RESUMO
Proliferative vitreoretinopathy (PVR) is a severe, vision-threatening disorder characterized by the fibrous membrane formation that leads to tractional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up-regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αV-mediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFß2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR.
Assuntos
Moléculas de Adesão Celular/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Adulto , Idoso , Animais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Several retinal ischemic diseases can cause neovascular glaucoma (NVG). Trabeculectomy with mitomycin C (MMC) is a relatively better treatment modality in the management of eyes with NVG than other glaucoma surgeries. The aim of this study was to investigate the factors that may influence the outcome of trabeculectomy with MMC for NVG. METHODS: Forty-nine NVG eyes from 43 patients (26 males and 17 females) underwent primary trabeculectomy with MMC. The mean follow-up period was 16.8 ± 8.1 months (range, 6 to 34 months). Twenty-one eyes of 21 patients received intravitreal bevacizumab (IVB) 3.6 ± 1.8 days before trabeculectomy with MMC. A Kaplan-Meier survival-curve analysis was used to summarize the cumulative probability of success. We examined the relationship between the surgical outcome and the following surgical factors: gender, age, history of panretinal photocoagulation, history of cataract surgery, history of vitrectomy, preoperative IVB, NVG in the fellow eye, and postoperative complications (hyphema, choroidal detachment, and formation of fibrin) by multivariate analysis. RESULTS: The survival rate was 83.7% after 6 months, 70.9% after 12 months, and 60.8% after 24 months. The Kaplan-Meier survival curves showed no significant difference in the survival rate between the eyes with preoperative IVB (n = 21) and the eyes without preoperative IVB (n = 28) (p = 0.14). The multiple logistic regression analysis showed that postoperative hyphema (odds ratio, 6.54; 95% confidence interval, 1.41 to 35.97) was significantly associated with the surgical outcome (p = 0.02). CONCLUSIONS: Postoperative hyphema was significantly correlated with the outcome of trabeculectomy for NVG. There was no significant association between preoperative IVB and postoperative hyphema or the results of trabeculectomy.
Assuntos
Glaucoma Neovascular/cirurgia , Hifema/etiologia , Hemorragia Pós-Operatória/etiologia , Trabeculectomia/efeitos adversos , Adulto , Idoso , Inibidores da Angiogênese/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Feminino , Seguimentos , Glaucoma Neovascular/diagnóstico , Glaucoma Neovascular/fisiopatologia , Humanos , Hifema/diagnóstico , Hifema/epidemiologia , Incidência , Pressão Intraocular , Injeções Intravítreas , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Hemorragia Pós-Operatória/epidemiologia , Hemorragia Pós-Operatória/prevenção & controle , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida/tendências , Fatores de Tempo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Acuidade VisualRESUMO
Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1ß (MIP-1ß) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1ß in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1ß was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1ß reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1ß-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1ß is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1ß induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases.
Assuntos
Células da Medula Óssea/fisiologia , Linhagem da Célula , Quimiocina CCL4/fisiologia , Monócitos/fisiologia , Oxigênio/toxicidade , Doenças Retinianas/fisiopatologia , Neovascularização Retiniana/etiologia , Animais , Movimento Celular , Quimiocina CCL4/análise , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR5/análise , Retina/química , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-beta is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-beta is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-beta's importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-beta2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of alpha-smooth muscle actin (alpha-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-beta blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted alpha-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-beta in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-beta and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Actinas/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Coelhos , Vitreorretinopatia Proliferativa/patologia , Cicatrização/fisiologiaRESUMO
