Elevated Levels of Hepatokine Angiopoietin-like 3 Correlate Paradoxically with Hypocholesterolemia and Hemolysis in Sickle Cell Anemia

Sickle cell disease (SCD) is characterized by chronic hemolytic anemia and vaso-occlusion. Hypocholesterolemia has been long recognized in SCD patients, and while it has been postulated that it is secondary to increased erythropoiesis, these mechanisms are not fully understood. Despite their name, angiopoietin-like proteins (ANGPTLs) are family of regulators of angiogenesis unable to bind to receptors classically targeted by angiopoetins. ANGPTL3 and ANGPTL4 are also inhibitors of lipoprotein lipase (LPL), and have become pharmacological targets in the treatment of dyslipidemia. We hypothesized that ANGPTL levels would be abnormal in SCD and could contribute to the understanding of lipid dysregulation. We aimed to determine, compare and correlate the circulating concentrations of ANGPTL3 and ANGPTL4 with hemolysis, lipid metabolism and endothelial dysfunction biomarkers in patients with sickle cell anemia (HbSS), hemoglobin SC disease (HbSC), and healthy individuals (HbAA). Thirty-six HbSS patients (age range 18-55, 21 men), 19 HbSC patients (HbSC) (age range 29-68, 8 men), and 31 HbAA controls (age range 18-66, 23 men) were enrolled in this study. Exclusion criteria were pregnancy, history of blood transfusion or sickle cell pain crisis in the past 3 months. Peripheral blood samples were collected for complete blood counts, biochemistry tests (lactate dehydrogenase [LDH], bilirubin, cholesterol and low and high density fractions, oxidized LDL [oxLDL], hemopexin), and determination of ANGPTL3, ANGPTL4, and soluble vascular cell adhesion molecule-1 (sVCAM-1) levels as marker of endothelial dysfunction. Differences in the intensity of hemolytic anemia between the two groups of SCD were confirmed, as well as decrease in total cholesterol (AA 176±32, SC 141±28 and SS 124±33mg/dL, p<0.0001), HDL (AA 46±11, SC 45±9, SS 37±11mg/dL, p<0.001) and LDL (AA 97±30, SC 76±34 or SS 63±27mg/dL, p<0.0001), as expected. Levels of both ANGPTL3 and ANGPTL4 were significantly higher in both SCD groups than in controls (ANGPTL3: AA 106±30, SC 147±37 and SS 155±58ng/mL, p<0.0001; ANGPTL4: AA 29±23ng/mL, SC 49±52ng/mL, SS 52±39ng/mL, p=0.04). While ANGPTL4 correlated only with LDH and HDL, ANGPTL3 correlated positively with reticulocyte count, LDH, total bilirubin, sVCAM-1, and the degree of LDL oxidation as estimated by oxLDL/LDL ratio, and negatively with hemoglobin, hemopexin, total cholesterol, and LDL levels. Our findings are surprising because the increase in AGPTLs should enhance inhibition of lipoprotein lipase, which is expected to cause hypercholesterolemia. It is therefore possible that hemolytic stimulus is able to override a feedback controlled by the liver between the amount of circulating lipoproteins and the need for LPL inhibition. This may also at least partially support the assumption that the plasma cholesterol pool is consumed for the synthesis of new red blood cells, so inhibition of LPL in this setting could be a compensatory mechanism. Nevertheless, the lack of significant differences between HbSS and HbSC suggests that erythropoietic drive is not the only determining factor. ANGPTL3 has been reported to be elevated in atherosclerosis and in rheumatic disease, so low grade chronic inflammation affecting the endothelium, along with a tendency towards more LDL oxidation could explain the positive correlation between sVCAM-1 and ANGPTL3. It is most remarkable that only ANGPTL3 correlated with hemolysis. ANGPTL3 is exclusively produced by the liver, while ANGPTL4 can be synthesized by adipose tissue, muscle, kidney, heart and thyroid, suggesting the stimulus for its overproduction generated by hemolysis is mostly sensed by hepatocytes. Considering the negative correlation with hemopexin, one possibility is that heme-hemopexin complexes taken up by liver cells and subsequent heme oxygenase induction may be involved in upregulation of ANGPTL3. Our data reveal that the production of lipoprotein lipase inhibitors is dysregulated in SCD-associated hypocholesterolemia, with a paradoxical increase in ANGPTLs which is most likely due to a liver response to hemolysis. Further studies should determine whether this mechanism is compensatory or driven by excessive heme production.