Copper, Lysyl Oxidase Activity, and Collagen in Aqueous Humour of Primary Glaucoma: An Association with Clinical Parameters.

INTRODUCTION: To measure copper (Cu), lysyl oxidase (LOX) activity, and collagen levels in aqueous humour (AH) of primary glaucoma patients and correlate with clinical parameters. METHODS: 120 patients with 40 each of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG), and cataract controls were recruited in this case-control study. AH samples were collected during the trabeculectomy and cataract surgeries. Cu levels were measured using an atomic absorption spectrophotometer. LOX unit activity was determined by Amplex Red assay and collagen concentration by Sirius red assay. RESULTS: Significantly higher levels of Cu expressed as median (IQR) µmol/L were observed in POAG (p = 0.008) and PACG (p = 0.005) compared to controls. The LOX activity was increased in POAG and PACG (p = 0.04) compared to controls represented as median (IQR) µmol/min. The collagen levels given as median (IQR) mg/ml showed an insignificant increase in POAG and PACG compared to controls (p = 0.78). The LOX unit activity was correlated with visual field index (VFI), which showed a significant increase with the progression of the diseases (p < 0.05), whereas Cu levels were negatively correlated with LOX activity in AH. Cu and LOX activity showed weak correlation with YAG peripheral iridotomy (YAGPI), duration of anti-glaucoma medications, and highest preoperative intraocular pressure. CONCLUSION: Elevated Cu and LOX activity was observed in both POAG and PACG groups compared to controls. LOX activity showed notable increase with VFI as the severity of the disease. Although Cu levels are increased in glaucoma, it's insufficient to significantly increase the activity of LOX.

Anti-glaucoma medications lowered decorin and altered profibrotic proteins in human tenon's fibroblasts.

Long term exposure to anti-glaucoma medications (AGMs) leads to an increase in extracellular matrix (ECM) accumulation in primary glaucoma patients. This study aims to evaluate the effect of topical AGMs in primary human tenon's fibroblasts (HTFs) and analyze the expression of profibrotic and anti-fibrotic proteins. Primary HTFs were cultured from patients undergoing cataract (control) and trabeculectomy. The different types of AGMs in single/multiple combinations (BB, PG, AA, CAI, CH, combinations of 3- PG + AA + CAI, 4A- BB + PG + AA + CAI, 4B- BB + PG + CAI + CH and 5- BB + PG + AA + CAI + CH) on chronic exposure were tested for cell viability using MTT assay and morphological alterations. Profibrotic proteins mainly SPARC, LOXL2, COL1A1 and anti-fibrotic DCN were analyzed in treated HTFs using q-PCR and ELISA. Sirius red staining and collagen gel contraction (CGC) assay were performed to assess collagen synthesis and the contractility of HTFs, respectively. Except for AA and CH, the other AGMs at a higher concentration were found to decrease the cell viability of HTFs. The morphology of HTFs were altered on exposure to BB, CH and AA; Profibrotic proteins i.e., SPARC, LOXL2 and COL1A1 were significantly increased (p < 0.05) on exposure to a combination of AGMs with TGF-ß1, whereas the anti-fibrotic DCN expression was significantly lowered (p < 0.05) in single/multiple AGM exposure. Sirius red staining showed increased collagen synthesis with combinations of AGMs with TGF-ß1. Meanwhile, HTFs showed increased collagen gel contraction with TGF-ß1, CAI and CH. This study reveals that altered profibrotic proteins, with significantly lowered DCN on chronic exposure of AGMs in HTFs.

RAGE silencing deters CML-AGE induced inflammation and TLR4 expression in endothelial cells.

