Retinal Capillary Basement Membrane Research Articles

Ten differentially expressed proteins in the tear film of dogs with diabetic retinopathy

AbstractPurposeA common early lesion of diabetic retinopathy in human patients and a diverse range of animal models is retinal capillary basement membrane thickening occur within 6 months of induction of diabetes. This hallmark pathology of diabetic microvascular disease is used in the evaluation of drug‐mediated modulation of diabetic retinopathy in a range of animal models. Moreover results from published clinical practice have shown that more than 22% of dogs with naturally contracted diabetes mellitus develop ophtalmoscopic signs of retinal hemorrhages or microaneurysms in a relatively short time from onset of the disease. Therefore, we hypothesize that changes in the retina in such dogs results in changes in the protein profile of tear fluid, that can be of the diagnostic or therapeutic value and can contribute to the better understanding the pathophysiology mechanisms underlying that disease. The objective of this study was to identify the protein content of tear film in dogs with naturally contracted diabetes mellitus with and/ without retinopathy, and its comparison with the content of control group. That approach results with mapping out the comprehensive proteome of tear film in diabetic dog and the may contribute to development a noninvasive method to detect potential protein candidates for diabetic retinopathy biomarkers in tear film.MethodsTear film was collected with Schirmer strips from the canine diabetic patients with/ and without retinopathy and sex‐ and age‐matched control group.The study was conducted on 24 dogs (8 dogs in each group).Two‐dimensional electrophoresis was performed followed by MALDI‐TOF mass spectrometry identification of differential proteins. Quantitative analysis of the differentiating electrophoretic spots was done with 2Delta software. Metabolic pathways associated with those proteins were identified with Panther GO software.ResultsAltogether, 5 significantly differentially expressed proteins were identified. Of those, 4 were downregulated, and 1 was upregulated in the tear film of diabetic patients with retinopathy. Pathway analysis revealed that the proteins were associated with multiple pathways involved in biological regulation, cellular and metabolic process and cellular component organization.ConclusionsTear film protein analysis provides a novel way to screen diabetic retinopathy biomarkers in dogs.

Retinal capillary basement membrane thickening: Role in the pathogenesis of diabetic retinopathy.

Vascular basement membrane (BM) thickening has been hailed over half a century as the most prominent histological lesion in diabetic microangiopathy, and represents an early ultrastructural change in diabetic retinopathy (DR). Although vascular complications of DR have been clinically well established, specific cellular and molecular mechanisms underlying dysfunction of small vessels are not well understood. In DR, small vessels develop insidiously as BM thickening occurs. Studies examining high resolution imaging data have established BM thickening as one of the foremost structural abnormalities of retinal capillaries. This fundamental structural change develops, at least in part, from excess accumulation of BM components. Although BM thickening is closely associated with the development of DR, its contributory role in the pathogenesis of DR is coming to light recently. DR develops over several years before clinical manifestations appear, and it is during this clinically silent period that hyperglycemia induces excess synthesis of BM components, contributes to vascular BM thickening, and promotes structural and functional lesions including cell death and vascular leakage in the diabetic retina. Studies using animal models show promising results in preventing BM thickening with subsequent beneficial effects. Several gene regulatory approaches are being developed to prevent excess synthesis of vascular BM components in an effort to reduce BM thickening. This review highlights current understanding of capillary BM thickening development, role of BM thickening in retinal vascular lesions, and strategies for preventing vascular BM thickening as a potential therapeutic strategy in alleviating characteristic lesions associated with DR.

Osteopontin Upregulates Col IV Expression by Repressing miR-29a in Human Retinal Capillary Endothelial Cells

