High glucose induced inflammation is inhibited by copper chelation via rescuing mitochondrial fusion protein 2 in retinal pigment epithelial cells.

Altered trace element homeostasis is associated with diabetic complications, and studies have shown elevated copper levels in the serum of individuals with type 1 & 2 diabetes. Copper chelation has been shown to be beneficial by preventing or reversing diabetic organ damage and developing as a new treatment strategy for treating diabetic complications. Diabetic retinopathy is the major vision-threatening complication of diabetes. Recent studies have reported copper to be elevated in the serum of patients with diabetic retinopathy. Here in this study, we attempt to unravel the role of copper chelator penicillamine in retinal pigment epithelial cells exposed to high glucose (HG) and copper as a model for diabetic retinopathy. We have found that high glucose by itself and along with copper alters the mitochondrial morphology, reduces the expression of the mitochondrial fusion protein 2 (MFN2), and induces endoplasmic reticulum (ER) stress and inflammation. Copper chelation with penicillamine reduced all these changes in mitochondria, thereby rescuing the cells from mitochondrial damage and inflammation.

Structure based design of inhibitory peptides targeting ornithine decarboxylase dimeric interface and in vitro validation in human retinoblastoma Y79 cells.

Polyamine synthesis in human cells is initiated by catalytic action of Ornithine decarboxylase (ODC) on Ornithine. Elevated levels of polyamines are manifested proliferating cancer cells and are found to promote tumour cell adhesion. Di-flouro methyl orninthine is a known inhibitor of ODC, however its usage is limited due its low affinity quick clearance and incompetent cellular uptake, thus posing a need for potential inhibitors. Currently, peptides are substituting drugs, as these are highly selective, specific and potent. Hence, in this study, the interacting interfaces of native homodimeric form of ODC and its heterodimer with Antizyme were probed to design inhibitory peptides targeting ODC. The designed peptides were validated for structural fitness by extensive molecular dynamics simulations and Circular dichroism studies. Finally, these peptides were validated in Y79 retinoblastoma cells for impact on ODC activity, cytotoxicity cell cycle and cell adhesion. On collective analysis, Peptide3 (Pep 3) and Peptide4 (Pep 4) were found to be potentially targeting ODC, as these peptides showed significant decrease in intracellular polyamine levels, cell adhesion and cell cycle perturbation in Y79 cells. Thus, Pep 3 and Pep 4 shall be favourably considered as therapeutic agents for targeting ODC mediated proliferation in retinoblastoma.Communicated by Ramaswamy H. Sarma.

Copper mediates mitochondrial biogenesis in retinal pigment epithelial cells.

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 µM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1ß and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1ß mRNA showed an increase in choroid-RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.

Antidiabetic effect of free amino acids supplementation in human visceral adipocytes through adiponectin-dependent mechanism.

BACKGROUND & OBJECTIVES: : Amino acids are general nutrients having anti-diabetic property. The present study was undertaken to investigate the mechanism of anti-diabetic effects of amino acids in human visceral adipocyte cells in high glucose environment. METHODS: : Experiments were carried out in human visceral adipocytes. Adiponectin (APN) siRNAs were designed using Ambion tools. APN mRNA expression was quantified using real-time polymerase chain reaction, and protein level was studied using ELISA. AMP-activated kinase (AMPK) activity was measured and glucose uptake by 2-deoxyglucose uptake method. RESULTS: : Amino acids (proline and phenylalanine) exposure to adipocytes significantly (P <0.01) increased APN mRNA by 1.5-folds when compared to control whereas proline increased APN secretion by 10.6-folds (P <0.01), phenylalanine by 12.7-folds (P <0.001) and alanine by 6.3-folds (P <0.01). Free amino acid-induced AMPK activity and glucose uptake were decreased with the transient knockdown of APN. INTERPRETATION & CONCLUSIONS: : Antidiabetic effect of the tested amino acids was exhibited by increased glucose uptake through the AMPK pathway by an APN-dependent mechanism in human visceral adipocytes. This should be tested and confirmed in in vivo system. Newer treatment modalities with amino acids which can enhance glucose uptake and APN secretion can be developed as drug for treating both diabetes and obesity.

Free amino acids hydroxyproline, lysine, and glycine promote differentiation of retinal pericytes to adipocytes: A protective role against proliferative diabetic retinopathy.

