Novel transgenic mouse models develop retinal changes associated with early diabetic retinopathy similar to those observed in rats with diabetes mellitus.

Retinal capillary pericyte degeneration has been linked to aldose reductase (AR) activity in diabetic retinopathy (DR). Since the development of DR in mice and rats has been reported to differ and that this may be linked to differences in retinal sorbitol levels, we have established new murine models of early onset diabetes mellitus as tools for investigating the role of AR in DR. Transgenic diabetic mouse models were developed by crossbreeding diabetic C57BL/6-Ins2(Akita)/J (AK) with transgenic C57BL mice expressing green fluorescent protein (GFP), human aldose reductase (hAR) or both in vascular tissues containing smooth muscle actin-α (SMAA). Changes in retinal sorbitol levels were determined by HPLC while changes of growth factors and signaling were investigated by Western Blots. Retinal vascular changes were quantitatively analyzed on elastase-digestion flat mounts. Results show that sorbitol levels were higher in neural retinas of diabetic AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors VEGF, IGF-1, bFGF and TGFß, as well as signaling changes in P-Akt, P-SAPK/JNK, and P-44/42 MAPK. Increased loss of nuclei per capillary length and a significant increase in the percentage of acellular capillaries presented in 18 week old AK-SMAA-GFP-hAR mice. These changes are similar to those observed in streptozotocin-induced diabetic rats. Retinal changes in both mice and rats were prevented by inhibition of AR. These studies confirm that the increased expression of AR in mice results in the development of retinal changes associated with the early stages of DR that are similar to those observed in rats.

Multifunctional redox modulator prevents blast-induced loss of cochlear and vestibular hair cells and auditory spiral ganglion neurons.

Blast wave exposure, a leading cause of hearing loss and balance dysfunction among military personnel, arises primarily from direct mechanical damage to the mechanosensory hair cells and supporting structures or indirectly through excessive oxidative stress. We previously reported that HK-2, an orally active, multifunctional redox modulator (MFRM), was highly effective in reducing both hearing loss and hair cells loss in rats exposed to a moderate intensity workday noise that likely damages the cochlea primarily from oxidative stress versus direct mechanical trauma. To determine if HK-2 could also protect cochlear and vestibular cells from damage caused primarily from direct blast-induced mechanical trauma versus oxidative stress, we exposed rats to six blasts of 186 dB peak SPL. The rats were divided into four groups: (B) blast alone, (BEP) blast plus earplugs, (BHK-2) blast plus HK-2 and (BEPHK-2) blast plus earplugs plus HK-2. HK-2 was orally administered at 50 mg/kg/d from 7-days before to 30-day after the blast exposure. Cochlear and vestibular tissues were harvested 60-d post-exposure and evaluated for loss of outer hair cells (OHC), inner hair cells (IHC), auditory nerve fibers (ANF), spiral ganglion neurons (SGN) and vestibular hair cells in the saccule, utricle and semicircular canals. In the untreated blast-exposed group (B), massive losses occurred to OHC, IHC, ANF, SGN and only the vestibular hair cells in the striola region of the saccule. In contrast, rats treated with HK-2 (BHK-2) sustained significantly less OHC (67%) and IHC (57%) loss compared to the B group. OHC and IHC losses were smallest in the BEPHK-2 group, but not significantly different from the BEP group indicating lack of protective synergy between EP and HK-2. There was no loss of ANF, SGN or saccular hair cells in the BHK-2, BEP and BEPHK-2 groups. Thus, HK-2 not only significantly reduced OHC and IHC damage, but completely prevented loss of ANF, SGN and saccule hair cells. The powerful protective effects of this oral MFRM make HK-2 an extremely promising candidate for human clinical trials.

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) activity 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 h in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galactose 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-ß, 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 osmotically 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-ß and the altered cytotoxic signaling that is observed during sugar cataract formation.

Combined antioxidants and anti-inflammatory therapies fail to attenuate the early and late phases of cyclodextrin-induced cochlear damage and hearing loss.