BACKGROUND: While statins have an anti-angiogenic property, their underlying mechanisms are not fully understood. We investigated intracellular mechanisms of simvastatin-mediated reduction in VEGF-induced signalings. METHODS: The effects of simvastatin on cell proliferation and viability were evaluated by [(3)H]-thymidine incorporation in retinal endothelial cells (RECs) and cell counting. The impact of simvastatin on VEGF-induced phosphorylation of p44/42 mitogen-activated protein (MAP) kinase, myosin light chain (MLC), and VEGF-receptor (VEGFR) 2 were examined by Western blotting. Involvement of the mevalonate pathway in VEGF-induced signaling was also examined. RESULTS: Simvastatin (1 and 10 microM) suppressed VEGF-induced RECs proliferation in a concentration-dependent manner, without affecting cell viability. Simvastatin significantly inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream mediators, p44/42 MAP kinase and MLC. Mevalonate completely reversed VEGF-induced VEGFR2 phosphorylation, but only partially reversed the phosphorylation of p44/42 MAP kinase and MLC. CONCLUSION: These data indicate that simvastatin exerts its anti-angiogenic effects through the reduction of VEGFR2 phosphorylation in RECs at least in part. However, there seems to be both mevalonate-dependent and independent pathway in simvastatin's anti-angiogenic property.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Western Blotting , Bovinos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). In this study, vitreous samples from PDR or PVR patients caused significantly stronger contraction of hyalocyte-containing collagen gels, an in vitro model of cicatricial contraction, compared with those from nonproliferative controls. We elucidated the critical role of transforming growth factor-beta (TGF-beta) in the contractile effect and its underlying mechanisms mediating cicatricial contraction in proliferative vitreoretinal diseases at least in part. Fasudil, a potent and selective Rho-kinase (ROCK) inhibitor, almost completely blocked collagen gel contraction induced by vitreous samples. In addition, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes in vivo. Rho/ROCK pathway, considered to be a key downstream mediator of TGF-beta and other contractile-inducing factors, might become a unique therapeutic target for the treatment of proliferative vitreoretinal diseases.
Assuntos
Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/fisiopatologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/fisiopatologia , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , CoelhosRESUMO
BACKGROUND: To evaluate the effect of tamponade by room air after vitrectomy for the treatment of idiopathic macular hole (MH). METHODS: There were 156 eyes of 151 patients studied. The patients' ages ranged from 35 to 88 years old (mean: 65.1 years). After conventional pars plana vitrectomy with internal limiting membrane peeling, fluid air exchange was performed using 20% SF(6) (Gas group: 91 eyes) or room air (Air group: 65 eyes). Surgical outcomes were retrospectively analyzed. RESULTS: Mean preoperative hole diameter was 352 microm in the Gas group and 370 microm in the Air group (P = 0.558). The closure rate of all cases was 91.0% after first surgery and 98.7% at last follow-up. The primary closure rate was 90.1% in the Gas group after 7.44 +/- 1.66 (mean +/- SD) days prone positioning period, and 92.3% in the Air group after 3.83 +/- 0.97 days of prone positioning. There was significant difference in prone positioning period (P < 0.0001), but not in the first closure rate (P = 0.132). CONCLUSION: This study suggests that room air may have an equivalent tamponade effect, in spite of the shorter prone positioning period, than SF(6) after MH surgery.
Assuntos
Ar , Perfurações Retinianas/cirurgia , Hexafluoreto de Enxofre/administração & dosagem , Vitrectomia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Decúbito Ventral , Perfurações Retinianas/fisiopatologia , Estudos Retrospectivos , Acuidade Visual/fisiologiaRESUMO
PURPOSE: To investigate the anatomical features of vitreoretinal interface in eyes with asteroid hyalosis (AH) with optical coherence tomography (OCT) and intravitreal triamcinolone acetonide (TA) during vitreous surgery. METHODS: This study was an interventional clinical case series. Records relating to ten eyes from ten patients who underwent a TA-assisted vitrectomy for the treatment of diverse vitreoretinal diseases complicated with AH. The posterior vitreoretinal interface was examined by preoperative OCT and by intraoperative visualization of posterior vitreous cortex utilizing TA. RESULTS: In eight of ten AH eyes, preoperative OCT revealed abnormal vitreoretinal adhesions. In four of these eight eyes, posterior vitreoschisis could be seen on OCT. In the other four of these eight eyes, a clear no posterior vitreous detachment (PVD) pattern could be seen on OCT. Although posterior vitreous cortex could not be clearly identified with preoperative OCT in two of ten AH eyes, a complete PVD was refuted by intraoperative visualization of the posterior vitreous cortex with TA identical to the other eight eyes. CONCLUSION: These results indicate that complete PVD appears to be unlikely to occur in eyes with AH. In addition, spontaneous PVD in eyes with AH might lead to vitreoschisis or residual whole layer or posterior vitreous cortex, possibly due to anomalous vitreoretinal adhesion.
