The retinal microvasculature of spontaneously diabetic BB rats: structure and luminal surface properties

Endothelial cell permeability and luminal surface anionic sites were studied in the retinal microvasculature of spontaneously diabetic rats. Horseradish peroxidase (HRP) was used as a tracer of pinocytotic transport, and cationized ferritin (CF) was used as a marker of luminal surface anionic sites. Diabetic and control rats were injected with HRP, and their retinas were fixed. Retinal tissue sections were then incubated in CF, reacted to visualize HRP, and prepared for quantitative electron microscopic analysis. In both control and diabetic rats treated with serotonin and histamine antagonists to prevent HRP-induced vascular changes, the endothelium formed a barrier to the tracer. Pinocytotic uptake was relatively low in most vessels. Reaction product was restricted to pinocytotic vesicles, tubular cisternae, and multivesticular bodies. HRP uptake appeared high in some of the deep capillaries of the diabetic retinas as compared with that of the controls, but the difference was not statistically significant. HRP-induced transendothelial permeability was observed in both control and diabetic rats when serotonin and histamine antagonist pretreatment was omitted. CF studies showed anionic sites in four luminal surface microdomains in control and diabetic endothelial cells. CF-binding, anionic sites were present on the plasma membrane, on all coated vesicles, on some uncoated vesicles, and on most diaphragms of uncoated vesicles. Plasma membrane binding was sparse and patchy in some diabetic vessels, especially in the deep vessels of rats that were not treated with the serotonin and histamine antagonists. However, statistical analysis showed similar numbers of plasma membrane binding sites in diabetic and control rats pretreated with serotonin and histamine antagonists. Our data suggest that the retinal microvasculature in diabetic rats remains normal in terms of permeability and luminal membrane anionic charge.     

The binding of cationic probes to apical and basal surfaces of rat lung capillary endothelium and of endothelial cells in tissue culture

The reasons for greater lung vascular permeability to anionic macromolecules are not understood. In order to determine whether the luminal or abluminal surfaces of lung capillary endothelial cells differ with respect to surface charge, we compared the binding of cationic ferritin, an electron dense probe, with these cell surfaces in lung capillaries. Because lung capillaries are not normally permeable to cationic ferritin, lungs were examined from rats with increased permeability edema caused by pretreatment with alpha-naphthylthiourea (ANTU). We found that more cationic ferritin particles bound to the luminal than to the abluminal surfaces of lung capillary endothelium. In order to determine whether this was due to inaccessibility of cationic ferritin to the lung interstitium, we also compared cationic ferritin binding to the apical and basal surfaces of bovine calf aortic and main pulmonary arterial endothelial cells in tissue culture. We found that more cationic ferritin bound to the apical than to the basal surface of the cultured cells. The binding of cationic ferritin to cultured endothelial cells was due to charge since native, anionic ferritin did not bind to either surface and binding was decreased by neuraminidase pretreatment of cultures. Cultures incubated with thiourea, another thiocarbamide that causes increased permeability edema in vivo, also showed greater binding of cationic ferritin to the apical cell surface, suggesting that the differences seen in vivo were not due to thiocarbamide injury. However, another cationic probe, ruthenium red, bound to both the apical and basal surfaces of cultured endothelial cells. These results suggest that the basal endothelial cell surface does not lack anionic sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced negative surface charge on arterial endothelium of diabetic rats

Summary Cationized ferritin binding was used to measure negative surface charge on endothelium of large arteries in streptozotocin-induced diabetic rats and normal control rats. The negative charge was significantly lower in the diabetic animals (p<0.01). This change, possibly related to glycosylation, may lead to altered vascular permeability and may be of importance in the vascular pathology of diabetes.     

