Elastin modulation and modification by homocysteine: a key factor in the pathogenesis of Pseudoexfoliation syndrome?

BACKGROUND: Pseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF. This study is aimed to probe Hcy-mediated alterations in elastin expression. METHODOLOGY: Lens level of Hcy (total Hcy (tHcy)), mRNA expression of Eln, CBS and MTR in lens capsule, protein expression of elastin in aqH were estimated by enzyme immunoassay, quantitative PCR and western blot, respectively in PXF, PXF with glaucoma (PXF-G) cases, in comparison with cataract-alone disease controls. Human lens epithelial cells (hLECs) were exposed to Hcy and homocysteine thiolactone (HCTL) to evaluate elastin expression in vitro. Furthermore, elastin recombinant protein was incubated with Hcy and HCTL to assess secondary and tertiary structural modifications based on circular dichroism spectroscopy, spectrophotometric and SEM studies. RESULTS: The lens tHcy was significantly high in PXF (p=0.02) and PXF-G (p=0.009). Eln expression was elevated in PXF and PXF-G (p=0.0007). Elastin level in aqH was elevated in PXF (p=0.01) and PXF-G (p=0.002). Hcy (200 µM) and HCTL (1 µM) promoted elastin expression at mRNA level by 36-fold (p=0.02) and 10-fold (p=0.05), respectively, and at protein level by nearly two-fold in cultured hLECs. Secondary structure changes in elastin protein caused by Hcy were evident from 34.11% drop in α-helix and 6.17% gain in ß-sheet. Fluorescence, spectral assays and SEM analyses showed aggregation and amyloid formation of elastin with homocysteinylation. CONCLUSION: The study reveals that lens accumulation of Hcy associated with hyperhomocysteinaemia is characteristic of PXF that augments elastin expression. Hcy causes structural changes promoting elastin aggregation, thereby contributing to defective elastin in PXF and PXF-G.

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.

Impact of epigallo catechin-3-gallate on acetylcholine-acetylcholine esterase cycle in aged rat brain.

Neurotransmission plays an important role in communication of messages in brain. Cholinergic alterations during aging are associated with learning and memory. Neurotransmitters and enzymes that influence these neurotransmitters are significant in age-associated memory. Neurotransmitters like acetylcholine, serotonin and dopamine levels were studied. Kinetics of acetylcholine esterase was studied. There was an alteration in km and Vm values which was brought back to near-normalcy by EGCG. Behavioural changes were assessed by radial maze experiment. EGCG, a good neuroprotective drug proved to alleviate the behavioural alterations in aged rat brain. Acetylcholine esterase was partially purified from rat brain and assayed in vitro. Several modifiers like EGCG and donepezil were added in silico and the activity of the enzyme was calculated. EGCG increased the activity when compared to negative control, donepezil. Using bioinformatics tools EGCG, acetylcholine and donepezil were docked with acetylcholine esterase. EGCG formed a good docking-complex with the enzyme. Thus, it shall be hypothesized that the neuroprotective activity of EGCG might be due to its influence on cholinergic neurotransmission thereby improving the cognitive functions of the brain.