Dysfunction of Bone Marrow Endothelial Progenitor Cells from Subjects with Poor Graft Function Following Allogeneic Hematopoietic Stem Cell Transplantation Can be Improved By Atorvastatin

Background

Poor graft function (PGF) is a serious complication post allo-HSCT. The definition is a hypo- or aplastic bone marrow with 2 or 3 of the following: (1) neutrophils ≤0.5×10E+9/L; (2) platelets ≤20×10E+9/L; and/or (3) hemoglobin concentration ≤70 g/L for at least 3 consecutive days after day +28 post-HSCT. The mechanisms of PGF remain poorly understood. Murine studies suggest that endothelial progenitor cells (EPCs) are preferential supporting cells for hematopoietic stem cells (HSCs) in the bone marrow (BM) microenvironment. In addition, our previous prospective, nested case-control study found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PGF after allo-HSCT. However, little is known about the functional role of BM EPCs and how to improve impaired BM EPCs in PGF.

Atorvastatin is widely used in the treatment of dyslipidemia and associated vascular abnormalities. Furthermore, atorvastatin has been reported to improve the mobilization and function of circulating EPCs in a number of diseases, such as heart disease, diabetes and others. Nevertheless, no previous studies have focused on the roles of atorvastatin on bone marrow-derived EPCs in subjects with poor graft function following allo-HSCT.

Aims

To evaluate the function of BM EPCs in subjects with PGF. Moreover, to investigate the effect of atorvastatin on the number and function of cultivated BM EPCs derived from subjects with PGF and its underling molecular mechanisms.

Methods

Three cohorts were included: subjects with PGF (N=23), subjects with good graft function (N=23), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=23). Atorvastatin (0.5nM,50nM,500nM) was administrated to the 5 day cultivated BM EPCs from subjects with PGF until tested on day 7. The number and functions of CD34(+)/CD133(+)/KDR(+)EPCs were evaluated by flow cytometry, cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, tube formation test and apoptosis. Reactive Oxygen Species (ROS) level was evaluated by flow cytometry. Cell proliferation was determined by cell counting kit-8 assay. Protein expression for ERK,JNK, p38, Akt was measured by flow cytometry and western blots. EPCs-CD34+ co-culture system and Colony-forming unit (CFU) assays were used to evaluate the supporting effect that atorvastatin exposure on hematopoietic progenitors 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 proliferation, migration, angiogenesis, and higher levels of reactive oxygen species and apoptosis, were revealed in subjects with PGF. Activation of p38 and its downstream transcription factor cAMP-responsive element-binding protein (CREB) were detected in BM EPCs from subjects with PGF. Furthermore, the number and function of BM EPCs derived from subjects with PGF were enhanced by atorvastatin treatment in vitro through down-regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. The colony-forming unit plating efficiency in CD34+ BM cells was also improved by atorvastatin when co-cultured with BM EPCs from subjects with PGF. Atorvastatin demonstrated similar improvement effects on the number and function of bone marrow EPCs as ROS inhibitor and p38 inhibitor from subjects with PGF.

Conclusion

In summary, dysfunctional BM EPCs were observed in subjects with PGF. Atorvastatin treatment in vitro quantitatively and functionally improved BM EPCs derived from subjects with PGF through down-regulation of the p38 MAPK pathway. These data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in subjects with PGF post-allotransplant.