Oxidative damage in cerebral vessels of diabetic db/db mice

BACKGROUND: Oxidative stress in diabetes mellitus has recently received increasing attention as it has been proven to be associated with the development of diabetic vascular complications. Our aim was to examine whether microvascular changes, including oxidative damage, were induced in the brains of diabetic animals. METHODS: The expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, the binding of cationized ferritin, a marker for evaluating endothelial glycocalyx, to the endothelial cells of capillaries and vascular permeability of intravenously injected horseradish peroxidase were examined in the cortices of 12- and 20-week-old db/db and db/+m mice. RESULTS: Immunostaining for 8-OHdG was clearly seen in the vessels of the cortex of 20-week-old db/db mice, but was hardly seen in those of mice in the other groups. The immunopositive area of 8-OHdG was significantly increased in the cortex of 20-week-old db/db mice compared with that of 20-week-old db/+m mice. No extravasated leakage of horseradish peroxidase was seen in any groups of mice, while the numbers of cationized ferritin particles binding to the endothelial cells was significantly decreased in 12- and 20-week-old db/db mice compared with that of db/+m mice at the same age, respectively. CONCLUSION: These findings suggest that changes in endothelial glycocalyx are induced in db/db mice and, in addition, the long-term diabetic condition of these mice induces oxidative DNA damage to the cerebral vessels.     

Alterations of glomerular basement membrane charge and structure in diabetic nephropathy

Summary We examined glomerular basement membrane anionic site distribution identified by cationic gold in seven patients with insulin-dependent and four patients with non-insulin-dependent diabetes mellitus, presenting a spectrum of clinical and glomerular changes. Anionic sites were investigated by pretreatment of tissue with glycosaminoglycan-degrading enzymes prior to cationic gold staining. The distribution of chondroitin sulphate proteoglycans — a previously unrecognized glomerular basement membrane component — and type IV collagen was examined by immunoelectron microscopy to identify structural changes in the basement membrane. Findings were compared with those of non-diabetic patients showing minor proteinuria and morphologically normal glomerular basement membranes. Two patients, originally diagnosed as having diabetic nephropathy were also examined at 19 weeks and 5 years after renal transplantation. Characteristic redistribution of type IV collagen and chondroitin sulphate proteoglycans was noted in thickened glomerular basement membrane segments (>400 nm) of diabetic patients and those with renal transplants. Extension of anionic sites deep into the glomerular basement membrane at pH 2.5, together with loss of interna sites at pH 5.8 is unique to diabetic nephropathy. Reduced charge density was apparent in some patients due to thickening of the glomerular basement membrane, although the number of anionic sites per unit length of membrane was actually increased. Thus, charge aberration in diabetic nephropathy is due to displacement rather than loss of anionic sites. Removal of more than 90% of these sites by heparitinase, confirms their association with heparan sulphate proteoglycans. Similar derangement of anionic sites in all patients with diabetic nephropathy irrespective of the degree of proteinuria, suggests that a heparan sulphate proteoglycan-related charge barrier plays a minor role in controlling permeability of the diabetic glomerular basement membrane.Abbreviations BSA Bovine serum albumin - CG cationic gold - CSPG chondroitin sulphate proteoglycans - GAG glycosaminoglycan - GBM glomerular basement membrane - HSPG heparan sulphate proteoglycans - LRE lamina rara externa - LRI lamina rara interna - PCI protein:creatinine index     

Anionic sites in diabetic basement membranes and their possible role in diffusion barrier abnormalities in the BB-rat

Summary Basement membrane anionic sites, thought to be responsible for charge selective permeability barriers, were investigated in retinal, endoneurial, and muscle capillary basement membranes and in Bruch's membrane of diabetic, and age- and sex-matched non-diabetic BB-rats using an ultrastructural quantitative histochemical technique. Six months of diabetes was associated with significant basement membrane thickening which was linearly related to a decrease in anionic site density suggesting a relative loss of proteoglycans. Calculation of anionic sites per unit length of basement membrane, reflecting their absolute number, revealed a significant loss in basement membrane, constituting part of normal blood-tissue barrier systems such as retinal and endoneurial capillary basement membranes, and the basement membrane of the retinal pigment epithelium. The absolute number of anionic sites in normally permeable microvessels, such as those of muscle and choriocapillaries, was unaltered by diabetes. We conclude that this specific loss of anionic sites in basement membranes of tissues affected by chronic diabetic complications may in part be responsible for permeability abnormalities seen in these tissues.     

