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Shervi Lie, Liam M Ashander, Binoy Appukuttan, Justine R Smith; Expression of Angiogenic Regulators by Human Retinal Cells Infected with Toxoplasma gondii: Understanding the Clinical Course of Ocular Toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1891.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal infection with the parasite, T. gondii, induces substantial inflammation and results in sizeable scarring. However, unlike other inflammatory posterior segment diseases that involve scarring, ocular toxoplasmosis is rarely complicated by neovascularization. To understand this phenomenon, we investigated the expression of angiogenic regulators, VEGF-A and -B, PEDF and TSP-1, and transcription factor, HIF-1α, by retinal cells that modulate the development of neovessels.
The natural T. gondii strain, GT-1, was maintained in tachyzoite form by culture in fibroblasts. Monolayers of human retinal pigment epithelial (ARPE-19) cells or human Mueller (MIO-M1) cells were infected with freshly egressed tachyzoites (MOI 5:1) in DMEM with up to 5% FBS or cultured with medium alone. Cell invasion and intracellular replication were confirmed by microscopy, and parasite viability was determined by plaque assay. Total RNA or protein was isolated at 4 and 24 hours post-infection (n=6/condition) and analyzed by qRT-PCR (RPLP0 and PPIA as references) or Western blot (β-actin as reference), respectively. Statistical significance was defined by p<0.05 on t-test.
VEGF-A mRNA was significantly induced 1.9- to 2.4-fold in infected ARPE-19 cells compared to controls at 4 and 24 hours post-infection; isoforms 121, 165 and 189 increased (≥2.6-fold) at 4 and/or 24 hours. PEDF and TSP-1 mRNA rose significantly (≥2.2 fold) at one or both time points. Consistently, HIF-1α protein increased by 24 hours in infected cells. Simultaneously, VEGF-B mRNA did not change. In MIO-M1 cells, infection with T. gondii significantly elevated PEDF mRNA at 4 hours (1.8-fold), but induced no rise in VEGF-A and its isoforms, VEGF-B or TSP-1 at the tested time points. Parasite viability was >15% for all infections, consistent with published measurements for natural isolates.
Although infection with T. gondii increased expression of pro-angiogenic VEGF-A isoforms in human retinal pigment epithelial cells, these changes were balanced by high expression of anti-angiogenic PEDF and TSP-1, and lack of rise in VEGF-B, as well as increased expression of PEDF by human Mueller cells. Our findings may explain the rare occurrence of neovascularization in ocular toxoplasmosis, in comparison to other inflammatory posterior segment diseases associated with scarring.
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