RESUMEN
The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.
Asunto(s)
Factor Inductor de la Apoptosis/fisiología , Neovascularización Coroidal/fisiopatología , Neovascularización de la Córnea/fisiopatología , Animales , Apoptosis/fisiología , Factor Inductor de la Apoptosis/deficiencia , Trasplante de Médula Ósea/métodos , Coroides/lesiones , Coroides/ultraestructura , Neovascularización Coroidal/etiología , Neovascularización Coroidal/patología , Neovascularización de la Córnea/inducido químicamente , Neovascularización de la Córnea/patología , Células Endoteliales/patología , Endotelio Vascular/patología , Angiografía con Fluoresceína , Rayos Láser , Leucocitos/patología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Mutantes , Factor A de Crecimiento Endotelial VascularRESUMEN
Diapedesis is a dynamic, highly regulated process by which leukocytes are recruited to inflammatory sites. We reported previously that removal of sialyl residues from PMNs enables these cells to become more adherent to EC monolayers and that sialidase activity within intracellular compartments of resting PMNs translocates to the plasma membrane following activation. We did not identify which surface adhesion molecules were targeted by endogenous sialidase. Upon activation, ß2 integrin (CD11b/CD18) on the PMN surface undergoes conformational change, which allows it to bind more tightly to the ICAM-1 and ICAM-2 on the EC surface. Removal of sialyl residues from CD18 and CD11b, by exogenous neuraminidase or mobilization of PMN sialidase, unmasked activation epitopes, as detected by flow cytometry and enhanced binding to ICAM-1. One sialidase isoform, Neu1, colocalized with CD18 on confocal microscopy. Using an autoperfused microflow chamber, desialylation of immobilized ICAM-1 enhanced leukocyte arrest in vivo. Further, treatment with a sialidase inhibitor in vivo reversed endotoxin-induced binding of leukocytes to ICAM-1, thereby suggesting a role for leukocyte sialidase in the cellular arrest. These data suggest that PMN sialidase could be a physiologic source of the enzymatic activity that removes sialyl residues on ß2 integrin and ICAM-1, resulting in their enhanced interaction. Thus, PMN sialidase may be an important regulator of the recruitment of these cells to inflamed sites.
Asunto(s)
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Quimiotaxis de Leucocito , Epítopos/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Neuraminidasa/metabolismo , Neutrófilos/enzimología , Antígeno CD11b/inmunología , Antígenos CD18/inmunología , Humanos , Cadenas beta de Integrinas , Neutrófilos/inmunología , Unión ProteicaRESUMEN
Metabolic syndrome (MetS) is a prevalent and complex disease, characterized by the variable coexistence of obesity, dyslipidemia, hyperinsulinaemia, and hypertension. The alarming rise in the prevalence of metabolic disorders makes it imperative to innovate preventive or therapeutic measures for MetS and its complications. However, the elucidation of the pathogenesis of MetS has been hampered by the lack of realistic models. For example, the existing animal models of MetS, i.e., genetically engineered rodents, imitate certain aspects of the disease, while lacking other important components. Defining the natural course of MetS in a spontaneous animal model of the disease would be desirable. Here, we introduce the Nile grass rat (NGR), Arvicanthis niloticus, as a novel model of MetS. Studies of over 1100 NGRs in captivity, fed normal chow, revealed that most of these animals spontaneously develop dyslipidemia (P<0.01), and hyperglycemia (P<0.01) by 1 yr of age. Further characterization showed that the diabetic rats develop liver steatosis, abdominal fat accumulation, nephropathy, atrophy of pancreatic islets of Langerhans, fatty streaks in the aorta, and hypertension (P<0.01). Diabetic NGRs in the early phase of the disease develop hyperinsulinemia, and show a strong inverse correlation between plasma adiponectin and HbA1c levels (P<0.01). These data indicate that the NGR is a valuable, spontaneous model for exploring the etiology and pathophysiology of MetS as well as its various complications.
