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Unique responses of stem cell-derived vascular endothelial and mesenchymal cells to high levels of glucose.

Keats E, Khan ZA - PLoS ONE (2012)

Bottom Line: Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs.Interestingly, MPCs showed a transient reduction in growth upon glucose challenge.The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
Diabetes leads to complications in selected organ systems, and vascular endothelial cell (EC) dysfunction and loss is the key initiating and perpetuating step in the development of these complications. Experimental and clinical studies have shown that hyperglycemia leads to EC dysfunction in diabetes. Vascular stem cells that give rise to endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) represent an attractive target for cell therapy for diabetic patients. Whether these vascular stem/progenitor cells succumb to the adverse effects of high glucose remains unknown. We sought to determine whether adult vascular stem/progenitor cells display cellular activation and dysfunction upon exposure to high levels of glucose as seen in diabetic complications. Mononuclear cell fraction was prepared from adult blood and bone marrow. EPCs and MPCs were derived, characterized, and exposed to either normal glucose (5 mmol/L) or high glucose levels (25 mmol/L). We then assayed for cell activity and molecular changes following both acute and chronic exposure to high glucose. Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs. The adult blood-derived EPCs were also resistant to the effects of glucose in terms of growth. Acute exposure to high glucose levels increased caspase-3 activity in EPCs (1.4x increase) and mature ECs (2.3x increase). Interestingly, MPCs showed a transient reduction in growth upon glucose challenge. Our results also show that glucose skews the differentiation of MPCs towards the adipocyte lineage while suppressing other mesenchymal lineages. In summary, our studies show that EPCs are resistant to the effects of high levels of glucose, even following chronic exposure. The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.

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Cells were cultured in 5 mmol/L (control) or 25 mmol/L (high glucose; HG) glucose throughout the completion of each cellular activity assay.In all graphs, values indicate mean ± SEM. (A) Growth of abEPCs was assessed as cell viability over a 12 day period in high serum media (EBM2/20% FBS). A spike in activity occurred in the hyperglycemic group at day 6 (*p<0.05 compared to cells in control media). However, abEPCs showed no significant differences between control and HG-treated groups over long-term culture. (B) Caspase-3 activity level was measured in abEPCs and HDMECs exposed to control or HG media (in EBM-2/1% FBS) for 24 hours [*p<0.05 compared to respective control media; †p<0.05 compared to HDMECs in HG media; n = 3]. (C) A 24-hour migration assay was performed on abEPCs, assessed as their ability to migrate through an 8-µm pore with bFGF as the chemoattractant. HG had no effect on the migratory abilities of abEPCs, cbEPCs, and HDMECs. (D) Growth of bmMPCs was assessed as cell viability over a 12 day period in high serum media (DMEM/20% FBS). HG-bmMPCs showed significantly less growth at day 1 as compared to control (*p<0.05). However, growth over long-term culture appeared to be unaffected by HG. (E) Migration of bmMPCs was assessed in the presence of 10% FBS as the chemoattractant. Exposure of bmMPCs to HG significantly reduced the migratory ability towards FBS (*p<0.05).
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pone-0038752-g002: Cells were cultured in 5 mmol/L (control) or 25 mmol/L (high glucose; HG) glucose throughout the completion of each cellular activity assay.In all graphs, values indicate mean ± SEM. (A) Growth of abEPCs was assessed as cell viability over a 12 day period in high serum media (EBM2/20% FBS). A spike in activity occurred in the hyperglycemic group at day 6 (*p<0.05 compared to cells in control media). However, abEPCs showed no significant differences between control and HG-treated groups over long-term culture. (B) Caspase-3 activity level was measured in abEPCs and HDMECs exposed to control or HG media (in EBM-2/1% FBS) for 24 hours [*p<0.05 compared to respective control media; †p<0.05 compared to HDMECs in HG media; n = 3]. (C) A 24-hour migration assay was performed on abEPCs, assessed as their ability to migrate through an 8-µm pore with bFGF as the chemoattractant. HG had no effect on the migratory abilities of abEPCs, cbEPCs, and HDMECs. (D) Growth of bmMPCs was assessed as cell viability over a 12 day period in high serum media (DMEM/20% FBS). HG-bmMPCs showed significantly less growth at day 1 as compared to control (*p<0.05). However, growth over long-term culture appeared to be unaffected by HG. (E) Migration of bmMPCs was assessed in the presence of 10% FBS as the chemoattractant. Exposure of bmMPCs to HG significantly reduced the migratory ability towards FBS (*p<0.05).