The Nε-(carboxymethyl)lysine (CML), the predominant advanced glycation end products (AGEs) in diabetes and its RAGE induced cytokine release has been well explored. But the CML mediated multiple AGEs receptor expression is still not understood and the role played by RAGE silencing in modulating CML generated pro-inflammatory cytokines in micro and macrovascular endothelial cells is yet to be studied. HUVEC and HREC cells were exposed with CML for 24 h. RAGE, AGER1, AGER2, Gal-3, TLR4, TLR2, CD36, FEEL-1, FEEL-2, and chemokine HMGB1 were quantified by either qPCR/western blotting. The receptor's expression was also determined in control vs diabetic retina. Expression of pro-inflammatory genes, ROS, and mitochondrial membrane potential change were assessed using ELISA, DCFDA, and JC-1 method respectively. RAGE expression was silenced either by Si-RAGE or neutralising antibody with anti-RAGE and expression of other AGE receptors, adaptors, and signalling pathway were studied compared with Si-Control. CML activated RAGE, TLR4, HMGB1(p < 0.001) and Gal-3 (p < 0.05) expression in both micro and macro vascular cells. Cadaveric diabetic retinal tissues also showed increased expression of RAGE, TLR4 and HMGB1 (p < 0.05). RAGE silencing significantly reduced TLR4, HMGB1 (p < 0.05) expression and inhibited the phosphorylation of NFκB and ERK1/2 in both these cells. The TLR4 adaptors MyD88 and TIRAP (p < 0.05) showed down regulation on RAGE silencing. This study shows CML induces AGE receptors expression as observed in diabetic retina and RAGE silencing down regulated TLR4 signalling and cytokine release by partly modulating TLR4 adaptors which needs further validation. From this study we speculate targeting the TLR4 adaptors like MyD88 and TIRAP can be a potential therapeutic target for reducing diabetic induced vascular complications.

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.

Lowered Decorin With Aberrant Extracellular Matrix Remodeling in Aqueous Humor and Tenon's Tissue From Primary Glaucoma Patients.

Purpose: To evaluate the inflammatory cytokine, growth factors, extracellular matrix (ECM) remodeling genes, profibrotic and antifibrotic molecules in patients undergoing glaucoma filtration surgery (GFS). Additionally, the effect of preoperative antiglaucoma medications (AGMs) and postoperative bleb status were related to these parameters. Methods: Tenon's tissue and aqueous humour (AH) were collected from 207 patients undergoing GFS with primary open-angle glaucoma (POAG) (n = 77), primary angle-closure glaucoma (PACG) (n = 62), and cataract controls (n = 68). Monocyte chemoattractant protein-1 (MCP-1), connective tissue growth factor (CTGF), transforming growth factor ß1/2 (TGF-ß1/2), lysyl oxidase (LOX), lysyl oxidase L2 (LOXL2), elastin (ELN), collagen type 1 α 1 (COL1A1), secreted protein acidic and rich in cysteine (SPARC), α-smooth muscle actin (α-SMA), and decorin (DCN) were determined in tenon's tissue by real-time PCR and in AH using ELISA. Results: A significant increase was observed in the transcripts of MCP-1, TGF-ß2, and SPARC in POAG and PACG (P < 0.05); CTGF, TGF-ß1, LOX, LOXL2, ELN, COL1A1, and α-SMA in PACG (P < 0.05) compared with control. DCN transcript was significantly decreased in POAG and PACG (P < 0.05) compared with control. The protein levels of CTGF, TGF-ß1/ß2, ELN, SPARC, and LOXL2 was significantly elevated in POAG and PACG (P < 0.05); DCN was decreased (P < 0.05) compared with control. These parameters showed significant association with duration of preoperative AGMs and postoperative bleb status. Conclusions: This study demonstrates increased expression of growth factors and ECM molecules, both at protein and transcript levels in GFS patients. A decreased DCN in AH seems striking, and if restored might have a therapeutic role in minimizing postoperative scarring to improve GFS outcome.

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.

Chronic Exposure to Chewing Tobacco Induces Metabolic Reprogramming and Cancer Stem Cell-Like Properties in Esophageal Epithelial Cells.

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.

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.

Somatic Mutations Profile of a Young Patient With Metastatic Urothelial Carcinoma Reveals Mutations in Genes Involved in Ion Channels.

Background: Urothelial carcinoma is the most common malignancy of the bladder and is primarily considered as a disease of the elderly. Studies that address bladder tumor occurrence in young age groups are rare. Case Presentation: A 19-year-old male presented with a gross total painless hematuria. A histology after biopsy revealed a high-grade transitional cell carcinoma with lymph node metastasis. The patient succumbed to the disease on day 72 of the treatment. Here, we used whole-exome sequencing of a paired tumor-normal sample to identify the somatic mutations and the possible targets of treatment. Result: We predicted eight potential driver mutations (TP53 p.V157L, RB1 c.1498+1G>T, MED23 p.L1127P, CTNND1 p.S713C, NSD1 p.P2212A, MED17 p.G556V, DPYD p.Q814K, and SPEN p.S1078*). In addition, we predicted deleterious mutations in genes involved in the ion channels (CACNA1S p.E1581K, CACNG1 p.P71T, CACNG8 p.G404W, GRIN2B p.A1096T, KCNC1 p.G16V, KCNH4 p.E874K, KCNK9 p.R131S, P2RX7 p.A296D, and SCN8A p.R558H). Conclusions: Most likely, mutations in genes involved in ion channels may be responsible for the aggressive behavior of a tumor. Ion channels are the second largest class of drug targets, and may thus serve as a putative potential therapeutic target in advanced stage urothelial carcinoma.

