Abstract 4373706: In vivo conversion of ferric heme to NO-ferroheme has vasodilatory and anti-inflammatory effects

Abstract

Background: Increased levels of cell-free hemoglobin (Hb) and ferric heme in plasma is vasculopathic and contributes to morbidity and mortality in hemolytic and non-hemolytic diseases, including sepsis. Nitric oxide (NO) signals through soluble guanylate cyclase (sGC) to induce vasorelaxation and is rapidly scavenged by plasma Hb/heme. Dysregulated NO signaling and increased oxidative stress underlie sickle cell disease (SCD) pathology and treatments that limit heme toxicity and promote NO signaling are needed. We have discovered a fast catalyzed reductive nitrosylation reaction that converts heme into NO-ferroheme, protecting NO from Hb scavenging and when in complex with albumin, elicits sGC-dependent vasorelaxation. Hypothesis: We hypothesize that reductive nitrosylation can be coopted as a therapeutic to convert pro-oxidative, pro-inflammatory plasma free heme to anti-inflammatory, vasodilatory NO-ferroheme. Approach: NO-ferroheme was measured by ozone-based chemiluminescence in mouse plasma following intravenous infusion of heme, NO donor and reductants to determine if NO-ferroheme is formed in vivo and the effects on mean arterial pressure (MAP) were assessed. To determine therapeutic potential, mice were treated with NO-ferroheme albumin following induction of LPS-mediated lung injury. Total cell extravasation, neutrophil response, and the expression of inflammatory cytokines were assessed. Results: Infusion with heme, NO donor (papaNONOate) and glutathione (GSH) or ascorbate yielded detectable levels of NO-ferroheme (ascorbate - 83.7 nM ± 3 7.7 nM; GSH - 57.2 nM ± 29.8 nM) with low levels of S-nitrosothiols in mouse plasma. Infusion of heme triggered acute hypertension, while infusion of NO donor, ascorbate and heme had a vasodilatory effect (MAP -5 mmHg from baseline). Treatment with 1 umol/kg NO-ferroheme albumin following LPS-mediated lung injury resulted in decreases in total cell extravasation, neutrophil to monocyte ratio and expression of IL-6 , Cxcl9, and Cxcl10 . Conclusions: Free heme is converted to NO-ferroheme in vivo in the presence of excess NO and reductant causing vasorelaxation, reversing the hypertensive effects of heme. Treatment with NO-ferroheme albumin is anti-inflammatory in an LPS model of acute lung injury. Conversion of pro-oxidative and pro-inflammatory heme into anti-inflammatory NO-ferroheme shows promise as a novel strategy to curtail heme-mediated injury in hemolytic conditions.