AIM: This study was conducted to estimate the aminoacid levels in the vitreous of patients with proliferative diabetic retinopathy, and to correlate it with the adiponectin levels. Secondly to test if these amino acids can alter or induce adiponectin levels and its related factors in retinal cells like pericyte as an in vitro model. METHODS: All human studies were done as per declaration of Helsinki with institutional approval and after obtaining consent from participating individuals. The vitreous amino acids were estimated in PDR (Proliferative diabetic retinopathy) and MH (Macular Hole) as disease control using HPLC. Bovine retinal pericytes (BRP) were cultured in DMEM/F12 medium and treated with 0.5 mM of any one of the individual amino acids (proline, hydroxyproline, phenylalanine, alanine, serine, glycine, lysine, isoleucine or valine) along with 100 nM insulin for 14 days in high glucose (25 mM) condition. The mRNA expression profile of adipogenic markers (such as Pref1, APN, ZAG and PPARγ), angiogenic markers (VEGF, MMP-2 and MMP-9, TGF-ß) and antioxidant markers (Nrf2 and UCP-2) were evaluated by qPCR. Adipogenesis was further confirmed by adipogenesis assay, secretion of adiponectin in medium and triglyceride accumulation by Oil red O staining in Bovine retinal pericytes. RESULTS: Amino acids valine (p < 0.004), isoleucine (p < 0.0007), leucine (p < 0.022), serine (p < 0.0007), glycine (p < 0.001), alanine (p < 0.017), phenylalanine (p < 0.013), and lysine (p < 0.001) were significantly elevated in the vitreous of PDR group (n = 30) when compared to macular hole (n = 20). There was a significant positive correlation between serine (p < 0.021), alanine (p < 0.00016), phenylalanine (p < 0.04), isoleucine (p < 0.023), leucine (p < 0.043), and lysine (p < 0.026) with adiponectin level in the vitreous. The amino acids hydroxyproline, proline, lysine, glycine and alanine induced the triglyceride accumulation and expression of Adiponectin. VEGF and MMP-9 expression was decreased with all the amino acids treated and PEDF was significantly increased with phenylalanine treatment. TGFß mRNA expression showed a significant decrease with proline, alanine, glycine, lysine and isoleucine. The Nrf2 expression was significantly increased in alanine and serine when compared to control. The UCP-2 gene showed a significant increase in proline and lysine treatment. DISCUSSION AND CONCLUSION: Our results suggest that amino acids hydroxyproline, proline, lysine, glycine and alanine which are elevated in the PDR vitreous show a tendency to induce adipogenic effects in retinal pericytes by triggering the accumulation of triglycerides and adiponectin. Hence we hypothesize that these aminoacids when elevated along with insulin and glucose can induce metabolic changes in pericytes. The functional implications of these changes tend to be protective as it increases the antioxidant potential and decreases the angiogenesis markers which are potentially pathogenic.

Insights on ornithine decarboxylase silencing as a potential strategy for targeting retinoblastoma.

Ornithine Decarboxylase (ODC) is a key enzyme involved in polyamine synthesis and is reported to be up regulated in several cancers. However, the effect of ODC gene silencing in retinoblastoma is to be understood for utilization in therapeutic applications. Hence, in this study, a novel siRNA (small interference RNA) targeting ODC was designed and validated in Human Y79 retinoblastoma cells for its effects on intracellular polyamine levels, Matrix Metalloproteinase 2 & 9 activity and Cell cycle. The designed siRNA showed efficient silencing of ODC mRNA expression and protein levels in Y79 cells. It also showed significant reduction of intracellular polyamine levels and altered levels of oncogenic LIN28b expression. By this study, a regulatory loop is proposed, wherein, ODC silencing in Y79 cells to result in decreased polyamine levels, thereby, leading to altered protein levels of Lin28b, MMP-2 and MMP-9, which falls in line with earlier studies in neuroblastoma. Thus, by this study, we propose ODC silencing as a prospective strategy for targeting retinoblastoma.

Copper transporters and chaperones: Their function on angiogenesis and cellular signalling.

