Flavonoids as inhibitors of lens aldose reductase.

Flavonoids are effective inhibitors of lens aldose reductase. Quercetin, quercitrin, and myricitrin are significantly more potent than the previously known aldose reductase inhibitors. The inhibitory activity is of the noncompetitive type. In addition, quercitrin effectively blocks polyol accumulation in intact rat lenses incubated in medium containing high concentration of sugars.

Diabetic cataracts and flavonoids.

Oral administration of quercitrin, an inhibitor of aldose reductase, leads to a significant decrease in the accumulation of sorbitol in the lens of diabetic Octodon degus. The onset of cataract is effectively delayed when quercitrin is continuously administered. Thus in these diabetic animals, as in galactosemic rats, the use of an effective aldose reductase inhibitor impedes the course of cataract development. These observations support the hypothesis that in diabetes, as in galactosemia, aldose reductase plays a key role in initiating the formation of lens opacity.

Retinal capillaries: basement membrane thickening by galactosemia prevented with aldose reductase inhibitor.

A twofold thickening of capillary basement membranes of rat retinas resulting from dietary galactose was prevented by sorbinil, an inhibitor of aldose reductase. Since the basement membrane thickening was ultrastructurally similar to that typical of diabetic retinopathy, it may indicate changes in vessel permeability and susceptibility to hemorrhage. Galactosemic rats should be useful models for studying basement membrane-related complications of diabetes and for examining the potential biochemical regulation of basement membrane synthesis by aldose reductase inhibitors.

Cross-linking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen.

In a dye-sensitized photooxidation system, lens crystallin polypeptides become cross-linked, and a blue fluorescence that is associated with the proteins is produced. These changes are similar to those seen in vivo in the aging human lens. Evidence implicating singlet oxygen as the causative agent of the effects in vitro is presented, and the possibility that this species may play a role in aging and cataractogenesis in vivo is discussed.

Polyol accumulation in galactosemic and diabetic rats: control by an aldose reductase inhibitor.

An orally active inhibitor of aldose reductase, 1,3-dioxo-1H-benz[de]-isoquinoline-2(3H)acetic acid (AY-22,284), prevented cataractous changes in cultured lenses exposed to high concentrations of galactose. When given orally, AY-22,284 markedly decreased the accumulation of polyols in the lenses and sciatic nerves of galactosemic rats and rats with streptozotocin-induced diabetes. In addition, treatment of galactosemic rats with AY-22,284 effectively suppressed the formation of cataracts.

Localization of aldose reductase in the human eye.

The presence of the enzyme aldose reductase is increasingly being linked to diabetic complications. The distribution of this enzyme in human cornea, lens, retina, and optic nerve has been studied using specific antibodies against purified human placental aldose reductase raised in both rabbit and goat. The antisera from both animals gave equal, specific reactions. In frozen sections of ocular tissues, significant aldose reductase localization was reproducibly demonstrated in the endothelium and epithelium of the cornea and in the basal cell layers of the conjunctiva. In the lens, staining was observed in the epithelium and superficial lens fibers. In retinal sections, the presence of aldose reductase was demonstrated in the Mueller's cells, especially near the inner limiting membrane. It was also found in some ganglion and cone cells. In the optic nerve, positive staining was observed in the axon. All other cells of the tissues examined revealed only weak, nonspecific staining.

Prevention of basement membrane thickening in retinal capillaries by a novel inhibitor of aldose reductase, tolrestat.

A diabetic-like thickening of retinal capillary basement membranes induced in rats fed for 207 consecutive days a diet containing 50% galactose was prevented by the addition to the diet of tolrestat, a potent, structurally novel inhibitor of aldose reductase. Analysis of electron micrographs (X 25,000) of capillaries from the outer plexiform layer of the retina by computer planimetry showed that the basement membranes were approximately twofold thicker in rats fed galactose than in those fed either a standard diet or a diet containing galactose and tolrestat in doses of 43 or 57 mg/kg/day. The thickening of basement membranes in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes typical of diabetic microangiopathy, such as multilamination and the formation of vacuoles and dense inclusions. Therefore, the galactosemic rat represents a useful model for studying basement membrane-related complications of diabetes and their possible prevention by aldose reductase inhibitors.

