Abstract WMP112: Cyb5r3 Provides Resilience Against Cerebral Infarct And Hemorrhagic Transformation In Mice Following Stroke

Abstract

Introduction: Cytochrome B5 reductase 3 (CYB5R3) is a heme reductase, ubiquitously expressed in red cells and somatic cells. We discovered a novel role for smooth muscle cell (SMC) CYB5R3 in reducing oxidized heme (Fe 3+ ) of soluble guanylate cyclase back to its reduced form (Fe 2+ ) to enable nitric oxide signaling for cyclic guanosine monophosphate production and vessel dilation. We have published higher systemic and pulmonary artery pressures in mice with conditional SMC-specific CYB5R3 knockdown. While there are over 40 CYB5R3 variants, the T117S loss-of-function (~50%) variant is interesting as it is enriched (0.23 minor allele) in persons of African ancestry. We hypothesized that the T117S variant increases risk for stroke with poor outcomes. Methods: We studied transient middle cerebral artery occlusion (50 min) in mice with CRISPR-Cas9 induced T117S CYB5R3 (T117S), Cre/Lox induced endothelial cell-specific CYB5R3 knockdown (EC-KD), Cre/Lox induced SMC-specific CYB5R3 knockdown (SMC-KD), global CYB5R3 overexpression (OE), and wild-type (WT) mice; all were adult males on a C57Bl6 background. Cerebral infarct volume (mm 3 ) was determined 24 hr later using 2,3,5-Triphenyltetrazolium chloride (TTC) and ImageJ. Results: Infarct volume was larger in T117S (132 vs 71; P=0.002) versus WT controls, which SMC-KD replicated (123 vs 70; P=0.01) and OE reversed (49 vs 100; P=0.02); EC-KD had little effect (88 vs 72). Hemorrhagic transformation was greatest in EC-KD (7/8 vs 5/9) and SMC-KD (3/6 vs 1/6), whereas there was no effect for T117S or OE. Conclusion: A congenital global point mutation causing CYB5R3 loss-of-function (T117S) enhances area at risk for cerebral infarct after stroke, while conditional deletion of EC and SMC CYB5R3 contribute to hemorrhagic transformation. Determining T117S carrier status of patients may help predict stroke risk and allow CYB5R3-targeted precision therapy approaches to stroke management.