Up-regulation of miR-210 by vascular endothelial growth factor in ex vivo expanded CD34+ cells enhances cell-mediated angiogenesis

Abstract

Ex vivo culture has been proposed as a means to augment and repair autologous cells in patients with chronic diseases, but the mechanisms governing improvement in cell function are not well understood. Although microRNAs (miRs) are increasingly appreciated as key regulators of cellular function, a role for these factors in CD34+ cell-mediated angiogenesis has not been elucidated. Vascular endothelial growth factor (VEGF) was previously shown to induce expression of certain miRs associated with angiogenesis in endothelial cells and promote survival and number of vascular colony forming units of haematopoietic stem cells (HSCs). We sought to evaluate the role of VEGF in expansion and angiogenic function of CD34+ cells and to identify specific miRs associated with angiogenic properties of expanded cells. Umbilical cord blood CD34+ cells were effectively expanded (18- to 22-fold) in culture medium containing stem cell factor (SCF), Flt-3 ligand (Flt-3), thrombopoietin (TPO) and interleukin-6 (IL-6) with (postEX/+VEGF) and without VEGF (postEX/noVEGF). Tube formation in matrigel assay and tissue perfusion/capillary density in mice ischaemic hindlimb were significantly improved by postEX/+VEGF cells compared with fresh CD34+ and postEX/noVEGF cells. MiR-210 expression was significantly up-regulated in postEX/+VEGF cells. MiR-210 inhibitor abrogated and 210 mimic recapitulated the pro-angiogenic effects by treatment of postEX/+VEGF and postEX/noVEGF cells respectively. Collectively, these observations highlight a critical role for VEGF in enhancing the angiogenic property of expanded cells, and identify miR-210 as a potential therapeutic target to enhance CD34+ stem cell function for the treatment of ischaemic vascular disease.

Fig 1

Fold increase in cell number after expansion with and without VEGF. Data presented as fold increase in cell number compared to preEX cells (adjusted to 1); P = 0.001.

Fig 2

Representative flow cytometry data of preEX (fresh CD34+), postEX/+ VEGF, and postEX/noVEGF cells. Comparison of flow cytometry data showed decrease in CD34, CD133, and c-Kit in post-expansion cells, but no indication of differentiation into monocytic or lymphocytic lineages (CD11b, CD3 or CD19). There was low expression of CXCR4 and VEGFR2 in preEX cells and further decrease in both expansion groups. P <0.01 for CD34, CD133, c-Kit, CD11b, CD14 and CD45, P < 0.05 for CD3, CD19, and VEGFR2, and P > 0.05 for CXCR4. * and ** represents post-hoc P values of <0.05 and <0.01, respectively.

Fig 3

Enhanced HUVEC in vitro tube formation by CD34+ cells expanded in VEGF-enriched medium. Pre-or post- expanded CD34+ cells were washed and incubated with HUVEC in 96-well plate coated with matrigel. Upper panel shows representative images of different groups (×40; scale bar = 100 μm). Lower panel represents percentage increase in number of branch points compared to HUVEC alone. Data show significant increase in tube formation in postEX/+VEGF group (P < 0.0001). *, *** represents post-hoc P values of <0.05, and <0.001 respectively.

Fig 4

CD34+ cells expanded in VEGF-enriched medium promote tissue perfusion and capillary density in mice ischaemic hindlimb. PBS or 2.5 × 10⁴ cells were injected into ischaemic limb. Graphs show significant increase in tissue perfusion (A; P < 0.0001) and capillary density (B; P < 0.0001) in postEX/+VEGF group. Representative images of tissue perfusion and capillary density in calf muscle assessed by CD31staining (×200; scale bar = 200 μm) are shown in the right upper and lower panels, respectively. *** represents post-hoc P value of <0.001.

Fig 5

Expression profile of various miRs associated with angiogenesis. Representative graph of quantitative PCR data for selected miRs in PreEX (fresh CD34+), PostEX/+VEGF, and PostEX/noVEGF cell groups (n = 3 donors). P <0.01 for miR-17, 18a, 19b-1, 20a, 92a-1 and 210, P < 0.05 for miR-19a, 126 and 296 and P > 0.05 for miR-130. * and ** represents post-hoc P values of <0.05 and <0.01 respectively. RNU48 used as reference gene.

Fig 6

MiR-210 is essential for the pro-angiogenic effects of expanded CD34+ cells on HUVEC in vitro tube formation assay. Post-expansion cells were transfected with miR-210 inhibitor or mimic, washed and incubated with HUVEC in matrigel coated 96-well plate. MiR-210 inhibitor resulted in significant decrease in number of branch points by cells expanded with or without VEGF (A: P < 0.0001 and B: P < 0.0001), whereas 210-mimic enhanced tube formation by postEX/noVEGF cells. Upper panel shows representative images of different groups (×40; scale bar = 100 μm). Lower panel represents percentage increase in number of branch points compared to HUVEC alone. **, *** represents post-hoc P values of <0.01, and <0.001 respectively.

Fig 7

MiR-210 promotes tissue perfusion and capillary density by expanded CD34+ cells in mice ischaemic hindlimb. Expanded cells were transfected for 48 hrs with either miR-210 inhibitor or mimic on day 5. After washing, 2.5 × 10⁴ cells were injected into ischaemic limb. MiR-210 inhibition abrogated tissue re-perfusion (A, upper panel) and capillary density (A, lower panel) in postEX/+VEGF group, whereas mimic significantly improved tissue re-perfusion (B, upper panel) and capillary density (B, lower panel) in postEX/noVEGF. Representative images of tissue perfusion and capillary density in calf muscle assessed by CD31staining (×200; scale bar = 200 μm) are shown in the right upper and lower panels, respectively.