Abstract
Purpose :
It is known that in proliferative retinopathies hypoxia increases VEGF expression and other growth factors that have an important modulatory role in neovascularization and neurodegeneration development. In this pathological scenario, Müller glial cells (MGCs) participate in retinal homeostasis maintenance by eliminating protein aggregates, activating stress proteins and detoxifying mechanisms. The purpose of this study is to explore in two experimental models (in vivo and in vitro) whether autophagy flux in MGCs is modified under hypoxic conditions.
Methods :
In vitro: MIO-M1, a human immortalized cell line of GMC, were pre incubated with chloroquine 10 uM and exposed to CoCl2 250 uM or incubated in a cell chamber with 0,1%O2 for 24hs.Westen blot, qPCR and immunofluorescence analysis were performed(N=3). In vivo: Oxygen induced retinopathy (OIR) mice model. C57/BL6 mice exposed to 75% O2 from postnatal day (P) 7 to 12, were brought to room air (RA) for additional five(P17) or nine days(P26). Age-matched mice maintained in RA were used as control (N=4).An intraperitoneal injection of leupeptin 40mg/kg was administrated before sacrifice to block autophagy flux. 1 or 2 way ANOVA was used for statistical analysis.
Results :
Western blot and immunofluorescence analysis revealed increased levels of LC3B II and p62/SQTSM1(p=0,01) but reduced colocalization of LC3B and lysotracker. No significant changes in MAP-1(p=0,38), ATG5(p=0,69) nor BECLIN-1(p=0,94) transcrips were observed. Similar results were obtained when MIO-M1 were exposed to 0,1%O2. In addition, western blot of neural retinas showed significant changes in autophagy flux and glutamine syntethase, total caspase 3 and stress protein levels at p12, p17 and p26 in OIR mice respect to controls(p<0,05). By immunofluorescence staining a distinct pattern of LC3B was visualized at the inner limiting membrane in end feet of MGCs.
Conclusions :
Altogether the findings indicate that hypoxia is able to modify the autophagy flux of MGCs during ischemic retinopathies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.