microvascular endothelial cells. A lead compound inhibited angiogenesis in vitro without inducing apoptosis and had efficacy in the oxygen-induced retinopathy model in vivo.
Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Isoflavonas/química , Isoflavonas/farmacologia , Retina/efeitos dos fármacos , Neovascularização Retiniana/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Retina/citologia , Retina/patologia , Neovascularização Retiniana/patologiaRESUMO
Retinopathies in human and animal models have shown to occur through loss of pericytes resulting in edema formation, excessive immature retinal angiogenesis, and neuronal apoptosis eventually leading to blindness. In recent years, the concept of regenerating terminally differentiated organs with a cell-based therapy has evolved. The cells used in these approaches are diverse and include tissue-specific endogenous stem cells, endothelial progenitor (EPC), embryonic stem cells, induced pluripotent stem cells (iPSC) and mesenchymal stem cells (MSC). Recently, MSC derived from the stromal fraction of adipose tissue have been shown to possess pluripotent differentiation potential in vitro. These adipose stromal cells (ASC) have been differentiated in a number of laboratories to osteogenic, myogenic, vascular, and adipocytic cell phenotypes. In vivo, ASC have been shown to have functional and phenotypic overlap with pericytes lining microvessels in adipose tissues. Furthermore, these cells either in paracrine mode or physical proximity with endothelial cells, promoted angiogenesis, improved ischemia-reperfusion, protected from myocardial infarction, and were neuroprotective. Owing to the easy isolation procedure and abundant supply, fat-derived ASC are a more preferred source of autologous mesenchymal cells compared to bone marrow MSC. In this review, we present evidence that these readily available ASC from minimally invasive liposuction will facilitate translation of ASC research into patients with retinal diseases in the near future.
RESUMO
Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Isoflavonas/síntese química , Isoflavonas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Eritropoetina/metabolismo , Humanos , Isoflavonas/química , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved "b" wave amplitude (as measured by electroretinogram) within 1-3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.
Assuntos
Tecido Adiposo/citologia , Transplante de Células , Retinopatia Diabética/terapia , Animais , Apoptose , Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Glucose/farmacologia , Humanos , Injeções Intravítreas , Ratos , Vasos Retinianos/fisiopatologia , Células Estromais/efeitos dos fármacos , Células Estromais/transplante , Fatores de TempoRESUMO
Genomic studies of the pediatric ocular tumor retinoblastoma are paving the way for development of targeted therapies. Robust model systems such as orthotopic xenografts are necessary for testing such therapeutics. One system involves bioluminescence imaging of luciferase-expressing human retinoblastoma cells injected into the vitreous of newborn rat eyes. Although used for several drug studies, the spatial and temporal development of tumors in this model has not been documented. Here, we present a new model to allow analysis of average luciferin flux ([Formula: see text]) through the tumor, a more biologically relevant parameter than peak bioluminescence as traditionally measured. Moreover, we monitored the spatial development of xenografts in the living eye. We engineered Y79 retinoblastoma cells to express a lentivirally-delivered enhanced green fluorescent protein-luciferase fusion protein. In intravitreal xenografts, we assayed bioluminescence and computed [Formula: see text], as well as documented tumor growth by intraocular optical coherence tomography (OCT), brightfield, and fluorescence imaging. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated in orthotopic xenografts. By OCT, xenografts were dense and highly vascularized, with well-defined edges. Small tumors preferentially sat atop the optic nerve head; this morphology was confirmed on histological examination. In vivo, [Formula: see text] in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of [Formula: see text] modeling and in vivo intraocular imaging allows both quantitative and high-resolution, non-invasive spatial analysis of this retinoblastoma model. This technique will be applied to other cell lines and experimental therapeutic trials in the future.
