Deborah Carper

Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis.

Many sight-threatening diseases have two critical phases, vessel loss followed by hypoxia-driven destructive neovascularization. These diseases include retinopathy of prematurity and diabetic retinopathy, leading causes of blindness in childhood and middle age affecting over 4 million people in the United States. We studied the influence of ω-3- and ω-6-polyunsaturated fatty acids (PUFAs) on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model of oxygen-induced retinopathy. We show that increasing ω-3-PUFA tissue levels by dietary or genetic means decreased the avascular area of the retina by increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization. The bioactive ω-3-PUFA-derived mediators neuroprotectinD1, resolvinD1 and resolvinE1 also potently protected against neovascularization. The protective effect of ω-3-PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-α. This inflammatory cytokine was found in a subset of microglia that was closely associated with retinal vessels. These findings indicate that increasing the sources of ω-3-PUFA or their bioactive products reduces pathological angiogenesis. Western diets are often deficient in ω-3-PUFA, and premature infants lack the important transfer from the mother to the infant of ω-3-PUFA that normally occurs in the third trimester of pregnancy. Supplementing ω-3-PUFA intake may be of benefit in preventing retinopathy.

Hypertonic stress induces αB-crystallin expression

Alpha B-crystallin, a major lens protein, was induced in primary cultures of dog lens epithelial cells and glomerular endothelial cells when they were grown under conditions of hypertonic stress. With Western blot analysis using a specific alpha B-crystallin antibody, we observed a significant increase in the concentration of alpha B-crystallin protein in cells grown for 4-6 days in media supplemented with 150 mM NaCl or 250 mM cellobiose. These supplements increased the osmolarity of the medium from 300 to 550-600 mosmol kg-1. Alpha B-crystallin mRNA was also increased reaching a maximum four-fold increase in lens and 16-fold increase in kidney cells within 1-2 days. These studies demonstrate a type of regulation of alpha B-crystallin expression in cells from lenticular and non-lenticular tissues.

Aldose reductase and ϱ-crystallin belong to the same protein superfamily as aldehyde reductase

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH‐dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ‐crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ‐crystallin are members of a superfamily of related proteins.

A superfamily of NADPH-dependent reductases in eukaryotes and prokaryotes

Aldose reductase (AR) is implicated in some of the disabling complications of diabetes, including neuropathy, retinopathy and cataracts. Our studies are aimed at further clarifying the role of AR in diabetes and facilitating the design of new classes of potent, specific AR inhibitors by gaining an understanding of the protein structure of AR. To this end, we have determined the complete protein sequence of rat lens AR using cDNA analysis and primer extension of mRNA. By comparing protein sequences, we have found that the structural relatedness (41% to 57%) among the vertebrate proteins, aldose reductase, aldehyde reductase, prostaglandin F synthase and the frog lens protein rho-crystallin can now be extended to prokaryotes by the inclusion of Corynebacterium 2,5-diketo-D-gluconate reductase. This more distantly related protein shares 30-40% identity with the vertebrate enzymes. Sequence alignments reveal that 18% of the amino acids are completely conserved in all members of the superfamily, many of them in clusters, suggesting that they mark important structural features such as the nucleotide binding site and substrate binding site. rho-Crystallin, which is structurally related to this superfamily of NADPH-dependent reductases, does not appear to reduce PGH2, PGD2, xylose or glyceraldehyde to any appreciable extent. It does, however, bind NADPH.

Osmotic Response Element Is Required for the Induction of Aldose Reductase by Tumor Necrosis Factor-α

Induction of aldose reductase (AR) was observed in human cells treated with tumor necrosis factor-α (TNF-α). AR protein expression increased severalfold in human liver cells after 1 day of exposure to 100 units/ml TNF-α. An increase in AR transcripts was also observed in human liver cells after 3 h of TNF-α treatment, reaching a maximum level of 11-fold at 48 h. Among the three inflammatory cytokines: TNF-α, interleukin-1, and interferon-γ, TNF-α (100 units/ml) gave the most induction of AR. Differences in the pattern of AR induction were observed in human liver, lens, and retinal pigment epithelial cells with increasing concentrations of TNF-α. A similar pattern of AR promoter response was observed between TNF-α and osmotically stressed human liver cells. The deletion of the osmotic response element (ORE) abolished the induction by TNF-α and osmotic stress. A point mutation that converts ORE to a nuclear factor-κB (NF-κB) sequence abolished the osmotic response but maintained the TNF-α response. Electrophoretic gel mobility shift assays showed two NF-κB proteins, p50 and p52, capable of binding ORE sequence, and gel shift Western assay detected NF-κB proteins p50 and p65 in the ORE complex. Inhibitors of NF-κB signaling, lactacystin, and MG132 abolished the AR promoter response to TNF-α.