Assuntos
Oftalmopatias/diagnóstico , Doenças Retinianas/diagnóstico , Corpo Vítreo/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Glucocorticoides , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia de Coerência Óptica , Triancinolona Acetonida , VitrectomiaRESUMO
The critical association of connective tissue growth factor (CTGF), which is thought to be one of the downstream mediators of transforming growth factor-beta (TGF-beta), with vitreoretinal diseases remains to be clarified. In the current study, we first demonstrated the correlation between the concentrations of TGF-beta2 as well as CTGF in the vitreous and CTGF gene regulation in cultured hyalocytes. Concentrations of TGF-beta2 and CTGF in the vitreous from patients with proliferative vitreoretinal diseases were significantly higher than in those with nonproliferative diseases, and there was a positive correlation between their concentrations (r = 0.320, P < 0.01). Cultured hyalocytes expressed CTGF mRNA, which was enhanced in the presence of TGF-beta2, associated with nuclear accumulation of Smad4. TGF-beta2-dependent Smad4 translocation and CTGF gene expression were mediated through Rho kinase and at least partially via p38 mitogen-activated protein kinase. Finally, fasudil, a Rho kinase inhibitor already in clinical use, inhibited both Smad4 translocation and CTGF gene expression. In conclusion, combined effects of TGF-beta2 and CTGF appear to be involved in the pathogenesis of proliferative vitreoretinal diseases. Hyalocytes may be a possible source of CTGF and thus might play a role in vitreoretinal interface diseases. Furthermore, Rho kinase inhibitors might have therapeutic potential to control fibrotic disorders in the eye.
Assuntos
Proteínas Imediatamente Precoces/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Doenças Retinianas/fisiopatologia , Fator de Crescimento Transformador beta2/fisiologia , Corpo Vítreo/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Fator de Crescimento do Tecido Conjuntivo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lisofosfolipídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/genética , Vitrectomia , Corpo Vítreo/fisiopatologia , Quinases Associadas a rhoRESUMO
PURPOSE: Vascular endothelial growth factor (VEGF) plays a pivotal role in pathological angiogenesis. In this study, we addressed the therapeutic potential of fasudil, a potent Rho-kinase inhibitor, for VEGF-elicited angiogenesis and also for the intracellular signalings induced by VEGF. METHODS: In vitro, the inhibitory effects of fasudil on the VEGF-dependent VEGF receptor 2 (VEFGR2 or KDR), extracellular signal-related kinase (ERK) 1/2, Akt and myosin light chain (MLC) phosphorylation, as well as on the migration and proliferation of bovine retinal microvascular endothelial cells (BRECs) were analyzed with Western blotting, [3H]-thymidine uptake, and modified Boyden chamber assay. VEGF-elicited in vivo angiogenesis was analyzed with a mouse corneal micropocket assay coembedded with or without fasudil. RESULTS: VEGF caused enhanced MLC phosphorylation of BRECs, which was almost completely attenuated by 10microM fasudil. VEGF-dependent phosphorylation of ERK1/2 and Akt were also partially but significantly attenuated by treatment with fasudil without affecting VEGFR2 (KDR) phosphorylation. Moreover, both VEGF-induced [3H]-thymidine uptake and the migration of BRECs were significantly inhibited in the presence of fasudil. Finally, VEGF-elicited angiogenesis in the corneal micropocket assay was potently attenuated by coembedding with fasudil (P < 0.01). CONCLUSIONS: These findings indicate that fasudil might have a therapeutic potential for ocular angiogenic diseases. The antiangiogenic effect of fasudil appears to be mediated through the blockade not only of Rho-kinase signaling but also of ERK and Akt signaling.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Inibidores da Angiogênese/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Western Blotting , Bovinos , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neovascularização da Córnea/prevenção & controle , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: To investigate the intracellular events in retinal glial cells exposed to indocyanine green (ICG) and brilliant blue G (BBG). METHODS: The human Müller cell line MIO-M1 was exposed to a low dose (0.25 mg/mL) and a clinical dose (2.5 mg/mL) of ICG and a clinical dose (0.25 mg/mL) of BBG for 15 minutes, respectively. To quantify the proliferation and viability of the cells, [(3)H]-thymidine