Amelioration of Diabetes-Associated Abnormalities in the Vitreous Fluid by an Inhibitor of Albumin Glycation

Abstract

purpose. Albumin modified by Amadori glucose adducts is a plasma-borne factor that activates cell signaling pathways, modulates the expression of growth factors and cytokines, and participates in the pathogenesis of microvascular complications of diabetes. In the present study, streptozotocin diabetic rats were treated with an orally administered compound that inhibits the nonenzymatic glycation of albumin to evaluate whether increased glycated albumin contributes to diabetes-associated abnormalities in the vitreous fluid.

methods. Vitreous obtained from age-matched nondiabetic and streptozotocin-diabetic rats, half of which received the test compound 2-(3-chlorophenylamino) phenylacetic acid (23CPPA) by oral gavage for 26 weeks, was analyzed by immunoassay for pigment epithelium-derived factor (PEDF), vascular endothelial growth factor (VEGF) and glycated albumin content, by measurement of thiobarbituric acid reactive substances (TBARs) for lipid peroxide products and by colorimetric assay for hyaluronan content.

results. Compared with that of nondiabetic controls, vitreous of diabetic rats contained decreased PEDF, increased VEGF, higher VEGF/ PEDF ratio, and elevated levels of TBARs, glycated albumin, and hyaluronan. These changes were significantly attenuated in rats treated with test compound despite the presence of marked hyperglycemia.

conclusions. Results indicate that inhibiting the formation of glycated albumin, which is increased in diabetes, ameliorates vitreous changes in angiogenic and metabolic factors associated with the development of diabetic retinopathy. The observed improvement in vitreous alterations associated with reductions in glycated albumin suggests that elevated levels of glycated albumin play a retinopathogenic role in diabetes that is operative and that can be therapeutically addressed independently of glycemic status.

Recent studies have shown that the vitreous manifests changes in angiogenic and metabolic factors concordant with abnormalities in the retinal microvasculature that participate in the pathogenesis of diabetic retinopathy. ^{1 2 3 4} Retinal neovascularization occurs toward the vitreous compartment, with microproliferation and migration of cells onto the posterior vitreous cortex, giving rise to the vitreal presence of the angiogenic vascular endothelial growth factor (VEGF) ^{5 6 7 8} and the antiangiogenic serine protease inhibitor pigment epithelium-derived factor (PEDF). ^{9 10 11 12} Decreased expression of PEDF and increased VEGF expression by retinal cells in diabetes is reflected in vitreous samples, which contain reduced levels of PEDF and elevated levels of VEGF, from patients with proliferative diabetic retinopathy and from genetically diabetic db/db mice compared with specimens from nondiabetic counterparts. ^{1 2 3 4} These reciprocal changes of increased expression of VEGF and VEGF receptors and decreased PEDF are believed to causally contribute to increased retinal vascular permeability and neovascularization in diabetes. ^{1 2 3 7 13 14} The observations that PEDF reduces VEGF-induced retinal vascular hyperpermeability ¹⁵ and downregulates the expression of VEGF ¹⁴ and that exogenously administered PEDF inhibits retinal angiogenesis ^{11 12} implicate decreased PEDF as mechanistically involved in VEGF overexpression. Changes in vitreous constituents, growth factors, and cytokines may exacerbate diabetic retinopathy, ^{2 16 17} but they may also represent developing pathophysiology at an early stage, as suggested by the findings that VEGF is increased in vitreous and in nonvascular cells of diabetic eyes without overt retinopathy. ^{4 18 19} Increased VEGF and decreased PEDF also have been found in the aqueous humor of patients with diabetes, ²⁰ even those with no or mild retinopathy. ²¹  

Among factors in the diabetic milieu that participate in the pathogenesis of the microvascular complications of diabetes, the elevated levels of Amadori-modified glycated albumin figure prominently. Increased concentrations of this plasma-borne factor, which associate independently with microvascular complications of diabetes in humans, ^{22 23 24} influence cell signaling pathways and molecular mediators that causally contribute to the development of diabetic retinopathy and nephropathy. In retinal pigment epithelial cells, a major source of growth factors and chemokines that play a pathogenetic role in diabetic retinopathy, glycated albumin increases the production of the inflammatory cytokines interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 and activates the mitogen-activated protein kinase (MAPK) pathway, protein kinases, JAK, and the transcription factor nuclear factor (NF)-κB. ^{25 26 27 28 29} Additionally, glycated albumin is toxic to retinal pericytes and induces pericyte death. ³⁰ In glomerular mesangial, endothelial, and epithelial cells, Amadori-modified glycated albumin upregulates the fibrogenic transforming growth factor (TGF)-β1 system, stimulates the production of matrix proteins, activates protein kinase (PK)C-β1 and extracellular signal-related kinase (ERK), and decreases nephrin expression. ^{31 32 33 34 35 36} In hyperglycemic genetically diabetic db/db mice, inhibiting the nonenzymatic glycation of albumin decreases microalbuminuria, restores glomerular nephrin, downregulates renal TGF-β1 and VEGF overexpression, and ameliorates matrix accumulation, glomerular histomorphometric changes, and the development of renal insufficiency ^{37 38 39}  

