Assessment of the angiogenic proteins placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1) in patients with hereditary spherocytosis

Abstract Background: Hereditary spherocytosis (HS) is a rare genetic disorder characterized primarily by hemolytic anemia, jaundice, splenomegaly and spherocytosis. Because of abnormalities in the coding of red blood cell (RBC) membrane skeleton protein genes, the respective protein synthesis is altered, leading to partial loss of the RBC membrane's function and to RBC becoming spherical. Placental Growth Factor (PlGF) is a member of the Vascular Endothelial Growth Factor (VEGF) family and is associated with inflammation and with pathologic angiogenesis. PlGF is released from marrow erythroid cells. Soluble fms-like Tyrosine Kinase 1 (sFlt-1) is a soluble form of VEGF Receptor-1 (VEGF-R1), which binds to and sequesters circulating free VEGF and free PlGF. PlGF and VEGFR-1 are key molecules in regulating the angiogenic switch during pathological conditions maintaining the proangiogenic and antiangiogenic balance. Perturbations of this balance, which is expressed as sFLT1/PlGF ratio, can be associated with various degrees of hemolysis and endothelial dysfunction, which are common pathologies in patients with hemoglobinopathies and which can induce pulmonary hypertension (PHT). In this context, we aimed to investigate if circulating levels of PlGF and sFLT-1 proteins are affected in patients with HS and to explore potential link with genotype-phenotype expression, erythroid marrow activity and iron metabolism. Patients and Methods: Sixty-one patients with HS (M/F 33/28) of median age: 11.8 years (2 months-62.9 years) were included in the study. A positive Eosin-Maleimide-test and/ or genetic analysis via targeted Next-Generation-Sequencing (MiSeq, Ilumina) confirmed the diagnosis. Among them, 15/61 had undergone splenectomy. Along with standard laboratory parameters, we measured the sera concentrations of PlGF, sFLT-1, Erythropoietin (Epo), soluble Transferrin Receptors, (sTfR), Hepcidin-25 and Growth Differentiation Factor-15 (GDF-15) by means of immunoenzymatic assays. We also calculated the sFLT1/PlGF ratio and its log expression, as well as the Reticulocyte Production Index (RPI). Results: Genetic analysis revealed a Pathogenic/Likely Pathogenic variant in 80.3% of the patients. ANK1 variants accounted for 27.9% of the patients, SPTB for 37.7% and SLC4A1 for 14.8%. We found that: a) in patients with HS the PlGF levels were lower and sFLT-1 levels were higher compared to the reported levels for apparently healthy individuals (mean/SD: 10.2±6.3pg/mL vs 18±10.5pg/mL, p<0.01, and 442±85.1pg/mL vs 385±49.4pg/Ml, p<0.01, respectively). This results in a significantly higher calculated ratio of the two proteins logsFLT-1/PlGF 1.74±0.34 (ranged from 1.23-2.72) in these patients; b) the splenectomized patients with HS have lower PlGF levels compared to non-splenectomized patients 5.4±2.9pg/mL vs 11.5±6.4pg/mL, respectively (p<0.001), while sFLT-1 levels do not differ significantly between splenectomized/non-splenectomized patients, p>0.116; c) PlGF levels in patients with HS correlated significantly positive with RDW r=0.460 p<0.001, RPI r=0.385 p=0.002, Epo r=0.410 p=0.001, sTfR r=0.435 p<0.001, GDF-15 r=0.380 p=0.028; d) No correlations were found between sFLT-1 levels and the other parameters measured and e) Stratified analysis was performed according to splenectomy status and no significant difference was found between genotype and angiogenic markers (PIGF, Sflt-1, sFLT1/PlGF ratio). Conclusions: To our knowledge this is the first study on circulating PlGF and sFLT-1 levels in patients with HS. We demonstrated that, even though levels of both proteins are altered compared to reference values, patients with HS maintain overall the proangiogenic and antiangiogenic balance, with a slightly shift towards the antiangiogenic condition. Further work is required to evaluate the consequences of these alterations in the pathogenesis of HS and the development of complications like thromboembolism and pulmonary hypertension.