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

De Falco E, Avitabile D, Totta P, Straino S, Spallotta F, Cencioni C, Torella AR, Rizzi R, Porcelli D, Zacheo A, Di Vito L, Pompilio G, Napolitano M, Melillo G, Capogrossi MC, Pesce M - J. Cell. Mol. Med. (2009)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

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.

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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.
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fig03: 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.

Mentions: Diabetes has been reported to inhibit human EPCs differentiation [12, 17] and defects in CXCR4 signalling are known to jeopardize EPCs’ angiogenic properties [34, 35]. Therefore, we examined the effect of diabetes on SDF-1-directed EPC differentiation into endothelial cells, and tested whether culture in hyperglycaemia mimics DM effects on c-kit+ cell differentiation. We have previously described that SDF-1 enhances mouse BM c-kit+ cells endothelial differentiation through increased stem cell adhesion to FN and collagen I [11]. Under our experimental conditions the majority (>95%) of adherent cells differentiated and expressed factor VIII (vWF), KDR, CD31 and were also positive for acetylated LDL-DiI uptake [11] (Fig. S5). Therefore, an increase in differentiation was indicated by a higher number of cells adherent to the FN-coated glass chamber slide and not by an increase in the number of cells positive for endothelial cell markers. It was then quantitatively examined the adherence/differentiation of c-kit+ cells into endothelial cells as determined by acetylated LDL-DiI uptake [36]. In the absence of exogenous SDF-1, the basal level of endothelial adhesion/differentiation was similar in c-kit+ cells isolated from both experimental groups. In contrast, upon exposure to SDF-1 adhesion/differentiation was significantly higher in c-kit+ cells from normal than from DM mice. Moreover, stimulation by inactivated SDF-1 (SDF-1 B) failed to induce adhesion/differentiation of cells isolated both from normoglycaemic and DM mice (Fig. 3A).


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

De Falco E, Avitabile D, Totta P, Straino S, Spallotta F, Cencioni C, Torella AR, Rizzi R, Porcelli D, Zacheo A, Di Vito L, Pompilio G, Napolitano M, Melillo G, Capogrossi MC, Pesce M - J. Cell. Mol. Med. (2009)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516496&req=5

fig03: 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.
Mentions: Diabetes has been reported to inhibit human EPCs differentiation [12, 17] and defects in CXCR4 signalling are known to jeopardize EPCs’ angiogenic properties [34, 35]. Therefore, we examined the effect of diabetes on SDF-1-directed EPC differentiation into endothelial cells, and tested whether culture in hyperglycaemia mimics DM effects on c-kit+ cell differentiation. We have previously described that SDF-1 enhances mouse BM c-kit+ cells endothelial differentiation through increased stem cell adhesion to FN and collagen I [11]. Under our experimental conditions the majority (>95%) of adherent cells differentiated and expressed factor VIII (vWF), KDR, CD31 and were also positive for acetylated LDL-DiI uptake [11] (Fig. S5). Therefore, an increase in differentiation was indicated by a higher number of cells adherent to the FN-coated glass chamber slide and not by an increase in the number of cells positive for endothelial cell markers. It was then quantitatively examined the adherence/differentiation of c-kit+ cells into endothelial cells as determined by acetylated LDL-DiI uptake [36]. In the absence of exogenous SDF-1, the basal level of endothelial adhesion/differentiation was similar in c-kit+ cells isolated from both experimental groups. In contrast, upon exposure to SDF-1 adhesion/differentiation was significantly higher in c-kit+ cells from normal than from DM mice. Moreover, stimulation by inactivated SDF-1 (SDF-1 B) failed to induce adhesion/differentiation of cells isolated both from normoglycaemic and DM mice (Fig. 3A).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

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.

Show MeSH
Related in: MedlinePlus