Protection against methylglyoxal-derived AGEs by regulation of glyoxalase 1 prevents retinal neuroglial and vasodegenerative pathology.

AIMS/HYPOTHESIS: Methylglyoxal (MG) is an important precursor for AGEs. Normally, MG is detoxified by the glyoxalase (GLO) enzyme system (including component enzymes GLO1 and GLO2). Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using a transgenic rat model that overexpresses GLO1, we investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy. METHODS: Transgenic rats were developed by overexpression of full length GLO1. Diabetes was induced in wild-type (WT) and GLO1 rats and the animals were killed after 12 or 24 weeks of hyperglycaemia. N ε)-(Carboxyethyl)lysine (CEL), N(ε)-(carboxymethyl)lysine (CML) and MG-derived-hydroimidazalone-1 (MG-H1) were determined by immunohistochemistry and by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS). Müller glia dysfunction was determined by glial fibrillary acidic protein (GFAP) immunoreactivity and by spatial localisation of the potassium channel Kir4.1. Acellular capillaries were quantified in retinal flat mounts. RESULTS: GLO1 overexpression prevented CEL and MG-H1 accumulation in the diabetic retina when compared with WT diabetic counterparts (p < 0.01). Diabetes-related increases in Müller glial GFAP levels and loss of Kir4.1 at the vascular end-feet were significantly prevented by GLO1 overexpression (p < 0.05) at both 12- and 24-week time points. GLO1 diabetic animals showed fewer acellular capillaries than WT diabetic animals (p < 0.001) at 24 weeks' diabetes. CONCLUSIONS/INTERPRETATION: Detoxification of MG reduces AGE adduct accumulation, which, in turn, can prevent formation of key retinal neuroglial and vascular lesions as diabetes progresses. MG-derived AGEs play an important role in diabetic retinopathy.

Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products.

AIMS/HYPOTHESIS: The impact of AGEs and advanced lipoxidation end-products (ALEs) on neuronal and Müller glial dysfunction in the diabetic retina is not well understood. We therefore sought to identify dysfunction of the retinal Müller glia during diabetes and to determine whether inhibition of AGEs/ALEs can prevent it. METHODS: Sprague-Dawley rats were divided into three groups: (1) non-diabetic; (2) untreated streptozotocin-induced diabetic; and (3) diabetic treated with the AGE/ALE inhibitor pyridoxamine for the duration of diabetes. Rats were killed and their retinas were evaluated for neuroglial pathology. RESULTS: AGEs and ALEs accumulated at higher levels in diabetic retinas than in controls (p < 0.001). AGE/ALE immunoreactivity was significantly diminished by pyridoxamine treatment of diabetic rats. Diabetes was also associated with the up-regulation of the oxidative stress marker haemoxygenase-1 and the induction of glial fibrillary acidic protein production in Müller glia (p < 0.001). Pyridoxamine treatment of diabetic rats had a significant beneficial effect on both variables (p < 0.001). Diabetes also significantly altered the normal localisation of the potassium inwardly rectifying channel Kir4.1 and the water channel aquaporin 4 to the Müller glia end-feet interacting with retinal capillaries. These abnormalities were prevented by pyridoxamine treatment. CONCLUSIONS/INTERPRETATION: While it is established that AGE/ALE formation in the retina during diabetes is linked to microvascular dysfunction, this study suggests that these pathogenic adducts also play a role in Müller glial dysfunction.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE).

AIMS/HYPOTHESIS: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia. METHODS: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting. RESULTS: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia. CONCLUSIONS/INTERPRETATION: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.

Advanced glycation end products accumulate in the reproductive tract of men with diabetes.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility.

Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products.