Aldose reductase and p-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 p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.

Role of aldose reductase in the development of diabetes-associated complications.

The aldose reductase-initiated intracellular accumulation of polyols has been clearly shown to have a hyperosmotic effect on the lens of experimental animals. Similar localized osmotic changes resulting in the onset of pathology may also occur in the nerve, corneal epithelium, and retinal pericytes. Except for experimental diabetic cataracts, however, the exact mechanism by which aldose reductase is involved in these diabetes-associated complications remains to be clarified.

Immunohistochemical localization for aldose reductase in diabetic lenses.

Sugar cataract formation has been demonstrated to result from lenticular sorbitol accumulation. In the lens, the activity of aldose reductase has been observed to increase with the onset of diabetes, while the activity of sorbitol dehydrogenase decreases. This shift in activities of these two Sorbitol Pathway enzymes favors the increased accumulation of sorbitol. Immunohistochemical studies with antibodies prepared against purified rat lens aldose reductase reveal a striking increase in immunoreactive positive staining for aldose reductase in lenses from diabetic rats. Two weeks after the onset of diabetes, increased immunohistochemical staining for aldose reductase appears beneath the epithelial region where water cleft formation occurs, and the intensity of this staining increases with the formation of vacuoles. By 6-8 weeks, the presence of large vacuoles and areas of liquifaction containing dense immunoreactive stain can be observed. Examination of human cataractous lenses with antibodies prepared against purified human placenta aldose reductase suggest similar increases in immunoreactive staining in the human diabetic lens. Cataractous lenses from diabetic patients revealed increased immunoreactive staining for aldose reductase, which was associated with the presence of vacuoles in both the anterior or posterior superficial cortical layers. Examination of similar vacuole containing regions from non-diabetic cataractous lenses revealed no increase in immunoreactive staining for aldose reductase. These results suggest that the enhanced activity of aldose reductase observed in diabetes is due to an increased amount of enzyme, rather than enzyme activation.

Reversal of galactose cataract with Sorbinil in rats.

Sorbinil, a potent aldose reductase inhibitor, can effectively block the progression of a galactose cataract even though the cataractous process is well underway. The prevention of dulcitol accumulation by Sorbinil is just as effective in reversing the cataract as the removal of galactose from the diet. The progression and reversal of the cataract were followed by ophthalmoscopy and histology. The results also further support the concept that in galactosemia the cataract is not caused by the toxic effects of galactose per se but by the consequence of the aldose reductase reaction.

Synthesis of gamma crystallin by a cloned cell line from Nakano mouse lens.

A cloned cell line was derived from a culture of Nakano mouse lens epithelial cells. The cloned cells grew vigorously and produced large numbers of lentoid bodies. Sodium dodecyl sulfate (SDS) and non-SDS slab-gel electrophoresis of the soluble proteins from the cultured cell revealed protein bands identical in pattern to those of purified gamma crystallin. Antibody to mouse gamma crystallin reacted to the soluble protein fraction of the cultured cells, indicating the synthesis in culture of gamma crystallin by this cloned cell line.

Altering the course of cataracts in diabetic rats.

A potent new aldose reductase (AR) inhibitor was effective in preventing cataractous changes in diabetic rats. Untreated diabetic rats developed early lens changes by 3 weeks and dense nuclear opacities by 6 to 9 weeks. In contrast, diabetic rats treated with the AR inhibitor showed no lens changes during the 5-month period of the experiment.

The accumulation of myoinositol and rubidium ions in galactose-exposed rat lens.

When rat lens is incubated in 30 mM galactose overnight, the extent of accumulation of rubidium ions (Rb) and myoinositol (MI) are affected, as well as the Na-K ATPase activity. Rb accumulation and Na-K ATPase activity are only slightly affected compared to the dramatic drop in MI accumulation. These changes are completely abolished by sorbinil, which blocks polyol formation, or by rendering the galactose medium hypertonic to offset the osmotic effect of polyol formation. On the other hand, the addition of excess MI to the galactose medium had no effect on correcting these changes. The results obtained are consistent with the polyol-osmotic theory of sugar cataract formation.