Copper, although known as a micronutrient, has a pivotal role in modulating the cellular metabolism. Many studies have reported the role of copper in angiogenesis. Copper chaperones are intracellular proteins that mediate copper trafficking to various cell organelles. However, the role and function of copper chaperones in relation to angiogenesis has to be further explored. The intracellular copper levels when in excess are deleterious and certain mutations of copper chaperones have been shown to induce cell death and influence various cellular metabolisms. The study of these chaperones will be helpful in understanding the players in the cascade of events in angiogenesis and their role in cellular metabolic pathways. In this review we have briefly listed the copper chaperones associated with angiogenic and metabolic signalling and their function.

Localization of Human Copper Transporter 1 in the Eye and its Role in Eales Disease.

PURPOSE: Copper (Cu) is an essential trace element; however excess is toxic due to the pro-oxidant activity. Increased intracellular Cu levels in vitreous and monocyte were reported in Eales disease (ED) previously. Copper transporter1 (CTR1) maintains copper homeostasis and hence, we studied the presence of CTR1 in ocular tissues and its role in ED. METHODS: Real-time PCR, ELISA and Western blot experiments were performed in donor eyeballs tissues and PBMCs isolated from controls and ED. Immunostaining were performed for CTR1 from donor eyeballs and one ED case. RESULTS: CTR1 protein was expressed in all ocular tissues. PBMCs showed a three-fold increase in CTR1 protein in ED when compared with controls. Retinal sections from ED patients also revealed increased CTR1 protein expression in retinal tissues, compared with control. CONCLUSIONS: CTR1 was significantly increased in ED when compared with controls, indicating its considerable role in the ED pathology.

Effect of adiponectin on expression of vascular endothelial growth factor and pigment epithelium-derived factor: an in vitro study.

AIM: This study was carried out to identify the role of adiponectin (APN) in modulating the expression of vascular endothelial growth factor (VEGF) and pigment epithelial-derived factor (PEDF) in relation to ocular angiogenesis. MATERIALS AND METHODS: Human retinal pigment epithelial cell lines (ARPE-19) were cultured in Dulbeco's minimum essential medium with 10% fetal bovine serum (FBS) and exposed to varying concentrations of recombinant adiponectin (5-5 ng/ml) for 1 h. Analysis of VEGF and PEDF mRNA was done by reverse transcriptase and further quantified by quantitative polymerase chain reaction. VEGF and PEDF protein expression were studied using enzyme-linked immune sorbent assay (ELISA). STATISTICAL ANALYSIS: Unpaired Student's t-test was used to analyze the significance. P < 0.05 was accepted as statistically significant. RESULTS: ARPE cells exposed to APN showed decreased expression of VEGF mRNA, protein whereas PEDF protein is unaltered and PEDF mRNA was increased. CONCLUSION: Our in vitro study on ARPE exposed to APN showed a negative correlation with VEGF levels. Thus indicating the protective role for APN in angiogenesis-related diseases.

Tolerance of ARPE 19 cells to organophosphorus pesticide chlorpyrifos is limited to concentration and time of exposure.

Age related macular degeneration is a blinding disease common in elder adults. The prevalence of age related macular degeneration has been found to be 1.8% in the Indian population. Organophosphates are widely used insecticides with well documented neurological effects, and the persistent nature of these compounds in the body results in long term health effects. Farmers exposed to organophosphorus pesticides in USA had an earlier onset of age related macular degeneration when compared to unexposed controls. A recent study found significant levels of an organophosphate, termed chlorpyrifos, in the blood samples of Indian farmers. Therefore, in understanding the link between age related macular degeneration and chlorpyrifos, the need for investigation is important. Our data show that ARPE-19 (retinal pigment epithelial cells) exhibit a cytoprotective response to chlorpyrifos as measured by viability, mitochondrial membrane potential, superoxide dismutase activity, and increased levels of glutathione peroxidase and reduced glutathione, after 24 h exposure to chlorpyrifos. However, this cytoprotective response was absent in ARPE-19 cells exposed to the same range of concentrations of chlorpyrifos for 48 h. These results have physiological significance, since HPLC analysis showed that effects of chlorpyrifos were mediated through its entry into ARPE-19 cells. HPLC analysis also showed that chlorpyrifos remained stable, as we recovered up to 80% of the chlorpyrifos added to 6 different ocular tissues.

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice.

Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvß(5) integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.

Decorin derived antiangiogenic peptide LRR5 inhibits endothelial cell migration by interfering with VEGF-stimulated NO release.