Circulating glycated albumin and glomerular anionic charges

Aiming to discern the mechanisms by which circulating glycated albumin alters the glomerular filtration properties that lead to glomerular dysfunction in diabetes, the authors studied the distribution and densities of anionic charges through the rat glomerular wall upon intravascular infusion of Amadori products, as well as in various conditions of increased glomerular permselectivity. Polylysine-gold was used as the probe to reveal the anionic charges. The study was carried on renal tissue sections of bovine serum albumin (BSA)- and glycated BSA-injected, normoglycemic animals. Results were generated through morphometrical evaluations of the gold labeling. Changes in glomerular anionic distribution were corroborated on renal tissue sections of short- and long-term diabetic rats and of normal newborn rats, situations known for abnormal glomerular filtration. Altered renal function in these conditions was clearly associated with changes in glomerular anionic charges. On the other hand, the infusion of glycated albumin in the circulation of normal rats, though altering glomerular filtration properties, did not modify the distribution and density of the polylysine-gold labeling through the glomerular basement membrane. Thus, anionic charges seem not to be the factor involved in the early changes of glomerular permeability induced by circulating glycated albumin.     

Distribution and movement of anionic cell surface sites in cultured human vascular endothelial cells.

Cell surface anionic sites on primary and transformed cultures of human vascular endothelium were studied using cationized ferritin (CF) as an ultrastructural marker. The native distribution of anionic sites on the upper (free) surfaces of cells, fixed in situ with glutaraldehyde, was uniform. Binding of the polycationic ligand, CF, in unfixed cells induced redistribution of anionic sites. Rapid formation of discrete patches of CF particles was followed by reappearance of binding between patches, movement of surface-bound CF into intercellular clefts, and endocytosis, over the next 30 min. Aldehyde-fixed cells, detached from the culture surface, bound CF on both upper and lower surfaces. The distribution and mobility of negatively charged membrane components in vascular endothelium may have relevance for thrombosis, atherogenesis, and vascular permeability.     

Differentiated distribution of the cell surface charge on the alveolar-capillary unit: Characteristic paucity of anionic sites on the air-blood barrier

The distribution of the surface charge on the cells which constitute the air-blood barrier was investigated by perfusing cationized ferritin (CF) into the vasculature or into the airways of the mouse lung. Binding of CF is selective and defines highly differentiated domains. The most salient finding is that the air-blood barrier proper that includes type I epithelial cell and part of the corresponding avesicular area of the endothelial cell has very few or lacks anionic sites. In the vesicular area of endothelial cells the plasma membrane binds CF homogenously, with the exception of the membrane of plasmalemmal vesicles and transendothelial channels and their associated diaphragms. In contradistinction to the luminal surface of the type I epithelial cell, which is virtually devoid of anionic sites, CF decorates heavily the luminal surface of type II epithelial cells, up to the level of the junction with type I epithelial cells. Extruded lamellar bodies and tubular myelin also binds CF, presumably due to the phosphate groups of the dipalmitoyl phosphatidylcholine. The cell surface of the alveolar macrophages has relatively few binding sites for CF, but significant internalization of the latter occurs at early time intervals. The preferential distribution of anionic sites on cell surfaces of the alveolar-capillary unit may be influential in the transport of molecules and gases across various regions of the air-blood barrier.     

