Abstract WMP83: The Pentose Phosphate Pathway is Crucial for Combating Heme-Induced Ferroptosis in Red Blood Cells After ICH

Abstract

Intracerebral hemorrhage (ICH) occurs upon blood vessel rupture, allowing blood into brain tissue. Red blood cells (RBCs) make up 99%+ of blood cells. Brain-trapped RBCs undergo phagocytosis; however, many RBCs lyse prior to being phagocytosed due to their abundance. RBC lysis releases hemoglobin and toxic heme, driving oxidative stress especially in lipid-rich plasma membranes (PM), causing tissue damage and ferroptosis. Few studies have explored RBC fate in ICH, but unraveling the factors governing RBC lysis may yield ICH therapies.In in vitro human and murine RBC cultures, we observed RBCs are exquisitely sensitive to heme-induced death with increased oxidized fatty acids (FAs), a ferroptosis marker (Fig 1A-B). The ferroptosis inducer Erastin validated RBC susceptibility to ferroptosis (Fig 1C). Heme-induced cell death was countered by β-mercaptoethanol (β-ME), N-acetylcysteine (NAC), ferrostatin-1 (Fer-1) and Vitamin E (Vit. E), ferroptosis inhibitors, confirming RBC death was due to ferroptosis (Fig 1D-G). Heme and Erastin induced RBC ferroptosis differs from eryptosis, RBC specific programmed cell death, as it isn't Ca++ dependent (Fig1H&I). Unlike other cells, RBCs solely rely on the Pentose Phosphate Pathway (PPP) for NADPH production for oxidation defense. PPP inhibition with Dehydroisoandrosterone (DHEA) and treatment with ThioNa, an NADP+ synthesis inhibitor, both heightened heme-induced RBC ferroptosis (Fig 1J-L). These data support the PPP's role against heme-driven ferroptosis. Heme induced RBC ferroptosis may trigger a chain reaction of RBC lysis and ongoing heme release. Notably, 400 million people face G6PD deficiency, the rate-limiting PPP enzyme, leading to NADPH depletion in RBCs with enhanced susceptibility to oxidation. Hence, in addition to identifying the PPP in RBCs resistance to heme toxicity, our findings could aid in identifying a population that is particularly vulnerable to the devastating effects of ICH.