gms | German Medical Science

Background: Autophagy dysfunction in RPE (retinal pigment epithelium) cells can lead to the accumulation of toxic material, oxidative stress and inflammation, all of which may contribute to the development and progression of age-related macular degeneration (AMD). RPE cells are essential in maintaining retinal health and photoreceptor survival. AMD represents a complex, multifactorial disease, including RPE dysfunction and cell death. The dysfunction of RPE autophagy results in in the accumulation of toxic products, such as drusen, in the subretinal space, which in turn can further damage the surrounding cells. In our inflammatory porcine ex-vivo model of AMD, we observed that this inflammatory environment massively disrupts the autophagy-ability of the RPE within a very short time-frame.

Methods: Primary porcine RPE cells were cultured as functional monolayers for 4–5 weeks and then co-cultured with retinal explants for 2 days. TER measurements as well as analysis of inflammatory cytokines and autophagy markers (ELISA, qRT-PCR) and apoptosis level (Caspase 3/7 activity) were performed. Further, autophagosome expression, stress fibres (β-Actin) and neutral lipid deposition (LipidGreen2) were monitored by specific stainings.

Results: A vast increase of TNF-β (3,7-fold%, p<0.01), IL-6 (20-fold%, p<0.05), IL-1β (5,8-fold%, p<0.01), INF-y was observed in the RPE cells as well as in the retinal explant. mRNA expression of the autophagy markers Beclin and Sequestom was reduced, as well as autophagosomes (3-fold, p<0.0001). Apoptosis induction was demonstrated with significantly induced caspase 3/7-activity. Neutral lipids were accumulated, and the number of stress fibres was increased.

Conclusions: RPE cells react very sensitive to an inflammatory environment, resulting in barrier loss, apoptotic cell death and dysfunctional autophagy. Based on this model, the pathogenesis of RPE in inflammatory diseases like AMD can be further explored and therapeutic options investigated.