It has been suggested that increased production of nitric oxide (NO), a potent endothelium-derived vasodilator, may be responsible for increased blood flow in the retinal and renal vascular beds in early diabetes. However, NO-mediated vasodilation has been reported as impaired in diabetes, and there is evidence that the synthesis and release of NO by the vascular endothelium may be flawed in this condition. We examined the effect of high ambient glucose and exposure to exogenous glycated proteins on NO synthesis in cultured retinal microvascular endothelial cells (RMECs), using a polarographic sensor to measure released NO gas. Nitrite (the stable end product of the reaction between NO and molecular oxygen) was measured in tissue culture supernatants. The expression of vascular endothelial constitutive nitric oxide synthase (eNOS), which is responsible for NO synthesis in endothelial cells, was studied by Western blot analysis and Northern hybridization experiments. A dose-dependent reduction of NO synthesis by RMECs occurred 5 days after exposure to 15 and 25 mmol/l glucose, and concomitantly we found that accumulation of nitrite in culture supernatants of high-glucose exposed cells was also reduced. Coincubation of endothelial cells with inhibitors of protein kinase C (PKC) increased the accumulation of nitrite but did not restore it to the levels obtained when cells were cultured in 5 mmol/l glucose. The expression of eNOS by RMECs was markedly reduced by 5 days of exposure to 25 mmol/l glucose and glycated albumin. This study implicates the PKC pathway, which is known to be upregulated on exposure to high ambient glucose concentrations, as a possible factor in the inhibition of eNOS expression in RMECs. This study also suggests that glycated proteins may be involved in the pathogenesis of vascular endothelial dysfunction by modulating the nitric oxide synthase (NOS)/NO pathway in retinal vascular endothelial cells.

Gremlin gene expression in bovine retinal pericytes exposed to elevated glucose.

AIM: To assess the influence of high extracellular glucose on the expression of the bone morphogenetic protein (BMP) antagonist, gremlin, in cultured bovine retinal pericytes (BRPC). METHODS: BRPC were cultured under conditions of 5 mM and 30 mM d-glucose for 7 days and total RNA was isolated. Gremlin mRNA levels were correlated, by RT-PCR, with other genes implicated in the pathogenesis of diabetic retinopathy and the signalling pathways in high glucose induced gremlin expression were probed using physiological inhibitors. Gremlin expression was also examined in the retina of streptozotocin induced diabetic mice. RESULTS: High glucose stimulated a striking increase in BRPC gremlin mRNA levels in parallel with increases in mRNA for the growth factors vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and connective tissue growth factor (CTGF) and changes in other genes including fibronectin and plasminogen activator inhibitor-1 (PAI-1). High glucose triggered gremlin expression was modulated by anti-TGFbeta antibody, by the uncoupler of oxidative phosphorylation, CCCP, and by inhibition of MAP-kinase (MAPK) activation. Striking gremlin expression was observed in the outer retina of diabetic mice and also at the level of the vascular wall. CONCLUSIONS: Gremlin gene expression is induced in BRPC in response to elevated glucose and in the retina of the streptozotocin induced diabetic mouse. Its expression is modulated by hyperglycaemic induction of the MAPK, reactive oxygen species, and TGFbeta pathways, all of which are reported to have a role in diabetic fibrotic disease. This implicates a role for gremlin in the pathogenesis of diabetic retinopathy.

Characterisation of the advanced glycation endproduct receptor complex in the retinal pigment epithelium.

AIMS: Advanced glycation endproducts (AGEs) accumulate with ageing and may have a significant impact on age related dysfunction of the retinal pigment epithelium (RPE). Many of the cellular effects of AGEs in other cell types are mediated through AGE binding proteins. The aim of this study was to characterise the AGE receptor complex in RPE cells in vitro and to focus on the role of the R3 component (galectin-3) as the primary effector of the complex. METHODS: Primary cultures of bovine RPE cells and the human D407 RPE cell line were exposed to AGE modified albumin. Receptor expression was determined using mRNA analysis by quantitative real time RT-PCR and protein characterisation by western blotting. Immunocytochemical analysis examined the cellular localisation of the various components of the AGE receptor complex. The role of the galectin-3 receptor component was examined by transfection and overexpression using the D407 cell line and analysis of soluble AGE-R3 by ELISA. RESULTS: All three components of the AGE receptor complex were expressed by bovine and human RPE cells. AGE exposure upregulated two components of the receptor complex and also induced significant RPE expression of VEGF mRNA (p<0.05). RPE D407 cells stably transfected to overexpress galectin-3 showed less VEGF induction. In non-transfected RPE which were exposed to AGEs, there was less soluble galectin-3 protein released into the medium (p<0.05), a response that was not evident in transfected cells. CONCLUSION: A conserved AGE receptor complex is evident in primary cultures of bovine RPE cells and also in a human cell line. These cells show a pathological response to AGE exposure, an effect which appears to be modulated by the galectin-3 component of the receptor complex.

