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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 48 hours (h), 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.
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