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Objectives: The objectives of this study were to (i) evaluate the prevalence of pre-iridal monocellular and fibrovascular membranes in canine globes affected with congenital glaucoma associated with anterior segment dysgenesis (ASD), primary glaucoma associated with goniodysgenesis (GD), and secondary glaucoma, and (ii) examine the associations between monocellular and fibrovascular membranes by breed, gender, age and histopathologic ocular changes on light microscopic examination. Methods: Records of dogs who had eyes enucleated due to blindness and uncontrolled glaucoma were reviewed. Glaucoma was categorized clinically and histologically into three groups: congenital/ASD, primary/GD, and secondary glaucoma. The presence or absence and type of pre-iridal membrane (monocellular or fibrovascular) and other intraocular histologic findings were reviewed and compared statistically for each group. Results: In total, 108 canine globes (101 dogs) were included. Pre-iridal monocellular membranes were identified with light microscopy in 10 out of 19 congenital/ASD, 29 out of 40 primary, and 23 out of 49 secondary glaucoma globes. Fibrovascular membranes were observed in 3 out of 19 congenital/ASD, 9 out of 40 in primary, and 24 out of 49 secondary glaucoma globes. There were no associations between the type of membrane and breed, gender, or age. Peripheral anterior synechiae were more common in globes with fibrovascular membranes, and uveal atrophy was more common in globes with monocellular membranes. Conclusion: Pre-iridal monocellular membranes are common in all types of canine glaucoma. They are identified with light microscopy most easily in cases of primary glaucoma, and they are masked by pre-iridal fibrovascular membranes in other forms of glaucoma.
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AIM: To assess the effects of intravitreal ranibizumab (IVR) on angiogenesis and glial activity of the fibrovascular membrane (FVM) in patients with proliferative diabetic retinopathy (PDR). METHODS: Forty-two eyes from 42 patients with PDR requiring vitrectomy were included and divided into two groups: control group (n=16) did not receive IVR, while IVR group (n=26) underwent IVR 5d before vitrectomy. FVM specimens were collected by the same surgeon during the interventions. Histopathological morphology was examined by hematoxylin-eosin (H-E) staining and cell densities in the FVM was assessed. Microvessels were outlined by immunohistochemical staining of CD31 and microvessel density (MVD) assessed as an index of FVM angiogenesis. Dual-color immunofluorescence staining, and confocal microscopy was used to detect co-localization and relative expression levels of glial fibrillary acidic protein (GFAP) and α-smooth muscle actin (α-SMA) as markers of glial-mesenchymal transition (GMT). The GMT index (GI; ratio of relative GFAP/α-SMA expression) was used to semi-quantify the degree of GMT or glial activity of FVMs. RESULTS: H-E staining showed similar vascularization in both groups, with microvessels and scattered stromal cells in the matrix. Infiltrated cell densities did not differ significantly between the two groups (P>0.05). The MVD of the IVR group (130.62±15.46/mm2) was significantly lower than that of the controls (142.25±19.16/mm2, P<0.05). In both groups, all sections were strongly immunostained for GFAP and α-SMA. The Pearson's correlation coefficients (PCC) of intensity of automated pixel count of two markers indicated GFAP and α-SMA co-stained well and GMT participated in the remolding of FVMs in PDR. The mean relative GFAP expression in the IVR group was significantly lower, whereas that of α-SMA was significantly higher than in controls (P<0.05). GI in the IVR group (1.10±0.10) was significantly lower than in the controls (1.21±0.12, P<0.05). CONCLUSION: IVR can reduce angiogenesis, glial activity of FVM and promote glial-fibrotic transformation by reducing MVD and promoting GMT but does not decrease the cell density in patients with PDR.