Niemann-Pick C1 (NPC1) is a fatal neurodegenerative disease caused by aberrant cholesterol metabolism. The progression of the disease can be slowed by removing excess cholesterol with high-doses of 2-hyroxypropyl-beta-cyclodextrin (HPßCD). Unfortunately, HPßCD causes hearing loss; the initial first phase involves a rapid destruction of outer hair cells (OHCs) while the second phase, occurring 4-6 weeks later, involves the destruction of inner hair cells (IHCs), pillar cells, collapse of the organ of Corti and spiral ganglion neuron degeneration. To determine whether the first and/or second phase of HPßCD-induced cochlear damage is linked, in part, to excess oxidative stress or neuroinflammation, rats were treated with a single-dose of 3000 mg/kg HPßCD alone or together with one of two combination therapies. Each combination therapy was administered from 2-days before to 6-weeks after the HPßCD treatment. Combination 1 consisted of minocycline, an antibiotic that suppresses neuroinflammation, and HK-2, a multifunctional redox modulator that suppresses oxidative stress. Combination 2 was comprised of minocycline plus N-acetyl cysteine (NAC), which upregulates glutathione, a potent antioxidant. To determine if either combination therapy could prevent HPßCD-induced hearing impairment and cochlear damage, distortion product otoacoustic emissions (DPOAE) were measured to assess OHC function and the cochlear compound action potential (CAP) was measured to assess the function of IHCs and auditory nerve fibers. Cochleograms were prepared to quantify the amount of OHC, IHC and pillar cell (PC) loss. HPßCD significantly reduced DPOAE and CAP amplitudes and caused significant OHC, IHC and OPC losses with losses greater in the high-frequency base of the cochlea than the apex. Neither minocycline + HK-2 (MIN+ HK-2) nor minocycline + NAC (MIN+NAC) prevented the loss of DPOAEs, CAPs, OHCs, IHCs or IPCs caused by HPßCD. These results suggest that oxidative stress and neuroinflammation are unlikely to play major roles in mediating the first or second phase of HPßCD-induced cochlear damage. Thus, HPßCD-induced ototoxicity must be mediated by some other unknown cell-death pathway possibly involving loss of trophic support from damaged support cells or disrupted cholesterol metabolism.

Autoimmunity in membranous nephropathy targets aldose reductase and SOD2.

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Multifunctional Redox Modulators Protect Auditory, Visual, and Cognitive Function.

Significance: Oxidative stress contributes to vision, hearing and neurodegenerative disorders. Currently, no treatments prevent these disorders; therefore, there is an urgent need for redox modulators that can prevent these disorders. Recent Advances: Oxidative stress is associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species, metal dyshomeostasis, and mitochondrial dysfunction. Here, we discuss the role that oxidative stress and metal dyshomeostasis play in hearing loss, visual impairments, and neurodegeneration and discuss the benefits of a new class of multifunctional redox modulators (MFRMs) that suppress sensory and neural degeneration. MFRMs not only reduce free radicals but also independently bind transition metals associated with the generation of hydroxyl radicals. The MFRMs redistribute zinc from neurotoxic amyloid beta zinc (Aß:Zn) complexes to the cytoplasm, facilitating the degradation of Aß plaques by matrix metalloprotease-2 (MMP-2). Although MFRMs bind copper (Cu1+, Cu2+), iron (Fe2+, Fe3+), zinc (Zn2+), and manganese (Mn2+), they do not deplete free cytoplasmic Zn+2 and they protect mitochondria from Mn+2-induced dysfunction. Oral administration of MFRMs reduce ROS-induced cataracts, protect the retina from light-induced degeneration, reduce neurotoxic Aß:Zn plaque formation, and protect auditory hair cells from noise-induced hearing loss. Critical Issues: Regulation of redox balance is essential for clinical efficacy in maintaining sensory functions. Future Directions: Future use of these MFRMs requires additional pharmacokinetic, pharmacodynamics, and toxicological data to bring them into widespread clinical use. Additional animal studies are also needed to determine whether MFRMs can prevent neurodegeneration, dementia, and other forms of vision and hearing loss.

Diabetic cataracts: mechanisms and management.

Diabetes mellitus is associated with a 5-fold higher prevalence of cataracts, which remains a major cause of blindness in the world. Typical diabetic cataracts contain cortical and/or posterior subcapsular opacities. Adult onset diabetic cataracts also often contain nuclear opacities. Mechanisms of diabetic cataractogenesis have been studied in less detail than those of other diabetic complications. Both animal and human studies support important contribution of increased aldose reductase activity. Surgical extraction is the only cure of diabetic cataract today. An improved understanding of pathogenetic mechanisms, together with finding effective therapeutic agents, remain highest priority for diabetic cataract-related research and pharmaceutical development.