Excessive angiogenesis plays critical roles in many human diseases including cancer. We have previously shown that human decorin derived 26 amino acids peptide Leucine Rich Repeat 5 (LRR5) inhibits multiple aspects of angiogenesis including vascular endothelial growth factor (VEGF) stimulated migration of endothelial cells (ECs). In this study, we have characterized the molecular mechanism of LRR5 which reveals that its anti-migratory effect on ECs is mediated by inhibiting VEGF-stimulated endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. LRR5 carried out this function through signaling pathways that involves PI3 kinase and Akt, but not ERK. This anti-NO release effect is mediated by the C-terminal 13 amino acids of LRR5, correlating with the anti-migratory function of this region.

The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor.

Angiogenesis is critical for tumour growth and metastasis where factors that regulate this process are potential targets for development of anti-cancer drugs. In this study, we show that the first TSR domain of the extracellular matrix protease ADAMTS5, unlike the second TSR, has anti-angiogenic activities where it inhibits endothelial cell tube formation on Matrigel, reduces cell proliferation and attachment, while promoting cell apoptosis and migration, all in a dose-dependent manner. Furthermore, it influences the architecture of endothelial cells by disrupting actin stress fibres and reducing focal adhesions, likely via suppressing RhoA activation. TSR1 of ADAMTS5 is therefore a novel anti-angiogenic peptide and could serve as a prototype for future development into anti-cancer drugs.

Histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish.

Histone deacetylases (HDACs) are key transcription regulators that function by deacetylating histones/transcription factors and modifying chromatin structure. In this work, we showed that chemical inhibition of HDACs by valproic acid (VPA) led to impaired liver development in zebrafish mainly by inhibiting specification, budding, and differentiation. Formation of exocrine pancreas but not endocrine pancreas was also inhibited. The liver defects induced by VPA correlate with suppressed total HDAC enzymatic activity, but are independent of angiogenesis inhibition. Gene knockdown by morpholino demonstrated that hdac3 is specifically required for liver formation while hdac1 is more globally required for multiple development processes in zebrafish including liver/exocrine pancreas formation. Furthermore, overexpression of hdac3 but not hdac1 partially rescued VPA induced small liver. One mechanism by which hdac3 regulates zebrafish liver growth is through inhibiting growth differentiation factor 11 (gdf11), a unique target of hdac3 and a member of the transforming growth factor beta family. Simultaneous overexpression or morpholino knockdown showed that hdac3 and gdf11 function antagonistically in zebrafish liver development. These results revealed a novel and specific role of hdac3 in liver development and the distinct functions between hdac1 and hdac3 in zebrafish embryonic development.

Developing antiangiogenic peptide drugs for angiogenesis-related diseases.

Angiogenesis is regulated by stimulators and inhibitors and involve multiple biological processes including endothelial cell proliferation, migration, cell-cell and cell-matrix adhesion, assembly into tube structures as well as apoptosis. Designing and developing peptides for therapeutic application to inhibit angiogenesis is an important area in antiangiogenic drug development. Small peptides have advantages over proteins for therapeutic application, due to their stability, solubility, increased bio-availability and lack of immune response in the host cell. Endogenous protein angiogenesis stimulators and inhibitors hold vital information for designing antiangiogenic peptides for drug development. These proteins function through their interaction with extracellular matrix molecules, cell surface receptors, proteases, as well as growth factors and cytokines. Conserved domains such as thrombospondin type 1 repeats (TSRs), kringle domains as well as critical amino acid residues present in these domains are involved in their functions. By exploiting these properties, several small peptides have been designed, synthetically made and being tested for therapeutic efficacy. Peptides derived from type 1 repeat of thrombospondin, alpha 4 and beta 1 chains of laminin, arginine rich N terminus of endostatin, leucine rich repeat 5 of decorin, pigment epithelium derived factor and N terminal of parathyroid hormone are examples of small antiangiogenic peptides derived from endogenous proteins. Such bioactive peptides are further modified physico-chemically to increase their potency and stability. In addition, phage-display library screening and combinatorial approach are also in use to identify novel antiangiogenic peptides targeting tumour and various proteins. This review will provide a comprehensive summary of the current status of the antiangiogenic peptides and their relevance for drug designing and development. Several critical issues that need to be resolved in translating this concept into clinical practice are also discussed.

