The anti-inflammatory and neuroprotective activity of novel hybrid molecule SA-10 in in vitro ischemic models

Abstract

Purpose : Embolism and/or high intraocular pressure lead to poor retinal circulation, reactive oxygen species (ROS) production, microglial activation and retinal ganglion cell (RGC) death. Previously we reported, a small hybrid molecule, SA-10 with both anti-oxidant and nitric oxide donating activity protect retina in a mouse model of ischemia/reperfusion (I/R) injury. Here, we determined the anti-inflammatory and angiogenic effects of SA-10 using in vitro primary human endothelial and rat retinal neuronal cells.

Methods :
Tube formation assay: Human umbilical vascular endothelial cells (HUVECs) were seeded on Matrigel with H₂O₂ (200 µM) and with or without SA-10. Formation of microtubules were captured at 12h and analyzed by Angiogenesis analyzer on ImageJ. I/R cell assays: Primary and immortalized (R28) rat retinal mixed neurons (Oxygen Glucose Deprivation model) and primary rat retinal microglial cells (activated with TII: TNF-α IL-β and IFN-g) were used with or without SA-10 (1.0, 10, 100uM). ROS scavenging (DCFDA) and cytoprotective (LDH) activities were measured at 24h. Inhibition of microglial activation by SA-10 was evaluated by measuring inflammatory cytokines from cell supernatant using multiplex ELISA. Data represented as Mean ± SEM. N = 4.

Results : In HUVECs, SA-10 dose dependently increased microtubule formation with an EC₅₀ of 0.125 µM. SA-10 (10uM) significantly attenuated cell death in both microglia and R28 cells (43% vs 13% and 52 % vs 17% respectively) and decreased ROS (68% to 38%) production in retinal microglial cells. OGD induced cell death is attenuated in primary retinal neurons (66% vs 10%) by SA-10. In microglia after TII insult, there was significant increase in inflammatory cytokines IL-4, IL-1β, IL-6, TNF-α and decrease in anti-inflammatory cytokine IL-10 as compared to control. SA-10 (10uM) significantly decreased (2-3-fold) all inflammatory cytokines and increased IL-10 (2.5-fold).

Conclusions : Our results are consistent with our hypothesis that the compound SA-10 is protective to retinal neurons and microglia by decreasing oxidative stress, inflammation with possibility to improve retinal blood perfusion by forming new blood vessels. We predict SA-10 as a possible therapeutic candidate in treating I/R injury in retina and diseases associated with microglia activation and inflammation.

This is a 2021 ARVO Annual Meeting abstract.