Abnormal synthesis of extracellular matrix (ECM), especially collagen type IV (Col IV), in human retinal capillary endothelial cells (HRCECs) and resultant basement membrane (BM) thickening is the most prominent and characteristic feature of early diabetic retinopathy (DR). Osteopontin (OPN) has been shown to play an important role in the pathogenesis of DR and specifically, found to be critically involved in diabetic nephropathy, as it can upregulate many factors, like collagen IV. However, the precise role of OPN in the pathogenesis of DR and the underlying mechanisms remain unclear. In this study, 51 differentially expressed microRNAs (miRNAs; 42 miRNAs upregulated and 9 miRNAs downregulated) were first identified in retina of streptozotocin (STZ)-induced diabetic mice with DR. Among these miRNAs, we identified miRNA (miR)-29a as a prominent miRNA that targeted and directly downregulated Col IV expression through database prediction and dual-luciferase reporter assay, which was further confirmed in HRCECs using miR-29a mimic, miR-29a inhibitor, and pre-miR-29a transfection. Furthermore, OPN upregulated Col IV expression via a miR-29a-repressed pathway in HRCECs. Taken together, these results provided a miR-29a-repressing mechanism through which OPN plays roles in abnormal synthesis of Col IV in HRCECs and resultant BM thickening, contributing to the pathogenesis of DR.

Decreased lysyl oxidase level protects against development of retinal vascular lesions in diabetic retinopathy

Retinal capillary basement membrane (BM) thickening is closely associated with the development of vascular lesions in diabetic retinopathy. Thickened capillary BM can compromise blood-retinal-barrier characteristics and contribute to retinal vascular permeability, a significant clinical manifestation of diabetic retinopathy. We have previously shown that high glucose increases the expression and activity of lysyl oxidase (LOX), a crosslinking enzyme, in retinal endothelial cells. Additionally, concomitant with overexpression of LOX, increased vascular permeability was observed in diabetic rat retinas. However, it is unknown whether decreasing LOX overexpression may have protective effects against development of retinal vascular lesions in diabetes. To investigate whether reduced LOX level protects against diabetes-induced development of retinal vascular lesions characteristic of diabetic retinopathy, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, LOX +/− mice, and STZ-induced diabetic LOX +/− mice were used for this study. Diabetes was maintained for 16 weeks; at the end of the study, retinas were assessed for LOX protein level by Western Blot (WB) analysis, and retinal capillary networks were isolated using retinal trypsin digestion and stained with hematoxylin and periodic acid Schiff to identify the number of acellular capillaries (AC) and pericyte loss (PL). In parallel, TUNEL assay was performed on retinal trypsin digests (RTDs) to detect cells undergoing apoptosis in the retinal capillary networks. Retinal vascular permeability was analyzed following FITC-dextran injection in retinal whole mounts. A significant increase in LOX expression was detected in the diabetic retinas compared to those of the WT control retinas, and as expected, a significant decrease in LOX expression in the diabetic LOX +/− retinas was observed compared to those of the diabetic retinas. RTD images showed significantly increased AC and PL counts in the retinas of diabetic mice compared to those of the WT control mice. Importantly, the number of AC and PL was significantly decreased, as was retinal vascular permeability in the retinas of the diabetic LOX +/− mice compared to those of the diabetic mice. Results suggest that decreasing diabetes-induced LOX overexpression may have protective effects against the development of vascular lesions characteristic of diabetic retinopathy. Therefore, LOX overexpression may be a potential target in preventing retinal vascular cell loss and excess permeability associated with diabetic retinopathy.

Fenofibrate Ameliorates Oxidative Stress-Induced Retinal Microvascular Dysfunction in Diabetic Rats

ABSTRACTPurpose: The aim of this study is to investigate the effects of fenofibrate (FA) on microvascular dysfunction in the retina of diabetic rats, and to determine the underlying mechanism.Methods: Streptozotocin (STZ)-induced diabetic rats and control rats were used in this study. A subgroup of STZ-induced diabetic rats was treated orally with vehicle or FA (100 mg/kg/day) for 24 weeks along with regular monitoring of body weight and serum parameters. At the end of the 24-week treatment, retinal vascular permeability was quantified by confocal microscopy using Evans blue as a tracer. Retinal capillary basement membrane thickness (BMT) was examined by transmission electron microscopy. The retinal reactive oxygen species (ROS) level was quantified by flow cytometry using a fluorescent probe. Levels of vascular endothelial growth factor (VEGF), nuclear facto (NF)-κB (p65), and thioredoxin interacting protein (TXNIP) were measured by Western blotting or enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction.Results: Retinal vascular permeability and BMT were significantly increased in diabetic rats compared to in non-diabetic control rats. Diabetes also increased the retinal ROS levels. These effects were associated with increased levels of VEGF, phosphorylation of p65(P-p65), and TXNIP. FA significantly ameliorated the retinal vascular permeability, alleviated retinal BMT, and reduced retinal ROS level. Consistent with these effects, FA also decreased VEGF and P-p65 expression and, at the same time, decreased TXNIP expression.Conclusions: FA prevents the retinal microvascular dysfunction induced by diabetes, likely by restoring VEGF and P-p65 levels, and possibly by reducing oxidative stress and TXNIP expression.