incorporation was measured and cell numbers were counted 24 hours after treatment. Cell morphology was evaluated using phase-contrast microscopy and transmission electron microscopy. The effects of ICG and BBG on phosphorylation of p38 MAPK and cleavage of caspase-9 and caspase-3 were examined by Western blot. RESULTS: ICG and BBG significantly reduced [(3)H]-thymidine incorporation in MIO-M1 cells compared with the vehicle-treated controls (P < 0.01). Cell number significantly decreased after exposure to ICG at 2.5 or 0.25 mg/mL (P < 0.01) but did not decrease after exposure to BBG at 0.25 mg/mL. Transmission electron microscopy revealed apoptotic changes only in the ICG-treated cells. Prominent p38 MAPK phosphorylation was observed in the presence of ICG, even at the low concentration and within a short time exposure; however, no apparent enhancement was observed in the presence of 0.25 mg/mL BBG. Furthermore, ICG, but not BBG, induced the cleavage of caspase-9 and caspase-3, which was inhibited by an inhibitor of p38 MAPK. CONCLUSIONS: ICG is toxic to retinal glial cells because it induces apoptosis, involving induction of the caspase cascade through p38 MAPK phosphorylation. In contrast, BBG does not cause apoptosis and thus could be a safer adjuvant during vitreoretinal surgery.
Assuntos
Verde de Indocianina/toxicidade , Neuroglia/efeitos dos fármacos , Retina/efeitos dos fármacos , Corantes de Rosanilina/toxicidade , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Caspase 9/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de Fase , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Retina/metabolismo , Retina/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia. METHODS: In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. RESULTS: Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 µmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 µmol/L and 6.8 to 14.8 µmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion. CONCLUSIONS: Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.
Assuntos
Endotélio Vascular/citologia , Isoquinolinas/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Sulfonamidas/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Endotélio Vascular/efeitos dos fármacos , Humanos , Hipóxia/tratamento farmacológico , Técnicas In Vitro , Isoquinolinas/administração & dosagem , Isoquinolinas/análise , Camundongos , Camundongos Endogâmicos C57BL , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Soluções Oftálmicas , Fosforilação/efeitos dos fármacos , Retina/química , Sulfonamidas/administração & dosagem , Sulfonamidas/análise , Espectrometria de Massas em Tandem , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: Plasma kallikrein is a serine protease and circulating component of inflammation, which exerts clinically significant effects on vasogenic edema. This study examines the role of plasma kallikrein in VEGF-induced retinal edema. METHODS: Intravitreal injections of VEGF and saline vehicle were performed in plasma prekallikrein-deficient (KLKB1-/-) and wild-type (WT) mice, and in both rats and mice receiving a selective plasma kallikrein inhibitor, VA999272. Retinal vascular permeability (RVP) and retinal thickness were measured by Evans blue permeation and optical coherence tomography, respectively. The retinal kallikrein kinin system was examined by Western blotting and immunohistochemistry. Retinal neovascularization was investigated in KLKB1-/- and WT mice subjected to oxygen-induced retinopathy. RESULTS: Vascular endothelial growth factor-induced RVP and retinal thickening were reduced in KLKB1-/- mice by 68% and 47%, respectively, compared to VEGF responses in WT mice. Plasma kallikrein also contributes to TNFα-induced retinal thickening, which was reduced by 52% in KLKB1-/- mice. Systemic administration of VA999272 reduced VEGF-induced retinal thickening by 57% (P < 0.001) in mice and 53% (P < 0.001) in rats, compared to vehicle-treated controls. Intravitreal injection of VEGF in WT mice increased plasma prekallikrein in the retina, which was diffusely distributed throughout the inner and outer retinal layers. Avascular and neovascular areas induced by oxygen-induced retinopathy were similar in WT and KLKB1-/- mice. CONCLUSIONS: Vascular endothelial growth factor increases extravasation of plasma kallikrein into the retina, and plasma kallikrein is required for the full effects of VEGF on RVP and retinal thickening in rodents. Systemic plasma kallikrein inhibition may provide a therapeutic opportunity to treat VEGF-induced retina edema.
