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

Dramatic Resolution of Recalcitrant Cystoid Macular Edema after Concurrent Intravitreal Injection of Bevacizumab and Triamcinolone

Background: Cystoid macular edema (CME), a common complication of branch retinal vein occlusion (BRVO), is associated with a significant vision loss. Anti-vascular endothelial growth factor (anti-VEGF) therapy is the gold standard of treatment, while grid macular photocoagulation has also been used as an adjuvant in patients with CME secondary to BRVO. More recent efforts were successful by the use of intravitreal triamcinolone acetonide. We proposed a concurrent use of intravitreal triamcinolone acetonide and intravitreal bevacizumab in the treatment of CME secondary to BRVO. Case presentation: We described an 82-year-old female with a BRVO in the right eye who developed associated CME. Repeated injections of intravitreal bevacizumab and modified grid macular laser treatment were ineffective. A concurrent treatment with intravitreal bevacizumab and triamcinolone acetonide resulted in complete and dramatic resolution of CME with a favorable visual outcome. Optical Coherence Tomography (OCT) demonstrated a significant decrease in central subfield thickness (CST) from 764μm to 253μm, without any post-procedure complications or recurrence of macular edema with complete recovery of visual acuity at 6-month follow-up. Conclusion: Early concurrent treatment with intravitreal anti-VEGF therapy (e.g. intravitreal bevacizumab) and intravitreal triamcinolone acetonide is likely to be more effective than intravitreal anti-VEGF agents alone or grid macular photocoagulation in the management of CME associated with BRVO

Limitations in assessing nerve growth factor levels in aqueous humor samples from human eyes

<p>Abstract</p> <p>Background</p> <p>Nerve growth factor (NGF) helps in the healing and survival of ganglion cells, photoreceptors, and optic nerve after injury and has been implicated to have a role in pathophysiology of glaucoma. So far, in animal studies, injury to iris in vitro has revealed an increase in NGF levels in aqueous. There is a great interest in investigating the levels of NGF in human aqueous in glaucomatous eyes, as suggested by animal studies, to gain a better understanding of the pathophysiology of glaucoma.</p> <p>Findings</p> <p>In this study, we examined the presence of NGF levels in aqueous humor collected from human eyes and the limitations in determining the NGF levels in human samples. NGF was assessed by ELISA immunoassay in undiluted aqueous samples collected from 32 consecutive patients undergoing surgery for cataract (control) or primary open angle glaucoma (POAG). Recombinant NGF was used as positive control. NGF levels were below undetectable levels in aqueous humor from eyes with POAG and controls by immunoassay. Less than 10% of samples had detectable NGF levels and these were considered outliers.</p> <p>Conclusion</p> <p>Our result highlights the undetectable levels of NGF in human aqueous samples.</p

Freezing adversely affects measurement of vascular endothelial growth factor levels in human aqueous samples

Sankarathi Balaiya Sandeep Grover Ravi K Murthy Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Aqueous levels of vascular endothelial growth factor (VEGF) can be a surrogate marker of intraocular VEGF activity and a measure of efficacy of anti-VEGF treatment in a variety of vasoproliferative retinal disorders, including diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusion. Measurement of the VEGF level may be adversely affected by premeasurement variables, such as freezing and delay, in sample analysis. We aim to evaluate the effect of storage and delayed measurement of human aqueous VEGF levels in these conditions.Methods: Aqueous samples collected from patients receiving intravitreal injection of bevacizumab for various retinal diseases were divided into two groups. In Group 1, the VEGF levels were analyzed on the same day; in Group 2, the VEGF levels were analyzed after 21 days of freezer storage (-80&amp;deg;C) using immunobead assay. Statistical comparison using a paired t-test was performed between the two groups.Results: Thirty-one aqueous humor samples were collected, and the VEGF concentration for fresh samples was 7.8 &amp;plusmn; 5.9 pg/mL (mean &amp;plusmn; SD) compared to 6.5 &amp;plusmn; 6.0 pg/mL in frozen samples, resulting in a statistically significant difference (P = 0.03).Conclusions: Accurate measurement of the VEGF level is a vital component of clinical decision-making. Delayed analysis of VEGF levels in aqueous samples may result in significant sample degradation and lower levels of measured VEGF.Keywords: VEGF level, aqueous humor, immunobead assay, VEGF storag