Curcumin Alleviates Diabetic Retinopathy in Experimental Diabetic Rats

Purpose: To investigate the potential protective effects of curcumin on the retina in diabetic rats. Methods: An experimental diabetic rat model was induced by a low dose of streptozotocin combined with a high-energy diet. Rats which had blood glucose levels ≥11.6 mmol/L were used as diabetic rats. The diabetic rats were randomly divided into 3 groups: diabetic rats with no treatment (DM), diabetic rats treated with 100 mg/kg curcumin (DM + Cur 100 mg/kg), and diabetic rats treated with 200 mg/kg curcumin (DM + Cur 200 mg/kg). Curcumin was orally administered daily for 16 weeks. After 16 weeks of administration, the rats were euthanized, and eyes were dissected. Retinal histology was examined, and the thickness of the retina was measured. Ultrastructural changes of retinal ganglion cells, inner layer cells, retinal capillary, and membranous disks were observed by electron microscopy. Malondialdehyde, superoxide dismutase, and total antioxidant capacity were measured by ELISA. Expression levels of vascular endothelial growth factor (VEGF) in retina tissues were examined by immunohistochemical staining and ELISA. Expression levels of Bax and Bcl-2 in retina tissues were determined by immunohistochemical staining and Western blotting. Results: Curcumin reduced the blood glucose levels of diabetic rats and decreased diabetes-induced body weight loss. Curcumin prevented attenuation of the retina in diabetic rats and ameliorated diabetes-induced ultrastructure changes of the retina, including thinning of the retina, apoptosis of the retinal ganglion cells and inner nuclear layer cells, thickening of retinal capillary basement membrane and disturbance of photoreceptor cell membranous disks. We also found that curcumin has a strong antioxidative ability in the retina of diabetic rats. It was observed that curcumin attenuated the expression of VEGF in the retina of diabetic rats. We also discovered that curcumin had an antiapoptotic effect by upregulating the expression of Bcl-2 and downregulating the expression of Bax in the retina of diabetic rats. Conclusions: Taken together, these results suggest that curcumin may have great therapeutic potential in the treatment of diabetic retinopathy which could be attributed to the hypoglycemic, antioxidant, VEGF-downregulating and neuroprotection properties of curcumin.

Identification of structural alerts for substances with PBT/vPvB potential

Rats fed a high-galactose diet develop marked thickening of their retinal capillary basement membranes. The effect is prevented if the animals also receive the aldose reductase inhibitor sorbinil. The effect does not appear to be due to aldose reductase itself, since immunoreactive aldose reductase has not been found in the retinal microvasculature of the rat but rather to a related enzyme with similar substrate specificity. The detailed biochemical mechanism for basement membrane thickening is obscure, involving an alteration of the extracellular matrix, where aldose reductase and similar enzymes have not been described; osmotic damage to the microvascular cells, such as has been described following aldose reductase-induced sugar alcohol accumulation in lens epithelial cells, is not apparent in diabetic or galactosemic animals. It is possible that concentrations of intracellular sugar alcohols that do not substantially change the osmolarity of the cell cytosol alter intracellular enzyme activities. This, in turn, could affect the biosynthesis of extracellular matrix macromolecules, as suggested, for example, by the hypothesis of Rohrbach et al, based on studies of a basement membrane-producing tumor implanted in diabetic mice, which proposes that the hyperglycemia of diabetes mellitus causes a reduced synthesis of the heparan sulfate BM-1 proteoglycan with a subsequent overproduction of type IV collagen. This and other hypotheses of basement membrane thickening can be tested in diabetic or galactosemic rats, some of which receive aldose reductase inhibitors, or in retinal microvascular pericytes and endothelial cells grown in culture.