Topical KINOSTAT™ ameliorates the clinical development and progression of cataracts in dogs with diabetes mellitus.

OBJECTIVE: To determine whether topical administration of the aldose reductase inhibitor Kinostat™ can ameliorate the onset or progression of cataracts in dogs with naturally occurring diabetes mellitus (DM). MATERIALS AND METHODS: A randomized, prospective, double-masked placebo control pilot study was conducted with 40 dogs newly diagnosed with DM with no or minimal lens changes. Twenty-eight dogs received Kinostat™ and 12 dogs received placebo. PROCEDURES: Owners administered the agent into both eyes three times daily for 1 year and compliance was monitored with log sheets. Complete ophthalmic examinations were performed on dilated eyes at the time of enrollment and 1, 2, 3, 6, and 12 months into treatment. Cataract severity was assessed on a scale of 0-3. At 12 months, full bloodwork, including HbA1C and blood Kinostat™ levels were performed. RESULTS: After 12 months of treatment, the cataract score in the placebo group significantly increased with seven dogs (14 eyes) developing mature cataracts, two dogs (4 eyes) developing cortical opacities, and one dog (2 eyes) developing equatorial vacuoles with mild punctate cortical opacities. In contrast, the cataract score in the Kinostat™ treated dogs was significantly less with seven developing anterior equatorial vacuoles, two developing incipient anterior cortical cataracts, and four developing mature cataracts. In fact, the cataract scores of the Kinostat™ group at 12 months did not significantly increase from the score at the time of enrollment. The HbA1C values between the two groups after 12 months of treatment were similar, and no blood levels of Kinostat™ were found in any enrolled dog. CONCLUSION: The onset and/or progression of cataracts in dogs with DM can be significantly delayed by topical administration of Kinostat™.

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.

Cellular osmolytes reduce lens epithelial cell death and alleviate cataract formation in galactosemic rats.

PURPOSE: Many cataractogenic stresses also induce endoplasmic reticulum (ER) stress in lens epithelial cells (LECs), which appears to be one of the universal inducers of cell death. In galactosemic rats, activation of ER stress results in the activation of the unfolded protein response (UPR)-dependent death pathway, production of reactive oxygen species (ROS), and cell death. All are induced and precede cataract formation. Cellular osmolytes such as 4-phenylbutyric acid (PBA), trimethylamine N-oxide (TMAO), and tauroursodeoxychoric acid (TUDCA) are known to suppress the induction of ER stress. We investigated whether these small molecules prevent cataract formation in galactose-fed rat lenses. METHODS: Cultured LECs were treated with galactose and each cellular osmolyte. Sprague-Dawley rats were fed a 50% galactose chow for 15 days with or without cellular osmolyte treatment. Similarly, selenite was injected subcutaneously into rats with or without cellular osmolytes. Calcein AM and ethidium homodimer-1 (EthD) were used to detect live and dead cells, respectively. The cellular osmolytes, PBA, TMAO, and TUDCA were tested for their ability to suppress LEC death and cataract formation. RESULTS: Cellular osmolytes rescued cultured human LECs which were treated with the ER stressors. We administered these osmolytes either orally or by injection into galactosemic Sprague-Dawley rats. These rats had significantly reduced LEC death and partially delayed hypermature cataract formation. Since the UPR was not activated in cultured LECs treated with selenite, we used the selenite nuclear cataract as a UPR-independent death pathway control. In selenite-induced nuclear cataract in rats, cellular osmolytes did not prevent LEC death and did not alleviate cataract formation. CONCLUSIONS: These results further establish that ER stress and LEC death play a vital role in certain types of cataract formation. In addition, cellular osmolytes may be potential prophylactic drugs for some types of cataracts.

Evaluation of the vascular targeting agent combretastatin a-4 prodrug on retinal neovascularization in the galactose-fed dog.