Detection of Proteins Associated with Extracellular Matrix Regulation in the Aqueous Humour of Patients with Primary Glaucoma.

Purpose: The protein composition of aqueous humour (AH) has held significant relevance and remains to be the prime sample in the discovery of biomarkers in glaucoma. The purpose of this study is to analyze the AH protein concentrations in primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) and further examine the proteome changes compared to cataract control. Methods: AH was collected from 90 POAG, 72 PACG, 78 cataracts (controls) in this study. The total protein was quantified using Bradford's assay. Samples were subjected to trypsin digestion followed by liquid chromatography-mass spectrometry (LC-MS) for proteomic studies (n = 3 per group). The extracellular matrix has a major influence on the AH outflow, and the regulator proteins osteopontin (OPN), cathepsin D, and cystatin C detected by mass spectrometry are validated in AH samples by Western blot and turbidimetric immunoassay. Results: We observed a significant increase in protein levels of POAG (p = .0009); interestingly, a similar increase in PACG compared to cataract (p < .0001) and POAG (p = .02). Proteomics analysis identified 184, 190, and 299 proteins in control, POAG and PACG. OPN was increased in POAG (p = .0319) and PACG (p = .0103) compared to control. The precursor form of cathepsin D was increased in POAG and decreased in PACG, though not significant compared to control. Cystatin C was also increased in both POAG (p = .0310) and PACG (p = .0125) compared to control. Conclusion: In this study, we report for the first time that PACG cohort had higher total protein compared to controls. A qualitative comparison of proteomes revealed increased numbers of proteins identified in PACG. We assume that elevated levels of OPN and cystatin C in POAG and PACG along with altered cathepsin levels may contribute to ECM aberration in glaucoma.

Effect of brilliant Blue-G on cellular stress response in retinal pigment epithelial cells: In vitro.

To assess the cellular stress evoked by exposure of Brilliant Blue-G (BBG), adult retinal pigment epithelial (ARPE-19) cells were treated with various dilutions of BBG in balanced salt solution plus (BSS-PLUS) with and without endoillumination (Alcon Constellation Vision System). The treatments lasted for acute periods of 2 and 5 min. MTT and presto blue assays were performed to assess the changes in cell viability; reactive oxygen species (ROS) production was quantified by DCFDA (dichlorofluorescin diacetate) assay, and the expression of inflammatory stress and endoplasmic reticulum (ER) genes were quantified by qPCR. We observed no reduction in cell viability at 2 min of dye treatment with and without endoillumination while at 5 min exposure, a reduction in cell viability at all concentrations of the dye was observed compared to control. Though there was an increase in ROS with endoillumination, it was insignificant. There was no change in the mRNA expression of TNF-α while that of GRP78, and inflammatory genes viz. IL-8, IL-1ß showed a significant increase at 0.5 mg/ml dye with endoillumination. BBG reduced cell viability with increasing concentration and time. The undiluted concentration of the dye results in inflammatory stress compared to the diluted formulations. Interestingly, increased GRP78 at undiluted concentration indicates a protective response in cells exposed to light. However, further studies are needed to evaluate the effect of cellular stress on the visual outcome. We infer that the commercially available formulation of BBG is safe for the RPE, at the recommended dose for a short duration however its toxicity to other cell types need to be addressed.

A neoepitope derived from a novel human germline APC gene mutation in familial adenomatous polyposis shows selective immunogenicity.