Endothelin expression in ocular tissues of diabetic and insulin-treated rats.

PURPOSE: The endothelins are potent vasoactive peptides that are widely distributed in ocular tissues. There is evidence linking the endothelins to vascular dysfunction in diabetic microangiopathy. Thus, the synthesis and distribution of endothelin-1 (ET-1) and endothelin-3 (ET-3) were studied in the retinas of diabetic and nondiabetic animals. METHODS: Levels of ET-1 and ET-3 were determined by radioimmunoassay in ocular tissues of normal rats, and in rats with streptozotocin-induced diabetes of 6 and 12 weeks' duration, insulin-treated and untreated. In a separate cohort of similarly treated animals, retinal vascular trypsin digest preparations were immunostained, using antibodies raised against ET-1 and ET-3. RESULTS: Ocular ET-1 levels were elevated twofold in diabetic animals that received insulin treatment for 7 days when compared with levels in normal rats. Insulin treatment for 10 days before death caused a fourfold elevation of ET-1 in ocular tissues. Endothelin-1 was also increased in 12-week-old diabetic animals and in those maintained on insulin throughout their period of diabetes. Immunofluorescence to anti-ET-1 within the capillary bed and veins of the retina in diabetic insulin-treated animals was elevated when compared with digests from normal litter-matched control animals. Ocular tissue ET-3 levels were unaffected by diabetes. CONCLUSIONS: Overall ocular and retinal tissue levels of ET-1 were selectively elevated by diabetes and insulin treatment, suggesting that the endothelins may be involved in the pathogenesis of diabetic retinal microangiopathy.

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

PURPOSE: The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). METHODS: Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. RESULTS: Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. CONCLUSION: These results illustrate the internalization and intracellular trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.

Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy.

PURPOSE: Advanced glycation end products (AGEs) form irreversible cross-links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS: By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGEs were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS: There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS: This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.

Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR.

PURPOSE: To determine whether continuous monitoring of SYBR Green I fluorescence provides a reliable and flexible method of quantitative RT-PCR. Our aims were (i) to test whether SYBR Green I analysis could quantify a wide range of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs. METHODS: Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler (Roche). Retinal VEGF mRNA levels were measured in a murine model of oxygen-induced retinopathy during vaso-obliterative and hypoxic phases. RESULTS: This technique was able to detect as few as 10 control template copies, with quantitative data available routinely for 1000 or more copies. The levels of retinal VEGF mRNA expression followed the hypoxia-induced pattern determined previously by conventional methods. All gene-specific primer pairs which amplify a specific product by conventional PCR were successfully converted to SYBR Green analysis, including those for housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), cyclophilin, and acidic ribosomal phosphoprotein PO (ARP/36B4) and for 28S rRNA. In each case melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification product. CONCLUSIONS: The sequence-independent detection of DNA with SYBR Green I means that it can be used to quantify the amplification of any cDNA using gene-specific primers. This rapid and flexible method is ideally suited for researchers in vision science wishing to quantify mRNAs from many different genes because it does not require investment in gene-specific hybridization probes.

The effect of triclabendazole ("Fasinex") on protein synthesis by the liver fluke, Fasciola hepatica.

