Dysfunction of Bone Marrow Endothelial Progenitor Cells from Subjects with Prolonged Isolated Thrombocytopenia Following Allogeneic Hematopoietic Stem Cell Transplantation Can be Improved By N-Acetyl-L-Cysteine

Background

Prolonged isolated thrombocytopenia (PT) is a frequent complication with low platelet count or dependence on platelet transfusions for more than 60 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PT increased risk of bleeding and had poor overall survival. However, the exact mechanisms underlying PT remain unclear. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) not only support hematopoietic stem cells (HSCs) but also play an important role in differentiation and proliferation of megakaryocytes (MKs), thereby enhancing platelet production in BM microenvironment. In addition, our previous study found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PT after allo-HSCT. However, little is known about the functional role of BM EPCs and BM HSCs in patients with PT after allo-HSCT. In addition, in vivo and in vitro studies reported that the anti-oxidant -N-acetyl-L-cysteine (NAC) repaired the endothelial functions and enhanced engraftment of HSCs through prevention of exogenous ROS accumulation.Nevertheless, whether in vitro treatment of NAC is able to improve the functions of BM EPCs from patients with PT has never been investigated.

Aims

To evaluate the function of BM EPCs and HSCs in patients with PT. Moreover, to investigate the effect of NAC on the number and function of the cultivated BM EPCs in patients with PT and its underlying molecular mechanisms.

Methods

Three cohorts were included: patients with PT (N=25), patients with good graft function (N=25), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=25). BM mononuclear cells were cultivated in fibronectin pre-coated plates with EGM-2-MV-SingleQuots and 10% fetal bovine serum at 37°C for 7 days and characterized as EPCs by CD34(+)/CD133(+)/KDR(+) using flow cytometry. The number and functions of EPCs were evaluated by flow cytometry, cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining,migration, tube formation test and apoptosis. ROS level was evaluated by flow cytometry. Protein expression for ERK,JNK, p38, Akt was measured by flow cytometry and western blots. 1mM NAC was administrated to cultivated BM EPCs from patients with PT 24 hours before tested on day 7. EPCs-MKs co-culture system and Colony-forming unit (CFU)-MKs assays were used to evaluate the supporting effect that NAC exposure on MKs in vitro.

Results

Human BM EPCs was characterized by the spindle shape and expression of CD34, CD309 and CD133 at day 7 of cultivation. Dysfunctional BM EPCs, which were characterized by impaired migration, angiogenesis, and higher levels of reactive oxygen species(ROS) and apoptosis, were revealed in patients with PT. Activation of p38 was detected in BM EPCs from patients with PT. Furthermore, there was no significantly difference in functions of CD34+ cells between patients with PT and GGF, except higher level of ROS and lower number of CFU-GEMM, suggesting an impaired process of megakaryocytopoiesis in patients with PT. Furthermore, the number and function of BM EPCs derived from patients with PT were enhanced by NAC treatment in vitro through down-regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. The platelet number and percentage and functions of MKs characterized by apoptosis, ploidy and CFU-MKs were further improved by NAC in BM MKs and EPCs co-culture system from patients with PT. NAC demonstrated similar improvement effect on the number and function of BM EPCs as p38 inhibitor from patients with PT.

Conclusion

The dysfunction of BM EPCs in patients with PT was improved by in vitro treatment of NAC through down-regulation of the p38 MAPK pathway, which further augment megakaryocytopoiesis and platelet production in patients with PT. These data suggest that NAC may serve as an effective therapeutic option to improve platelet production through repair of BM EPCs in patients with PT. Further prospective clinical trials will be conducted in the future.