Mentions: We wanted to know whether acute and chronic exposure to high levels of glucose would alter abEPC growth. We cultured abEPCs for a period of 12 days in EBM-2/20% FBS supplemented with either 5 mmol/L glucose (control) or 25 mmol/L glucose (high glucose, HG). We chose to culture the cells in normal serum levels because this setting would be reminiscent of the in vivo conditions, and allow us to study the effect of chronically elevated glucose levels without the confounding toxicity associated with serum-free media. Our results show that the growth of abEPCs, and even HDMECs, is not affected by high levels of glucose (Figure 2A). An increase in total cell number was noted at day 6 in the abEPCs. However, this increase was normalized (relative to control media containing normal glucose levels) by day 12. We then plated a high density of cells and assessed the cell capacity for survival in depleted media (1% FBS), and how the addition of high glucose might impede this process. Addition of high glucose in 1% serum media reduced the cell number at 24 hours but had no effect at day 4 (data not shown). To confirm that the reduction in cell number was due to apoptosis, we measured caspase-3 activity in the abEPCs and HDMECs. Our results show a slight but significant increase in caspase-3 activity in abEPCs (1.4x increase as compared to control media without high glucose) (Figure 2B). Interestingly, the same conditions led to a 2.3x increase in caspase-3 activity in the mature HDMECs (Figure 2B). As the cells were plated under identical conditions (media, cell density, plate coating), these data suggest that abEPCs are resistant to glucose-induced toxicity as compared to the mature HDMECs.


Unique responses of stem cell-derived vascular endothelial and mesenchymal cells to high levels of glucose.

Keats E, Khan ZA - PLoS ONE (2012)

Cells were cultured in 5 mmol/L (control) or 25 mmol/L (high glucose; HG) glucose throughout the completion of each cellular activity assay.In all graphs, values indicate mean ± SEM. (A) Growth of abEPCs was assessed as cell viability over a 12 day period in high serum media (EBM2/20% FBS). A spike in activity occurred in the hyperglycemic group at day 6 (*p<0.05 compared to cells in control media). However, abEPCs showed no significant differences between control and HG-treated groups over long-term culture. (B) Caspase-3 activity level was measured in abEPCs and HDMECs exposed to control or HG media (in EBM-2/1% FBS) for 24 hours [*p<0.05 compared to respective control media; †p<0.05 compared to HDMECs in HG media; n = 3]. (C) A 24-hour migration assay was performed on abEPCs, assessed as their ability to migrate through an 8-µm pore with bFGF as the chemoattractant. HG had no effect on the migratory abilities of abEPCs, cbEPCs, and HDMECs. (D) Growth of bmMPCs was assessed as cell viability over a 12 day period in high serum media (DMEM/20% FBS). HG-bmMPCs showed significantly less growth at day 1 as compared to control (*p<0.05). However, growth over long-term culture appeared to be unaffected by HG. (E) Migration of bmMPCs was assessed in the presence of 10% FBS as the chemoattractant. Exposure of bmMPCs to HG significantly reduced the migratory ability towards FBS (*p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368917&req=5