PURPOSE: Combretastatin A-4 (CA-4) is a vascular targeting agent known to rapidly shut off blood flow in new vessels and, as a result, regress neovascularization. In this pilot study, the ability of CA-4 to modify retinal neovascularization, which results in altered retinal vessel blood flow and retinal permeability, was evaluated in aphakic long-term galactose-fed beagles, an animal model that develops diabetes-like retinal neovascularization. METHODS: Two (2) groups of aphakic dogs, each group comprised of 4 galactose-fed dogs and 2 age-matched controls dogs, were utilized. Each group initially received the combretastatin A-4-phosphate prodrug (CA-4P) as either sub-Tenon's injections, administered at the corneoscleral junction, or intravitreal injections. Six (6) weeks after this treatment, all dogs also received systemic (intravenous) injections of CA-4P. Retinal vascular changes were monitored at 2-week intervals by fluorescein angiography. RESULTS: All galactose-fed dogs demonstrated the presence of retinal neovascular lesions by fluorescein angiograms. Fluorescein leakage or perfusion through neovascular vessels was not altered by either sub-Tenon's, intravitreal, or systemic CA-4P administration. Whereas CA-4P was well tolerated by the healthy eyes of the control animals, its administration to some galactose-fed dogs was associated with corneal edema and increases in intraocular pressure following sub-Tenon's and intraocular injections. CONCLUSIONS: Neovascularization in the galactose-fed dog progresses over a period of years, similar to that observed with clinical diabetic retinopathy. The failure of CA-4P to ameliorate neovascularization suggests that chronic, long-term administration may be required to destroy the slowly growing retinal endothelial cells.

Age-dependent retinal capillary pericyte degeneration in galactose-fed dogs.

The galactose-fed beagle develops diabetes-like microvascular changes that are histologically and clinically similar in appearance to all stages of human diabetic retinopathy. This animal model is extremely useful for evaluating drugs for the treatment of diabetic retinopathy; however, the time required to develop the various retinal lesions (24-72 months for background to the proliferative stage) may be considered prohibitive. Retinal vascular changes begin with an initial degeneration of capillary pericytes, which has been linked to the aldose reductase catalyzed formation of galactitol. Because aldose reductase-linked sugar cataract formation is known to be age dependent, with the onset and severity of cataract higher in younger diabetic and galactose-fed animals, retinal capillary changes in the eyes of initially 2- versus 9-month-old beagles fed a diet containing 30% galactose were compared. Eyes were enucleated after 36 months of galactose feeding, the intact retinal capillaries were isolated by trypsin digestion, and defined retinal regions were evaluated by computer image analysis. Nicotinamide adenine dinucleotide phosphate-dependent reductase activity, using DL-glyceraldehyde and D-xylose as substrates, was also compared in the lenses and whole retinas of eyes from the 2- and 9-month-old beagles. Significantly (P

Topical aldose reductase inhibitor formulations for effective lens drug delivery in a rat model for sugar cataracts.

PURPOSE: In the topical delivery of drugs to the lens, drug retention on the eye surface is considered to be important because increased retention on the ocular surface should lead to increased ocular absorption of a drug through the cornea into the aqueous humor and subsequently the lens. The aim of this study was to investigate whether increasing the viscosity of a topical aldose reductase inhibitor suspension increases the lenticular bioavailability of the inhibitor and whether such a formulation can arrest sugar cataract formation. METHODS: Five topical suspensions of 3% 2-methylsorbinil (2-MS) were prepared using (1) hydroxypropyl methylcellulose (HPMC, 0.5% w/v), (2) xanthan gum (0.5% w/v), (3) gellan gum (0.5% w/v), (4) carbopol (0.25% w/v), and (5) carbopol (0.25% w/v)--hydroxypropyl methylcellulose (HPMC) (0.25% w/v). Viscosity measurements were conducted with a viscometer. Lenticular levels of 2-MS were determined in the lenses from young Sprague Dawley rats receiving 1 drop of selected topical suspension twice-daily for 7 days. The efficacy of the suspensions to arrest sugar cataract formation was evaluated by administering the suspensions for 21 days to similar rats fed a diet containing 50% galactose. Lens changes were examined by portable slit lamp following mydriasis. RESULTS: Lenticular levels of 2-MS was highest in rats administered suspensions containing 0.25% carbopol + 0.25% HPMC as vehicles followed by 0.5% gellan gum, 0.5% HPMC, 0.25% carbopol, and 0.5% xanthan gum. All untreated rats fed a 50% galactose diet developed hypermature cataracts within 15 days; however, none of the topical treated rats demonstrated cortical vacuole formation after 21 days of galactose feeding. CONCLUSIONS: In the suspensions examined, no direct relationship between the lenticular drug levels of 2-MS and either viscosity or pH of the vehicles were observed. The observed arrest of sugar cataract formation indicated that therapeutically adequate lenticular levels of 2-MS were provided by all topical suspensions.

