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Pazopanib Selectively Inhibits Choroidal Vascular Endothelial Cell Proliferation and Promotes Apoptosis
Bharani Mynampati1, Moises Enghelberg1, Kakarla V Chalam1
1Department of Ophthalmology, Loma Linda University School of Medicine, Loma Linda, California, USA.
Abstract:
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.
Insights
Pazopanib effectively inhibits choroidal vascular endothelial cell proliferation in exudative age-related macular degeneration (AMD) models. This study establishes pazopanib as a potential therapeutic agent by demonstrating its dose-dependent reduction in cell viability and increase in apoptosis.
Area of Science:
- Ophthalmology
- Oncology
- Cell Biology
Background:
- Exudative age-related macular degeneration (AMD) involves choroidal neovascularization (CNV) driven by vascular endothelial growth factor (VEGF).
- Current treatments for exudative AMD include anti-VEGF monoclonal antibodies, but novel therapeutic targets are being explored.
- Pazopanib, a tyrosine kinase inhibitor, targets VEGF and platelet-derived growth factor receptors, suggesting potential anti-angiogenic properties.
Purpose of the Study:
- To investigate the inhibitory effects of pazopanib on VEGF-stimulated choroidal vascular endothelial cell (CVEC) proliferation in vitro.
- To determine a safe and effective dosage range for pazopanib in this context.
- To evaluate pazopanib's impact on CVEC viability, apoptosis, and reactive oxygen species (ROS) production.
Main Methods:
- VEGF-enriched CVECs were treated with escalating doses of pazopanib (10, 50, 100, and 250 µM).
- Cell proliferation was assessed using the WST-1 assay.
- Cell viability was measured by trypan blue exclusion assay, and intracellular caspase 3 levels and ROS were quantified.
Main Results:
- Pazopanib significantly decreased CVEC proliferation in a dose-dependent manner, with notable inhibition at higher concentrations.
- Cell viability decreased significantly with increasing pazopanib doses, particularly at 100 and 250 µM.
- Intracellular caspase 3 levels, an indicator of apoptosis, increased significantly with pazopanib treatment, suggesting induction of programmed cell death.
Conclusions:
- Pazopanib demonstrates potent in vitro inhibitory effects on VEGF-induced CVEC proliferation and viability.
- The findings suggest pazopanib's potential as a therapeutic agent for conditions involving aberrant angiogenesis, such as exudative AMD.
- Further research is warranted to explore pazopanib's efficacy and safety in vivo for treating neovascular eye diseases.
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