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PURPOSE: Diabetic retinopathy is a leading cause of vision impairment. Surging diabetic population and poor visual care raises the need for better diagnostic tools. Hence, it is worthwhile to look for biomarkers associated with the disease pathogenesis. Periostin and tenascin-C are matricellular proteins mediating fibrillogenesis in retinopathy. Their serum levels and association with the presence and severity of retinopathy in diabetics is of importance to be explored. METHODS: The study involved two groups of type 2 diabetes patients, 38 controls without retinopathy and 38 cases with retinopathy. We obtained serum sample and performed biochemical autoanalysis for routine parameters. Special parameters periostin, tenascin-C, and C-peptide were estimated by ELISA. RESULTS: Periostin and tenascin-C were significantly elevated in the retinopathy group. Periostin progressively increased among subgroups. C-peptide decreased significantly in retinopathy group and had a negative correlation with duration of DM, duration of retinopathy, HbA1c and tenascin-C. We observed a positive correlation for periostin and tenascin-C with duration of diabetes. The AUC for C-peptide was the highest (0.750) amongst our parameters. HOMA 2 (%B) index was significantly lower in retinopathy group. CONCLUSIONS: Serum Levels of PO and TnC increased in retinopathy. As the disease advances, periostin level increases, indicating continuing fibrosis and fibrovascular membrane formation. Periostin and tenascin-C increase with duration of retinopathy whereas levels of C-peptide decrease. C-peptide has a better differentiating potential for DR from DM. Reduced insulin production as indicated by declined HOMA 2-%BETA in retinopathy favors hyperglycemia and chronic inflammatory state for the disease progression.
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Diabetes Mellitus Tipo 2 , Retinopatía Diabética , Péptido C , Diabetes Mellitus Tipo 2/complicaciones , Retinopatía Diabética/metabolismo , Fibrosis , Humanos , Tenascina/metabolismoRESUMEN
PURPOSE: The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting. MATERIALS AND METHODS: Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring ß-tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients. RESULTS: Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs. CONCLUSION: Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR.
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Diabetes Mellitus , Retinopatía Diabética , Animales , Retinopatía Diabética/metabolismo , Humanos , Ratones , Proyección Neuronal , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Cuerpo Vítreo/metabolismoRESUMEN
OBJECTIVES: (i) To evaluate immunohistochemical labeling of pre-iridal monocellular and fibrovascular membranes and (ii) describe the light and scanning electron microscopic (SEM) characteristics of these membranes in glaucomatous and normal/control canine globes. MATERIALS AND METHODS: All globes were evaluated with light microscopy. Immunohistochemical labeling for CD18, Smooth muscle actin (SMA), and CD117 was completed on 40 canine globes with congenital/anterior segment dysgenesis-associated glaucoma (n = 10), primary/goniodysgenesis-associated glaucoma (n = 10), secondary glaucoma (n = 10), and normal/control globes (n = 10). SEM was completed on 10 globes: 5 with monocellular membranes, 3 with fibrovascular membranes, and 2 without a histologically detectable membrane. RESULTS: Monocellular membranes were detected in all normal/control globes with light microscopy and appeared to be morphologically very similar to those in diseased globes. CD18 labeling was detected in 9/10 monocellular membranes in normal/control globes, 15/23 monocellular, and 7/8 fibrovascular membranes in globes with glaucoma. SMA and CD117 labeling was not detected in monocellular membranes of normal/control globes. SMA was expressed in 10/23 monocellular and 7/8 fibrovascular membranes of glaucomatous globes. CD117 was expressed in 7/23 monocellular and 5/8 fibrovascular membranes of glaucomatous globes. SEM of monocellular membranes revealed a continuous sheet of mostly spindle cells and few individual round cells that extended over the anterior iris face in normal/control and all glaucomatous globes. CONCLUSION: Pre-iridal monocellular membranes are a normal component of the anterior iris surface, and CD18 immunoreactivity suggests some cells within these are of leukocytic origin. SMA and CD117 labeling of monocellular membranes in glaucomatous, but not normal/control globes, suggest metaplastic cellular change secondary to intraocular pathology related to glaucoma.
