ALBERT

Description: Abstract 1537 Poster Board I-560 Sickle cell disease (SCD) is characterized by intravascular hemolysis generating cell-free hemoglobin at concentrations that exceed the scavenging capabilities of heme-binding plasma proteins. Heme is a major source of oxidative stress, which is widely known to increase vascular permeability and cause tissue edema. Barrier disruptive effects of oxidative stress can however be counterbalanced by adaptive antioxidant defenses. Hitherto, the identity of specific antioxidant enzymes and cognate mechanisms protecting individual organs from oxidative stress in SCD remains poorly defined. In this study, microarray analysis was performed using non-toxic concentrations of hemin to analyze mechanisms of antioxidant defense by the endothelium. Multiple candidate antioxidant enzymes were identified each differentially elevated in several major organs in anemic SCD mice compared to non-anemic heterozygote and hemizygote littermates. Remarkably, none of the antioxidants was elevated in the brain of sickle mice. Moreover, the antioxidant phenotype in the kidney, spleen and liver of sickle mice were predominantly acute while the sickle lung was characterized by a predominantly chronic antioxidant phenotype. This latter finding was confirmed in SCD patients with chronic lung disease. Antioxidant enzyme activity was significantly higher in the lungs of adult sickle mice aged 3-6 months than in younger mice aged 4-6 weeks (p=0.004). However, this enhanced antioxidant activity declined significantly in middle-age mice 11-13 months old (p=0.005). Vascular permeability assessed by extravsasation of Evans blue dye was normal in the brain of sickle mice of all ages in agreement with our data indicating absence of oxidative stress in this organ. On the contrary, vascular permeability in the lung, kidney and heart of adult sickle mice was abnormally high. This abnormality deteriorated significantly exclusively in the lung (p= 0.04) but not in the heart or kidney in middle-age mice. Increased lung permeability in middle-age sickle mice was confirmed by overt tissue edema determined by lung wet/dry weight ratios. Our study has shown for the first time that the antioxidant response to the systemic chronic hemolysis of SCD is organ-specific. Furthermore, we have identified decline of antioxidant reserve concomitant with tissue edema as potential age-dependant risk factors for fatal lung complications in SCD. Finally, our data provide a framework to develop targeted antioxidant therapies to preserve organ function in SCD. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1538 Poster Board I-561 Circulating plasma hemoglobin contributes to major vasculopathies including pulmonary hypertension in patients who have sickle cell disease (SCD). There is an emerging concept that such vasculopathies are relatively mild because of activation of several cytoprotective pathways in SCD. The biochemical profile of plasma and the transcriptome of peripheral blood cells in patients who have SCD offer indirect support for this concept. Indeed, heme oxygenase-1 (HO-1), an acute phase enzyme that degrades heme into intermediates and byproducts with vasculoprotective properties is markedly elevated in mononuclear leukocytes of patients who have SCD. Nonetheless, the scope of the cytoprotective mechanisms of the lung and other organs impacted directly by sickle vasculopathies remain poorly appreciated. We previously identified an array of cytoprotective enzymes in lung endothelial cells chronically exposed to non-toxic concentrations of hemin in vitro. In this study, we examined the expression of NAD(P)H oxidase and candidate cytoprotective enzymes in two models of transgenic mice with SCD, and examined HO-1 expression in sickle chronic lung disease. Although NAD(P)H oxidase catalyzes reactive oxygen species generation by heme and is responsible for increased adhesion of leukocytes to the endothelium in SCD mice, there was no elevation of any of its subunits (gp91Phox and p22Phox, p47Phox, p67Phox and p40 Phox) in sickle mice lungs compared to hemizygote control mice lungs. Quantitative RT-PCR analysis revealed unexpectedly no difference in HO-1 mRNA level in sickle and non-sickle control lungs. On the contrary, analysis of the same tissues showed significantly higher NAD(P)H quinone oxidoreductase-1 (NQO1) mRNA level in both Berkeley and Townes knock-in sickle mice compared to controls (p