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Rapamycin and Resveratrol Modulate the Gliotic and Pro-Angiogenic Response in Müller Glial Cells Under Hypoxia
Paula V Subirada1,2, María V Vaglienti1,2, Mariana B Joray3,4
1Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Bioquímica Clínica, Córdoba, Argentina.
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.
Insights
Hypoxia in retinal diseases increases autophagy in Müller glial cells (MGCs). Rapamycin treatment reduced gliosis and blood vessel formation, suggesting a protective role in retinal injury.
Area of Science:
- Ophthalmology and Molecular Biology
- Cellular response to hypoxia and reoxygenation
- Autophagy and glial cell function in retinal disease
Background:
- Hypoxia and reoxygenation are common in retinal diseases, involving Müller glial cells (MGCs) in injury and repair.
- Pharmacological strategies aim to modulate MGCs' pro-angiogenic and gliotic responses for faster recovery.
- Understanding autophagy flux variations under hypoxia is crucial for developing new treatments.
Purpose of the Study:
- To investigate autophagy flux changes in MGCs under hypoxia and hypoxia-reoxygenation.
- To evaluate the effects of autophagy inducers (Rapamycin, Resveratrol) on MGCs' gliotic and pro-angiogenic responses.
- To analyze the impact of MGCs' conditioned media on endothelial cell tubulogenesis.
Main Methods:
- MGCs were exposed to gas (1% O2) and chemical (CoCl2) hypoxia, followed by reoxygenation.
- Autophagy flux was assessed by measuring LC3B II and p62 protein levels.
- Gliosis markers (GFAP, Vimentin) and angiogenic factors (VEGF, PEDF) were analyzed.
- Endothelial cell (EC) tubulogenesis was evaluated using MGCs' supernatants.
Main Results:
- Hypoxia and hypoxia-reoxygenation increased LC3B II and p62 levels in MGCs.
- Reoxygenation after hypoxia upregulated LC3B II but decreased cell survival.
- Hypoxia increased GFAP expression; Rapamycin reduced GFAP levels.
- MGC supernatants from hypoxic conditions with autophagy inducers inhibited EC tubulogenesis.
- Rapamycin reduced VEGF mRNA, while Resveratrol increased PEDF mRNA.
Conclusions:
- Autophagy flux variations under hypoxia/reoxygenation act as a protective mechanism against retinal injury.
- Rapamycin treatment demonstrated beneficial effects by decreasing MGC gliosis and inhibiting neovascularization.
- Modulating autophagy in MGCs presents a promising therapeutic strategy for retinal diseases.
