Endothelial differentiation of bone marrow mesenchyme stem cells applicable to hypoxia and increased migration through Akt and NFκB signals

Abstract

Background: Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) are used to repair hypoxic or ischemic tissue. However, the underlining mechanism of resistance in the hypoxic microenvironment and the efficacy of migration to the injured tissue are still unknown. The current study aims to understand the hypoxia resistance and migration ability of MSCs during differentiation toward endothelial lineages by biochemical and mechanical stimuli.

Method: MSCs were harvested from the bone marrow of 6-8-week-old Sprague-Dawley rats. The endothelial growth medium (EGM) was added to MSCs for 3 days to initiate endothelial differentiation. Laminar shear stress was used as the fluid mechanical stimulation.

Results: Application of EGM facilitated the early endothelial lineage cells (eELCs) to express EPC markers. When treating the hypoxic mimetic desferrioxamine, both MSCs and eELCs showed resistance to hypoxia as compared with the occurrence of apoptosis in rat fibroblasts. The eELCs under hypoxia increased the wound closure and C-X-C chemokine receptor type 4 (CXCR4) gene expression. Although the shear stress promoted eELC maturation and aligned cells parallel to the flow direction, their migration ability was not superior to that of eELCs either under normoxia or hypoxia. The eELCs showed higher protein expressions of CXCR4, phosphorylated Akt (pAkt), and endogenous NFκB and IκBα than MSCs under both normoxia and hypoxia conditions. The potential migratory signals were discovered by inhibiting either Akt or NFκB using specific inhibitors and revealed decreases of wound closure and transmigration ability in eELCs.

Conclusion: The Akt and NFκB pathways are important to regulate the early endothelial differentiation and its migratory ability under a hypoxic microenvironment.

Keywords: Akt; Endothelial differentiation; Hypoxia; Migration; Nuclear factor-κB; Stem cell.

Fig. 1

Surface CD markers were characterized by flow cytometry in fibroblasts (F), bone marrow mesenchymal stem cells (M), and early endothelial lineage cells (E) (a). Fibroblasts, but not mesenchymal stem cells (MSC) or early endothelial lineage cells (eELC), showed apoptotic morphology in phase images after treating with desferrioxamine (DFO) for 48 hr (b). The DFO-increased apoptotic (Annexin V(+)/PI(−)) and dead (PI(+)) somatic fibroblasts were quantified by flow cytometry (c) and confirmed by the protein expression of cleaved PARP (d). Scale bar: 200 μm. *Significant from the same cell under regular culture conditions, p < 0.05. PARP poly-ADP ribose polymerase, PI propidium iodide

Fig. 2

DFO treatment enhanced gene expression of Flk and vWF (a). eELCs showed higher wound closure ability (%) than MSCs in normoxia (DFO = 0), and hypoxia (DFO) further increased the closure performance in a dose-dependent manner (b). CXCR4 gene expression was increased after treating eELCs with DFO for 24 hr (c). Scale bar: 200 μm. *Significant from the same cell under regular culture condition (without DFO), p < 0.05; #significant from MSCs under the same treatment, p < 0.05. DFO desferrioxamine, MSC mesenchymal stem cells, eELC early endothelial lineage cells, vWF von Willebrand factor, SDF-1 stromal cell-derived factor-1, CXCR4 C-X-C chemokine receptor type 4

Fig. 3

Mature endothelial characteristics were observed after the eELCs were subjected to laminar shear stress (LSS) for 24 hr. The PECAM-1 gene (a) and cellular alignment in responding to the flow direction (b) were increased under LSS. However, the Boydon chamber assay demonstrated decreases of cell transmigration after LSS stimulation (c). Scale bar: (b) 200 μm; (c) 400 μm. #Significant from static eELC under hypoxia (DFO, 50 μM), p < 0.05. DFO desferrioxamine, MSC mesenchymal stem cells, eELC early endothelial lineage cells

Fig. 4

CXCR4 protein expression was increased in eELCs under both normoxia (without DFO) and hypoxia (with DFO) conditions (a). Phosphorylated Akt (pAkt) was increased in MSCs under DFO treatment (b). eELCs showed higher protein expressions of pAkt, NFκB p65, and IκBα than MSCs regardless of DFO treatment. *Significant from MSCs under regular culture condition, p < 0.05. DFO desferrioxamine, MSC mesenchymal stem cells, eELC early endothelial lineage cells, CXCR4 C-X-C chemokine receptor type 4

Fig. 5

Inhibition of Akt and NFκB were administrated by applying LY294002 (LY) and PDTC, respectively. CXCR4 expression in eELCs was inhibited by both LY and PDTC (a). The nuclear translocation of NFκB was inhibited by PDTC treatment under hypoxia and was totally abolished when treating with LY in both normoxia and hypoxia conditions (b). The increase of wound closure in eELCs under hypoxia (DFO) was inhibited by LY and almost abolished by PDTC treatment (c). The increase of transmigration ability in hypoxic eELCs was also inhibited by either LY or PDTC inhibitor (d). Scale bar: (c) 200 μm; (d) 400 μm. *Significant from eELCs under regular culture condition (without DFO), p < 0.05; #significant from eELCs under hypoxia (DFO, 50 μM), p < 0.05. AMD AMD3100, CXCR4 C-X-C chemokine receptor type 4, DFO desferrioxamine, PARP poly-ADP ribose polymerase, PDTC pyrrolidinedithiocarbamate, Ct control, Veh vehicle