Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus

Abstract

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor-1 (SDF-1) plays a key role in bone marrow (BM) c-kit(+) stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM-derived c-kit(+) cells and on their response to SDF-1. Acute hindlimb ischemia was induced in streptozotocin-treated DM and control mice; circulating c-kit(+) cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c-kit(+) cells as well as SDF-1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM-derived c-kit(+) cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF-1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF-1 ability to induce differentiation of c-kit(+) cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c-kit(+) cells from normoglycaemic mice failed to differentiate in response to SDF-1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c-kit(+) cell number following hindlimb ischemia and inhibits SDF-1-mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM-derived c-kit(+) cells.

Figure 1

Diabetes impairs blood flow recovery in response to hindlimb ischemia and c-kit⁺ cell number in the systemic circulation. (A) Laser Doppler perfusion imaging of normoglycaemic and DM mice before and at 14 and 21 days following acute hindlimb ischemia. Perfusion index was calculated by normalizing the colour intensity of the ischemic versus non ischemic limb in the same animal. Arrows indicate the ischemic limb. Note the impaired perfusion recovery in DM. (B) Average perfusion index evaluated by laser Doppler perfusion imaging and expressed as the ratio between ischemic and non-ischemic paw in normoglycaemic (black bars; n= 3–7) and DM (open bars; n= 3–12) mice. The response profile was significantly different between normal and DM mice (anovaP < 0.01). Post hoc analysis for pairwise comparisons between DM and control animals demonstrated significant differences in perfusion index at day 21 (P < 0.05) and day 28 (P < 0.05). Student’s t-test showed significant differences at 14, 21 and 28 days after ischemia (P < 0.05) (C) C-kit⁺ cells in the systemic circulation expressed as% of PB-MNCs. Prior to femoral artery dissection there were more c-kit⁺ cells in normoglycaemic (black bars; n= 12) than in DM (open bars; n= 11) mice (Student’s t-test; P < 0.05). Moreover, after acute ischemia the transient increase in c-kit⁺ cells was more pronounced and sustained in normoglycaemic (n= 8–14 at each time-point) than in DM (n= 8–14 at each time-point) mice (anovaP < 0.01); post hoc analysis for pairwise comparisons demonstrated a significant difference at day 7 (P < 0.05). PB c-kit⁺ cells were expressed as percentage of 5 × 10⁴ PB-MNCs as evaluated by flow cytometry. Prior to femoral artery dissection total PB-MNCs number was 2.85 × 10⁶± 1.27 × 10⁶ in normoglycaemic (n= 4) and 2.81 × 10⁶± 0.59 × 10⁶ in DM (n= 4) mice (P= n.s.); at different time-points after ischemia there continued to be no difference in PB-MNCs number between normoglycaemic and DM mice.

Figure 2

CD34⁺ and KDR⁺ cells in the bone marrow and peripheral blood of control and diabetic mice. (A) Percentages of BM-derived c-kit⁺, c-kit⁺/CD34⁺ and c-kit⁺/KDR⁺ cells purified for c-kit antigen by MACS (n= 3). (B) Histogram showing percentage of CD34⁺, KDR⁺ and CD34⁺/KDR⁺ in the BM of control (black bars; n= 9) and DM (open bars; n= 11) mice. (C) Histogram showing percentage of CD34⁺, KDR⁺ and CD34⁺/KDR⁺ in the PB of control (black bars; n= 10) and DM (open bars; n= 9) mice. Result of statistical analysis by anova and post hoc test is indicated by P-value above bars.

Figure 3

DM reduces BM-derived c-kit⁺ cell adhesion/differentiation into endothelial cell. BM-derived c-kit⁺ cell adhesion/differentiation into endothelial lineage was determined by Ac-LDL-DiI uptake. (A) SDF-1 enhanced c-kit⁺ cells adhesion/differentiation into endothelium when cells were obtained from normoglycaemic mice (black bars; n= 7, Student’s t-test P < 0.05) but had no effect when cells were obtained from DM mice (open bars; n= 7, Student’s t-test, P= n.s.). Inactive SDF-1 (SDF-1 B) failed to induce cell adhesion/differentiation in both experimental groups. The response to SDF-1 differed between normoglycaemic- and DM-derived cells (P-value for anova and post hoc test is shown in the figure above the bar graph). (B) C-kit⁺ cells from DM mice (open bars), expanded for 1 week in stem span medium supplemented with IL-3, IL-6, Flt3-L and SCF, and subsequently kept in differentiation medium for another week, augmented their adhesion/differentiation into endothelial cells in the presence of SDF-1 (n= 8, Student’s t-test P < 0.05). A similar response to SDF-1 was observed in c-kit⁺ cells from normoglycaemic mice (black bars) (n= 8, Student’s t-test P < 0.05). Interestingly, under these experimental conditions, the response to SDF-1 was similar between the two experimental groups (P-value [n.s.] for anova and post hoc test is shown). (C) C-kit⁺ cells from control (black bars; n= 4) and DM mice (open bars; n= 4) were expanded for 1 week in hyperglycaemic medium and then shifted to hyperglycaemic differentiation medium for another week (see ‘Materials and methods’). C-kit⁺ cells from normoglycaemic mice exposed to hyperglycaemia failed to enhance adhesion/differentiation towards the endothelial lineage in response to 100 ng/ml SDF-1 and this effect was comparable to that of cells from DM mice.

Figure 4

Diabetes impairs SDF-1-induced c-kit⁺ cell adhesion/differentiation into endothelium and AKT phosphorylation. (A) BM-derived c-kit⁺ cells were obtained from normoglycaemic mice and cultured in RPMI containing 5 mM glucose for 7 days. SDF-1 enhanced Ac-LDL-DiI⁺ cell number and this effect was abolished by the PI3K/AKT inhibitor LY294002 (n= 3 for each group). This effect was comparable to that of DM shown in Fig. 3A. Statistical significance was evaluated by anova and post hoc analysis; P-values are reported above the bar graph. (B, C) Western blot analysis shows that SDF-1 enhanced AKT phosphorylation in BM-derived mononuclear cells obtained from normoglycaemic mice (black bars in C); this effect was already evident at 1 min. and remained elevated up to 10 min. after the exposure to the chemokine (Student’s t-test P < 0.05). In contrast, SDF-1 had no effect on AKT phosphorylation in BM-derived mononuclear cells obtained from DM mice (open bars in C). Significance was evaluated also by anova and post hoc analysis; n= 4 for each group, P-values are reported above the bar graph. (D, E) SDF-1 effect on AKT phosphorylation in c-kit⁺ cells from normal and DM mice. (D) shows representative flow cytometry plots of pAKT levels in c-kit⁺ cells. SDF-1 enhanced c-kit⁺/pAKT⁺ cell fraction in the control population (normal); in contrast it had no effect on cells obtained from DM mice (diabetes). Average results are shown in (E). SDF-1 increased c-kit⁺/pAKT⁺ cell percentage in total BM cells from normal mice after 10 min. of exposure to the chemokine (n= 3, Student’s t-test P < 0.05). In contrast, AKT phosphorylation was impaired in c-kit⁺ cells from DM mice (n= 3, Student’s t-test, P= n.s.). The response to SDF-1 differed between the two experimental groups (ANOVA, P < 0.001; post hoc P-value is indicated above the bar graph).