The unfolded protein response in lens epithelial cells from galactosemic rat lenses.

PURPOSE: Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats. METHODS: Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. RESULTS: Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation. CONCLUSIONS: The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.

Structure of asteroid bodies in the vitreous of galactose-fed dogs.

PURPOSE: Asteroid hyalosis is a condition in which white spherical particles (asteroid bodies) are suspended in the vitreous, usually in the dependent part of the vitreous. These particles seldom cause serious visual symptoms; however, their presence can be a source of irritation. It has been suggested, but not confirmed, that asteroid hyalosis may be associated with systemic diseases such as diabetes, hyperlipidemia, or hypertension. Studies indicate that these particles are composed of lipid material and calcium; however, the specific composition and structure of asteroid bodies remains unknown. We have observed that asteroid hyalosis occurs in galactose-fed dogs, and this represents the first animal model which consistently forms this vitreal condition. The purpose of this study was to identify the main structural component of the asteroid bodies present in the vitreous of these dogs. METHODS: Vitreous humor containing asteroid bodies was collected and frozen from long-term galactose-fed beagles and from age-matched normal controls where asteroid bodies were absent. A portion of the frozen vitreous was sent out for elemental analysis. Thawed vitreous samples were sonicated with HPLC grade water and the aqueous layer was extracted three times with chloroform and then three times with n-butanol. The three organic layers from each extraction were combined and the solvents removed in vacuo. The residue from each extraction was re-dissolved in methanol and analyzed by electrospray ionization mass spectrometry (ESI-MS). RESULTS: Vitreous-containing asteroid bodies had significantly higher levels of calcium and phosphorus. Negative mode ESI-MS analysis of the n-butanol extracts from vitreous samples with and without asteroid bodies were similar with both containing a predominant peak with a mass to charge ratio (m/z) of 538.4. However, similar analyses of the chloroform extracts indicated that three peaks with m/z values of 547.1, 690.5, and 1430.6 were present only in vitreous samples containing asteroid bodies. Subtraction analysis indicated that the m/z of 690.5 peak corresponded to the main component present. This peak was identified and confirmed to be the quasimolecular ion of 1,2-dipalmitoyl-glycero-3-phosphoethanolamine (DPPE). CONCLUSIONS: Based on the current belief that asteroid bodies are composed of lipid-calcium complexes, we propose that the main component of asteroid hyalosis in the galactose-fed dog is a quasimolecular ion of DPPE in which two molecules of DPPE are complexed through their phosphates groups with calcium.

Induction of endoplasmic reticulum stress in retinal pericytes by glucose deprivation.

Diabetic retinopathy is one of the major microvascular complications associated with diabetes mellitus, and the selective degeneration of retinal capillary pericytes is considered to be a hallmark of early retinopathy. Because glucose fluctuations commonly occur in diabetes, we hypothesized that these fluctuations will increase the endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR) in retinal pericytes. To study whether ER stress and the UPR can be induced in retinal pericytes, rat retinal capillary pericytes were cultured in different concentrations of glucose. Hypoglycemia but not hyperglycemia was found to activate UPR-specific enzymes in pericytes. Strong UPR activation leading to apoptosis was also observed when pericytes were cultured in glucose concentrations that were reduced from high to low or no glucose. These results indicate that induction of UPR is related not only to absolute concentrations but also to a shifting from higher to lower concentrations of glucose.

Effects of topical administration of an aldose reductase inhibitor on cataract formation in dogs fed a diet high in galactose.