These considerations, coupled with the demonstration that diabetic rodents manifest diabetes-associated vitreal and retinal abnormalities at an early stage in the development of retinal microvasculature pathology, ^{4 40} prompted the present study in which we examined the effects of a compound that inhibits albumin glycation on biochemical abnormalities in the vitreous of experimentally diabetic rats. The compound, 2-(3-chlorophenylamino) phenylacetic acid (23CPPA), contains an anionic side chain acetyl group that interacts with positively charged, potentially glycatable lysine amino groups in the albumin protein and that has been shown to decrease the formation of albumin modified by Amadori glucose adducts and to reduce elevated levels of glycated albumin in diabetic rodents independently of any effect on hyperglycemia. ^{37 38 39} We report that chronic administration of 23CPPA to rats with streptozotocin-induced diabetes attenuates vitreal changes associated with the dysregulation of retinal angiogenesis. 

Materials and Methods

Diabetes was induced by intravenous injection (50 mg/kg) of streptozotocin (Sigma, St. Louis, MO) into the tail veins of male Wistar rats (Harlan, Indianapolis, IN) aged 6 weeks and weighing between 120 and 140 g. Animals with plasma glucose concentrations ≥ 15 mM within 1 week after the induction of diabetes were included in the study. Age-, weight-, and sex-matched Wistar rats served as nondiabetic controls. The diabetic rats were divided into two groups, one of which received the glycation inhibitor 23CPPA ^{37 38 39} by oral gavage at a total dosage of 15 mg/kg/d from age 7 weeks through age 33 weeks, with the other group serving as diabetic controls. Commercial rodent chow and water were provided ad libitum, and animals were monitored for blood glucose and weight. All diabetic rats received long-acting insulin (Lantos; Aventis, Bridgewater, NJ) every other day with adjustments of dosage to prevent ketoacidosis, improve survival, and keep animals on a positive growth curve. The rats were housed in a temperature-controlled facility, and the Institutional Animal Care and Use Committee approved all procedures. Rats were killed at the conclusion of the 6-month experimental period, eyes were harvested, and the vitreous was collected by aspiration and stored at −70°C until analysis. Experiments performed in this study adhered to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. Nonophthalmic tissue was harvested for other investigations. 

PEDF and VEGF were measured by immunoassay according to the instructions provided by the manufacturers (PEDF ELISA; BioProducts, Middletown, MD, and VEGF ELISA; Ray Biotech, Norcross, GA). Lipid peroxide products were determined by the TBAR assay using, as standard, the formation of malondialdehyde from 1,1,3,3-tetramethoxypropane and normalizing results to albumin content measured by immunoassay specific for rat albumin according to the manufacturer’s instructions (Nephrat; Exocell, Philadelphia, PA). Total protein was measured by the bicinchoninic acid colorimetric (BCA; Pierce, Rockford, IL) method. Hyaluronan levels were measured in a microplate assay using 3-hydroxydiphenyl as the coloring reagent. ⁴¹ Samples were incubated with 10 μg hyaluronic acid-specific hyaluronidase (hyaluronate lyase; Sigma) for 20 hours at 37°C, followed by the removal of protein with 10% trichloroacetic acid before quantification of uronic acid in the microplate procedure. Glycated albumin was measured according to the manufacturer’s instructions (Glycaben; Exocell) by an immunoassay using monoclonal antibody specific for Amadori-modified epitopes in glycated albumin. Data were analyzed by means for analysis of variance (ANOVA) among the three experimental groups. 

Results

General characteristics of the experimental animals at the conclusion of the study period are summarized in Table 1 . Diabetic rats weighed significantly less than nondiabetic controls, but body weights and blood glucose levels did not differ in diabetic controls compared with diabetic rats receiving 23CPPA. The growth curves and glucose levels in the animals in this study are similar to those reported by others using streptozotocin diabetic rats receiving insulin without or with coadministration of an agent that does not affect hyperglycemia. ⁴² Plasma concentrations of glycated albumin were significantly higher in diabetic than in nondiabetic controls and were significantly reduced, though not normalized, in diabetic rats receiving test compound, consistent with the direct effect of 23CPPA on albumin glycation despite prevailing hyperglycemia. 

