Robert N. Frank

Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats

Abstract.Aims/hypothesis: A strong positive correlation has been found between lipid peroxidation product and vascular endothelial growth factor concentrations in the vitreous of patients with proliferative diabetic retinopathy. To establish a causal relation between diabetes-associated enhanced oxidative stress and vascular endothelial growth factor production, we evaluated two antioxidants, dl-α-lipoic acid and taurine, on retinal vascular endothelial growth factor protein and mRNA expression and on parameters of oxidative stress in streptozotocin-diabetic rats. Methods: Our experiments were on control rats and streptozotocin-diabetic rats with a 6-week duration of diabetes, treated with or without dl-α-lipoic acid (100 mg · kg–1· d–1, i. p.) or taurine (1 % in the diet) starting from induction of diabetes. Vascular endothelial growth factor protein in retinal homogenates was assessed by sandwich ELISA with an affinity-purified polyclonal antibody and vascular endothelial growth factor mRNA by ribonuclease protection assay. Retinal lipid peroxidation products i. e. malondialdehyde plus 4-hydroxyalkenals were quantified with n-methyl-2-phenylindole. Retinal reduced and oxidized glutathione, ascorbate, dehydroascorbate, and sorbitol pathway intermediates were measured spectrofluorometrically, and taurine by reverse-phase HPLC. Results: Vascular endothelial growth factor protein concentration (means ± SD) was increased in diabetic rats compared with control rats (33 ± 7 vs 19 ± 5 pg/mg total protein, p < 0.01) This increase was attenuated by taurine (26 ± 8, p < 0.05) and prevented by dl-α-lipoic acid (21 ± 4, p < 0.01). Vascular endothelial growth factor mRNA abundance was reduced by 1.4-fold in diabetic rats compared with control rats and this decrease was attenuated but not completely prevented by both antioxidants. Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats compared with control rats, and both antioxidants arrested accumulation of lipid peroxidation products. Taurine, reduced glutathione, oxidized glutathione, ascorbate, dehydroascorbate and sorbitol pathway intermediate concentrations as well as oxidized glutathione/reduced glutathione and dehydroascorbate/ascorbate ratios were similar in control and diabetic rats treated with or without taurine. Conclusion/interpretation: Oxidative stress is directly involved in up regulation of vascular endothelial growth factor protein in the retina during early diabetes. [Diabetologia (2001) 44: 1102–1110]

On the pathogenesis of diabetic retinopathy.

Recent investigations of retinal vascular cells in tissue culture, animal models, and diabetic human subjects suggest several potential pathogenetic mechanisms for diabetic retinopathy. These include the enzyme aldose reductase, which appears to be responsible for basement membrane thickening in galactosemic rats (since the lesion is prevented by an aldose reductase inhibitor), and a picture, in galactosemic dogs, that closely resembles early, background diabetic retinopathy; insulin, which stimulates, and elevated glucose levels, which inhibit in vitro proliferation of retinal pericytes. Various hormones, including the sex hormone, the insulin-like growth factors and, perhaps independently, growth hormones, may influence the later stages of diabetic retinopathy. Chronic hyperglycemia appears to be the primary pathogenetic agent in diabetic retinopathy as well as in other complications of diabetes, but the different rates of onset and progression of these complications suggest that glucose acts through different biochemical pathways that are probably under different genetic control. Finally, the locus of the primary biochemical lesion in diabetic retinopathy may reside in the neuronal or glial cells of the retina, with the retinal blood vessels only secondarily involved.

On the Pathogenesis of Diabetic Retinopathy: A 1990 Update

Although most investigators now agree that chronic hyperglycemia is the basis for diabetic retinopathy, this has not been proven definitively. Even if chronic hyperglycemia is the initial common pathway leading to retinopathy and other complications of diabetes, it appears to act by different mechanisms in different tissues. The enzyme, aldose reductase, may play a major role in the development of diabetic retinopathy, but contradictory evidence exists. At the present time, results of the only study of aldose reductase inhibition and diabetic retinopathy reported in humans were negative. Another mechanism worthy of consideration is nonenzymatic glycation (glycosylation) of proteins, but there is no direct evidence of a causal role in diabetic retinopathy. Several growth factors have been identified in the retina that may promote neovascularization, and at least two inhibitors may prevent the process. There is evidence to support a role for basic and, perhaps, acidic fibroblast growth factors in retinal vasoproliferation. Transforming growth-factor beta, a peptide produced by capillary pericytes and smooth muscle cells and activated by the interaction of these cells with vascular endothelial cells, appears to be an important inhibitor of neovascularization, as is the vascular basement membrane.

