Pazopanib Selectively Inhibits Choroidal Vascular Endothelial Cell Proliferation and Promotes Apoptosis

Exudative age related macular degeneration (AMD) is related to active choroidal neovascularization (CNV) and formation of disciform scars. Vascular endothelial growth factor (VEGF) mediated choroidal vascular endothelial cell (CVECs) proliferation is characteristic of CNV. Intravitreal injections of bevacizumab, ranibizumab and aflibercept (anti-VEGF monoclonal antibodies) are used to treat exudative AMD. Pazopanib, a tyrosine kinase inhibitor, inhibits neovascularization through blockade of intracellular tyrosine kinase VEGF receptor and platelet-derived growth factor receptor. In this in vitro investigation, we evaluated the inhibitory consequences of escalating doses of pazopanib on proliferation of VEGF-enriched CVECs to establish a safe dosage range. VEGF (50 ng/mL) enriched CVECs were treated with escalating doses of pazopanib (10, 50,100 and 250 µM). Cell proliferation rates (WST-1 assay), cell viability (trypan blue exclusion assay), and reactive oxygen species (ROS) levels were measured at 48h, 72h and 1 week. Intracellular caspase 3 levels and morphological changes were recorded. VEGF enriched CVECs showed a significant decrease in cell proliferation rates after one week of treatment with increasing doses of pazopanib (10, 50,100 and 250 µM) treatment i.e. 87.8%, 43.0%, 38.1% and 9.3% compared to controls (p<0.001). Similarly, trypan blue exclusion assay revealed a decrease in cell viability as 81.8%, 81.0%, 53.4% and 8.7%, respectively (p<0.05). Further, pazopanib actively inhibited proliferation of VEGF-enriched CVECs, with 1.32, 1.92, 1.92 and 4.1-fold increase (p<0.01) in intracellular caspase 3 levels. VEGF-enriched CVECs treated with escalating doses of pazopanib decreased cell viability and increased caspase 3 levels in a time and dose dependent manner

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