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Membrana Celular/ultraestructura , Enfermedades de los Perros/patología , Endotelio Vascular/ultraestructura , Glaucoma/veterinaria , Iris/ultraestructura , Actinas/ultraestructura , Animales , Antígenos CD18 , Perros , Membrana Epirretinal/patología , Glaucoma/patología , Inmunohistoquímica/veterinaria , Microscopía Electrónica de Rastreo/veterinariaRESUMEN
PURPOSE: The aim of this study was to investigate the relationship between erythropoietin rs1617640 polymorphism and proliferative diabetic retinopathy (PDR) in Slovenian subjects with type 2 diabetes mellitus. The second aim was to find whether erythropoietin expression in fibrovascular membranes varies among individuals carrying different genotypes of the rs1617640. METHODS: This was a retrospective cross-sectional study based on 797 unrelated Slovenian (Caucasian) participants with type 2 diabetes mellitus. The study group consisted of 217 cases with PDR and 580 controls without clinical signs of diabetic retinopathy. Each subject was genotyped for rs1617640 polymorphism. Fibrovascular membranes from 27 subjects who underwent vitreoretinal surgery were analysed with immunohistochemistry. We searched for expression of erythropoietin, its cognate receptor and for a pan-endothelial marker CD-34. RESULTS: Our results show that subjects carrying a minor GG genotype had significantly higher risk for PDR in both unadjusted (p = 0.02) and adjusted (p = 0.04) recessive genetic models. Subjects with the GG genotype had a 1.6-fold increased risk of developing PDR compared to subjects carrying the major T allele. In fibrovascular membranes from subjects with PDR, the mean number of cells expressing EPO was significantly higher in G allele carriers compared to the homozygotes for the common T allele. CONCLUSION: In Slovenian subjects with type 2 diabetes mellitus, a significant increased risk of PDR was found in GG carriers of the erythropoietin gene rs1617640 polymorphism.
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ADN/genética , Diabetes Mellitus Tipo 2/complicaciones , Retinopatía Diabética/genética , Eritropoyetina/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Alelos , Estudios Transversales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/epidemiología , Retinopatía Diabética/etiología , Eritropoyetina/metabolismo , Femenino , Estudios de Seguimiento , Frecuencia de los Genes , Genotipo , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Eslovenia/epidemiología , Factores de Tiempo , Adulto JovenRESUMEN
The transparency of the eye can be disturbed by several eye diseases. It has recently been reported that periostin plays pivotal roles in the pathogenesis of several eye disease, such as diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma, pterygia, corneal dystrophy, and chronic ocular allergic diseases. In these diseases, formation of fibro (vascular) tissue plays an important role. Gene expression profiling of human retinal fibro (vascular) membrane revealed significant up-regulation of periostin. The expression of periostin after environmental perturbations, including IL-4 and/or IL-13 induction, can alter normal physiological interactions among fibroblasts, macrophages and ECM protein in the eye. Modulating the expression of periostin by low-molecular weight compounds, antibodies or RNAi directed against the molecule could be a novel therapeutic strategy for inhibiting the progression of those periostin-involved eye diseases.
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Moléculas de Adhesión Celular/fisiología , Oftalmopatías/fisiopatología , Retinopatía Diabética , Humanos , Degeneración Macular , Interferencia de ARNRESUMEN
Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-ß2 (TGF-ß2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases.