Familial adenomatous polyposis (FAP) is an inherited condition arising from genetic defects in the Adenomatous polyposis coli (APC) gene. Carriers with mutations in the APC gene develop polyps in the colon and rectum which if not managed, transition into colon cancer. In this study, we identified a novel germline mutation in the APC gene in members of an FAP-affected (Familial adenomatous polyposis) family. This unique heterozygous variant (c.735_736insT; p.Ser246PhefsTer6) was identified in ten out of twenty six family members, ranging in age from 6 to 60 years. Polyps were detected in six of the ten individuals (35-60 years) carrying this mutation. The remaining four members (6-23 years) remain polyp free. A significant fraction of FAP affected individuals eventually develop colon cancer and therapeutic interventions to prevent cancer progression remain elusive. To address this issue, we sought to determine if peptides derived from the novel APC mutation could induce a cytotoxic T cell response, thereby qualifying them as vaccine candidates. Peptides harboring the variant amino acids were first interrogated in silico for their immunogenicity using a proprietary neoepitope prioritization pipeline, OncoPeptVAC. A single 9-mer peptide was predicted to be immunogenic. Remarkably, CD8+ T cells isolated from either an FAP+/ APCmut individual, or from a FAP-/ APCmut individual, failed to respond to the peptide, whereas those from either an unaffected family member (FAP-/ APCwt) or from healthy unrelated donors, showed a robust response, suggesting that CD8+ T cells from individuals carrying this germline APC mutation have been tolerized to the mutation. Furthermore, experimental testing of six additional reported APC gene mutation-derived peptides revealed one of the six to be immunogenic. While not all APC mutant peptides are inmmunogenic, a few qualify as vaccine candidates offering novel treatment opportunities to patients with somatic APC gene mutations to delay/treat colorectal cancer.

A cancer vaccine approach for personalized treatment of Lynch Syndrome.

Lynch syndrome (LS) is a cancer predisposition disorder wherein patients have a 70-80% lifetime risk of developing colorectal cancers (CRC). Finding germline mutations in predisposing genes allows for risk assessment of CRC development. Here we report a germline heterozygous frame-shift mutation in the mismatch repair MLH1 gene which was identified in members of two unrelated LS families. Since defects in DNA mismatch repair genes generate frame-shift mutations giving rise to highly immunogenic neoepitopes, we postulated that vaccination with these mutant peptide antigens could offer promising treatment options to LS patients. To this end we performed whole-exome and RNA seq analysis on the blood and tumour samples from an LS-CRC patient, and used our proprietary neoepitope prioritization pipeline OncoPeptVAC to select peptides, and confirm their immunogenicity in an ex vivo CD8+ T cell activation assay. Three neoepitopes derived from the tumour of this patient elicited a potent CD8+ T cell response. Furthermore, analysis of the tumour-associated immune infiltrate revealed CD8+ T cells expressing low levels of activation markers, suggesting mechanisms of immune suppression at play in this relapsed tumour. Taken together, our study paves the way towards development of a cancer vaccine to treat or delay the onset/relapse of LS-CRC.

Inhibition of angiogenesis in endothelial cells by Human Lysyl oxidase propeptide.

Angiogenesis is a critical process involved in normal physiology. Pathological angiogenesis is observed in vascular diseases and neoplasia. The propeptide domain of LOX (LOX-PP) has been shown to inhibit tumorigenesis in various cancers. In this study, we explored the role of both overexpressed and recombinant LOX-PP in naïve human umbilical vein endothelial cell with the addition of vascular endothelial growth factor (VEGF). Primarily, we observed a significant reduction in the angiogenesis signaling pathways upon LOX-PP overexpression by proteomic analysis. Further functional analysis showed that the VEGF induced cell proliferation, migration, adhesion and tube formation was inhibited by LOX-PP. Moreover, LOX-PP arrested cells at S-phase, reduced F-actin levels and decreased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK). The anti-angiogenic effect of LOX-PP was further confirmed by the reduction in the vascular network formation in chick chorioallantoic membrane (CAM). These results indicate that inhibition of angiogenesis events is not only achieved by overexpressing LOX-PP but also by addition of rLOX-PP. Taken together our findings discovered the anti-angiogenic role of LOX-PP in endothelial cells which suggests that harnessing this potential can be a promising strategy to inhibit angiogenesis.

Molecular alterations associated with chronic exposure to cigarette smoke and chewing tobacco in normal oral keratinocytes.

Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.

Hyperglycemia induced early growth response-1 regulates vascular dysfunction in human retinal endothelial cells.