Retinopathy in Juvenile-onset Type I Diabetes of Short Duration

We evaluated the prevalence and severity of diabetic retinopathy in 173 juvenile-onset, type I diabetic subjects and 78 nondiabetic controls of similar age, race, and sex distribution by stereoscopic fundus photography and fluorescein angiography, performed by a standardized protocol and evaluated by five expert, masked observers. The overall prevalence of retinopathy was 18% in the diabetic group and 0% in the controls. Retinopathy prevalence increased with duration of diabetes in the diabetic group, with a prevalence of 1% from 0–4 yr after diagnosis, 25% after 5–9 yr, and 67% 10–16 yr after onset of the systemic disease. There was an independent association with age, with little retinopathy before age 15 and a 48% prevalence in older persons. Retinopathy was also found to be independently associated with the following: diabetic “control,” evaluated semiquantitatively but on a masked basis; lens opacities; and frequency of daily insulin injections. Among the 166 diabetic subjects who had both angiography and photography, a retinopathy prevalence of 17% was detected by angiography and 11% by photography. This difference was statistically significant (P = 0.01). This study provides baseline data for use in estimating sample size in controlled trials of therapeutic measures to prevent retinopathy in juvenile diabetic populations. The study also supports the hypothesis that long-term hyperglycemia as well as changes (possibly hormonal in nature) associated with puberty are causally related to diabetic retinopathy.

A model of subretinal neovascularization in the pigmented rat.

We produced krypton laser photocoagulation lesions of mild to moderate whiteness in the posterior retinas of one eye of 23 pigmented rats, and identical appearing argon laser burns in the fellow eyes. We observed foci of subretinal neovascularization, histopathologically markedly similar to that which occurs in several human retinal diseases, in the krypton laser treated eyes of 6 of the 14 rats that were followed for one to three months after photocoagulation. No such lesions were observed in the argon laser treated fellow eyes, nor in krypton or argon laser treated eyes examined earlier than one month after photocoagulation. The photocoagulation damaged only the choriocapillaris, the retinal pigment epithelium (RPE), and the photoreceptor layer. In the acute lesions, we did not observe ruptures in Bruchs membrane. The neovascularization was surrounded by multiple layers of RPE cells, a histopathologic finding that has also been reported in some human eyes with subretinal neovascularization in age-related macular degeneration. These observations suggest that the RPE cells may be modifying the proliferative behavior of adjacent choroidal capillaries. This model differs from previous models of subretinal neovascularization in primates, and may be useful for additional studies of this important pathological process.

ATP causes retinal pericytes to contract in vitro

We evaluated the contractility of bovine retinal microvascular pericytes in culture by permeabilizing the cells with 0.1% Triton X-100 and measuring their response to MgATP. Sequential photographs of the cells were taken over 20 min and their surface areas were measured. Our study directly demonstrates that pericytes are contractile cells, which respond to MgATP in a dose-dependent fashion over a relatively short time course (minutes). Pericytes did not contract in response to GTP, pyrophosphate or beta, gamma-methylene ATP. Immunofluorescence study showed the presence of muscle actin in Triton X-100-treated cells before and after contraction, indicating preservation of this cytoskeletal protein even after treatment with the detergent. Similar experiments on human umbilical vein endothelial cells, bovine lens epithelial cells and human retinal pigment epithelial cells showed that these cells were significantly less contractile than retinal pericytes. That pericytes show substantial contraction over a short time course indicates that these cells may play a major role in regulating blood flow in the microcirculation.