Induction of aldose reductase expression in rat kidney mesangial cells and Chinese hamster ovary cells under hypertonic conditions.

Rat kidney cortex mesangial cells (MES) and Chinese hamster ovary cells (CHO) responded to hypertonicity (600 mosmol/kg) in culture by accumulating sorbitol. The accumulation of sorbitol was due to increased aldose reductase (AR) activity, apparently brought about by increased levels of AR mRNA and protein. The levels of AR mRNA increased approximately 60-fold in MES cells and 30-fold in CHO cells by 24 h in culture media (300 mosmol/kg supplemented with 150 mM NaCl, 600 mosmol/kg total). AR activity also markedly increased (14- to 16-fold above control), but MES took 4 days and CHO 6 days to reach this maximum. Other osmolytes, raffinose and sorbitol (at concentrations of 250 to 300 mM) elicited the same response as that of 150 mM NaCl. These data show that AR expression is induced in MES and CHO cells under hypertonic conditions. Of special interest is the induction of large amounts of AR in rat kidney cortex mesangial cells, a target tissue of diabetes and a site where excessive accumulation of sorbitol is suspected to be a critical factor in diabetic nephropathy.

Characterization of mRNA and genes for aldose reductase in rat

Aldose reductase (AR; E.C. 1. 1. 1. 21) has been implicated in a variety of diabetic complications. To investigate the expression of this enzyme in target tissues susceptible to such complications, mRNA encoding AR was characterized by Northern blot hybridization in various tissues and cultured cell preparations. The size of mRNA for AR (approximately 1500 bases) was in good agreement with the size determined by sequence analysis. A cDNA probe for AR from rat lens hybridized to the same size species of RNA isolated from cultured dog lens epithelial cells, cultured human retinal capillary pericytes (mural cells), and Y 79 human retinoblastoma cells. In rat tissues, a substantial amount of mRNA was expressed not only in lens, but also in retina, sciatic nerve and kidney medulla. AR mRNA seemed to be less abundant in rat skeletal muscle and brain, and was scarcely present in liver. Furthermore, Southern blot analysis of rat genomic DNA indicated that there are multiple sequences related to that for AR, probably indicating the existence of a multi-gene family.

Rapid purification of human placental aldose reductase.

Abstract Aldose reductase (alditol:NADP oxidoreductase EC 1.1.1.21) an enzyme in the polyol pathway which has been implicated in the pathogenesis of diabetic complications has been purified from the human placenta. Using affinity chromatography the enzyme can be obtained with apparent homogeneity in three steps. An initial 30–70% ammonium sulfate fraction was placed on a 4-carboxybenzaldehyde-coupled AH-Sepharose 4B column where upon elution with 0.1 m Na,K-phosphate buffer, pH 6.2, an enzyme fraction separated from the major protein peak. Chromatography of this enzyme fraction on an Amicon Matrex Orange A dyeligand column and elution with phosphate buffer containing 0.1 m m NADPH yielded aldose reductase of high purity. Antibodies raised in rabbits against the purified enzyme gave an apparent single line of identity with cither crude or purified aldose reductase.

Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens

Abstract Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano. Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.

Increase in aldose reductase mRNA in dog lens epithelial cells under hypertonic conditions

Aldose reductase (AR) mRNA levels increase when dog lens epithelial cells are exposed to hypertonic conditions. Hybridization of mRNA to an AR cDNA, using Northern and slot blots, showed that AR mRNA is elevated at least fourfold when primary dog lens epithelial cells are grown in media (300 mosmol kg-1) supplemented with 150 mM NaCl (600 mosmol kg-1 final). A time course showed an increase in AR mRNA of approximately twofold by 24 hr with a maximum increase of between four- and eightfold by 48 hr. AR mRNA remained elevated for the duration of the experiment, 8 days. The addition of Tolrestat, an inhibitor of aldose reductase, had no effect on the increased level of AR mRNA in these hypertonically stressed cells. Cells grown in media supplemented with 250 mM sorbitol also showed a substantial increase in AR mRNA. These data indicate, as in other cell types, the lens, a target tissue of diabetes, responds to hypersomotic stress with an induction of AR expression and suggests that AR may play a role in intracellular osmotic regulation.