Assuntos
Edema Macular/metabolismo , Calicreína Plasmática/metabolismo , Retina/patologia , Animais , Western Blotting , Permeabilidade Capilar , Injeções Intravítreas , Edema Macular/induzido quimicamente , Edema Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Calicreína Plasmática/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/fisiopatologia , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/intoxicaçãoRESUMO
This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P < 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry-based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P < 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P < 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor-induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.
Assuntos
Complicações do Diabetes/etiologia , Sistema Calicreína-Cinina/fisiologia , Calicreínas/metabolismo , Cininas/metabolismo , Edema Macular/etiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Ratos , Vasos Retinianos/patologia , Corpo Vítreo/químicaRESUMO
PURPOSE: Leukocyte adhesion releases tumor necrosis factor (TNF)-α that contributes to endothelial damage in early diabetic retinopathy (DR). Rho/Rho-kinase (ROCK) signaling mediates retinal endothelial damage in early DR. However, whether ROCK regulates TNF-α-mediated diabetic vascular damage is unknown. Here, the contribution of ROCK to TNF-α-mediated microvascular damage is investigated. METHODS: In DR patients and nondiabetic control subjects, the levels of membranous (m) TNF-α on neutrophils, soluble (s) TNF-α and its receptors in sera, were measured. In cultured microvascular endothelial cells, phosphorylation of myosin phosphatase target protein (MYPT)-1, a downstream target of ROCK, was investigated with TNF-α or DR sera pretreatment. TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) phosphorylation were measured with and without ROCK inhibition by fasudil or ROCK-specific small-interfering RNA (siRNA). In isolated neutrophils from control subjects, MYPT-1 phosphorylation was investigated in the presence of TNF-α. The impact of ROCK inhibition by fasudil on TNF-α-induced integrin (CD18, CD11a, CD11b) and intracellular cytoskeletal changes were investigated. RESULTS: The serum levels of mTNF-α, sTNF-α, and its receptors were significantly elevated in DR patients. TNF-α as well as DR sera promoted MYPT-1 phosphorylation in endothelial cells, which was significantly reduced by anti-TNF-α neutralizing antibody. TNF-α-induced ICAM-1 expression, eNOS dephosphorylation, cytoskeletal changes, and CD11b/18 expression in neutrophils were significantly suppressed by fasudil as well as ROCK-specific siRNA. CONCLUSIONS: ROCK is a key mediator of TNF-α signaling in diabetic microvessels. The important role of TNF-α in early DR provides a new rationale for ROCK inhibition beyond the previously shown mechanisms.
Assuntos
Angiopatias Diabéticas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Quinases Associadas a rho/fisiologia , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Retinopatia Diabética/metabolismo , Células Endoteliais , Endotélio Vascular/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Neutrófilos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
PURPOSE: A timely regression of the hyaloid vascular system (HVS) is required for the normal ocular development. Although macrophages have a critical role in this process, the exact mechanism remains undetermined. Periostin is a matricellular protein involved in tissue and vascular remodeling. The purpose of our study was to determine whether periostin is involved in the HVS regression. METHODS: We used wild type (WT) and periostin knockout (KO) mice. Indocyanine green angiography and immunohistochemistry with isolectin B4 were used to evaluate the HVS regression. TUNEL-labeling was used to quantify the number of apoptotic hyaloid vascular endothelial cells. F4/80 and Iba-1 staining was performed to determine the number and location of macrophages in the vitreous. The location of periostin also was investigated by immunohistochemistry. To determine the functional role of periostin, the degree of adhesion of human monocytes to fibronectin was measured by an adhesion assay. RESULTS: The HVS regression and peak in the number of TUNEL-positive apoptotic endothelial cells were delayed in periostin KO mice. The number of F4/80 positive cells in the vitreous was higher in periostin KO mice. Only a small number of Iba-1-positive cells near the hyaloid vessels was co-stained with periostin, and peripheral blood monocytes were not stained with periostin. Adhesion assay showed that periostin increased the degree of attachment of monocytes to fibronectin. CONCLUSIONS: These results suggest that periostin, which is secreted by the intraocular macrophages, enhances the HVS regression by intensifying the adhesion of macrophages to hyaloid vessels.