Macrophage mediated damage to rat lenses in culture: a possible model for uveitis-associated cataract.

Rat lenses incubated in the presence of "activated" murine peritoneal macrophages are markedly impaired in their ability to accumulate certain radio-labeled compounds from the culture medium whereas incubation with resident macrophages has no such effect. The damage to the lens can be prevented in part by addition of certain antioxidants to the culture medium. The results suggest that mediators released by activated macrophages may be involved in initiation of the cataracts associated with chronic ocular inflammation.

Membrane glycoproteins of Philly mouse lens.

The Philly mouse is a new model for genetic cataracts, in which there is an apparent defect in lens membrane permeability. This abnormality results in electrolyte imbalance and lens hydration, typical of an osmotic cataract. Since membrane glycoproteins are believed to be involved in transport processes, we have studied the changes in these polypeptides in the Philly mouse lens during cataract development and compared them with those in the control Swiss-Webster mouse. The membrane glycoproteins were labeled by treatment with galactose oxidase and tritiated borohydride. Radioactivity was found to be incorporated into six glycoproteins of approximate molecular weights of 128 k, 103 k, 82 k, 71 k, 35 k, and 22 k daltons. The 35 k polypeptide is the major glycoprotein in the mouse lens membrane and shows increased incorporation of the tritium label with progression of the cataract. In contrast to the murine lens, the 35 k peptide could not be detected in rabbit lens membranes. Other changes observed in glycoproteins of the Philly mouse lens during cataract development were a loss of the 103 k and 71 k polypeptides and a corresponding increase in the 66 k polypeptide. These changes in glycoproteins may be related to the permeability changes and cataract development in the Philly mouse lens.

Galactose cataract prevention with sorbinil, an aldose reductase inhibitor: a light microscopic study.

Cataract formation in galactosemic rats was studied by ophthalmoscopy, slit-lamp biomicroscopy, and by light microscopy using plastic embedding with methacrylate. Untreated rats developed nuclear cataracts by 14 days and mature cataracts by 21 days. However, rats treated with the aldose reductase inhibitor sorbinil did not develop any cataractous change for up to 8 months of 50% galactose feeding and could not be distinguished from normal controls. This strongly suggests that aldose reductase is the common factor involved in the formation of sugar cataracts.

Aldose reductase localization in human retinal mural cells.

A hallmark of early diabetic retinopathy is the selective loss of the retinal mural cells (pericytes) from vessels. Using antibodies prepared against purified human placental aldose reductase, the presence of the enzyme aldose reductase can be demonstrated immunohistochemically in the cytoplasm of retinal mural cells of trypsin-digested human retinal vessels. This enzyme, which reduces various hexose sugars to their sugar alcohols, has been implicated in the pathogenesis of several diabetic complications.

Alterations of lens protein synthesis in galactosemic rats.

A differential effect on protein synthesis has been demonstrated in the lenses of galactosemic rats. During galactose cataract development the synthesis of lens crystallins is depressed, whereas that of noncrystallin proteins is unaffected. This effect correlates with the influx of Na+ and loss of K+ from the lens. Removal of the galactose diet results in a gradual recovering of crystallin synthesis to normal levels. In vitro the nuclear cataractous lenses leak crystallins into the media; however, upon 5 day's recovery no leak-out of crystallins could be detected. Both decreased synthesis and leak-out probably account for the marked loss of dry weight of cataractous lenses. These results support the hypothesis that crystallin synthesis may be affected by cation imbalance or changes concomitant with such an imbalance.

Prevention and reversal of galactose cataract in rats with topical Sorbinil.

Rats fed a 50% galactose diet were treated topically in one eye with 1% Sorbinil . The eye treated with Sorbinil remained clear during the following 4-week period. Unexpectedly, the lens of the untreated eye also maintained transparency. Histologically both lenses remained normal. Moreover, the reduced dulcitol levels in the lenses of both eyes were identical. These findings suggest that the effect of topically administered Sorbinil in galactosemic rats was mainly systemic rather than local. Confirmation of this came from the observations that the extent of inhibition of polyol synthesis in these rats was found to be similar in the sciatic nerve, blood, and lens. A reversal of the galactose cataracts also was affected by Sorbinil eye drop treatment.