Vitreous PEDF levels were significantly lower in diabetic than in nondiabetic control rats (4.6 ±1.4 vs. 15.3 ±3.0 ng/mL, respectively; P ≤ 0.05), whereas vitreous VEGF levels were higher in diabetic than in nondiabetic controls (167 ± 49 vs. 75 ± 10 pg/mL, respectively; P ≤ 0.05; Fig. 1 ). In contrast, compared with their untreated diabetic control counterparts, PEDF levels were higher (8.7 ± 2.0 ng/mL; P ≤ 0.05 compared with diabetic control) and VEGF levels were lower (105 ± 22 pg/mL; P ≤ 0.05 compared with diabetic control) in diabetic rats receiving test compound (Fig. 1) . The relative ratio of VEGF to PEDF was significantly higher (7.3 ± 1.9; P ≤ 0.05 vs nondiabetic control) in vitreous from diabetic than in nondiabetic control rats, assigned an arbitrary value of 1.0 (±0.2), consistent with a diabetes-associated imbalance between the angiogenic VEGF and the antiangiogenic PEDF (Fig. 1) . In contrast, the relative ratio of VEGF to PEDF in vitreous from diabetic rats treated long term with 23CPPA was reduced by 50% compared with that in diabetic controls (3.75 ± 0.6; P ≤ 0.05 vs. diabetic control; Fig. 1 ). 

Lipid peroxide products, which are increased in the vitreous of patients with proliferative diabetic retinopathy, ⁴³ were higher in vitreous from diabetic than in nondiabetic control rats (84 ± 3 vs. 62 ± 3 nmol/mg albumin, respectively; P ≤ 0.05), but levels in the vitreous from diabetic rats receiving test compound (65 ± 9 nmol/mg albumin) did not differ significantly from those in the nondiabetic controls (Fig. 2A) . Similarly, the concentration of glycated albumin was higher in diabetic than in nondiabetic vitreous (1.92 ± 0.37 vs. 0.71 ± 0.07 μg/mL, respectively; P ≤ 0.05) but was not significantly different in vitreous from diabetic rats receiving test compound (0.85 ± 0.10) compared with the nondiabetic controls (Fig. 2B) . Total concentrations of vitreous albumin did not differ significantly among the three experimental groups, and the changes in glycated albumin pertained when expressed as microgram per milligram vitreous albumin (4.75 ± 0.48, 12.8 ± 2.4, and 5.6 ± 0.70 μg/mg, respectively, in nondiabetic, diabetic control, and diabetic rats receiving 23CPPA). Compared with nondiabetic controls, total vitreous protein was higher in control and treated diabetic rats (263 ± 24, 445 ± 31, and 402 ± 29 μg/mL, respectively). Hyaluronan levels, which corroborated authenticity of the vitreous samples, were significantly higher in the vitreous of diabetic compared with nondiabetic controls (433 ± 84 vs. 56 ± 27 μg/mL, respectively; P ≤ 0.05) and were significantly lower in vitreous from rats receiving 23CPPA compared with the diabetic control rats (140 ± 55 μg/mL; P ≤ 0.05 vs. diabetic control; Fig. 2C ). 

Discussion

These results indicate that administration of an inhibitor of nonenzymatic glycation to streptozotocin-treated diabetic rats for 6 months ameliorates vitreal changes in angiogenic and metabolic factors associated with the development of diabetic retinopathy. Specifically, 23CPPA partially corrected or normalized the decreased PEDF levels and the increased levels of VEGF, lipid peroxide products, hyaluronan, and Amadori-modified glycated albumin found in diabetic control rats. These salutary effects occurred without change in glycemic status and in the presence of marked hyperglycemia. 

Vascular leakage in the choriocapillaris allows the passage of nonglycated and glycated albumin toward the retina, with accumulation in the extracellular spaces and the possibility of local formation of glycated albumin after leakage. ^{16 29} An increased expression of VEGF and VEGF receptors in diabetes could be expected to promote microvascular permeability and thereby enhance the likelihood of direct contact of plasma-borne glycated albumin with the outermost retinal layer, modulating the production of growth factors and cytokines by retinal pigment epithelial cells. ²⁹ Vitreal total protein concentration was significantly higher in diabetic than in nondiabetic controls, a finding that may reflect increased retinal microvascular leakage or that may reflect differences in diabetes in the content of intrinsic vitreous proteins that cannot be distinguished by generic colorimetric assay. On the other hand, total vitreal protein concentrations were not significantly different in diabetic rats receiving 23CPPA than in diabetic controls, possibly because of relative insensitivity of or the vitreal factors interfering with methods used for the measurement of total proteins. The observation that total vitreous concentrations of albumin, measured by specific immunoassay, did not differ in the three experimental groups suggests that microvascular leakage was not a main contributor to the increased protein or glycated albumin content in the vitreous of diabetic rats. The decreased vitreal glycated albumin in rats receiving 23CPPA is consistent with systemic and local inhibition of the nonenzymatic glycation of this protein, but the possibility of lessened retinal microvascular leakage in these animals cannot be excluded. 