Eales' disease: oxidant stress and weak antioxidant defence.

Eales' disease (ED) is an idiopathic retinal periphlebitis characterized by capillary non-perfusion and neovascularization. In addition to the existing system, a new staging system has been proposed by Saxena et al. Immunological, molecular biological and biochemical studies have indicated the role of human leucocyte antigen, retinal S antigen autoimmunity, Mycobacterium tuberculosis genome, free radical damage and possibly hyperhomocysteinemia in its etiopathogenesis, which appears multifactorial. Oxidant stress has been shown by increase in the levels of thiobarbituric acid reactive substances (lipid oxidation) in the vitreous, erythrocytes, platelets and monocytes. A decrease in vitamins E and C both in active and healed vasculitis, superoxide dismutase, glutathione and glutathione peroxidase showed a weakened antioxidant defence. Epiretinal membrane from patients of ED who underwent surgery showed, by immunolocalization, presence of carboxy methyl lysine, an advanced glycation end product formed by glycoxidation and is involved in angiogenesis. OH. free radical accumulation in monocytes has been directly shown by electron spin resonance spectrometry. Free radical damage to DNA and of protein was shown by the accumulation of 8 hydroxy 2 deoxyguanosine (in leucocytes) and nitrotyrosine (in monocytes), respectively. Nitrosative stress was shown by increased expression of inducible nitric oxide synthase in monocytes in which levels of iron and copper were increased while those of zinc decreased. A novel 88 kDa protein was found in serum and vitreous in inflammatory condition and had antioxidant function. Platelet fluidity was also affected. Oral, methotrexate in low dosage (12.5 mg/week for 12 weeks) as well as oral vitamin E (400 IU) and C (500 mg) daily for 8 weeks are reported to have beneficial effects.

Biochemistry of homocysteine in health and diseases.

The amino acid homocysteine (Hcy), formed from methionine has profound importance in health and diseases. In normal circumstances, it is converted to cysteine and partly remethylated to methionine with the help of vit B12 and folate. However, when normal metabolism is disturbed, due to deficiency of cystathionine-beta-synthase, which requires vit B6 for activation, Hcy is accumulated in the blood with an increase of methionine, resulting into mental retardation (homocystinuria type I). A decrease of cysteine may cause eye diseases, due to decrease in the synthesis of glutathione (antioxidant). In homocystinurias type II, III and IV, there is accumulation of Hcy, but a decrease of methionine, thus, there is no mental retardation. Homocysteinemia is found in Marfan syndrome, some cases of type I diabetes and is also linked to smoking and has genetic basis too. In hyperhomocysteinemias (HHcys), clinical manifestations are mental retardation and seizures (type I only), ectopia lentis, secondary glaucoma, optic atrophy, retinal detachment, skeletal abnormalities, osteoporosis, vascular changes, neurological dysfunction and psychiatric symptoms. Thrombotic and cardiovascular diseases may also be encountered. The harmful effects of homocysteinemias are due to (i) production of oxidants (reactive oxygen species) generated during oxidation of Hcy to homocystine and disulphides in the blood. These could oxidize membrane lipids and proteins. (ii) Hcy can react with proteins with their thiols and form disulphides (thiolation), (iii) it can also be converted to highly reactive thiolactone which could react with the proteins forming -NH-CO- adducts, thus affecting the body proteins and enzymes. Homocystinuria type I is very rare (1 in 12 lakhs only) and is treated with supplementation of vit B6 and cystine. Others are more common and are treated with folate, vit B12 and in selected cases as in methionine synthase deficiency, methionine, avoiding excess. In this review, the role of elevated Hcy levels in cardiovascular, ocular, neurologial and other diseases and the possible therapeutic measures, in addition to the molecular mechanisms involved in deleterious manifestations of homocysteinemia, have been discussed.

Plasma homocysteine and total thiol content in patients with exudative age-related macular degeneration.

