Category: Federal Forum Posters
Purpose: Among retina degeneration disease, Age-related macular degeneration (AMD) is an important cause of blindness in elder people over 65 years old. AMD can be classified into dry or wet type based on the lesion of drusen accumulation or choroid neovascularization (CNV), respectively. Understanding the mechanism that modulate the early stage of retinal pigment epithelium (RPE) dysfunction is strongly required for development of early detection and treatment of AMD. Oxidative stress was a well-known stress to induce AMD. Therefore, we chose oxidative low-density lipoprotein (oxLDL) as a stress source to treat with RPE.
Methods: The differentiation of human induced pluripotent stem cells to retinal pigment epithelial cell was modified according to Buchholz ’s paper. To evaluate the purity of RPE cells derived from hiPSCs, the specific markers of RPE cells such as retinal pigment epithelium-specific 65 kDa, Zonula occludens-1 (ZO-1) were measured by immunofluorescence and functional marker such as microphthalmia-associated transcription factor (MITF), tyrosinase were measured by western blot. We used MTT assay to distinguish the different effects between LDL and oxLDL. We added 100 and 300 μg/ml LDL or oxLDL then observed 24-72hrs. To clarify the plausible mechanisms involved in oxLDL caused RPE death, we used immunofluorescence to detect whether the apoptotic signaling molecules presented in the damaged RPE cells. To confirm whether oxLDL could induce RPE cell to secrete more VEGF, we used VEGF ELISA kit to measure the supernatant concentration of VEGF releasing from 100 and 300 μg/ml oxLDL treated RPE cells. To generate retina degeneration animal model, we Intravitreal injected VEGF at 100 ng/10μl combined with the retrobulbar injected oxLDL 500μg/ 50μl and then followed up for 2-4 weeks. Then we used fundus to check the phenotypes of whole eyes and optical coherence tomography (OCT) and fluorescein angiography (FAG) to confirm the integrity of retinal layers as well as determine whether the choroidal neovascularization was occurred.
Results: We observed our RPE had hexagonal morphology and pigmentation under microscope. To confirm RPE was successfully differentiated from hiPSCs, specific markers such as RPE65, ZO-1 were detected by immunofluorescence. We found the level of MITF and tyrosinase were highly expressed in RPE cells. These results suggest that RPE cells had been differentiated from hiPSCs successfully. Treatment of LDL had no effect on cell viability in RPE cells. Stimulation of oxLDL reduced cell viability dramatically and enhanced caspase-3 activation in RPE cells. Also, we observed that oxLDL stimulation increased AMD markers, Hsp 90, expression in RPE cells. These results suggest that oxLDL is a stress to induce RPE damage. On the other hand, the highly expressed level of VEGF secreted from 300 μg/ml oxLDL was also detected. It was match with wet type AMD. Therefore, we Intravitreal injected VEGF at 100 ng/10μl combined with the retrobulbar injected oxLDL 500μg/ 50μl to mimic a stress damage. Hemorrhage and neovascularization was detected by fundus, OCT and FAG. Therefore, we can confirm the effects of oxLDL on retina impairment and choroidal neovascularization.
Conclusion: Oxidative stress was a well-known stress to induce AMD. Therefore, we chose oxLDL as a stress source to treat with RPE. We successfully differentiated RPE and confirmed the specific markers expression. We also observed oxLDL could elevate a series of factors which were corresponded to lysosome dysfunction, or even apoptosis. Because of oxLDL induced VEGF secretion, it was a good source to induce wet type AMD-like animal model. In conclusion, oxLDL could induce the RPE morphological change, apoptosis, secret more VEGF and could be a stress to induce retina damage.