Introduction: Diabetes increases bleeding into the brain and mediates vasoregression leading to poor stroke recovery. The iron released from the bleeding is neurotoxic but how it affects the repair of damaged blood vessels is not known. Ferroptosis and necroptosis are recently identified forms of regulated cell death. While ferroptosis is an iron-dependent process, activation of toll like receptor (TLR) mediate necroptosis. We hypothesized that 1) iron activates TLR4 signaling through oxidative stress and reduces viability and migration of brain microvascular endothelial cells (BMVECs), and 2) necroptosis and ferroptosis contribute to BMVEC death after hypoxia/reoxygenation in diabetes.
Methods: To mimic increased HT (free iron) in diabetes, BMVECs isolated from diabetic rats were subjected to oxygen glucose deprivation (OGD) and iron (III) sulfate (0.1mM) with and without TLR4 neutralizing antibody (10ug/ml) or antioxidant N-acetyl cysteine (NAC; 1mM) for 6 hours, followed by 12 hours of reoxygenation. TLR4 and markers of necroptosis (RIPK3) and ferroptosis (citrate synthase; CS) were assessed by immunohistochemistry in brain sections of diabetic rats subjected to middle cerebral artery occlusion. In pilot experiments, BMVECS were treated with inhibitors of necroptosis (necrostatin; 100μM) or ferroptosis (ferrostatin-1(2μM)/TAK-242(10μM) during hypoxia and apoptosis (caspase-3)/necrosis (7AAD) were measured by flow cytometry.
Results: Presence of iron in OGD reduced viability (% of normoxia, 76.9±3.1 vs 97.4±4.0 OGD alone) and both TLR4 inhibition (114±8.0) and NAC (112±5.0) prevented this effect (n=3, p<0.05). OGD reduced migration (in %, 31.7±3.1 vs 42.7±3.4) and iron did not have an additive effect (29.1±3.5). Both TLR4 inhibition (47.0±3.1) and NAC (54.4±4.8) prevented this effect (n=3, p<0.05). CS, TLR4 and RIPK3 colocalized to the vasculature after stroke. Inhibition of necroptosis and ferroptosis reduced the number of apoptotic and necrotic cells while increasing the number of viable cells.
Conclusions: Necroptosis and ferroptosis contribute to endothelial cell death after hypoxia/reoxygenation via TLR4 activation. Inhibiting TLR4 signaling may prevent vasoregression and improve recovery after stroke in diabetes.