Protective effect of nitro-oleic acid on oxidative stress, gliosis and pro-angiogenic response in Müller glial cells

VAGLIENTI MV; SUBIRADA, PAULA V.; JORAY B; PAZ MC; BARCELONA PF; BONACCI GR; SANCHEZ MC

Congreso; ARVO Annual (Virtual) Meeting 2023; 2023

Inflammation and oxidative stress are involved in proliferative retinopathies (PR). PR occurs with increase of VEGF, alterations of retinal blood barrier, extravasation of α2-macroglobulin (α2M) and gliosis in Müller Glial cells (MGCs). Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) could be beneficial against oxidative stress, gliosis and pro-angiogenic response in MGCs.MIO-M1 cells were treated or not with NO2-OA, and HO-1 was measured by WB at 8 and 16h post-stimulus (N=4). MIO-M1 cells were incubated 1h with trigonelline (1 and 25μM) before adding NO2-OA (5μM) for 4h. The HO-1 mRNA expression was measured by qRT-PCR (N=3). 6h after NO2-OA treatment, MIO-M1 cells were challenged by PMA (1µM) or LPS (1ng/ml) for 30min and ROS levels were determined by DCF assay (N=3). Then, MIO-M1 cells were treated or not with NO2-OA for 30 min before adding α2M for 2 to 6h and the GFAP, Vimentin and HO-1 levels were measured by WB (N=3) whereas ROS levels were determined by DCF assay (N=3). In addition, MIO-M1 cells were incubated under hypoxic and proinflammatory (IL-1β) conditions with NO2-OA or vehicle, and the VEGF expression was determined by qRT-PCR (N=3). Finally, BAEC were seeded in Matrigel and treated with vehicle or NO2-OA (5µM) with the addition of VEGF (10ng/mL) (N=3). GraphPad Prism program was employed for statistical analysis.NO2-OA increased HO-1 protein expression at 8 and 16h post-stimulus (p<0.05), as well as of HO-1 mRNA expression at 4h (p<0.05) which was abrogated by inhibition of Nrf2 with trigonelline. PMA and LPS increased ROS levels in MIO-M1 cells compared with control (p< 0.001), whereas NO2-OA prevented the increase induced by both stimuli (p>0,05). α2M induced gliosis mediated by the increase of GFAP and vimentin expression (p< 0.001). In addition to this effect α2M increased ROS levels compared to control (p< 0.05). The treatment with NO2-OA before the 6 h with α2M reduced the GFAP and ROS levels to the control level (p>0,05) as well as the VEGF mRNA levels (p< 0.05). Furthermore, NO2-OA inhibited ECs tubulogenesis (p< 0.05) via Keap1/Nrf2 activation (p>0.05).Our results highlight NO2-OA protective effect on oxidative damage, gliosis and the exacerbated pro-angiogenic response in MGCs.