Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus

Abstract

Background: Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment.

Methods: Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed.

Results: In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro.

Conclusion: EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Conflict of interest statement

Competing Interests: The authors confirm the role of Pieter A. Doevendans as a PLOS ONE Editorial Board Member and would like to state that this does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. EPC levels and mobilization in diabetes.

Diabetic animals have reduced levels of Sca1+Flk-1+ EPC in peripheral blood under steady-state (baseline) conditions compared to controls. After daily injection with mobilizing cytokines G-CSF and SCF from day 0 to 4, a significant mobilization of EPC was observed. EPC levels returned to baseline after cessation of cytokine injection. In contrast, diabetic animals showed a diminished mobilization response. P<0.001 for interaction of time and diabetes in 2-way ANOVA. #P<0.05 compared to controls, ## P<0.01 compared to controls, *P<0.05 compared to baseline, ** P<0.01 compared to baseline

Figure 2. Diabetic mice do not recover after a 5-FU challenge.

After 5-FU injection, a reduction in both WBC (A) and platelets (B) was observed in control and diabetic animals, which was maximal at 7 days after injection. Control animals fully recovered with restoration of WBC levels and platelets, displaying a typical rebound thrombocytosis after recovery. In contrast, diabetic animals died between day 10 and 14 with severe peripheral blood leukopenia and thrombopenia.

Figure 3. Progenitor cell support by bone marrow stroma ex vivo or endothelial cells in vitro.

Plastic-adherent bone marrow stromal cells were grown to confluence from isolated bone marrow cell suspensions. Stromal layers from control (n = 10) and diabetic (n = 8) mice had a comparable morphology and growth pattern. Stromal layers were then used as feeder layer for human CD34+ HPC. (A)The number of CFU derived from 10-day co-cultures was significantly lower for diabetic stroma than for control stroma. Human CD34+ HPC were co-cultured with E4Orf1-transfected HUVEC for six weeks. At several time points, hematopoietic colonies (CFU) were counted. (B) Fewer CFU were obtained from CD34+ HPC co-cultured on endothelium in the presence of hyperglycemia than under control conditions. * P<0.05.