AMPK activation by omega-3 fatty acids protects the retina against ischemic insults

Author(s)

Marcella Neves Dátilo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2023-03-16

Examining board members

Jacqueline Mendonça Lopes de Faria; Dania Emi Hamassaki Britto; Maria Lucia Cardillo Corrêa Giannella; Mônica Barbosa de Melo; Sara Teresinha Olalla Saad

• Advisor: Jacqueline Mendonça Lopes de Faria

Abstract

Background: Ischemic retinopathies (IRs) are the main causes of severe visual alterations, which can lead to vision loss. Understanding the mechanisms related to disease development and elaborating new therapies to prevent progression are necessary. Several mechanisms are associated with the genesis of IRs, among them the increase in the inflammatory process, oxidative stress, and angiogenesis. Evidence suggests that activation of AMP-dependent protein kinase (AMPK) in retinal cells is able to reduce inflammation and oxidative stress. However, the role of the AMPK protein in IRs remains poorly understood. In addition, evidence also demonstrates the beneficial role of polyunsaturated fatty acids of the ?3 family (?3-PUFAs) in the development of retinopathies, but the mechanisms remain poorly understood. In relation to AMPK activation, studies suggest that nutrients such as ?3-PUFAs can act as protein activators, however, the role of the ?3-AMPK axis in retinal tissue remains unknown. Objective: Evaluate the possible beneficial effects of omega-3 in a model of ischemic retinopathy and whether these effects may depend on the activation of the AMPK protein. Methods: Primary human retinal microvascular endothelial cells (hRMECs) were exposed to a hypoxia mimetic (Dimethyloxalylglycine – DMOG, 400µm), in the presence or absence of ?3-PUFA (docosahexaenoic acid – DHA, 50µm), under silencing for the protein AMPK?. After treatments, ischemia parameters, the mesenchymal transition phenomenon, endothelial barrier integrity, angiogenesis, and AMPK protein activity were evaluated. After that, AMPK?2 knockout mice and their genetic controls (C57BL/J6) were distributed into the following experimental groups: the control group (CT); the group submitted to the oxygen-induced retinopathy (OIR) protocol, and the OIR+?3 group that received a maternal supplementation with 50µL of oil rich in ?3-PUFAs. At the end of the experimental period, ischemic and angiogenesis markers were evaluated using immunofluorescence or immunohistochemistry. Results: Cells exposed to DMOG treatment showed an increase in the expression of hypoxic markers, mesenchymal transition (vimentin), and disruption of the zonula junction protein Occludens-1 (ZO-1), along with an increase in cell migration and angiogenesis when compared to the control (CT) (p<0.05). DMOG exposure reduced threonine 172 (Thr172) phosphorylation of AMPK (pAMPKThr172). Interestingly, DHA treatment under ischemic conditions (DMOG+DHA) was able to restore pAMPKThr172 (p<0.05 vs DMOG). DHA treatment also increased the expression of its receptor (GPR120), the intracellular calcium concentration, and the expression of the AMPK upstream protein, CaMKK?. In addition, DHA treatment restored the alterations promoted by DMOG. However, under AMPK? silencing, the beneficial effects promoted by DHA were lost. In vivo, AMPK?2-/- and WT mice submitted to the OIR protocol showed similar morphological retinal changes. On the other hand, supplementation with oil rich in ?3-PUFAs reduced alterations promoted by ischemia only in WT animals, reinforcing the possible role of AMPK? in the beneficial effects of omega-3 fatty acids in an ischemic environment. Conclusion: Omega-3 fatty acids can activate the AMPK protein via GPR120-CaMKK? protecting the retina against ischemic insults. Furthermore, ?3-PUFAs effects seem to be dependent on the activation of the AMPK. Then, the evidence presented in this study may contribute to the development of a new therapeutic tool to control pathological angiogenesis in retinal ischemic diseases. However, further studies are required to confirm this original hypothesis (AU)

• FAPESP's process: 18/07398-3 - Involvement of AMPK in the retina of AMPK Knockout mice in experimental models of Diabetes and Proliferative Ischemic Retinopathy: in vivo and in vitro
• Grantee: Marcella Neves Dátilo
• Support Opportunities: Scholarships in Brazil - Doctorate