Assuntos
Neoplasias Oculares/patologia , Retinoblastoma/patologia , Animais , Linhagem Celular Tumoral , Neoplasias Oculares/diagnóstico por imagem , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Medições Luminescentes , Radiografia , Ratos , Retinoblastoma/diagnóstico por imagem , Tomografia de Coerência Óptica , Transplante HeterólogoRESUMO
Untreated HIV infection is associated with endothelial dysfunction and subsequent cardiovascular disease, likely due to both direct effects of the virus and to indirect effects of systemic inflammation on the vasculature. We have recently shown that treatment with the antiinflammatory agent pentoxifylline (PTX) improved in vivo endothelial function and reduced circulating levels of the inflammatory markers vascular cell adhesion molecule-1 (VCAM-1) and interferon-gamma-induced protein (IP-10) in HIV-infected patients. To delineate the mechanisms underlying this therapeutic effect, we tested whether clinically relevant concentrations of PTX suppress VCAM-1 or IP-10 release in cultivated human lung microvascular endothelial cells. Indeed, we found that tumor necrosis factor (TNF)-α-induced VCAM-1 was reduced with concentrations of PTX in the low nanomolar range, comparable to plasma levels in PTX-treated groups. We also investigated the effect of HIV proteins and found that HIV transactivator of transcription (HIV-Tat) and HIV-envelope-derived recombinant gp120 enhanced TNF-α-induced VCAM-1 gene expression in lung microvascular and coronary macrovascular endothelial cells, respectively. In addition, PTX and a NF-κB-specific inhibitor reduced this enhanced VCAM-1 gene induction in microvascular and macrovascular endothelial cells. These results provide novel insights in how the antiinflammatory agent PTX can directly reduce HIV-associated proinflammatory endothelial activation, which may underlie vascular dysfunction and coronary vascular diseases.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Anti-Inflamatórios/farmacologia , Quimiocina CXCL10/imunologia , HIV-1/imunologia , Pentoxifilina/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Células Cultivadas , Quimiocina CXCL10/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , HIV-1/efeitos dos fármacos , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacos , Vasodilatadores/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke-induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.
Assuntos
Citocinas/fisiologia , Proteínas de Neoplasias/fisiologia , Enfisema Pulmonar/genética , Proteínas de Ligação a RNA/fisiologia , Poluição por Fumaça de Tabaco/efeitos adversos , Adulto , Animais , Anticorpos Neutralizantes/uso terapêutico , Apoptose , Câmaras de Exposição Atmosférica , Bronquíolos/efeitos dos fármacos , Bronquíolos/patologia , Líquido da Lavagem Broncoalveolar/química , Caspase 3/toxicidade , Inibidores de Caspase , Citocinas/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Imunização Passiva , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas de Neoplasias/uso terapêutico , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Proteínas de Ligação a RNA/uso terapêutico , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fumar/efeitos adversos , Fumar/metabolismo , Adulto JovemRESUMO
PURPOSE: TNF-alpha is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF-alpha-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. METHODS: Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-alpha was visualized by immunofluorescence. The expression of TNF-alpha receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-alpha was determined by Western blot analysis. RESULTS: Exposure to TNF-alpha induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-alpha also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. CONCLUSIONS: TNF-alpha breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-alpha-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.
Assuntos
Endotélio Corneano/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Western Blotting , Caderinas/metabolismo , Bovinos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Impedância Elétrica , Endotélio Corneano/metabolismo , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína da Zônula de Oclusão-1 , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
EMAP II is an endothelial cell and monocyte activating proinflammatory cytokine, which has been demonstrated to induce endothelial cell apoptosis. In order to analyze its role in disease models linked to inflammation and endothelial cell death, we aimed to develop a neutralizing antibody against mouse EMAP II. Therefore, we generated rat monoclonal anti-mouse EMAP II antibodies by immunization with recombinant full length, mouse pro-EMAP II protein. We could identify by ELISA, hybridoma clones from fusion with mouse myeloma SP2/0 cells which produced antibodies recognizing both full length and mature EMAP II. We further characterized one antibody, M7/1 and demonstrated its ability to detect both EMAP II forms in Western blotting and to neutralize EMAP II directed migration of human peripheral blood monocytes as well as EMAP II induced apoptosis of tumor and endothelial cells. We conclude that this antibody can be useful to both target and analyze murine disease models, in which EMAP II may be involved.