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Moléculas de Adhesión Celular/genética , Neovascularización Coroidal/genética , Retinopatía Diabética/genética , Degeneración Macular/genética , Vitreorretinopatía Proliferativa/genética , Actinas/genética , Actinas/inmunología , Animales , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/inmunología , Neovascularización Coroidal/inmunología , Neovascularización Coroidal/patología , Neovascularización Coroidal/terapia , Retinopatía Diabética/inmunología , Retinopatía Diabética/patología , Retinopatía Diabética/terapia , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Degeneración Macular/inmunología , Degeneración Macular/patología , Degeneración Macular/terapia , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Retina/inmunología , Retina/patología , Transducción de Señal , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/inmunología , Vitreorretinopatía Proliferativa/inmunología , Vitreorretinopatía Proliferativa/patología , Vitreorretinopatía Proliferativa/terapia , Cuerpo Vítreo/inmunología , Cuerpo Vítreo/patologíaRESUMEN
PURPOSE: The aim of this study was to examine the role of the rs6060566 polymorphism of the reactive oxygen species modulator 1 (Romo-1) gene in the development of diabetic retinopathy (DR) in Caucasians with type 2 diabetes (T2DM). Moreover, another aim was to investigate the effect of Romo-1 genotypes on Romo-1 expression in fibrovascular membranes from patients with proliferative DR. METHODS: A total of 806 subjects with T2DM were enrolled in cross-sectional case-control study: 278 patients with DR and 528 subjects without clinical signs of DR. Genetical analysis was performed in 806 subjects with T2DM. Moreover, immunohistochemical analysis of 40 fibrovascular membranes of patients with proliferative DR was performed. The number of positive (labelled) cells per area - numerical areal density of the Romo-1-positive cells (the number of positive cells/mm(2) ) - was calculated. RESULTS: A significantly higher frequency of the CC genotype of the rs6060566 polymorphism of the Romo-1 gene was found in subjects with T2DM with DR compared to those without DR (odds ratio=3.3, 95% confidence interval=1.1-8.8; p = 0.024). Moreover, the Romo-1 C allele was found to effect Romo-1 expression in fibrovascular membranes of patients with proliferative DR. CONCLUSIONS: The rs6060566 polymorphism of the Romo-1 gene was found to be an independent risk factor for DR in Caucasians with T2DM. Moreover, the rs6060566 is most probably functional and its effect might be mediated through the increased expression of Romo-1 in the retina.
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Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Polimorfismo de Nucleótido Simple , Población Blanca/genética , Anciano , Glucemia/metabolismo , Estudios de Casos y Controles , Creatinina/sangre , Estudios Transversales , Diabetes Mellitus Tipo 2/etnología , Retinopatía Diabética/etnología , Femenino , Genotipo , Técnicas de Genotipaje , Hemoglobina Glucada/metabolismo , Humanos , Inmunohistoquímica , Lípidos/sangre , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Reacción en Cadena de la Polimerasa , Factores de RiesgoRESUMEN
PURPOSE: Diabetic retinopathy (DR) has features of chronic low-grade inflammation. The purpose of our study was to investigate whether the presence of inflammatory cells in fibrovascular membranes (FVMs) from patients with proliferative diabetic retinopathy (PDR) is associated with the activity of PDR and visual acuity improvement after vitreoretinal surgery. METHODS: Forty FVMs from 40 patients with PDR were obtained during vitrectomy, prepared by using the agar sandwich method, and examined using light microscope and immunohistochemistry methods to define the presence and density of inflammatory cells: CD45+ cells (leukocytes), CD4+ cells (T helper lymphocytes), CD8+ cells (T cytotoxic lymphocytes), CD19+ cells (B lymphocytes), and CD14+ cells (monocytes/macrophages). For each FVM, the inflammatory cell density defined as numerical areal density was calculated. The number of vessels was defined as the volume density of vessels. RESULTS: Among 40 patients with PDR, 33 patients had active PDR and 7 quiescent PDR. Significant differences in cell densities for CD4+, CD8+, and CD19+ cells were observed between patients with active and quiescent PDR. B lymphocytes were present in membranes of active PDR only. No correlation was observed between numerical areal density of inflammatory cells and the volume density of vessels. No association was found between visual acuity improvement after surgery and cell densities. CONCLUSIONS: Lymphocyte infiltration of FVMs might be associated with the activity of retinopathy but not with visual acuity improvement after surgery.