Case report on two diabetic donor eyes with no retinopathy: Clinicopathological and molecular studies.

We were intrigued to analyze donor eyes of two individuals without retinopathy even after 40 years of type 2 diabetes mellitus. Targeted molecular factors associated with angiogenesis and the key antioxidant enzymes in retinal tissue were analyzed. Accordingly PEDF, Adiponectin and Paraoxonase 2 showed augmented mRNA expression in both the retina with no significant change in VEGF expression. Vitreous showed increased PEDF protein in donor 1 and Adiponectin in donor 2 with no change in VEGF protein. This study highlights the profile of specific molecular factors that contribute to the non-development of diabetic retinopathy changes in these individuals.

Elemental concentrations in Choroid-RPE and retina of human eyes with age-related macular degeneration.

Heavy metals, metallic and toxic elements are reported to play an essential role in the complex multifactorial pathogenesis of age-related macular degeneration (AMD). This study was aimed to measure the concentrations of these elements in choroid-RPE and retina of human donor eyes with and without age-related macular degeneration associated changes. Human cadaver donor eyeballs were obtained from the CU Shah eye bank, Sankara Nethralaya Eye Hospital, India, after removal of the cornea. 39 control and 51 AMD donor eyes were used in this study. Alabama grading was done on the histopathological sections to identify early and late age-related macular degeneration changes. Concentrations of lead, cadmium, chromium, cobalt, nickel, arsenic and selenium were determined in choroid-RPE and retina using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Further, gene expression of oxidative stress-related genes, Nrf-2, HO-1, GCLC, GCLM, and detoxification related gene GSTpi was performed. The data were analyzed for statistical significance using Graph Pad® Prism 5 software. Donor eyes with early and late AMD had significantly higher levels of lead, cadmium, chromium, arsenic, and nickel in choroid-RPE and retina compared to the control eyes. Selenium was significantly increased in late AMD compared to control. No significant difference was observed in the levels of cobalt between eyes with and without AMD. Decreased transcript levels of oxidative stress-related genes were observed in the choroid-RPE and retinal tissues. Nrf-2 (p < 0.05), HO-1 and GCLC expressions were lowered in the retina of AMD, whereas GCLM and GSTpi expressions were decreased (p < 0.05) with an increase in HO-1 in choroid-RPE of AMD. This study provides evidence that alterations of the heavy metals and toxic elements along with oxidative stress may play a role in the pathogenesis of AMD.

Homocysteine & its metabolite homocysteine-thiolactone & deficiency of copper in patients with age related macular degeneration - A pilot study.

BACKGROUND & OBJECTIVES: Age related macular degeneration (ARMD) is a leading cause of blindness, particularly in persons above 60 yr of age. Homocysteine is implicated in many ocular diseases including ARMD. This study was undertaken to assess the status and relationship between plasma homocysteine, homocysteine - thiolactone, homocysteinylated protein and copper levels in patients with ARMD. METHODS: A total of 16 patients with ARMD and 16 age-matched controls were recruited for the study. Plasma glutathione, homocysteine, homocysteine - thiolactone and extent of homocysteine conjugation with proteins, copper and thiobarbituric acid reactive substances were measured. RESULTS: Homocysteine levels were elevated with increase in homocysteine-thiolactone, thiobarbituric acid reactive substances and a decrease of glutathione. The levels of homocysteinylated protein were elevated in ARMD. The elevated homocysteine, homocysteine-thiolactone correlated with the decrease in copper level. INTERPRETATION & CONCLUSIONS: Elevated homocysteine and its metabolite homocysteine-thiolactone and decreased levels of copper may play an important role in the pathogenesis of ARMD.

Increased homocysteine, homocysteine-thiolactone, protein homocysteinylation and oxidative stress in the circulation of patients with Eales' disease.

