ALBERT

Description: Abstract 4836 Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by 〉95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.

Description: Abstract 2135 Circulating heme is scavenged by multiple plasma proteins and delivered to the liver for degradation. We have recently demonstrated that heme scavenging is variably impaired in transgenic mice with sickle cell disease (SCD). In this study, we tested the hypothesis that excess protein-free plasma heme (PFPH) in the blood circulation, and excess scavenged protein-bound plasma heme (PBPH) destined for degradation cause different types of organ damage in SCD. Transgenic mice expressing exclusively human sickle hemoglobin (SS) were intravenously injected with a dose of heme (25 micromoles/kg) to elevate PBPH only, or with a dose (70 micromoles/kg) sufficient to raise PFPH. In agreement with our previous findings, PFPH was associated with severe lung injury and 100% lethality within 2 hours. This phenomenon occurred independent of any liver involvement. Modest elevation of circulating heme sufficient to raise PBPH only, increased alanine aminotransferase and aspartate aminotranferase 3- to 4-fold (P

Description: Abstract 2113 Sickle cell disease (SCD) is characterized by multiple exacerbating events that cause intravascular hemolysis. Heme released into the circulation is scavenged by multiple plasma proteins and delivered to the liver for degradation. Our recent data indicate that this process is impaired in SCD resulting in excess protein-free plasma heme (PFPH) that triggers a lethal form of acute lung injury (ALI) in mice. In this study, we tested the hypothesis that toll-like receptor 4 (TLR4) mediates heme-induced ALI. Wild-type and two TLR4 mutant strains (B6.B10ScN-Tlr4lps-del/JthJ and C3H/HeJ) were intravenously injected with a dose range of ferric heme (0–210 micromoles/kg) and respiratory function monitored using a pulse oximeter. Excess PFPH was associated with reductions in oxygen saturation (SpO2) and breath rate in the wild-type mice but not in the TLR4 variants. Lungs of heme-treated wild-type mice were congested, edematous, hemorrhagic, and had thickened alveolar walls, while no histological abnormalities were found in the TLR4 variants. All heme-treated wild-type mice succumbed within 2 hours, while all TLR4 variants survived. Transgenic mice expressing exclusively human sickle hemoglobin (SS) were intravenously injected with a small molecule TLR4 inhibitor (resatorvid/TAK-242), or a lipid vehicle prior to induction of lung injury with heme (35 micromoles/kg). TAK-242 preserved lung function in the majority of SS mice that failed to scavenge excess PFPH, while both SpO2 and breath rate deteriorated in vehicle treated mice. The unique response to heme by TAK-242 and vehicle-treated SS mice was supported by histological analysis and survival (TAK-242; 76.9% vehicle; 23.5%, n=13–17; log-rank survival test, p