PURPOSE: Exudative age-related macular degeneration (ARMD) is one of the debilitating ocular complications, which results in permanent blindness. Elevated homocysteine (Hcys) levels have been associated in the development of several vascular diseases. Vascular and oxidative stress theories have been implicated for the development of choroidal neovascularization in exudative ARMD. The aim of the present study was to investigate the possible role of plasma Hcys and thiol content (tSH) as a risk factor for the development of exudative ARMD. METHOD: A total of 16 patients with exudative ARMD and 20 age-matched controls were recruited for the study. Plasma Hcys levels were analysed using Reverse Phase High Performance Liquid Chromatography. Plasma glutathione (GSH) content was determined using o-phthalaldehyde (OPA) derivatization and subsequent detection by fluorimeter. Plasma tSH levels were determined by using thiol-specific reagent dithionitrobenzoic acid (DTNB) spectrophotometrically. RESULTS: Plasma Hcys levels in exudative ARMD were elevated three-fold (18+/-5.0 microM) when compared to healthy controls (6.7+/-1.8 microM). There was a two-fold decrease in the GSH and tSH in exudative ARMD when compared with controls. Negative correlation was observed between diminished tSH and Hcys levels (r=-0.4837, P=0.05). Similarly plasma Hcys levels negatively correlated with GSH content (r=-0.6620, P<0.05). CONCLUSION: Results from our present study revealed that there is an elevated Hcys level and diminished thiol pool content in exudative ARMD that are significant.

Primary vitrectomy for combined rhegmatogenous retinal detachment and choroidal detachment with or without oral corticosteroids: a pilot study.

PURPOSE: The occurrence of choroidal detachment (CD) in eyes with primary rhegmatogenous retinal detachment (RRD) is relatively uncommon (2%-4.5%). Recent reports suggest that primary vitrectomy yields better anatomic success than scleral buckling. However, for these inflamed eyes with low intraocular pressure, the influence of preoperative oral steroids on reattachment rates has not been elucidated yet. METHODS: Twenty eyes with combined RRD and CD that underwent primary vitrectomy were randomized to receive oral steroids (for 1 week) or no oral steroids before surgery. RESULTS: Preoperative clinical data such as mean age, lens status, Snellen visual acuity, duration of macular detachment, CD (size and extent), and retinal detachment characteristics (e.g., extent, number of retinal breaks, atrophic or tractional retinal break, size of retinal break, and location of retinal break) were similarly distributed in both groups. Single-operation anatomic success was 81.8% (9/11) among those patients who received preoperative oral steroids and was 66.7% (6/9) among those who did not receive preoperative oral steroids. After reoperation, anatomic success was 100% in both groups. The mean follow-up was 20.1 months. CONCLUSION: The results suggest that administration of oral steroids before primary vitrectomy in eyes with combined RRD and CD improves reattachment rates.

Amino acids differentially regulate insulin receptor tyrosine kinase and phosphatidyl inositol-3-OH-kinase activities in human monocytes exposed to high glucose concentration.

Chronic hyperglycemia and insulin resistance are the common factors involved in the development of vascular complications in diabetes mellitus (DM) patients. Since insulin signaling pathway has been shown to be regulated by nutritional supplements, in the present study, we investigated the possible effects of free amino acids, such as lysine, arginine and alanine and their mixture in modulating the insulin receptor tyrosine kinase (IRTK) and phosphatidyl inositol-3-OH-kinase (PI3K) activities and on the changes in actin dynamics in monocytes (MC), exposed to high glucose concentration (25 mM). IRTK and PI3K activities were markedly decreased in MC, incubated with 25 mM glucose. However, on treatment with amino acids, only lysine was effective in augmenting IRTK and PI3K activities in a dose-dependent manner. Arginine had marginal effect in promoting these activities. Equimolar mixture of amino acids showed marginal effect of augmenting only IRTK activity. Alanine had no effect. The F-actin filaments showed grossly diminished organization in the cells treated with 25 mM glucose alone, as assessed by specific binding to phalloidin-FITC, when compared with cells treated with 5 mM glucose. On the other hand, a significant improvement in the F-actin organization was observed in the cells co-incubated with 25 mM glucose and lysine. A possible molecular mechanism is the antiglycating effect of amino acids. The signal transduction starts with binding of ATP to lysine at position 1030 in the beta sub unit of the receptor. This lysine (1030) may be protected by the added lysine or to some extent arginine from glycation and loss of function. In summary, our findings suggest that the amino acids apart from their antiglycating property can also modulate/influence the activities of pivotal enzymes that are upstream in the insulin-mediated signal transduction pathway and bring down glucose.