Osmotic Stress, not Aldose Reductase Activity, Directly induces Growth Factors and MAPK Signaling changes during Sugar Cataract Formation

In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/ JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation

Orally Active Multi-Functional Antioxidants Delay Cataract Formation in Streptozotocin (Type 1) Diabetic and Gamma-Irradiated Rats

Age-related cataract is a worldwide health care problem whose progression has been linked to oxidative stress and the accumulation of redox-active metals. Since there is no specific animal model for human age-related cataract, multiple animal models must be used to evaluate potential therapies that may delay and/or prevent cataract formation.Proof of concept studies were conducted to evaluate 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8), multi-functional antioxidants that can independently chelate redox metals and quench free radicals, on their ability to delay the progression of diabetic "sugar" cataracts and gamma radiation-induced cataracts. Prior to 15 Gy of whole head irradiation, select groups of Long Evans rats received either diet containing compound 4 or 8, or a single i.p. injection of panthethine, a radioprotective agent. Compared to untreated, irradiated rats, treatment with pantethine, 4 and 8 delayed initial lens changes by 4, 47, and 38 days, respectively, and the average formation of posterior subcapsular opacities by 23, 53 and 58 days, respectively. In the second study, select groups of diabetic Sprague Dawley rats were administered chow containing compounds 4, 8 or the aldose reductase inhibitor AL1576. As anticipated, treatment with AL1576 prevented cataract by inhibiting sorbitol formation in the lens. However, compared to untreated rats, compounds 4 and 8 delayed vacuole formation by 20 days and 12 days, respectively, and cortical cataract formation by 8 and 3 days, respectively, without reducing lenticular sorbitol. Using in vitro lens culture in 30 mM xylose to model diabetic "sugar" cataract formation, western blots confirmed that multi-functional antioxidants reduced endoplasmic reticulum stress.Multi-functional antioxidants delayed cataract formation in two diverse rat models. These studies provide a proof of concept that a general cataract treatment focused on reducing oxidative stress instead of a specific mechanism of cataractogenesis can be developed

Effect of galactose diet removal on the progression of retinal vessel changes in galactose-fed dogs

PURPOSE. Feeding dogs a diet containing 30% galactose induces experimental galactosemia and results in the formation of diabetes-like microvascular lesions of the retina. The appearance and progression of these retinal lesions can be arrested in a dose-dependent manner by treating these dogs with aldose reductase inhibitors from the onset of galactosemia. To determine whether the elimination of galactosemia can also reduce the progression of retinal lesions, the galactose diet was removed from the galactosemic dogs after either the appearance of pericyte ghosts or formation of microaneurysms. METHODS. Ten control dogs were fed a normal diet, and 50 dogs were fed a diet containing 30% galactose. The galactose diet was removed from 15 dogs after 24 months, the time at which pericyte ghosts had previously been observed to develop, and from another 15 dogs after 31 months, when microaneurysms had previously been observed to develop. Eighteen dogs were continued on a galactose diet. Beginning at 24 months, eyes from each group were enucleated at approximately 6-month intervals. Changes in retinal lesions were quantified by computer image analyses. RESULTS. Significant (P Ͻ 0.05-0.01) increases in the endothelium-pericyte (E-P) ratio and decreases in pericyte density were observed with increased duration of galactose feeding. Although no reversal of retinal lesions occurred, differences in the progression of retinal lesions between the galactose-fed and galactose-deprived groups became evident after 12 to 24 months. CONCLUSIONS. Discontinuation of galactose in the diet at the initial stages of background retinopathy beneficially delays the progression of retinal lesions. (Invest Ophthalmol Vis Sci. 2002;43:1916 -1921 D iabetic retinopathy, a leading cause of blindness, is a major long-term complication of diabetes mellitus that is characterized by vascular changes of the retinal capillary bed. In its early, nonproliferative stage, these changes include the formation of microaneurysms, intraretinal hemorrhages, exudates and altered blood flow. In the later, proliferative stage, neovascularization occurs as retinal vessels break through the inner limiting membrane and grow into the vitreous. 1 A hallmark of this disease is the destruction of pericytes (mural cells) from retinal capillaries and the formation of pericyte ghosts. 2,3 Their loss is associated with vessel dilation, formation of microaneurysm, and endothelial cell proliferation. Retinal vessel changes similar to human retinopathy have been observed to occur in diabetic 4 -6 and galactose-fed Galactosemia is associated with a number of diabetes-like lesions. 10 Galactosemia, both hereditary and experimentally induced with galactose feeding, is associated with the rapid formation of galactitol and, to a lesser extent, the formation of galactonic acid and/or galactonolactone. 11-14 Long-term galactosemia is also associated with increased nonenzymatic glycation, basement membrane thickening, and biochemical changes that include decreased glutathione, taurine, and myoinositol levels, decreased adenosine triphosphate (ATP) and amino acid transport activity, increased PKC and VEGF levels, and altered membrane permeability. Formation of galactitol is catalyzed by the enzyme aldose reductase, and its intracellular accumulation precedes the biochemical changes associated with long-term galactosemia. The administration of aldose reductase inhibitors to animals from the onset of galactose feeding has been observed to reduce galactitol formation and prevent the subsequent formation of diabetes-like lesions. 10 However, aldose reductase inhibitors do not inhibit gulonic acid or formation of galactonolactone, suggesting that the formation of these latter two metabolic intermediates, unlike galactitol, do not contribute to the formation of diabetes-like lesions. 14 Studies using the galactose-fed dog model indicate that degeneration of retinal capillary pericytes is linked to the aldose reductase-catalyzed production of galactitol and that their loss precedes further vascular changes associated with retinopathy. In beagles fed a 30% galactose diet for 36 to 38 months, the onset and progression of destruction of pericytes and the formation of microaneurysms was reduced by the concomitant administration of the aldose reductase inhibitors sorbinil (S-6-fluoro-spirochroman-4-5Ј-imidazolidine-2Ј,4Јdione), its more potent 2-methyl analogue M79175 (2-methyl-6-fluorospirochroman-4-5Ј-imidazolidine-2Ј,4Јdione), or a combination of both inhibitors

