Evaluation of the role of lipoproteins in sickle cell disease

Sickle cell disease (SCD) is the major group of hemoglobin disorders found in Brazil, resulting from the HBB Glu6Val mutation in homozygosis or compound heterozygosis with other mutations in the beta globin gene. SCD is characterized by variable degrees of chronic hemolysis, vaso-occlusion, and end-organ damage. Due to hemolysis, hemoglobin released in circulation gets oxidized and generates free heme, which triggers inflammation and oxidative stress. However, several proteins are proposed to neutralize heme, such as hemopexin, albumin, ?1-microglobulin, and lipoproteins. Lipoproteins have a very high affinity for heme and can potentially defend the human body against free heme, but heme can also oxidize lipoproteins, which can cause tissue damage. Hypolipoproteinemia is often associated with hemolytic diseases, and it is unclear whether the mechanism for this involves heme biology. Other mediators of lipoprotein production are angiopoietin-like proteins (ANGPTLs), since ANGPTL3 and ANGPTL4 are inhibitors of lipoprotein lipase that hydrolyzes lipoproteins, but ANGPTL levels in SCD are largely unknown. In our study, we investigated the production of lipoproteins and their interaction with hemolysis and heme in SCD. We studied three groups of individuals: controls, patients with hemoglobin SC disease, a mild form of SCD, and patients with sickle cell anemia (SCA), the most severe type of SCD. In addition, we used endothelial cell models (HUVECs), sickle cell animal models (Townes and Berkeley) and models of acute hemolysis induced by phenylhydrazine (PHZ) in hypercholesterolemic (LDLr-/-) and normolipidemic (C57BL/6) mice. We found that abnormally low levels of lipoproteins in patients with SCD correlate with markers of hemolysis, particularly with hemopexin, the main specific heme scavenger. In isolated human lipoprotein particles, hemopexin concentration is higher in high-density lipoprotein (HDL), while heme is more associated with low density lipoprotein (LDL), and patients with SCA have depletion of hemopexin bound to HDL. Patients with SCA have high concentrations of ANGPTLs 3 and 4, which is paradoxical in patients with hypocholesterolemia and may suggest a compensatory mechanism driven by the effect of hemolysis in the liver. In addition, C57BL/6 mice treated with PHZ showed that acute intravascular hemolysis was not able to induce hypocholesterolemia. Endothelial cell cultures treated with LDL and hemin present reduced IL-6 production, but elevated IL-8 induction, suggesting ambiguous lipoprotein effects on the heme-induced endothelial response. On one hand, C57BL/6 mice and transgenic models for SCA display no hypocholesterolemia and treatment with a fat-rich diet does not increase cholesterol levels, suggesting significant differences between murine and human lipid metabolism. On the other hand, LDLr-/- mice with higher LDL had less anemia and lower levels of circulating heme after induction of acute hemolysis by PHZ than normocholesterolemic mice (C57BL/6), although lipoprotein oxidation and inflammatory response were increased. Our data contribute to a better understanding of lipoprotein biology in SCD, which may be applicable to other hemolytic diseases, and support further investigations into the mechanisms involved in the pathogenesis of hypocholesterolemia in patients with chronic hemolys (AU)