Evaluation of antioxidants and argpyrimidine in normal and cataractous lenses in north Indian population.

AIM: To assess the level of glutathione, thioltransferase, and argpyrimidine in nuclear and cortical cataractous lenses as well as in the clear lenses in the north Indian population. METHODS: Human cataractous lenses were collected from the patients who underwent extracapsular cataract extraction surgery; clear lenses were collected from the freshly donated eye bank eyes. Antioxidant molecules such as glutathione and thioltransferase enzyme activity were measured; simultaneously in these lenses a blue fluorophore argpyrimidine, an advanced glycation end (AGE) product level was assessed using high performance liquid chromatography (HPLC). RESULTS: The protein concentration was found to be present at higher levels in the control lenses compared to cataract lenses. A significant decrease in the glutathione level was observed in the nuclear cataractous lenses compared to cortical cataractous (P=0.004) and clear lenses (P≤0.005), but no significant change in the level of antioxidant enzyme thioltransferase was observed. Further, argpyrimidine a blue fluorophore (AGE) was found to be significantly higher in the nuclear cataract (P=0.013) compared to cortical cataract lenses. CONCLUSION: Antioxidants such as glutathione significantly decrease in age-related nuclear and cortical cataract and an AGE, argpyrimidine are present at significantly higher levels in nuclear cataract.

A silent mutation in human alpha-A crystallin gene in patients with age-related nuclear or cortical cataract.

A cataract is a complex multifactorial disease that results from alterations in the cellular architecture, i.e. lens proteins. Genes associated with the development of lens include crystallin genes. Although crystallins are highly conserved proteins among vertebrates, a significant number of polymorphisms exist in human population. In this study, we screened for polymorphisms in crystallin alpha A (CRYAA) and alpha B (CRYAB) genes in 200 patients over 40 years of age, diagnosed with age-related cataract (ARC; nuclear and cortical cataracts). Genomic DNA was extracted from the peripheral blood. The coding regions of the CRYAA and CRYAB gene were amplified using polymerase chain reaction and subjected to restriction digestion. Restriction fragment length polymorphism (RFLP) was performed using known restriction enzymes for CRYAA and CRYAB genes. Denaturing high performance liquid chromatography and direct sequencing were performed to detect sequence variation in CRYAA gene. In silico analysis of secondary CRYAA mRNA structure was performed using CLC RNA Workbench. RFLP analysis did not show any changes in the restriction sites of CRYAA and CRYAB genes. In 6 patients (4 patients with nuclear cataract and 2 with cortical cataract), sequence analysis of the exon 1 in the CRYAA gene showed a silent single nucleotide polymorphism [D2D] (CRYAA: C to T transition). One of the patients with nuclear cataract was homozygous for this allele. The in silico analysis revealed that D2D mutation results in a compact CRYAA mRNA secondary structure, while the wild type CRYAA mRNA has a weak or loose secondary structure. D2D mutation in the CRYAA gene may be an additional risk factor for progression of ARC.

Deprivation of bFGF Promotes Spontaneous Differentiation of Human Embryonic Stem Cells into Retinal Pigment Epithelial Cells.

BACKGROUND: The retinal pigment epithelium (RPE), an important tissue monolayer of retina, sustains visual function and retinal homeostasis. In disease conditions such as Retinitis Pigmentosa (RP) and Age related Macular degeneration (AMD), the integrity and functional capacity of RPE monolayer is compromised. Human embryonic stem cells derived RPE (hESC-RPE) is ideal for cell based therapy because of their ability to morphologically and functionally mimic native fetal and adult RPE. However protocols for optimum culture of hESC-RPE are not well established. AIM: To describe a simplified protocol for differentiating human embryonic stem cells (hESC) into retinal pigment epithelial cells. METHODS: hESC (WA09-DL-11) cell lines were grown with standard stem cell culture protocol. After cell colonies were established, basic fibroblast growth factor (bFGF) was deprived (day 0). hESC colonies expressing pigmentation were characterized for expression of RPE65 and Zonular occludens--1 (ZO-1) with immunocytochemistry on days 0, 36, 42, 56 and 70 and western blot analysis on days 0, 40, 48, 53 and 63. In addition, morphological assessment was conducted on transformed cells longitudinally. RESULTS: Pigmented cells were noted 36 days after deprivation of bFGF from growth media. Immunofluorescence demonstrated progressive up regulation of RPE specific proteins (ZO-1 & RPE 65). Immunofluorescence of ZO-1 (in pixels) was (3.08 ± 0.31) on day 42, (5.33 ± 0.89, p = 0.0001) on day 56 and (4.87 ± 0.57, p = 0.0011) on day 70. Similarly expression of RPE 65 was (2.44 ± 0.31) on day 42, which continued to increase (4.23 ± 0.60, p = 0.0011) on day 56 and (5.59 ± 0.36, p < 0.0001) on day 70. Protein expression patterns using western blot confirmed the trends seen in immunofluorescence. Western blot analysis of ZO-1 expression (in optical density unit) was 272.57 ± 31.75 on day 40, 4212.20 ± 911.31 (p = 0.0004) on day 48, 5182.43 ± 1230.38 (p = 0.030) on day 53 and 5848.76 ± 241.04 (p < 0.0001) on day 63. Protein expression of RPE 65 was 1607.64 ± 247.76 on day 40, 2448.07 ± 152.66 on day 48 and (2341.15 ± 52.84) on day 63. hESC-RPE cells displayed a series of specific morphological changes (cytoplasmic, nuclear pigmentary and cell shape) over the course of time frame. By day 70, cells with hexagonal pattern, dark dense nucleus and uniform cytoplasm were noted in densely pigmented RPE colonies. CONCLUSION: bFGF deprivation leads to successful differentiation of hESC into RPE cells. Longitudinal transformative changes were confirmed with measurement of ZO-1 and RPE 65, specific markers of RPE.