pone-0038752-g002: Cells were cultured in 5 mmol/L (control) or 25 mmol/L (high glucose; HG) glucose throughout the completion of each cellular activity assay.In all graphs, values indicate mean ± SEM. (A) Growth of abEPCs was assessed as cell viability over a 12 day period in high serum media (EBM2/20% FBS). A spike in activity occurred in the hyperglycemic group at day 6 (*p<0.05 compared to cells in control media). However, abEPCs showed no significant differences between control and HG-treated groups over long-term culture. (B) Caspase-3 activity level was measured in abEPCs and HDMECs exposed to control or HG media (in EBM-2/1% FBS) for 24 hours [*p<0.05 compared to respective control media; †p<0.05 compared to HDMECs in HG media; n = 3]. (C) A 24-hour migration assay was performed on abEPCs, assessed as their ability to migrate through an 8-µm pore with bFGF as the chemoattractant. HG had no effect on the migratory abilities of abEPCs, cbEPCs, and HDMECs. (D) Growth of bmMPCs was assessed as cell viability over a 12 day period in high serum media (DMEM/20% FBS). HG-bmMPCs showed significantly less growth at day 1 as compared to control (*p<0.05). However, growth over long-term culture appeared to be unaffected by HG. (E) Migration of bmMPCs was assessed in the presence of 10% FBS as the chemoattractant. Exposure of bmMPCs to HG significantly reduced the migratory ability towards FBS (*p<0.05).
Mentions: We wanted to know whether acute and chronic exposure to high levels of glucose would alter abEPC growth. We cultured abEPCs for a period of 12 days in EBM-2/20% FBS supplemented with either 5 mmol/L glucose (control) or 25 mmol/L glucose (high glucose, HG). We chose to culture the cells in normal serum levels because this setting would be reminiscent of the in vivo conditions, and allow us to study the effect of chronically elevated glucose levels without the confounding toxicity associated with serum-free media. Our results show that the growth of abEPCs, and even HDMECs, is not affected by high levels of glucose (Figure 2A). An increase in total cell number was noted at day 6 in the abEPCs. However, this increase was normalized (relative to control media containing normal glucose levels) by day 12. We then plated a high density of cells and assessed the cell capacity for survival in depleted media (1% FBS), and how the addition of high glucose might impede this process. Addition of high glucose in 1% serum media reduced the cell number at 24 hours but had no effect at day 4 (data not shown). To confirm that the reduction in cell number was due to apoptosis, we measured caspase-3 activity in the abEPCs and HDMECs. Our results show a slight but significant increase in caspase-3 activity in abEPCs (1.4x increase as compared to control media without high glucose) (Figure 2B). Interestingly, the same conditions led to a 2.3x increase in caspase-3 activity in the mature HDMECs (Figure 2B). As the cells were plated under identical conditions (media, cell density, plate coating), these data suggest that abEPCs are resistant to glucose-induced toxicity as compared to the mature HDMECs.

Bottom Line: Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs.Interestingly, MPCs showed a transient reduction in growth upon glucose challenge.The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
Diabetes leads to complications in selected organ systems, and vascular endothelial cell (EC) dysfunction and loss is the key initiating and perpetuating step in the development of these complications. Experimental and clinical studies have shown that hyperglycemia leads to EC dysfunction in diabetes. Vascular stem cells that give rise to endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) represent an attractive target for cell therapy for diabetic patients. Whether these vascular stem/progenitor cells succumb to the adverse effects of high glucose remains unknown. We sought to determine whether adult vascular stem/progenitor cells display cellular activation and dysfunction upon exposure to high levels of glucose as seen in diabetic complications. Mononuclear cell fraction was prepared from adult blood and bone marrow. EPCs and MPCs were derived, characterized, and exposed to either normal glucose (5 mmol/L) or high glucose levels (25 mmol/L). We then assayed for cell activity and molecular changes following both acute and chronic exposure to high glucose. Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs. The adult blood-derived EPCs were also resistant to the effects of glucose in terms of growth. Acute exposure to high glucose levels increased caspase-3 activity in EPCs (1.4x increase) and mature ECs (2.3x increase). Interestingly, MPCs showed a transient reduction in growth upon glucose challenge. Our results also show that glucose skews the differentiation of MPCs towards the adipocyte lineage while suppressing other mesenchymal lineages. In summary, our studies show that EPCs are resistant to the effects of high levels of glucose, even following chronic exposure. The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.

Show MeSH
Related in: MedlinePlus