Surface Charge of Red Blood Cells in Insulin-dependent Diabetic Patients with Incipient and Overt Nephropathy

The negative surface charge of red blood cells was studied in adults with insulin-dependent diabetes mellitus, using a chemical assay based on the binding of the cationic dye Alcian-blue 8GX to anionic sites on the cell surface. Twenty-one healthy non-diabetic adults, 21 normoalbuminuric diabetic patients, 25 diabetic patients with incipient nephropathy, and 19 diabetic patients with overt nephropathy were studied. The diabetic patients were matched for age, duration of diabetes, and glycaemic control. The red cell surface charge was nearly identical in the four groups studied. Our study does not suggest that loss of membrane negative charge is a generalized phenomenon in insulin-dependent diabetic patients with albuminuria.     

Permeability of small coronary arteries and myocardial injury in hypertensive diabetic rats

Damage to the coronary artery and myocardium is known to be more marked when hypertension is complicated by diabetes than when either is present alone. The reason is not clear, but one possible factor is vascular permeability. Therefore, we investigated vascular permeability using sodium fluorescein of 70 mg/kg body weight, in 12-week-old streptozotocin-induced diabetic spontaneously hypertensive rats. Non-diabetic spontaneously hypertensive rats, diabetic rats with normal blood pressure, and non-diabetic rats with normal blood pressure of the same age were used as controls and treated similarly. H-E and Azan staining were used to examine vascular abnormality and mycoardial damage. The diabetic spontaneously hypertensive rats showed marked sodium fluorescein in the coronary artery wall and surrounding area, demonstrating greater vascular permeability than non-diabetic spontaneously hypertensive rats, diabetic and non-diabetic normotensive rats. Heart weights and diameters of myocytes were smaller in the diabetic than in the non-diabetic spontaneously hypertensive rats. Diabetic spontaneously hypertensive rats also exhibited marked perivascular fibrosis and focal necrosis. Thus, increased vascular permeability may play a role in vascular and myocardial changes in diabetic spontaneously hypertensive rats.     

四周四氧嘧啶糖尿病大鼠主动脉超微结构和通透性变化

目的 了解糖尿病早期主动脉超微结构及通透性变化。方法 ４周四氧嘧啶糖尿病大鼠主动脉按透射电镜观察的要求进行固定、包埋、切片、染色及镧通透性检测的要求进行固定、染色、包埋和切片后,进行透射电镜观察。结果 糖尿病大鼠主动脉平滑肌细胞线粒体肿胀,有镧进入;内皮细胞紧密连接处有镧进入。结论 糖尿病早期主动脉平滑肌细胞膜及其线粒体膜通透性增加,线粒体肿胀,内皮通透性增加。     

Islet transplants in diabetic Lewis rats prevent and reverse diabetes-induced increases in vascular permeability and prevent but do not reverse collagen solubility changes

Summary The effects of islet transplantation on diabetes-induced increases in vascular permeability and collagen solubility were examined in new granulation tissue vessels and collagen formed after induction of streptozotocin diabetes in male Lewis rats. Albumin permeation was increased by 50% (p<0.001) and collagen solubility was decreased by 50% (p<0.001) in granulation tissue from untreated diabetic animals as compared with controls. The islet transplants reversed diabetes-induced vascular permeability increases in tissues formed prior to islet transplantation (tissue to blood isotope ratio =2.1±0.1 - SD for controls, 3.2±0.2 for diabetic rats and 2.0±0.2 for diabetic rats given islets) and prevented permeability increases in new tissues formed following transplantation (tissue to blood isotope ratio =2.1±0.1 for controls, 3.3±0.8 for diabetic rats and 1.9±0.2 for diabetic rats given islets). In contrast, while islet transplants prevented diabetes-induced decreased collagen solubility in tissues formed after transplantation (controls = 24%, diabetic rats =12%, and diabetic rats given islets =24%), collagen solubility in tissues formed prior to islet transplantation was virtually unaffected. These findings indicate that collagen changes induced by the diabetic milieu are not nearly as readily reversed by normalization of the diabetic milieu as (diabetes-induced) alterations in vascular functional integrity.     