Transmission Electron Microscopy Studies of Retinal Capillary Basement Membrane Thickening in a Galactose Induced Model of Diabetic Retinopathy

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

The effects of aminoguanidine on retinopathy in STZ-induced diabetic rats

BackgroundThe accumulation of advanced glycated end products (AGEs) in retinal blood vessels is one of the major etiological factors contributing to diabetic retinopathy. Aminoguanidine (AG) is one of the most extensively used inhibitors of AGEs formation. The aim of this study was to investigate whether AG could protect the development of diabetic retinopathy through inhibition of AGEs. MethodsRat diabetes was induced by intraperitoneal injection with streptozotocin (STZ). AG was given to rats in drinking water. Retina was extracted 3 and 6months following STZ and AG administration. Immunochemistry and transmission electron microscope were used to detect the expression of AGEs and retina morphology. ResultsExtensive staining of AGEs was detected in retinal blood vessels of 3- and 6-month diabetic rats, while no significant staining was found in the control non-diabetic retina or AG treated groups. Pericyte loss, endothelial cell proliferation, increased ratio of endothelial cells/pericytes, acellular capillaries and capillary occlusion were observed in the retina of 6-month diabetic rats. The increased electron density of retinal capillary basement membrane, mitochondrial swelling in pericytes and endothelial cells were also found in 6-month diabetic rats. The 3-month diabetic rats and the AG-treated rats did not have similar morphological changes compared to control group. The AGEs staining in AG-treated rats was still weakly positive. ConclusionsAGEs plays pivotal roles in diabetic retinopathy. AGE deposition occurs prior to retinal microvasculature changes. AG could prevent the onset and development of diabetic retinopathy through inhibition of AGEs.

Green Tea Prevents Hyperglycemia-Induced Retinal Oxidative Stress and Inflammation in Streptozotocin-Induced Diabetic Rats

Purpose: Our objective was to investigate the effect of green tea (GT) on diabetes-induced retinal oxidative stress and proinflammatory parameters in rats. Methods: Treatment (200 mg/kg body weight) was carried out for a period of 16 weeks in streptozotocin-induced diabetic rats and was evaluated for hypoglycemic, antioxidant [reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT)] and anti-inflammatory [tumor necrosis factor (TNF) α, vascular endothelial growth factor (VEGF)] activity. Histological changes were evaluated by transmission electron microscopy. Results: Retinal GSH levels were 1.5-fold lower in diabetic rats as compared to normal rats (p < 0.05). However, in GT-treated rats, retinal GSH levels were restored close to those of the normal group. The antioxidant enzymes SOD and CAT showed a more than 2-fold decrease in activity in diabetic retinae as compared to normal retinae (p < 0.05). Both SOD and CAT enzymatic activities were restored close to normal in the GT-treated group. Expression of proinflammatory parameters (TNF-α and VEGF) was significantly inhibited in GT-treated retinae as compared to diabetic retinae (p < 0.05). Moreover, GT treatment prevented retinal capillary basement membrane thickness. Conclusion: The beneficial effects of GT suggest its potential role in the prevention and treatment of diabetic retinopathy in human subjects.

Significant Retinal Capillary Basement Membrane Thickening in Hyperglycemic and Normoglycemic Diabetic-prone (DP) BB Wistar Rats

The diabetic-prone BioBreeding Wistar (BB/DP) rat is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 90% of the animals (BB/DPh) are hyperglycemic by 90–120 days of age, while the remaining ~10% (BB/DPn) and diabetes-resistant rats (BB/DR) are normoglycemic for life. The transmission electron microscope data from this study demonstrate expected significant age- and diabetes-related increases in retinal capillary basement membrane (RCBM) widths in (BB/DPh) rats relative to BB/DR animals. However, the data show, for the first time, an unexpected significant RCBM thickening in (BB/DPn) rats compared to BB/DR animals at 6 months and 1 year post-onset of hyperglycemia.