OBJECTIVE: To determine effects of a topical formulation of an aldose reductase inhibitor (ARI) on the development of sugar cataracts in dogs fed a diet high in galactose. Animals-Ten 6-month old Beagles. PROCEDURES: Dogs were fed a diet containing 30% galactose, and after 16 weeks, 6 dogs were treated topically with a proprietary ARI formulation and 4 dogs were treated with a placebo. Cataract formation was monitored by means of slit-lamp biomicroscopy and fundus photography. Dogs were euthanized after 10 weeks of treatment, and lenses were evaluated for degree of opacity, myo-inositol and galactitol concentrations, and concentration of the ARI. RESULTS: All dogs developed bilateral cortical opacities dense enough to result in a decrease in the tapetal reflex after being fed the galactose-containing diet for 16 weeks. Administration of the ARI arrested further development of cataract formation. In contrast, cataracts in the vehicle-treated dogs progressed over the 10-week period to the mature stage. Evaluation of the isolated lenses after 26 weeks of galactose feeding indicated that lenses from treated dogs were significantly less optically dense than lenses from control dogs. Lenticular myo-inositol concentration was significantly higher in the treated than in the control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that topical application of a proprietary ARI formulation may arrest or reverse the development of sugar cataracts in dogs fed a diet high in galactose. This suggests that this ARI formulation may be beneficial in maintaining or improving functional vision in diabetic dogs with early lens opacities.

Aldose reductase, ocular diabetic complications and the development of topical Kinostat(®).

Diabetes mellitus (DM) is a major health problem with devastating effects on ocular health in both industrialized and developing countries. The control of hyperglycemia is critical to minimizing the impact of DM on ocular tissues because inadequate glycemic control leads to ocular tissue changes that range from a temporary blurring of vision to permanent vision loss. The biochemical mechanisms that promote the development of diabetic complications have been extensively studied. As a result, a number of prominent biochemical pathways have been identified. Among these, the two-step sorbitol pathway has been the most extensively investigated; nevertheless, it remains controversial. To date, long-term pharmacological studies in animal models of diabetes have demonstrated that the onset and development of ocular complications that include keratopathy, retinopathy and cataract can be ameliorated by the control of excess metabolic flux through aldose reductase (AR). Clinically the alleles of AR have been linked to the rapidity of onset and severity of diabetic ocular complications in diabetic patient populations around the globe. In spite of these promising preclinical and human genetic rationales, several clinical trials of varying durations with structurally diverse aldose reductase inhibitors (ARIs) have shown limited success or failure in preventing or arresting diabetic retinopathy. Despite these clinical setbacks, topical ARI Kinostat(®) promises to find a home in clinical veterinary ophthalmology where its anticipated approval by the FDA will present an alternative treatment paradigm to cataract surgery in diabetic dogs. Here, we critically review the role of AR in diabetes mellitus-linked ocular disease and highlight the development of Kinostat(®) for cataract prevention in diabetic dogs. In addition to the veterinary market, we speculate that with further safety and efficacy studies in humans, Kinostat(®) or a closely related product could have a future role in treating diabetic keratopathy.

Diabetes can alter the signal transduction pathways in the lens of rats.

Diabetes is known to affect cataract formation by means of osmotic stress induced by activated aldose reductase in the sorbitol pathway. In addition, alterations in the bioavailability of numerous extralenticular growth factors has been reported and shown to result in various consequences. We have found that the basic fibroblast growth factor (bFGF) accumulates in the vitreous humor of 3- and 8-week diabetic rats. Consequently, the associating signal transduction cascades were severely disrupted, including upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and the common stress-associated mitogen-activated protein kinases p38 and SAPK/JNK. Conversely, under diabetic condition, we observed a dramatic inhibition of phosphatidylinositol-3 kinase activity in lenses obtained from the same animal. Rats treated with the aldose reductase inhibitor AL01576 for the duration of the diabetic condition showed that the diabetes-induced lenticular signaling alterations were normalized, comparable to controls. However, treatment of AL01576 in vitro was ineffective at normalizing the altered constituents in extracted diabetic vitreous after the onset of diabetes. The effect of AL01576 in the high galactose-induced cataract model in vitro was also examined. Administration of AL01576 to lens organ culture normalized the aberrant signaling effects and morphological characteristics associated with in vitro sugar cataract formation. In conclusion, our findings demonstrate diabetes-associated alterations in the lens signal transduction parameters and the effectiveness of AL01576 at normalizing such alterations. The causes for these alterations can be attributed to elevated vitreal bFGF in conjunction with osmotic stress and associated attenuation in redox status of the lens.

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-N,N'-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.