Abstract
Purpose:
Human Retinal Pigment Epithelium (hRPE) has been implicated in the pathogenesis of age related macular degeneration (AMD). Some patients with AMD also develop choroidal neovascularization. Since hRPE cells are also a known source of angiogenic factors, like VEGF, which play a role in proliferative retinal disease, inhibition of this synthesis may prevent or treat this condition. Therefore, since pazopanib (PZB) is a tyrosine-kinase inhibitor of VEGF-R1/VEGF-R2, we investigated if it has any effect on hRPE cell proliferation and VEGF synthesis.
Methods:
Primary differentiated hRPE cell cultures were established from non-pathologic human eyes obtained from the Michigan Eye Bank. Trypan blue exclusion method (T) was used to assess cell viability, and 3H-thymidine incorporation (3H-thy) was used to measure cell proliferation. Synthesis of VEGF was measured utilizing [(14)C]methionine immunoprecipitation. Nuclear staining studies were performed by DAPI. Student’s t-test was used to evaluate statistical significance, with p<0.05 considered significant.
Results:
Fetal Bovine Serum stimulated hRPE cell proliferation and maintained cell viability as shown by T and 3H-Thy. PZB inhibited hRPE cell proliferation in a dose dependent manner. PZB (10 mM) inhibited T (4.88±2.30 vs. 10.75± 2.20, cells per 0.1 µl ±SEM, p≤0.05, n=8). PZB (0-10 mM) also inhibited immunoprecipitated 14C-VEGF synthesis in a dose dependent manner. Qualitative studies using nuclear staining of hRPE cells showed that PZB altered hRPE morphology resulting in smaller dysplastic cells. Immunocytochemistry analysis demonstrated that PZB inhibits VEGF synthesis in hRPE cells.
Conclusions:
These studies demonstrate that PZB inhibits hRPE cell proliferation and inhibits VEGF synthesis in hRPE cells, and therefore it may be of therapeutic value in the prevention or treatment of blinding complications in proliferative vitreoretinal diseases.