BACKGROUND: Eales' disease (ED) is an idiopathic retinal vascular disorder. It presents with inflammation and neovascularization in the retina. Adult men, aged between 15 and 40 years are more susceptible than women. Homocysteine has been implicated in other ocular diseases including age-related macular degeneration (ARMD), central retinal vein occlusion (CRVO) and optic neuropathy. The present study investigates the role of homocysteine in ED. METHODS: Forty male subjects, 20 with ED and 20 healthy controls, were recruited to the study. Their blood samples were used to measure thiobarbituric acid reactive substances (TBARS), glutathione (GSH), homocysteine, homocysteine-thiolactone, extent of homocysteine conjugation with proteins and plasma copper concentration. RESULTS: In the ED group, plasma homocysteine (18.6 ± 1.77 µmol/L, P < 0.001) and homocysteine-thiolactone (45.3 ± 6.8 nmol/L, P < 0.0001) concentrations were significantly higher compared to homocysteine (11.2 ± 0.64 µmol/L) and homocysteine-thiolactone (7.1 ± 0.94 nmol/L) concentrations in control subjects. TBARS (P < 0.011) and protein homocysteinylation (P < 0.030) were higher in the ED group while GSH (5.9 ± 0.44 µmol/L, P < 0.01) and copper (6.6 ± 0.42 µmol/L, P < 0.001) were lower compared to GSH (8.1 ± 0.41 µmol/L) and copper (15.4 ± 0.73 µmol/L) concentrations in control subjects. CONCLUSIONS: Increased homocysteine, and its metabolite thiolactone, is associated with the functional impairment of protein due to homocysteinylation in ED.

Homocysteine levels in the vitreous of proliferative diabetic retinopathy and rhegmatogenous retinal detachment: its modulating role on lysyl oxidase.

PURPOSE: Homocysteine (Hcys), a well-known inducer of vascular endothelial cell damage has been associated with extracellular matrix changes. Lysyl oxidase (LOX) is a copper-dependent amine oxidase that initiates the covalent cross-linking of collagen and elastin in the extracellular matrix (ECM). LOX contributes to the structural integrity of the ECM, and low LOX activity could promote ECM disorganization. Hydroxyproline levels are used to predict collagen turnover status, and most of the endogenous hydroxyproline present in biological fluids is derived from the degradation of various forms of collagen. As Hcys is known to regulate ECM turnover and also inhibit LOX activity, the purpose of this study was to estimate the vitreous levels of Hcys in eyes with proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD) and to correlate the effect of Hcys, if any on LOX activity. METHODS: Undiluted human vitreous specimens obtained during vitreoretinal surgeries for PDR (n = 18) and RRD (n = 17) were used. Vitreous specimens from donor eyeballs were used as control (n = 19). Hcys was estimated by HPLC using a fluorescent detector. Hydroxyproline was estimated spectrophotometrically. RESULTS: The total vitreous Hcys level was found to be increased significantly in PDR (P = 0.011) and in RRD (P = 0.001) compared with that in control samples. Hydroxyproline was significantly increased in PDR (P = 0.049) and RRD (P = 0.007) compared with the level in control samples. There was a significant negative correlation between the Hcys level and the specific activity of LOX in PDR (P = 0.040) and in RRD (P = 0.029) CONCLUSIONS: This report shows that increased vitreous Hcys in PDR and RRD is associated with a significant decrease in LOX-specific activity along with an increase in collagen turnover.

Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.

PURPOSE: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Increase in Fe3+/Fe2+ ratio and iron-induced oxidative stress in Eales disease and presence of ferrous iron in circulating transferrin.

Eales disease was shown to be associated with oxidant stress and weak antioxidant defense. Total iron, ferrous iron, thiobarbituric acid reactive substances (TBARS), and ceruloplasmin were estimated in the blood samples of patients with inflammation, perivasculitis, and venous insufficiency. Levels of ferric iron were determined from the difference in those of total iron and ferrous iron. All biochemical parameters were estimated in age and sex-matched controls. Fe3+/Fe2+ ratio was greater in patients with Eales disease than in normal controls. Similar increase in TBARS and ceruloplasmin levels were noted. The patients were treated with prednisone and vitamins E and C, then Fe3+/Fe2+ ratios and TBARS decreased progressively; the active state of the disease was reduced to 16%. So Fe3+/Fe2+ ratio in addition to TBARS levels could be considered an index of the active state of the disease. Circulating transferrin in blood did contain ferrous iron. Binding of ferrous iron to transferrin was confirmed by estimating iron-binding capacity using ferrous ammonium sulphate.