Induction of intestinal glucose carriers in streptozocin-treated chronically diabetic rats

The maximal transport capacity (Vmax) for intestinal glucose absorption is increased in experimentally induced chronic diabetes mellitus. Using [3H]phlorizin radioautography, we examined the relation between this increase in transport Vmax and the number and distribution of sodium-glucose co-transporters on the luminal surface of rat ileum. Male Lewis rats were made diabetic with streptozocin. Ninety days later we measured 3-O-methyl-D-glucopyranose absorption and specific [3H]phlorizin binding to the ileal mucosa of the same rats. Net 3-O-methyl-D-glucopyranose flux was 6.9-fold greater in diabetic rats compared with age-matched controls. Specific binding of [3H]phlorizin to the luminal surface was 7.2-fold greater in the diabetic rats. Radioautography revealed that, in chronic diabetes, specific phlorizin binding extends into the midvillus region of the ileum, whereas in age-matched controls, it is confined to villus tips. We believe that, in untreated diabetes, a larger fraction of intestinal villus epithelial cells participate in glucose absorption.     

SPARC gene expression is increased in diabetes-related mesenteric vascular hypertrophy

The anti-adhesive extracellular matrix protein SPARC (secreted protein and rich in cysteine; osteonectin or BM-40) has been implicated in the regulation of matrix turnover, cell migration, and proliferation. The present study sought to examine whether modulation in the expression of this protein may play a role in diabetes-associated vascular remodeling. SPARC mRNA and protein were measured in mesenteric vessels of diabetic rats and controls. Hypertrophy of mesenteric vessels was noted after 3 and 32 weeks of diabetes as revealed by the increase in mesenteric vessel wet weight and an increased wall/lumen ratio. SPARC mRNA was sparsely present in intima and adventitia of control vessels. There was a marked increase in SPARC gene expression in the intima and adventitia of mesenteric vessels after 1, 3, and 32 weeks of diabetes. SPARC protein was demonstrated in the vessel wall in control animals and was increased in the mesenteric vessels of diabetic rats after 1 and 32 weeks of diabetes. Administration of the inhibitor of advanced glycation end-product formation, aminoguanidine, to diabetic rats attenuated both the hypertrophic response in mesenteric vessels and the overexpression of SPARC mRNA and protein without affecting glycemic control or food intake. In summary, diabetes-related mesenteric vascular hypertrophy is associated with an increase in SPARC expression in the vessel wall. The modulation of SPARC expression in mesenteric vessels of diabetic rats might be of pathogenetic significance in the development of vascular remodeling in diabetes.     

Rat strain-dependent susceptibility to ischemia-induced retinopathy associated with retinal vascular endothelial growth factor regulation

Vascular endothelial growth factor (VEGF) is a potent inflammation, vascular permeability, and angiogenic factor. Variations of the VEGF gene are implicated in the pathogenesis of diabetic retinopathy. Previous studies have shown that Brown Norway (BN) rats have higher retinal VEGF levels and more severe retinal vascular leakage than Sprague-Dawley (SD) rats in response to ischemia and diabetes. To investigate the molecular mechanism of vascular leakage in this animal model, F2 progeny were generated by crossbreeding BN and SD rats. Neonatal rats were exposed to hyperoxia to induce oxygen-induced retinopathy (OIR) models. The F2 rats in response to ischemia have shown a linear distribution of retinal VEGF levels, which is significantly and positively correlated to retinal vascular leakage. We identified a single nucleotide polymorphism (SNP) at upstream stimulating factor-binding site in the VEGF promoter region between BN and SD rats. No differences were found in retinal vascular permeability or VEGF levels between F2 rats with BN, SD, and BN/SD alleles of VEGF SNP. The increased retinal VEGF levels are correlated to ischemia-induced retinal vascular leakage in the OIR rat model. The VEGF mRNA and promoter are not responsible for increased retinal VEGF level and vascular permeability. The up-regulation of VEGF expression activated by a yet to be identified upstream factor or mediator affecting VEGF stability may be associated with a high susceptibility to retinal vascular leakage in BN rats.     