The effect of the active sulphoxide metabolite of the anthelmintic triclabendazole (TCBZ-SX, 15-50 micrograms ml-1) on the incorporation of radioactively labelled [14C] leucine by adult Fasciola hepatica tissue slices was measured by liquid scintillation counting. In addition, the ability of the microfilament-disrupting drug, cytochalasin B, and the microtubule-disrupting drug, tubulozole-C, to inhibit protein synthesis, was assessed by similar methods and compared with TCBZ-SX. The established protein synthesis inhibitors, cycloheximide and actinomycin D were used as positive controls. All the drugs showed a significant inhibition of protein synthesis, albeit to different extents; however, TCBZ-SX was the most potent, with no significant difference between its effect and that of cycloheximide or actinomycin D. Moreover, the concentration of TCBZ-SX, above 15 micrograms ml-1, had little further influence on incorporation of [14C] leucine. This investigation demonstrates the inhibitory effect of TCBZ-SX, cytochalasin B and tubulozole-C on protein synthesis in F. hepatica and confirms the qualitative observations made in several previous ultrastructural studies.

Characterisation of WE-14 in porcine ocular tissue.

WE-14 is derived from the cell-specific posttranslational processing of chromogranin A (CgA) in subpopulations of neuroendocrine cells and neurons. Region- and site-specific chromogranin A, pancreastatin and WE-14 antisera were employed to study the generation of WE-14 in porcine ocular tissues. No chromogranin A or pancreastatin immunostaining was detected in ocular tissue. Immunohistochemistry detected WE-14 immunostaining in a network of nerve fibre bundles and nerve fibres throughout the limbus, cornea, iris and ciliary body with sparse nerve fibres detected throughout the choroid and sclera. Retinal analysis detected intense WE-14 immunostaining in large ovoid cells in the ganglion cell layer with weak immunostaining in a population of small cells in the inner nuclear layer; weak immunostaining was detected within the fibre layers in the inner plexiform layer. Quantitatively, the highest WE-14 tissue concentration was recorded in aqueous retinal and corneal extracts with lower concentrations in the sclera, choroid and anterior uveal tissues. Chromatographic profiling resolved a minor chromogranin A-like immunoreactant and a predominant immunoreactant co-eluting with synthetic human WE-14. This is the first study to demonstrate that WE-14 is generated in neuronal fibres primarily innervating the anterior chamber and in select cell populations in the retina.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients.

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Diabetic retinopathy: morphometric analysis of basement membrane thickening of capillaries in different retinal layers within arterial and venous environments.

AIMS: To assess quantitatively variations in the extent of capillary basement membrane (BM) thickening between different retinal layers and within arterial and venous environments during diabetes. METHODS: One year after induction of experimental (streptozotocin) diabetes in rats, six diabetic animals together with six age-matched control animals were sacrificed and the retinas fixed for transmission electron microscopy (TEM). Blocks of retina straddling the major arteries and veins in the central retinal were dissected out, embedded in resin, and sectioned. Capillaries in close proximity to arteries or veins were designated as residing in either an arterial (AE) or a venous (VE) environment respectively, and the retinal layer in which each capillary was located was also noted. The thickness of the BM was then measured on an image analyser based two dimensional morphometric analysis system. RESULTS: In both diabetics and controls the AE capillaries had consistently thicker BMs than the VE capillaries. The BMs of both AE and VE capillaries from diabetics were thicker than those of capillaries in the corresponding retinal layer from the normal rats (p < or = 0.005). Also, in normal AE and VE capillaries and diabetic AE capillaries the BM in the nerve fibre layer (NFL) was thicker than that in either the inner (IPL) or outer (OPL) plexiform layers (p < or = 0.001). However, in diabetic VE capillaries the BMs of capillaries in the NFL were thicker than those of capillaries in the IPL (p < or = 0.05) which, in turn, had thicker BMs than capillaries in the OPL (p < or = 0.005). CONCLUSIONS: The variation in the extent of capillary BM thickening between different retinal layers within AE and VE environments may be related to differences in levels of oxygen tension and oxidative stress in the retina around arteries compared with that around veins.

Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs.

This study was undertaken to further characterise the fine structural changes occurring in the retinal circulation in early diabetes. The eyes of eight alloxan/streptozotocin and three spontaneously diabetic dogs were examined by trypsin digest and electron microscopy after durations of diabetes of between 1 and 7 years. Basement membrane (BM) thickening in the retinal capillaries was the only obvious fine structural change identified during the first 3 years of diabetes and was established within 1 year of induction. Widespread pericyte loss was noted after 4 years of diabetes and was paralleled by loss of smooth muscle (SM) cells, in the retinal arterioles. SM cell loss was most obvious in the smaller arterioles of the central retina. No microaneurysms were noted in the experimental diabetic dogs with up to 5 years' duration of diabetes but were widespread in a spontaneously diabetic animal at 7 years. This study has shown that SM cell loss, a hitherto unrecognised feature of diabetic microangiopathy, accompanies pericyte loss in the retinal circulation of diabetic dogs.

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments.

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p < or = 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.

Advanced glycation alters expression of the 67kDa laminin receptor in retinal microvascular endothelial cells.

The 67kDa laminin receptor (67LR) plays an important role in vascular cell function and dysfunction. The present study has examined 67LR expression in retinal microvascular endothelial cells after exposure to AGEs. Retinal microvascular endothelial cells were exposed to either AGE-BSA, or were grown on methylglyoxal-modified laminin or Matrigel and expression of 67LR analysed by Western Blotting and RT-PCR/Southern blotting. Western blotting of plasma membrane and RT-PCR/Southern blotting revealed a significant upregulation of 67LR protein/mRNA expression after exposure to AGEs (p<0.05-0.01). The results show that 67LR is upregulated in cells exposed to AGEs and suggests a previously unrecognised role for this receptor in retinal microvascular endothelial cell interaction with glycated substrates.

Endothelin-like immunoreactivity and receptor binding in the choroid and retina.

This study has examined the localisation and receptor-binding of the endothelins in retina and choroid of human and rat origin. Immunoreactivity to anti-ET1 and anti-ET3 was investigated in trypsin digests, frozen sections and ultrathin sections using immunocytochemistry and immunogold labelling techniques. In addition, receptor binding of 125I-ET1 and 125I-ET3 was visualised and quantified using autoradiography and image analysis. Intense immunoreactivity to anti-ET1 and anti-ET3 was observed in the photoreceptor inner segments and in the outer plexiform layer (OPL) of human and rat retina. Ultrastructural localisation using immunogold labelling confirmed the presence of ET1 and ET3 in the photoreceptor cells. In retinal vascular digests, ET1 was visualised in the arteries, arterioles and at the pre-arteriolar sphincters, however, immunoreactivity to anti-ET3 was absent in the retinal vasculature. Both ETA and ETB-type receptor binding sites to 125I-ET1 and 125I-ET3 were detected in the vascular smooth muscle of choroidal and retinal vessels with the former being predominant. Extravascular binding sites of the ETB-type were found in the ganglion cell layer.

Stereological estimation of Weibel-Palade bodies in the retinal vasculature of normal and diabetic dogs.

The absolute volume of Weibel-Palade (WP) bodies, the storage organelles of von Willebrand factor (vWF), was estimated by a stereological method in a known volume of central retina from normal and 5-year diabetic dogs. The results showed that the volume of WP bodies present in the endothelium of the retinal vasculature varies with blood vessel type and in diabetes. In both diabetic and normal dogs the endothelium of the retinal veins contained a higher volume of WP bodies than that of the retinal arteries. In dogs which had been diabetic for a duration of 5 years the volume of WP bodies present in the endothelium of retinal veins was significantly greater than in the endothelium of veins from the control animals. However, there was no significant difference in the volume of WP bodies present in the endothelium of retinal arteries or capillaries between the two groups of animals.