Asunto(s)
Modelos Animales de Enfermedad , Síndrome Metabólico , Animales , Diabetes Mellitus , Dislipidemias , Hígado Graso , Hiperglucemia , Síndrome Metabólico/complicaciones , Síndrome Metabólico/patología , Obesidad Abdominal , Ratas , Ratas EndogámicasRESUMEN
Recently we showed a critical role for Vascular Adhesion Protein-1 (VAP-1) in rodents during acute ocular inflammation, angiogenesis, and diabetic retinal leukostasis. However, the expression of VAP-1 in the human eye is unknown. VAP-1 localization was therefore investigated by immunohistochemistry. Five micrometer thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5 microg/ml), smooth muscle actin (1 microg/ml), CD31 or isotype-matched IgG at 4 degrees C. Subsequently, a secondary mAb was used for 30 min at room temperature, followed by Dako Envision + HRP (AEC) System for signal detection. The stained sections were examined using light microscopy and the signal intensity was quantified by two evaluators and graded into 4 discrete categories. In all examined ocular tissues, VAP-1 staining was confined to the vasculature. VAP-1 labeling showed the highest intensity in both arteries and veins of neuronal tissues: retina and optic nerve, and the lowest intensity in the iris vasculature (p < 0.05). Scleral and choroidal vessels showed moderate staining for VAP-1. VAP-1 intensity was significantly higher in the arteries compared to veins (p < 0.05). Furthermore, VAP-1 staining in arteries colocalized with both CD31 and smooth muscle actin (sm-actin) staining, suggesting expression of VAP-1 in endothelial cells, smooth muscle cells or potentially pericytes. In conclusion, immunohistochemistry reveals constitutive expression of VAP-1 in human ocular tissues. VAP-1 expression is nearly exclusive to the vasculature with arteries showing significantly higher expression than veins. Furthermore, VAP-1 expression in the ocular vasculature is heterogeneous, with the vessels of the optic nerve and the retina showing highest expressions. These results characterize VAP-1 expression in human ocular tissues.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Moléculas de Adhesión Celular/metabolismo , Ojo/irrigación sanguínea , Ojo/metabolismo , Actinas/metabolismo , Adulto , Anciano , Arterias/metabolismo , Coroides/irrigación sanguínea , Coroides/metabolismo , Endotelio Vascular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Técnicas para Inmunoenzimas , Iris/irrigación sanguínea , Iris/metabolismo , Masculino , Persona de Mediana Edad , Nervio Óptico/irrigación sanguínea , Nervio Óptico/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Vasos Retinianos/metabolismo , Esclerótica/irrigación sanguínea , Esclerótica/metabolismo , Venas/metabolismoRESUMEN
P-selectin glycoprotein ligand-1 (PSGL-1) is constitutively expressed on leukocytes and was thought to be down-regulated with cell activation. However, this work shows the surprising finding of functional PSGL-1 up-regulation during acute inflammation. PSGL-1 function was studied in our autoperfusion assay, in which blood from a mouse carotid flows through a microchamber coated with a fixed density of P-selectin. Under the inflammatory conditions--uveitis induced by systemic lipopolysaccharide injection--we recorded significantly reduced leukocyte rolling velocity, which suggests PSGL-1 up-regulation; however, flow cytometry showed reduced PSGL-1. When bound leukocytes were released from the vasculature by PSGL-1 blockade, a large peripheral blood leukocyte (PBL) population showed elevated PSGL-1, which could account for the reduced PSGL-1 in the remaining unbound population. In the eye, systemic blockade of PSGL-1 with a monoclonal antibody or recombinant soluble PSGL-1 drastically reduced the severe manifestations of uveitis. Furthermore, PSGL-1 blockade was significantly more effective in reducing retinal leukostasis than was P-selectin blockade. Our results provide surprising evidence for functional PSGL-1 up-regulation in PBLs during acute inflammation. The temporal overlap between PSGL-1 and P-selectin up-regulation reveals an as yet unrecognized collaboration between this receptor-ligand pair, increasing efficiency of the first steps of the leukocyte recruitment cascade.