Binding of charged ferritin to alveolar wall components and charge selectivity of macromolecular transport in permeability pulmonary edema in rats

Rat lungs were inflated and incubated in either anionic or cationic ferritin, and alveolar and capillary basement membranes were examined by electron microscopy. Cationic ferritin bound to heparan sulfate proteoglycans on the external surface of the alveolar basement membrane, whereas cationic ferritin bound to the lamina densa of the capillary basement membranes. Anionic and cationic ferritin was also perfused through the pulmonary circulation of lungs isolated from control rats and rats previously injected with alpha-naphthylthiourea, which produces permeability pulmonary edema. Neither anionic nor cationic ferritin leaked from the pulmonary capillaries in perfused controls; cationic, but not anionic, ferritin adhered to endothelial cell surfaces. In lungs with alpha-naphthylthiourea pulmonary edema, perfused for 2-15 minutes, anionic ferritin leaked from pulmonary capillaries into the alveolar interstitium and alveolar space, while cationic ferritin remained within the capillary lumen. Five times as much anionic ferritin appeared in the capillary basement membranes on the thick side of the alveolar wall, as in the alveolar basement membranes on the thin side of the alveolar wall. In alpha-naphthylthiourea lungs perfused for 45-60 minutes, cationic ferritin also leaked through the injured endothelium and bound twice as much to the alveolar as the capillary basement membranes. The negatively charged pulmonary capillary endothelium, the positively charged capillary basement membranes, and the negatively charged alveolar basement membranes may influence the transport of macromolecules from the pulmonary circulation in permeability pulmonary edema.     

Retina accumulates more glucose than does the embryologically similar cerebral cortex in diabetic rats

Aims/hypothesis. The retina is embryologically similar to cerebral cortex and the tissues of both are exposed to similar blood glucose concentrations. Nevertheless, in diabetes the retina develops metabolic abnormalities and microvascular lesions from which cerebrum seems relatively protected. We directly compared glucose concentrations and expression of GLUT-1 (the major carrier transporting glucose from blood into the neural retina and cerebrum) in the two tissues from normal and diabetic rats.¶Methods. Tissue and intracellular glucose were measured using two methods: direct assay of glucose and assay of Amadori products on intracellular proteins. The expression of GLUT-1 was measured using western blots in tissue and in the isolated endothelial luminal membrane of the two vascular beds.¶Results. Both methods assessing intracellular glucose indicate that intracellular concentrations of glucose in diabetes increased significantly in the retina but not in cerebral cortex. Concentrations of free glucose and Amadori product in retinas of diabetic animals were increased above normal by 334 % and 122 %, respectively, whereas there was no statistically significant increase in either parameter in the cerebral cortex of diabetic animals. In contrast to the observed increase in glucose in the retina in diabetes, expression of GLUT-1 on the luminal plasmalemma of the retinal vascular endothelium and in homogenates of whole retina decreased to a statistically significant extent (55 % and 36 %, respectively compared to normal). In the luminal cell membrane of the cerebral vasculature, diabetes did not decrease expression of GLUT-1 but tended to increase it slightly.¶Conclusions/interpretation. Even among tissues that do not require insulin for glucose uptake, tissue glucose concentration varies in diabetes. The greater increase in glucose concentration in retina than in cerebrum in diabetes probably contributes to the tissue differences in biochemical and histopathologic sequelae of the disease. The expression of GLUT-1 in the microvasculature is unlikely to account for the differences in tissue glucose between retina and cerebrum. [Diabetologia (2000) 43: 1417–1423]     

Mechanisms underlying endothelial dysfunction in diabetes mellitus

Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin- and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N:(G)-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91(phox) mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N:-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N:-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org.