Assuntos
Moléculas de Adesão Celular/genética , Células Endoteliais/fisiologia , Vasos Retinianos/crescimento & desenvolvimento , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/crescimento & desenvolvimento , Animais , Apoptose/fisiologia , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Fibronectinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Macrófagos/citologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/fisiologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Vasos Retinianos/fisiologia , Corpo Vítreo/fisiologiaRESUMO
OBJECTIVE: Plasma kallikrein (PK) has been identified in vitreous fluid obtained from individuals with diabetic retinopathy and has been implicated in contributing to retinal vascular dysfunction. In this report, we examined the effects of PK on retinal vascular functions and thickness in diabetic rats. RESEARCH DESIGN AND METHODS: We investigated the effects of a selective PK inhibitor, ASP-440, and C1 inhibitor (C1-INH), the primary physiological inhibitor of PK, on retinal vascular permeability (RVP) and hemodynamics in rats with streptozotocin-induced diabetes. The effect of intravitreal PK injection on retinal thickness was examined by spectral domain optical coherence tomography. RESULTS: Systemic continuous administration of ASP-440 for 4 weeks initiated at the time of diabetes onset inhibited RVP by 42% (P = 0.013) and 83% (P < 0.001) at doses of 0.25 and 0.6 mg/kg per day, respectively. Administration of ASP-440 initiated 2 weeks after the onset of diabetes ameliorated both RVP and retinal blood flow abnormalities in diabetic rats measured at 4 weeks' diabetes duration. Intravitreal injection of C1-INH similarly decreased impaired RVP in rats with 2 weeks' diabetes duration. Intravitreal injection of PK increased both acute RVP and sustained focal RVP (24 h postinjection) to a greater extent in diabetic rats compared with nondiabetic control rats. Intravitreal injection of PK increased retinal thickness compared with baseline to a greater extent (P = 0.017) in diabetic rats (from 193 ± 10 µm to 223 ± 13 µm) compared with nondiabetic rats (from 182 ± 8 µm to 193 ± 9 µm). CONCLUSIONS: These results show that PK contributes to retinal vascular dysfunctions in diabetic rats and that the combination of diabetes and intravitreal injection of PK in rats induces retinal thickening.
Assuntos
Calicreína Plasmática/metabolismo , Retina/metabolismo , Retina/patologia , Animais , Proteína Inibidora do Complemento C1/farmacologia , Inibidores Enzimáticos/farmacologia , Angiofluoresceinografia , Fluorofotometria , Hemodinâmica/efeitos dos fármacos , Humanos , Masculino , Calicreína Plasmática/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: To determine the vitreous levels of soluble vascular endothelial growth factor receptor (sVEGFR)-1 in patients with various vitreoretinal diseases, and to investigate its correlation with patients' age and the activity of proliferative diabetic retinopathy (PDR). METHODS: Vitreous fluid samples were obtained from 187 eyes of 170 patients who underwent vitrectomy for the treatment of idiopathic macular hole (MH, n=30), branch retinal vein occlusion (BRVO, n=37), central retinal vein occlusion (CRVO, n=27), diabetic macular oedema (DME, n=42) and PDR (n=51). The levels of sVEGFR-1 in the vitreous were measured by ELISA. RESULTS: The levels of sVEGFR-1 (pg/ml) were not significantly different among each disease examined (MH 3900.1 ± 1188.9, BRVO 3969.7 ± 1741.6, CRVO 4897.7 ± 1717.7, DME 3856.21 ± 1374.7, PDR 4212.3 ± 1474.9). There was a significant positive correlation between vitreous concentrations of sVEGFR-1 and patients' age (r=0.430, p<0.01). The sVEGFR-1 concentration in subjects with active PDR was significantly lower than in those with quiescent PDR (p<0.0001), even after being adjusted for age (p<0.0001). CONCLUSIONS: Vitreous concentrations of sVEGFR-1 increase with advancing age and are associated with quiescent rather than active PDR even after adjustment for age.