Increases in collagen type IV and laminin in galactose-induced retinal capillary basement membrane thickening—Prevention by an aldose reductase inhibitor

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet (P less than 0.001), or animals on a diet containing 30% galactose and 250 mg kg-1 of the aldose reductase inhibitor sorbinil (P less than 0.001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.

Measurement practices: methods for developing content-valid student examinations

Measurement experts generally agree that a systematic approach to test construction will probably result in an instrument with sound psychometric properties. One fundamental method is called the blueprint approach to test construction. A test blueprint is a tool used in the process for generating content-valid exams by linking the subject matter delivered during instruction and the items appearing on the test. Unfortunately, this procedure as well as other educational measurement practices is often overlooked. A survey of curriculum administrators at 144 United States and international medical schools was conducted to assess the importance and prevalence of test blueprinting in their school. Although most found test blueprinting to be very important, few require the practice. The purpose of this paper is to review the fundamental principals associated with achieving a high level of content validity when developing tests for students. The short-term efforts necessary to develop and integrate measurement theory into practice will lead to long-term gains for students, faculty and academic institutions.

Retinovitreal neovascularization in the royal college of surgeons rat

Retinovitreal blood vessels, which we have previously reported in the dystrophic retinas of approximately 20% of older (greater than 15 months of age) spontaneously hypertensive (SHR) rats, also occur in about 20% of retinal dystrophic Royal College of Surgeons (RCS) rats greater than 1 year of age. We have demonstrated previously that these vessels, in the SHR rat, have the anatomic characteristics of retinovitreal neovascularization as it has been described in vasoproliferative retinopathies in humans (1). We now provide strong evidence that the endothelial cells and pericytes of the retinovitreal vessels that occur in RCS rats are proliferating, since they demonstrate by autoradiography significantly increased nuclear labeling with [3H]-thymidine over intra-retinal or choroidal vessels, or the abnormal vessels that grow within the retinal pigment epithelium in these dystrophic retinas. One important difference between the neovascularization that occurs in these rats and that which is observed in various human retinal vascular diseases is the frequent association of the new vessels in dystrophic rat retinas with surrounding cords of proliferating retinal pigment epithelium. The retinovitreal vessels in these strains of dystrophic rats represent a new animal model that may be useful for studying the fundamental processes underlying new blood vessel growth in the retina.

Cerebral cortical capillary basement membrane thickening in galactosaemic rats

SummaryWistar-Kyoto rats fed a diet containing 30% by weight galactose for 15–21 months developed significant thickening of the endothelial basement membranes of capillaries from the frontal cortex of the cerebrum, by comparison with cerebral capillary basement membranes from animals on a standard diet (p<0.001), or animals receiving a diet containing 30% galactose together with 250 mg/kg diet of the aldose reductase inhibitor, Sorbinil (0.001<p<0.01). The effect was similar to that which we have reported previously in the retinal capillaries of these animals. Spontaneously hypertensive rats on the high-galactose diet showed modest cerebral capillary basement membrane thickening (0.02<p<0.05) only for one of the measurement protocols utilised, and the process was not prevented by Sorbinil. Biochemical assays of retina, cerebral cortex, and blood serum from Wistar-Kyoto and spontaneously hypertensive rats maintained on the Sorbinil regimen showed that the drug did cross the blood-retinal and blood-brain barriers. Similar to our previous study on the retinal capillaries, we observed no degeneration of pericyte or endothelial cell cytoplasm, and no alteration in the pericyte/endothelial cell nuclear ratio in the cerebral capillaries of galactosaemic animals, by comparison with controls. Based on immunocytochemical studies in the human retina, it has been claimed that aldose reductase is present in capillary pericytes but absent in the endothelial cells. However, we observe a considerably smaller pericyte/ endothelial cell nuclear ratio in the capillaries of the cerebral cortex of the rat, by comparison with those of the retina. Also, pericyte coverage of the cerebral cortical capillaries is much less than that of the retinal capillaries of these animals. Therefore, it appears that the biochemical process(es) responsible for basement membrane thickening are unlikely to reside within the pericytes.