Abstract

Subjects with sickle cell disease (SCD) have a chronic baseline inflammation and higher incidence of lung disease – airway hyper-reactivity predominates in children and restrictive lung disease in adults. The degree of inflammation, reflected in leukocytosis and leukocyte activation, correlates with higher mortality (Platt, NEJM, 1994). Stuart and colleagues have shown high plasma and urinary leukotriene (LT)-B4 at steady state, and even higher levels during acute sickle events and acute chest syndrome (J Lab Clin Med 2002). DeBaun and colleagues have shown high levels of urinary LT-E4 in this patient population (Am J Hematol, 2008). LT-B4 is one of the most potent activator of neutrophils, while cysteinyl leukotrienes (cys-LT), LT-C4, LT-D4 and LT-E4, promote bronchial smooth muscle constriction and asthma. LT are produced from arachidonic acid through a series of metabolic steps, with two critical steps catalyzed by five-lipoxygenase (5LO) and 5-lipoxygenase activating protein (FLAP). We have previously shown that that placenta growth factor (PLGF) is produced at high levels from the hyperplastic erythroid compartment in SCD and thalassemia and induces a strong proinflammatory cytochemokine response from monocytes (Perelman et al, Blood 2003; Selvaraj et al, Blood 2003). PLGF levels were high in subjects with SCD, and associated with activated mononuclear cells with augmented expression of pro-inflammatory cytochemokines (Perelman et al, Blood 2003). We now show PLGF induces FLAP expression. FLAP mRNA was significantly higher in peripheral blood mononuclear cells (PBM) of subjects with SCD at steady state, compared to healthy controls. PLGF activated PBM to increase FLAP expression and LT-B4 formation. It also increased Cys-LT production from PBM and the THP-1 monocytic cell line. PLGF-mediated increased FLAP expression was attenuated by PI-3 kinase inhibitor and PTEN; and by NADPH-oxidase inhibitor and p47phox siRNA in THP-1 cells. An in silico analysis of the FLAP promoter showed presence of hypoxia response elements, CEBP and NFκB sites. We found that FLAP induction by PLGF was mediated via HIF-1α. PLGF induced HIF-1α mRNA expression. Silencing with HIF-1α siRNA attenuated PLGF mediated FLAP expression, while over expression of HIF-1α had the converse effect. PLGF augmented FLAP-promoter luciferase activity by several folds, and this effect was abrogated upon mutation of hypoxia-response element (HRE), but not the NF-κB binding site in the FLAP proximal promoter. These studies indicate the role of HIF-1α motif in inducing FLAP expression, which was confirmed by electrophoretic mobility shift assays. Chromatin immunoprecipitation (ChIP) analysis further confirmed the role of HIF-1a in regulating PLGF-mediated FLAP expression. Our studies identify, for the first time, HIF-1α as a hypoxia-independent target of PLGF, which then upregulates FLAP and increases LT formation. Our studies define a mechanism of increased LT in SCD. The intrinsic elevated levels of PLGF seen in SCD could explain the increased leukotriene levels observed in patients with SCD, thereby contributing to the baseline inflammation and pulmonary pathology present in this disease. LT inhibitors are FDA approved in asthma, and could be used as a new modality to ameliorate inflammation and lung complications in SCD.

Disclosures: No relevant conflicts of interest to declare.

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