Asunto(s)
Endotoxinas/toxicidad , Glicoproteínas de Membrana/metabolismo , Regulación hacia Arriba/fisiología , Uveítis/inducido químicamente , Animales , Cámara Anterior/citología , Inflamación/metabolismo , Leucocitos/fisiología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Técnicas Analíticas Microfluídicas , Retina/citología , Uveítis/metabolismoRESUMEN
OBJECTIVE: Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy. Involvement of the Rho/Rho kinase (ROCK) pathway in diabetic microvasculopathy and therapeutic potential of fasudil, a selective ROCK inhibitor, are investigated. RESEARCH DESIGN AND METHODS: Localization of RhoA/ROCK and Rho activity were examined in retinal tissues of rats. Impact of intravitreal fasudil administration on retinal endothelial nitric oxide synthase (eNOS) and myosin phosphatase target protein (MYPT)-1 phosphorylation, intercellular adhesion molecule-1 (ICAM-1) expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from diabetic retinopathy patients or nondiabetic control subjects to cultured microvascular endothelial cells was quantified. The potential of fasudil for endothelial protection was investigated by measuring the number of adherent neutrophils and terminal transferase-mediated dUTP nick-end labeling-positive endothelial cells. RESULTS: RhoA and ROCK colocalized predominantly in retinal microvessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, whereas it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from diabetic retinopathy patients showed significantly higher adhesion to cultured endothelium and caused endothelial apoptosis, which was significantly reduced by fasudil. Blockade of the Fas-FasL interaction prevented endothelial apoptosis. The protective effect of fasudil on endothelial apoptosis was significantly reversed by Nomega-nitro-l-arginine methyl ester, a NOS inhibitor, whereas neutrophil adhesion remained unaffected. CONCLUSIONS: The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy. Fasudil protects the vascular endothelium by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury. ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Diabetes Mellitus Experimental/fisiopatología , Microvasos/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Microscopía Fluorescente , Microvasos/patología , Neutrófilos/citología , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Retina/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Neuroprotection can be achieved by preventing apoptotic death of postmitotic cells. Apoptotic death can occur by either a caspase-dependent mechanism, involving cytochrome c, apoptosis protease-activating factor-1 (Apaf-1), and caspase-9, or a caspase-independent mechanism, involving apoptosis-inducing factor (AIF). HIV protease inhibitors (PIs) avert apoptosis in part by preventing mitochondrial outer membrane permeabilization (MOMP), but the precise mechanism by which they work is not known. Here, we evaluated the impact of the PIs in a mouse model of retinal detachment (RD) in vivo and in murine primary retinal cell cultures in vitro. Oral administration of the PIs nelfinavir and ritonavir significantly inhibited photoreceptor apoptosis, while preventing the translocation of AIF from mitochondria to the nucleus as well as the activation of caspase-9. RD-induced photoreceptor apoptosis was similarly inhibited in mice carrying hypomorphic mutations of the genes encoding AIF or Apaf-1. Nelfinavir attenuated apoptosis as well as mitochondrial release of AIF and cytochrome c, and subsequent activation of caspase-9 in vitro, in photoreceptor cultures exposed to starvation or monocyte chemoattractant protein-1-stimulated (MCP-1-stimulated) macrophages. Our results suggest that the MOMP inhibition by PIs involved interruption of both caspase-dependent and caspase-independent apoptosis pathways and that PIs may be clinically useful for the treatment of diseases caused by excessive apoptosis.
Asunto(s)
Apoptosis , Citocromos c/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Mitocondrias/metabolismo , Animales , Caspasa 9/metabolismo , Quimiocina CCL2/metabolismo , Humanos , Ratones , Microscopía Fluorescente , Modelos Biológicos , Mutación , Nelfinavir/farmacología , Fármacos Neuroprotectores/farmacología , Retina/citologíaRESUMEN
Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -alpha, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Amina Oxidasa (conteniendo Cobre)/fisiología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/fisiología , Neovascularización Coroidal/prevención & control , Amina Oxidasa (conteniendo Cobre)/genética , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/genética , Movimiento Celular , Neovascularización Coroidal/etiología , Neovascularización Coroidal/patología , Neovascularización Coroidal/fisiopatología , Cartilla de ADN/genética , Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos/patología , Macrófagos/fisiología , Degeneración Macular/etiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: Azurocidin, released by neutrophils during leukocyte-endothelial interaction, is a main cause of neutrophil-evoked vascular leakage. Its role in the