Diabetic eNOS-Knockout Mice Develop Accelerated Retinopathy

Dysfunction of endothelial nitric oxide synthase (eNOS) has been implicated in the pathogenesis of diabetic vascular complications. This study was undertaken to determine the role of eNOS in the development of diabetic retinopathy (DR), by investigating the functional consequences of its deficiency in the diabetic state. Diabetes was induced in eNOS-knockout (eNOS(-/-)) and C57B/6 mice by streptozotocin (STZ) injection. Retinal vasculature was evaluated by albumin extravasation, to quantitatively measure vascular permeability, and by trypsin-digested retinal vascular preparations, to quantify acellular capillaries. Gliosis was evaluated by immunofluorescent techniques. Retinal capillary basement membrane thickness was assessed by transmission electron microscopy. Total retinal nitric oxide level was assessed by measuring nitrate/nitrite using a fluorometric-based assay, iNOS expression was examined by real-time PCR. Diabetic eNOS(-/-) mice exhibit more severe retinal vascular permeability than age-matched diabetic C57BL/6 mice, detectable as early as 3 weeks after diabetes induction. Diabetic eNOS(-/-) mice also show earlier onset and an increased number of acellular capillaries, sustained gliosis, and increased capillary basement membrane thickness. Total nitric oxide (NO) level was also increased, concomitant with elevated iNOS expression in diabetic eNOS(-/-) retina. Diabetic eNOS(-/-) mice exhibit A significantly wider range of advanced retinal vascular complications than the age-matched diabetic C57BL/6 mice, supporting the notion that eNOS-derived NO plays an essential role in retinal vascular function. This mouse model also faithfully replicates many of the hallmarks of vascular changes associated with human retinopathy, thus providing a unique model to aid in understanding the pathologic mechanisms of and to develop effective therapeutic strategies for diabetic retinopathy.

Ultrastructural Investigation of the Retinal Changes in Diabetic Rat (OLETF)

Purpose: To compare retinal ultra-structures of diabetic rats (OLETF, Otsuka Long-Evans Tokushima Fatty) with those of agematched non-diabetic rats (LETO, Long-Evans Tokushima Otsuka) using transmission electron micrography (TEM). Methods: The body weights and blood sugar levels of the OLETF rats and LETO rats (n=5) were measured at 10 and 50 weeks of age. Using a TEM, we compared the ultra-structural changes between the retinas of the 50-week-old OLETF and LETO rats. We analyzed the sizes of the pericytes and the thicknesses of the retinal capillary basement membranes between the two groups. Comparisons were made using a Scion Image. Results: The mean body weight and blood sugar levels of the 50-week-old OLETF rats were significantly higher than those of the LETO rats (p

Ultrastructural Features of Retinal Capillary Basement Membrane Thickening in Diabetic Swine

Purpose: To assess retinal capillary basement membrane thickening (BMT) in a swine model of type 1 diabetes. Materials and methods: Yorkshire pigs were rendered diabetic with streptozotocin and dyslipidemic with a high fat and cholesterol diet. At 18, 26, and 32 weeks of diabetes, the retina sections within 3 disc diameters from the optic disc were examined under transmission electron microscopy to evaluate the ultrastructural features of the capillary BM. Digital morphometric analysis was performed to measure BMT. Results: Diabetic swine had significantly thicker retinal capillary BMs compared to controls. Pigs that sustained diabetes for longer periods or experienced severe diabetes tended to have more BMT. Those pigs that did not sustain glucose levels above 200 mg/dL did not demonstrate thicker retinal capillary BMs. Characteristic ultrastructural features of diabetic vasculopathy observed included rarefaction as an early stage of Swiss cheese cavitation, lamellation with multiplication of electron dense layers, and fibrillar materials within capillary BM. Conclusions: Diabetic Yorkshire pigs develop characteristic features of an early retinal microvasculopathy fairly rapidly and may serve as a higher-order animal model for studies of type 1 diabetes.

