Karunakaran Coral

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors.

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P<0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride+total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.

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 enzymes 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.

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.

High Glucose Induced Differential Expression of Lysyl Oxidase and Its Isoform in ARPE-19 Cells

Purpose: Lysyl oxidase (LOX) stabilizes the extracellular matrix (ECM) by cross-linking collagen and elastin molecules. In proliferative diabetic retinopathy (PDR), there is ECM remodeling with neovascularization and basement membrane changes. While protease activities are well reported, the role of LOX in the pathogenesis of diabetic retinopathy is less studied. This study was done to see the effect of high glucose on the activity and expression of LOX and its isoforms in ARPE-19 cells. Materials and methods: ARPE-19 cells were exposed to high glucose up to 48 h, and LOX activity was determined by N-acetyl-3,7-dihydroxyphenoxazine assay. The mRNA expression of LOX and its isoforms was done by real-time PCR and the protein expression by ELISA. Immunohistochemistry for LOX was done in epiretinal membrane from PDR. Results: With an increase in glucose concentration LOX activity and protein was reduced significantly at 30 mM glucose at 48 h. mRNA expression of LOX, LOXL1, and LOXL2 varied with time and concentration of glucose. Vascular endothelial growth factor (VEGF) increased the LOX activity as well as the mRNA expression. Pigment epithelium-derived factor (PEDF) downregulated the mRNA expression of LOX, LOXL1, and LOXL2. The matrix metalloprotease (MMP) activity increased significantly with the increase in glucose concentration. The diabetic neovascular membrane showed increased immunostaining of LOX. Conclusions: This study suggests that although the LOX activity, which is composite of all the isoforms, was reduced under high glucose conditions, there was a differential mRNA expression with increased LOX and LOXL1 and decreased LOXL2 expression.

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.

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.

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

ABSTRACT 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 β (TGF-β) 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-β 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-β1 and TGF-β2 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-β in the aqueous humor of PXF cases is possibly associated with LOX regulation which needs further investigation.

Visual functions of workers exposed to organic solvents in petrochemical industries

Aim: The purpose of this study was to evaluate the visual functions of workers exposed to organic solvents in petrochemical industries. Materials and Methods: Thirty workers from the petroleum refinery and 30 age-matched controls (mean age) were recruited. Visual functions and occupational exposure levels were assessed among both the groups. Visual acuity, contrast sensitivity, color vision, and visual fields were evaluated at the workplace. The biological samples, namely blood and urine, were collected at the workplace and transported to the laboratory for analysis. The urinary excretion of hippuric and methylhippuric acid as well as creatinine was measured by high performance liquid chromatography. Results: The mean age of the workers and controls were 39.7 ± 7.6 years and 38.6 ± 8.1, years respectively. The mean years of experience of the workers were 15.6 ± 6.8 years. Visual acuity was >0.01 LogMAR among both the control and case groups. The contrast sensitivity was reduced at 12cpd among workers. Comparison between groups was done using independent sample t-test. The mean difference in color confusion index was 0.11 ± 0.05 (P = 0.037*). The mean difference in visual fields was −0.31 ± 0.36 dB (P = 0.933). The mean difference in urinary hippuric acid level (urinary metabolite of toluene) between the groups was 0.19 ± 0.96 g/g creatinine (P = 0.049FNx01). The mean difference in the excretion of methylhippuric acid (urinary metabolite of xylene) was 0.06 ± 0.04g/g creatinine (P = 0.154). We also found that exposure was a significant risk factor for color vision defect with an odds ratio of 4.43 (95% CI: 1.36–14.4); P = 0.013. Conclusion: The study results showed that contrast sensitivity and color vision were affected among workers in petrochemical industry.

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.

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.