Osmotic Stress, not Aldose Reductase Activity, Directly induces Growth Factors and MAPK Signaling changes during Sugar Cataract Formation

In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/ JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation

Orally Bioavailable Metal Chelators and Radical Scavengers: Multifunctional Antioxidants for the Coadjutant Treatment of Neurodegenerative Diseases

Neurodegenerative diseases are associated with oxidative stress that is induced by the presence of reactive oxygen species and the abnormal cellular accumulation of transition metals. Here, a new series of orally bioavailable multifunctional antioxidants (MFAO-2s) possessing a 2-diacetylamino-5-hydroxypyrimidine moiety is described. These MFAO-2s demonstrate both free radical and metal attenuating properties that are similar to the original published MFAO-1s that are based on 1-<i>N</i>,<i>N</i>′-dimethylsulfamoyl-1-4-(2-pyrimidyl)­piperazine. Oral bioavailability studies in C57BL/6 mice demonstrate that the MFAO-2s accumulate in the brain at significantly higher levels than the MFAO-1s while achieving similar neural retina levels. The MFAO-2s protect human neuroblastoma and retinal pigmented epithelial cells against hydroxyl radicals in a dose-dependent manner by maintaining cell viability and intracellular glutathione levels. The MFAO-2s outperform clioquinol, a metal attenuator that has been investigated for the treatment of Alzheimer’s disease

Failure of Oxysterols Such as Lanosterol to Restore Lens Clarity from Cataracts

The paradigm that cataracts are irreversible and that vision from cataracts can only be restored through surgery has recently been challenged by reports that oxysterols such as lanosterol and 25-hydroxycholesterol can restore vision by binding to αB-crystallin chaperone protein to dissolve or disaggregate lenticular opacities. To confirm this premise, in vitro rat lens studies along with human lens protein solubilization studies were conducted. Cataracts were induced in viable rat lenses cultured for 48 hours in TC-199 bicarbonate media through physical trauma, 10 mM ouabain as Na+/K+ ATPase ion transport inhibitor, or 1 mM of an experimental compound that induces water influx into the lens. Subsequent 48-hour incubation with 15 mM of lanosterol liposomes failed to either reverse these lens opacities or prevent the further progression of cataracts to the nuclear stage. Similarly, 3-day incubation of 47-year old human lenses in media containing 0.20 mM lanosterol or 60-year-old human lenses in 0.25 and 0.50 mM 25-hydroxycholesterol failed to increase the levels of soluble lens proteins or decrease the levels of insoluble lens proteins. These binding studies were followed up with in silico binding studies of lanosterol, 25-hydroxycholesterol, and ATP as a control to two wild type (2WJ7 and 2KLR) and one R120G mutant (2Y1Z) αB-crystallins using standard MOETM (Molecular Operating Environment) and Schrödinger’s Maestro software. Results confirmed that compared to ATP, both oxysterols failed to reach the acceptable threshold binding scores for good predictive binding to the αB-crystallins. In summary, all three studies failed to provide evidence that lanosterol or 25-hydroxycholesterol have either anti-cataractogenic activity or bind aggregated lens protein to dissolve cataracts