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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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Differentiation of bmMPCs into adipocytes.bmMPCs were cultured in 5 mmol/L (control glucose) or 25 mmol/L (high glucose; HG) glucose for 7 days prior to differentiation and assessed for induction of PPARγ2 and C/EBPα (A). HG-treated bmMPCs increased adipogenesis at day 7, as demonstrated by upregulated PPARγ2 levels (B) and C/EBPα (C). Analysis of cells at day 14 showed increased PPARγ2 upon differentiation but no differences between control- and HG-treated cells. C/EBPα levels (E), on the other hand, were significantly higher in HG-treated cells at day 14 as compared to control glucose treated cells [*p<0.05 compared to control media; †p<0.05 compared to cells treated with control glucose + differentiation media].
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pone-0038752-g005: Differentiation of bmMPCs into adipocytes.bmMPCs were cultured in 5 mmol/L (control glucose) or 25 mmol/L (high glucose; HG) glucose for 7 days prior to differentiation and assessed for induction of PPARγ2 and C/EBPα (A). HG-treated bmMPCs increased adipogenesis at day 7, as demonstrated by upregulated PPARγ2 levels (B) and C/EBPα (C). Analysis of cells at day 14 showed increased PPARγ2 upon differentiation but no differences between control- and HG-treated cells. C/EBPα levels (E), on the other hand, were significantly higher in HG-treated cells at day 14 as compared to control glucose treated cells [*p<0.05 compared to control media; †p<0.05 compared to cells treated with control glucose + differentiation media].

Mentions: We performed mesenchymal differentiation assays on the bmMPCs to assess their ability to differentiate into adipocytes, chondocytes, and osteocytes with the addition of high glucose. The cells were pre-treated with high levels of glucose for 7 days prior to culture in the differentiation media. qRT-PCR was used to examine the expression of specific transcription factors involved in the differentiation process (adipogenesis was assessed by C/EBPα and PPARγ2 (Figure 5A); chondrogenesis by Sox9, Nkx3.2, and Runx2 (Figure 6A); and osteogenesis by Runx2 and osterix/SP7 (Figure 7A). High glucose drastically increased the differentiation of bmMPCs into adipocytes, as assessed by C/EBPα and PPARγ2 induction at day 7 (Figure 5B and 5C). At day 14, PPARγ2 levels in cells exposed to high glucose were similar to cells in the normal glucose media (Figure 5D); however, C/EBPα levels remained significantly higher (Figure 5E). Next, we assayed for osteogenic differentiation by measuring levels of osteogenic transcription factors Runx2 and osterix/SP7 (Figure 6A). Analysis of cells exposed to the differentiation media at day 14 showed that high glucose prevented Runx2 induction (Figure 6B) and significantly reduced osterix/SP7 induction (Figure 6C). Lastly, we determined whether glucose regulates chondrogenesis in bmMPCs. Sox9 mRNA levels were found to be elevated in control cells exposed to the chondrogenic media at day 7, which was not observed in cells exposed to high levels of glucose (data not shown). We then measured these transcription factors at day 14. Surprisingly, we found that cells exposed to differentiation media alone (i.e. normal glucose levels), significantly downregulated early chondrogenesis genes Sox9 and Nkx3.2 (Figure 7A–C). At this time point, cells exposed to high levels of glucose exhibited significantly higher levels of both Sox9 and Nkx3.2 (Figure 7B and 7C). Recently, it has been shown that unlike the adipogenesis-specific and osteogenesis-specific transcription factors (determined above), Sox9 plays an essential stage-specific role in chondrogenesis [23]. To test whether high glucose may be delaying these differentiation steps (Figure 7A), we measured Runx2 (late marker of chondrogenesis) in our assay. Our results show that bmMPCs induce Runx2 at day 14, which coincides with repressed Sox9 and Nkx3.2 (Figure 7D). In contrast, cells exposed to high glucose showed significantly lower Runx2 levels.