Effects of Stobadine and Vitamin E in Diabetes-Induced Retinal Abnormalities: Involvement of Oxidative Stress

Because hyperglycemia-induced oxidative stress may be a cause of retinopathy, this study examined the hypothesis that administration of exogenous antioxidants, stobadine (ST) and vitamin E (vitE), can restore retinal abnormalities in experimental diabetes. Normal and streptozotocin (STZ)-induced male Wistar rats received daily intraoral doses of ST (24.7 mg/kg) and vitE (alpha-dl-tocopherol acetate, 400-500 IU/kg) individually or in combinations for 8 months. The biochemical parameters including aldose reductase enzyme (AR) activity and lipid peroxidation (MDA), and histopathological changes such as retinal capillary basement membrane thickness (RCBMT) and vascular endothelial growth factor (VEGF) expression were evaluated. A 37.99% increase in RCBMT was observed in rats after 8 months diabetes duration. The increase in RCBMT was 12.34% in diabetic rats treated with ST and 23.07% in diabetic rats treated with vitE. In diabetic rats treated with antioxidant combination, just a 4.38% increase was observed in RCBMT. The excess VEGF immunoreactivity and increased MDA and AR activity determined in diabetic retina were significantly attenuated by individual antioxidant treatments. Although both antioxidants decreased blood glucose, HbA1c, fructosamine and triglyceride levels in diabetic rats, poor glycemic control was maintained in all experimental groups during the treatment period. However, the antioxidant combination led to almost complete amelioration in retinal MDA and RCBMT in diabetic rats. The ability of antioxidant combination to arrest retinal abnormalities and lipid peroxidation even in the presence of poor glycemic control might advocate the key role of direct oxidative damage and the protective action of antioxidants in retinal alterations associated with diabetic retinopathy.

Effects of angiotensin-converting enzyme inhibitors and β-adrenergic blockers on retinal vascular endothelial growth factor expression in rat diabetic retinopathy

Vascular endothelial growth factor (VEGF) plays a pivotal role in diabetic retinopathy (DR) and hypertension has been identified as an independent risk factor for DR. The aim of the present study was to: (1) explore whether β-adrenergic blockers influence retinal VEGF expression; (2) determine the effect of angiotensin-converting enzyme inhibitors (ACEI) on retinal VEGF expression independently of their anti-hypertensive actions; and (3) investigate the correlation between retinal VEGF expression and changes in retinal capillary basement membrane thickness (BMT). Streptozotocin-induced diabetic rats and control animals were assigned at random to receive the β-adrenergic blocker propranolol, the ACEI fosenopril sodium, or vehicle for 24 weeks. Enzyme linked immunosorbent assay, immunohistochemistry, Western blot, and real-time reverse transcription-polymerase chain reaction were used to assess VEGF protein and mRNA expression. Computer-assisted morphometric measurements of transmission electron microscopy photographs were performed to evaluate BMT. Vitreous fluid and retinal VEGF protein and retinal VEGF mRNA expression were significantly higher in diabetic rats than in control rats, with a significant reduction in fosenopril sodium-treated diabetic rats ( p < 0.01). There was no significant difference in VEGF levels in diabetic rats and propranolol-treated diabetic rats ( p > 0.05), but there was a significant difference in VEGF protein and mRNA expression in propranolol-treated diabetic rats and fosenopril sodium-treated diabetic rats ( p < 0.01) without any significant difference in systolic blood pressure in the latter two groups ( p > 0.05). There was a significant correlation between the level of retinal VEGF expression and changes in retinal BMT ( p < 0.01). These findings suggest that the effect of ACEI on retinal VEGF expression is independent of their anti-hypertensive actions and that ACEI could offer particular benefits beyond blood pressure reduction in the treatment of DR with or without hypertension. β-adrenergic blockers had no influence on retinal VEGF expression in normal or diabetic rats.

Retinal capillary basement membrane thickness in diabetic mice genetically modified at the haptoglobin locus