The reason vitreal hyaluronan is elevated in diabetes and the mechanism by which 23CPPA reduces it in diabetic rodents have not been delineated. Hyaluronan in serum also has been reported to be higher in patients with diabetes than in patients without diabetes and to correlate with poor glucose control and diabetic angiopathy. ⁴⁴ Extracellular glycosaminoglycans modulate angiogenesis, with degradation products of nonsulfated hyaluronan being angiogenic and promoting endothelial cell proliferation, migration, and tube formation and with the native high-molecular weight form being antiangiogenic. ⁴⁵ Because the amount of vitreous from each animal in the present study was insufficient to assess intact hyaluronan versus smaller disaccharides, interpretation of the pathobiologic import of an elevated vitreous hyaluronan in diabetes is speculative. Nevertheless, because reactive oxygen species (ROS) stimulate hyaluronan degradation ⁴⁶ and ROS are increased in diabetic compared with nondiabetic vitreous, ^{4 46 47} the vitreous in diabetic animals likely contains increased levels of hyaluronan degradation products, creating an imbalance of antiangiogenic versus angiogenic influences related to this vitreous component. Glycated albumin-induced increases in ROS production may enhance the formation of AGE products, which promote photosensitization-induced acceleration of hyaluronan depolymerization. ⁴⁸ By decreasing ROS production, as evidenced by the reduced lipid peroxidation in the present study, 23CPPA may beneficially influence hyaluronan depolymerization through reduced formation of AGE products. 

The evolution of diabetic retinopathy encompasses an early loss of pericytes and endothelial cells in the retinal capillaries, dysregulated capillary blood flow, hypoxia, and abnormal expression of the molecular mediators PEDF and VEGF. Decreased PEDF and increased VEGF in the vitreous mirrors the abnormal retinal microvascular milieu and portends developing retinopathology. ^{4 40 49} Attenuation of these abnormalities with a glycation inhibitor suggests that this therapeutic approach may be of benefit in forestalling the evolution of diabetic retinopathy. Although progression to severe stages and blindness have been reduced with improved glycemic control and retinal laser photocoagulation, ⁵⁰ the need remains for innovative treatments specifically targeting factors underlying the pathogenesis of diabetic retinopathy ⁵¹ that can arrest its development at early stages and that operate independently of glycemic control. 

 Supported by National Institutes of Health DK Grants 62540 and 72587.

 Submitted for publication March 10, 2008; revised May 12, and June 6 and 24, 2008; accepted August 27, 2008.

 Disclosure: M.P. Cohen, Glycadia, Inc. (E, I), P; E. Hud, Glycadia, Inc. (E); V.-Y. Wu, Glycadia, Inc. (E); C.W. Shearman, Glycadia, Inc. (E)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Experimental Animal Data

	Nondiabetic n = 8	Diabetic Control n = 9	Diabetic-23CPPA n = 9
Body weight (g)	584 ± 12	366 ± 6*	390 ± 14
Blood glucose (mM)	7.0 ± 0.9	24.7 ± 0.6*	25.9 ± 1.0*
Glycated albumin (μg/mg albumin)	27.7 ± 2.5	47.5 ± 3.0*	36.4 ± 2.0, † , ‡

 Values are mean ± SEM.

*   P < 0.001 compared with nondiabetic control.

†   P < 0.01 compared with diabetic control.

‡   P < 0.02 compared with nondiabetic control.

Figure 1.

 

PEDF (A), VEGF (B), and VEGF/PEDF relative ratio (C) in the vitreous of rats in the three experimental groups described in Table 1 . Results represent mean ± SEM of levels measured by immunoassay. Relative ratios of VEGF to PEDF were calculated in comparison with the ratio in nondiabetic rats, assigned an arbitrary value of 1. Nondiab, nondiabetic controls; Db, diabetic controls; Db-Rx, diabetic rats receiving 23CPPA. *P ≤ 0.05 compared with nondiabetic controls. #P ≤ 0.05 compared with diabetic controls. @P ≤ compared with nondiabetic controls.

Figure 2.

 

TBARs (A), glycated albumin (B), and hyaluronan (C) in vitreous from rats in the three experimental groups described in Table 1 . Results represent mean ± SEM of TBARs assayed with MDA-oxidized standard and expressed as nmol/mg albumin, glycated albumin measured by monoclonal specific immunoassay, and hyaluronan measured after treatment with hyaluronic acid specific hyaluronidase. Nondiab, nondiabetic controls; Db, diabetic controls; Db-Rx, diabetic rats receiving 23CPPA. *P ≤ 0.05 compared with nondiabetic controls. #P ≤ 0.05 compared with diabetic controls.