Early growth response-1 (Egr-1) protein upregulation is reported in diabetes and vascular disorders. This study aims at deciphering its role in hyperglycemia induced changes of retinal endothelium. Human retinal endothelial cells (hRECs) were exposed to hyperglycemia (25mM) and normoglycemia (5.5mM). Gene silencing was done using siRNA against Egr-1. Transcript and protein level analysis of Egr-1 and gene targets were done using qPCR and immunoblotting respectively in hRECs, diabetic and nondiabetic human retina and immunofluorescence for localization in retinal sections. Hyperglycemia induced Egr-1 and vascular endothelial growth factor-A (VEGF-A) but not pigment epithelium derived factor (PEDF) in hRECs. Expression of Egr-1 repressor NGFI-A binding protein-2 (NAB-2) was unaltered. Egr-1 downstream gene targets, tissue factor (TF) and intercellular adhesion molecule-1 (ICAM-1) expression were increased in hRECs which was reduced by Egr-1 silencing in hyperglycemia. Diabetic retina, showed an increase in Egr-1, VEGF-A and gene target TF, ICAM-1 but not NAB-2 and PEDF similar to the changes seen in hyperglycemic hRECs. Hyperglycemic induction of Egr-1 and absence of NAB-2 repression in retinal endothelium, up-regulates downstream genes involved in pro-thrombotic and pro-inflammatory pathways linking Egr-1 in diabetes mediated vascular aberration of retina.

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.

Correlation of Aqueous Humor Lysyl Oxidase Activity with TGF-ß Levels and LOXL1 Genotype in Pseudoexfoliation.

PURPOSE: Pseudoexfoliation (PXF) is a microfibrillopathy involving disordered elastogenesis. Abnormal extracellular matrix (ECM) production underlies the pathophysiology of PXF. The enzyme Lysyl oxidase (LOX) and its isoforms are known to cross-link the elastin and collagen. Though the etiopathogensis of PXF is not well understood, studies report on the genetic risk involving LOXL1 gene. This study aims to screen LOXL1 coding variants rs1048661 and rs3825942 in the South Indian population and the implication of the single nucleotide polymorphism (SNP) with LOX activity. The levels of transforming growth factor ß (TGF-ß) in aqueous humor and its correlation with the LOX activity were also examined. METHODS: Blood, plasma, and aqueous aspirates were prospectively collected from PXF cases with and without glaucoma and cataract cases as controls. DNA was extracted from 48 PXF cases without glaucoma, 12 PXF cases with glaucoma, and 40 age-matched cataract-alone controls without PXF/glaucoma for analyzing LOX SNPs. LOX activity was measured in aqueous humor and plasma of 30 PXF cases without glaucoma, 24 age-matched cataract-alone controls without PXF/glaucoma, and 14 PXF cases with glaucoma. Protein levels of LOX, LOXL1, LOXL2, and total TGF-ß were estimated in plasma and aqueous humor by ELISA. RESULTS: The specific activity of LOX in aqueous humor was found to be significantly lowered in PXF cases compared with cataract-alone controls (p = 0.014). This decrease in LOX activity in PXF cases was associated with high-risk GG haplotype. However, this was not statistically significant and a larger sample size is warranted. TGF-ß1 and TGF-ß2 negatively correlated with LOX activity in aqueous humor (p = 0.028; p = 0.046, respectively). CONCLUSIONS: The LOXL1 SNPs, rs1048661 and rs3825942, are associated with PXF in the South Indian population correlating with lowered LOX activity in the aqueous humor. The increased level of total TGF-ß in the aqueous humor of PXF cases is possibly associated with LOX regulation which needs further investigation.

A molecular model of human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators.

Lysyl oxidase (LOX) is a copper dependent amine oxidase which catalyses the cross linking of collagen and elastin towards the maturation of extracellular matrix. The expression and activity of LOX is known to vary under pathological conditions such as tumorigenesis, hyperhomocysteinemia, copper deficiency diseases, pseudoexfoliation syndrome and proliferative diabetic retinopathy. Despite the implication of LOX in many diseases, there is inadequate information about its structure. Therefore, we describe a molecular model of Human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators. The predicted model was found to be highly plausible as per the stereochemistry checks. Further, Molecular Dynamics (MD) studies also inferred the stability of the predicted structure. We performed Induced Fit Docking (IFD) of LOX modulators to the predicted structure and also validated the molecular interactions in implicit solvent model by calculating Molecular Mechanics Generalized Born Surface Area (MMGBSA). The IFD results strongly reveal that aspartic acid residues in the catalytic cavity as the key players in establishing interactions with small molecules. The insights from this study will aid in better exploration of the structure-function relationship of LOX.