Individuals with diabetes mellitus (DM) homozygous for the haptoglobin (Hp) 1 allele are at decreased risk of retinopathy as compared to DM individuals with the Hp 2 allele. We sought to recapitulate these findings in DM mice genetically modified at the Hp locus. An early morphological characteristic of the microangiopathy seen in diabetic retinal disease is retinal capillary basement membrane (RCBM) thickening. RCBM thickness as assessed by electron microscopy was performed on a total of 12 eyes taken from three mice in each of the four study groups (three eyes from C57Bl/6 Hp 1 and C57Bl/6 Hp 2 mice with and without streptozotocin-induced diabetes). The non-parametric Kruskal-Wallis ANOVA test demonstrated that there was a highly significant difference between the four groups of mice (P < 0.0001). Mann-Whitney tests for specific pair-wise comparisons demonstrated that there was no significant difference in the RCBM thickness between Hp 1 and Hp 2 mice (p = 0.70) or between DM Hp 1 and non-DM Hp 1 mice (p = 0.42). However, induction of diabetes resulted in a marked increase in RCBM thickness in Hp 2 mice compared to non-DM Hp 2 mice (p = 0.0004) and compared to DM Hp 1 mice (p = 0.0005). A highly significant increase in RCBM thickness was observed in DM mice with the Hp 2 genotype. These data provide important support for association studies done in humans showing an increased prevalence of diabetic retinopathy in individuals with the Hp 2 genotype.

Effect of thalidomide and rosiglitazone on the prevention of diabetic retinopathy in streptozotocin-induced diabetic rats.

Our aim was to compare the therapeutic effect of thalidomide and rosiglitazone on the prevention of diabetic retinopathy in streptozotocin-induced diabetic rats. Male Holtzman rats of 6 to 8 weeks of age and weighing 170+/-30 g were randomly divided into four groups: control ( n=13), untreated diabetic ( n=17) and diabetic rats treated with thalidomide (200 mg kg(-1) day(-1)) ( n=8) or rosiglitazone (1 mg kg(-1) day(-1)) ( n=22) for 3 months. Diabetes was induced by streptozotocin with the rats having a body weight of 70 mg/kg. After treatment, vascular endothelial growth factor (VEGF) concentrations in ocular fluid were compared between the different groups, and retinal capillary basement membrane thickness was measured by electron microscopy. Higher VEGF concentrations in ocular fluid and thicker basement membranes were observed in untreated diabetic rats compared to the control rats. Similar VEGF concentrations and basement membrane thickness were observed for the thalidomide-treated group compared with the control group, whereas no difference in these parameters was observed between the rosiglitazone-treated rats and the control or untreated diabetic rats. Our findings confirm the association between VEGF concentrations and diabetic retinopathy as suggested by other investigators. Thalidomide, but not rosiglitazone, was associated with the inhibition of basement membrane thickening and the blockade of the increase of VEGF in ocular fluid, thus representing a potential therapeutic drug for the prevention of diabetic retinopathy.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug.

To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p< or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p< or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p< or =0.0001). This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Inhibition of advanced glycation end-products protects against retinal capillary basement membrane expansion during long-term diabetes.

The purpose of this study was to investigate the advanced glycation end-product (AGE)-inhibitory properties of aminoguanidine and to determine whether treatment in long-term diabetic rats can prevent basement membrane lesions of diabetic retinopathy. Four groups of male Wistar rats were studied: untreated diabetics injected with 45 mg/kg streptozotocin, aminoguanidine-treated diabetics, untreated controls, and aminoguanidine-treated controls. After 12 months' diabetes, the retinas from six animals were processed for electron microscopy or the retinal microvasculature was isolated using the trypsin digest technique. Stereological analysis was used to estimate quantitative ultrastructural changes in the retinal capillary-associated basement membrane. Serum AGEs were quantified by competitive AGE-ELISA, while microvascular-associated, immunoreactive AGEs were analysed on retinal trypsin digests. Aminoguanidine significantly reduced serum AGEs in the diabetic group (p < 0.001). In the retinal capillaries, there was a marked reduction in AGE immunoreactivity in the aminoguanidine-treated diabetics when compared with untreated diabetics. The surface area and absolute volume of the retinal capillary basement membrane were significantly increased in the diabetic rats when compared with non-diabetic controls (p < 0.001 and p < 0.001, respectively). Aminoguanidine treatment of diabetic rats protected against basement membrane expansion when compared with untreated diabetic counterparts. Aminoguanidine treatment prevents the development of diabetes-induced basement membrane expansion in retinal capillaries. The AGE inhibition properties of aminoguanidine suggest that AGEs play an important role in the complex pathogenesis of basement membrane thickening during diabetic retinopathy.