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

Keats E, Khan ZA - PLoS ONE (2012)

Differentiation of bmMPCs into adipocytes.bmMPCs were cultured in 5 mmol/L (control glucose) or 25 mmol/L (high glucose; HG) glucose for 7 days prior to differentiation and assessed for induction of PPARγ2 and C/EBPα (A). HG-treated bmMPCs increased adipogenesis at day 7, as demonstrated by upregulated PPARγ2 levels (B) and C/EBPα (C). Analysis of cells at day 14 showed increased PPARγ2 upon differentiation but no differences between control- and HG-treated cells. C/EBPα levels (E), on the other hand, were significantly higher in HG-treated cells at day 14 as compared to control glucose treated cells [*p<0.05 compared to control media; †p<0.05 compared to cells treated with control glucose + differentiation media].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368917&req=5

pone-0038752-g005: Differentiation of bmMPCs into adipocytes.bmMPCs were cultured in 5 mmol/L (control glucose) or 25 mmol/L (high glucose; HG) glucose for 7 days prior to differentiation and assessed for induction of PPARγ2 and C/EBPα (A). HG-treated bmMPCs increased adipogenesis at day 7, as demonstrated by upregulated PPARγ2 levels (B) and C/EBPα (C). Analysis of cells at day 14 showed increased PPARγ2 upon differentiation but no differences between control- and HG-treated cells. C/EBPα levels (E), on the other hand, were significantly higher in HG-treated cells at day 14 as compared to control glucose treated cells [*p<0.05 compared to control media; †p<0.05 compared to cells treated with control glucose + differentiation media].
Mentions: We performed mesenchymal differentiation assays on the bmMPCs to assess their ability to differentiate into adipocytes, chondocytes, and osteocytes with the addition of high glucose. The cells were pre-treated with high levels of glucose for 7 days prior to culture in the differentiation media. qRT-PCR was used to examine the expression of specific transcription factors involved in the differentiation process (adipogenesis was assessed by C/EBPα and PPARγ2 (Figure 5A); chondrogenesis by Sox9, Nkx3.2, and Runx2 (Figure 6A); and osteogenesis by Runx2 and osterix/SP7 (Figure 7A). High glucose drastically increased the differentiation of bmMPCs into adipocytes, as assessed by C/EBPα and PPARγ2 induction at day 7 (Figure 5B and 5C). At day 14, PPARγ2 levels in cells exposed to high glucose were similar to cells in the normal glucose media (Figure 5D); however, C/EBPα levels remained significantly higher (Figure 5E). Next, we assayed for osteogenic differentiation by measuring levels of osteogenic transcription factors Runx2 and osterix/SP7 (Figure 6A). Analysis of cells exposed to the differentiation media at day 14 showed that high glucose prevented Runx2 induction (Figure 6B) and significantly reduced osterix/SP7 induction (Figure 6C). Lastly, we determined whether glucose regulates chondrogenesis in bmMPCs. Sox9 mRNA levels were found to be elevated in control cells exposed to the chondrogenic media at day 7, which was not observed in cells exposed to high levels of glucose (data not shown). We then measured these transcription factors at day 14. Surprisingly, we found that cells exposed to differentiation media alone (i.e. normal glucose levels), significantly downregulated early chondrogenesis genes Sox9 and Nkx3.2 (Figure 7A–C). At this time point, cells exposed to high levels of glucose exhibited significantly higher levels of both Sox9 and Nkx3.2 (Figure 7B and 7C). Recently, it has been shown that unlike the adipogenesis-specific and osteogenesis-specific transcription factors (determined above), Sox9 plays an essential stage-specific role in chondrogenesis [23]. To test whether high glucose may be delaying these differentiation steps (Figure 7A), we measured Runx2 (late marker of chondrogenesis) in our assay. Our results show that bmMPCs induce Runx2 at day 14, which coincides with repressed Sox9 and Nkx3.2 (Figure 7D). In contrast, cells exposed to high glucose showed significantly lower Runx2 levels.

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