Abstract
Hypoxia and hypoxia-reoxygenation are frequently developed through the course of many retinal diseases of different etiologies. Müller glial cells (MGCs), together with microglia and astrocytes, participate firstly in response to the injury and later in the repair of tissue damage. New pharmacological strategies tend to modulate MGCs ability to induce angiogenesis and gliosis in order to accelerate the recovery stage. In this article, we investigated the variation in autophagy flux under hypoxia during 4 h, employing both gas culture chamber (1% O2) and chemical (CoCl2) hypoxia, and also in hypoxia-reoxygenation. Then, we delineated a strategy to induce autophagy with Rapamycin and Resveratrol and analysed the gliotic and pro-angiogenic response of MGCs under hypoxic conditions. Our results showed an increase in LC3B II and p62 protein levels after both hypoxic exposure respect to normoxia. Moreover, 1 h of reoxygenation after gas hypoxia upregulated LC3B II levels respect to hypoxia although a decreased cell survival was observed. Exposure to low oxygen levels increased the protein expression of the glial fibrillary acid protein (GFAP) in MGCs, whereas Vimentin levels remained constant. In our experimental conditions, Rapamycin but not Resveratrol decreased GFAP protein levels in hypoxia. Finally, supernatants of MGCs incubated in hypoxic conditions and in presence of the autophagy inductors inhibited endothelial cells (ECs) tubulogenesis. In agreement with these results, reduced expression of vascular endothelial growth factor (VEGF) mRNA was observed in MGCs with Rapamycin, whereas pigment epithelium-derived factor (PEDF) mRNA levels significantly increased in MGCs incubated with Resveratrol. In conclusion, this research provides evidence about the variation of autophagy flux under hypoxia and hypoxia-reoxygenation as a protective mechanism activated in response to the injury. In addition, beneficial effects were observed with Rapamycin treatment as it decreased the gliotic response and prevented the development of newly formed blood vessels.
Highlights
As part of the Central Nervous System, the retina is highly dependent on nutrients and bioactive molecules, among them oxygen (Country, 2017)
The quantitative analyses of LC3B II and p62 bands evidenced a significant increase in the autophagic protein levels since a concentration of 250 μM respect to vehicle (p = 0,027 for LC3B II and p = 0,031 for p62)
We have previously demonstrated in a mouse model of Oxygen-induced retinopathy (OIR) that Müller glial cells (MGCs) become reactive after the hyperoxic phase and modified the expression of stress and detoxifying proteins from postnatal days 12 to 26 in OIR mice (Ridano et al, 2017)
Summary
As part of the Central Nervous System, the retina is highly dependent on nutrients and bioactive molecules, among them oxygen (Country, 2017). Müller glial cells (MGCs), the most abundant macroglial cells of the retina, are known to collaborate in the immune response by secreting several cytokines, and to lead the vaso-proliferative response under hypoxia (Liu et al, 2014; Eastlake et al, 2016; Li et al, 2019). Many proteins participate in angiogenesis, including proangiogenic factors as the vascular endothelial growth factor (VEGF) and anti-angiogenic factors as pigment epitheliumderived factor (PEDF) (Eichler et al, 2004). These factors are secreted by many retinal cells, conditional VEGF KO mice revealed that the contribution of MGCs is indispensable for the vascular proliferation (Wang et al, 2010). A wide variety of compounds are able to modulate VEGF during pathological angiogenesis (Formica et al, 2021; Szymanska et al, 2021)
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