Endothelial Progenitor Cells in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH), a very rare disease, is characterized by hemolytic anemia, bone marrow failure, and venous thromboembolism. The disease is caused by somatic mutation of the X-linked gene PIG-A encoding a key enzyme responsible for the biosynthesis of the GPI-anchored proteins (GPI-APs), affecting hematopoietic stem cells (HSC). Venous thromboembolisms occur in unusual sites and the mechanism remains to be elucidated. A previous study showed an increase of endothelial cell activation markers in PNH patients (Helley et al., Haematologica 2010;95:574-581), however endothelial cells remain poorly studied in this disease. In polycythemia vera, associating JAK-2 mutation in HSC and thrombosis occurrence, Sozer and coworkers showed that liver endothelial cells bore the same JAK-2 mutation than the HSC (Sozer et al, Blood Cells, Molecules and Diseases, 2009;43:304-312). This suggests the existence of a common precursor between endothelial progenitor cells and HSC, the hemangioblast.

In PNH, PIG-A gene mutations might be present in endothelial cells. To test this hypothesis we have used endothelial progenitor cells circulating in blood, also called endothelial-colony forming cells (ECFC), as witnesses of endothelial cells status.

Peripheral blood mononuclear cells (PBMC) and neutrophiles (PMNL), from patients with classical PNH, were fractionated. PMNL were used to isolate DNA, while PBMC were plated on gelatine-coated plates to be cultured until appearance of ECFC colonies (usually 15–20 days), and then cultured to perform: (i) expression of GPI-anchored protein by flow cytometry; (ii) sequencing of the exons and flanking regions of the PIG-A gene from PMNL and ECFC.

Twelve PNH patients were enrolled (8 women, 4 men). Among them, 3 have venous thrombotic events (2 Budd-Chiari syndromes and one mesenteric veins thrombosis). ECFC colonies were obtained from 6 out of the 12 patients, a normal output due to the small number of ECFC in whole blood. Mutations identified by sequencing the PIG-A gene of PMNLs from these 6 patients are shown in Table 1, as well as clinical characteristics.

In 5 patients only (two with previous Budd-Chiari syndrome), we obtained sufficient amounts of ECFC to isolate DNA. None of the ECFC colonies bore the identified mutations (one exhibited a very faint peak of mutated nucleotide on electrophoregram but mRNA transcripts analysis from this ECFC colony revealed traces amounts of CD45 and CD11b mRNA, suggesting a contamination by leukocytes).

This is the first study reporting the absence of PIG-A gene mutation in ECFC from PNH patients, indicating that the mature endothelial cells are not bearing the PIG-A mutation. These cells are thus involved in thrombosis probably by free hemoglobin and thrombin cell activation, rather than by direct damage caused by PIG-A gene mutation. These results also indicate that, in PNH, the PIG-A gene mutation occurs most probably after the common precursor between endothelial progenitor cells and HSC.

Peffault de Latour:Alexion: Consultancy, Research Funding. Fischer:Alexion: Consultancy. Helley:Alexion: Consultancy.