retina, however, is unknown. METHODS: Brown Norway rats received intravitreal injections of azurocidin and vehicle control. Blood-retinal barrier (BRB) breakdown was quantified using the Evans blue (EB) dye technique 1, 3, and 24 hours after intravitreal injection. To block azurocidin, aprotinin was injected intravenously before the intravitreal injections. To investigate whether azurocidin plays a role in vascular endothelial growth factor (VEGF)-induced BRB breakdown, rats were treated intravenously with aprotinin, followed by intravitreal injection of VEGF(164). BRB breakdown was quantified 24 hours later. To investigate whether azurocidin may mediate BRB breakdown in early diabetes, aprotinin or vehicle was injected intravenously each day for 2 weeks to streptozotocin-induced diabetic rats, and BRB breakdown was quantified. RESULTS: Intravitreal injection of azurocidin (20 microg) induced a 6.8-fold increase in vascular permeability compared with control at 1-3 hours (P < 0.05), a 2.7-fold increase at 3 to 5 hours (P < 0.01), and a 1.7-fold increase at 24 hours (P < 0.05). Aprotinin inhibited azurocidin-induced BRB breakdown by more than 95% (P < 0.05). Furthermore, treatment with aprotinin significantly suppressed VEGF-induced BRB breakdown by 93% (P < 0.05) and BRB breakdown in early experimental diabetes by 40.6% (P < 0.05). CONCLUSIONS: Azurocidin increases retinal vascular permeability and is effectively blocked by aprotinin. The inhibition of VEGF-induced and early diabetic BRB breakdown with aprotinin indicates that azurocidin may be an important mediator of leukocyte-dependent BRB breakdown secondary to VEGF. Azurocidin may become a new therapeutic target in the treatment of retinal vascular leakage, such as during diabetic retinopathy.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Sanguíneas/farmacología , Barrera Hematorretinal/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Proteínas Portadoras/farmacología , Retinopatía Diabética/prevención & control , Vasos Retinianos/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/antagonistas & inhibidores , Aprotinina/farmacología , Proteínas Sanguíneas/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/etiología , Retinopatía Diabética/metabolismo , Azul de Evans/metabolismo , Masculino , Proteínas Quimioatrayentes de Monocitos/farmacología , Ratas , Ratas Endogámicas BN , Ratas Long-Evans , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
Early detection of ocular inflammation may prevent the occurrence of structural damage or vision loss. Here, we introduce a novel noninvasive technique for molecular imaging and quantitative evaluation of endothelial injury in the choriocapillaris of live animals, which detects disease earlier than currently possible. Using an established model of ocular inflammation, endotoxin-induced uveitis (EIU), we visualized the rolling and adhesive interaction of fluorescent microspheres conjugated to recombinant P-selectin glycoprotein ligand-Ig (rPSGL-Ig) in choriocapillaris using a scanning laser ophthalmoscope (SLO). The number of rolling microspheres in the choriocapillaris peaked 4-10 h after LPS injection. The number of the accumulated microspheres peaked 4 h after LPS injection in the temporal choriocapillaris and 4 and 36 h after LPS injection in the central areas around the optic disk. Furthermore, we semiquantified the levels of P-selectin mRNA expression in the choroidal vessels by reverse transcription-PCR and found its pattern to match the functional microsphere interactions, with a peak at 4 h after LPS injection. These results indicate that PSGL-1-conjugated fluorescent microspheres allow specific detection of endothelial P-selectin expression in vivo and noninvasive assessment of endothelial injury. This technique may help to diagnose subclinical signs of ocular inflammatory diseases.
Asunto(s)
Enfermedades de la Coroides/patología , Coroides/irrigación sanguínea , Endotelio/lesiones , Endotoxinas/efectos adversos , Oftalmoscopía/métodos , Uveítis/inducido químicamente , Animales , Diagnóstico por Imagen/métodos , Glicoproteínas de Membrana , Microesferas , Selectina-P/análisis , Selectina-P/genética , ARN Mensajero/análisis , RatasRESUMEN
Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age-related macular degeneration. Vascular adhesion protein-1 (VAP-1), a cell surface and soluble molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP-1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP-1 in an established model of ocular inflammation, the endotoxin-induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half-maximal inhibitory concentration (IC(50)) of 0.007 microM against human and 0.008 microM against rat SSAO, while its IC(50) against the functionally related monoamine oxidase (MAO) -A and MAO-B is >10 microM. In the retina, VAP-1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP-1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P-selectin expression. Our data suggest an important role for VAP-1 in the recruitment of leukocytes to the immune-privileged ocular tissues during acute inflammation. VAP-1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases.