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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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Characterization of human adult blood-derived EPCs (abEPCs) and bone marrow-derived MPCs (bmMPCs).(A) abEPCs (n = 9) and bmMPCs (n = 6) were derived from the mononuclear cell layer of adult peripheral blood samples and bone marrow samples, respectively. No significant change in the number of abEPC and bmMPC colonies was seen with the addition of high glucose (25 mmol/L) to the culture medium. (B) abEPCs were characterized through quantitative RT-PCR analysis for expression of known EC-markers: CD31, CD34, VE-cadherin, vWF, and VEGFR-2 [mRNA data normalized to 18S rRNA and presented relative to HDMECs; *p<0.05 compared to HDMECs; n = 3]. (C) abEPCs were further characterized through immunostaining for antibodies against cell surface markers CD31 and VE-cadherin, and intracellular marker vWF [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm]. (D) Similarly, qRT-PCR analysis of bmMPCs showed expression of mesenchymal markers: calponin, α-SMA, MHC, NG2, and PDGFR-β [data normalized to 18S rRNA and presented relative to uaSMCs; *p<0.05 compared to uaSMCs; n = 3]. (E) MPCs were immunostained for antibodies against membrane-bound proteins CD90, NG2, and PDGFR-β, and cytoplasmic protein α-SMA. [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm].
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pone-0038752-g001: Characterization of human adult blood-derived EPCs (abEPCs) and bone marrow-derived MPCs (bmMPCs).(A) abEPCs (n = 9) and bmMPCs (n = 6) were derived from the mononuclear cell layer of adult peripheral blood samples and bone marrow samples, respectively. No significant change in the number of abEPC and bmMPC colonies was seen with the addition of high glucose (25 mmol/L) to the culture medium. (B) abEPCs were characterized through quantitative RT-PCR analysis for expression of known EC-markers: CD31, CD34, VE-cadherin, vWF, and VEGFR-2 [mRNA data normalized to 18S rRNA and presented relative to HDMECs; *p<0.05 compared to HDMECs; n = 3]. (C) abEPCs were further characterized through immunostaining for antibodies against cell surface markers CD31 and VE-cadherin, and intracellular marker vWF [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm]. (D) Similarly, qRT-PCR analysis of bmMPCs showed expression of mesenchymal markers: calponin, α-SMA, MHC, NG2, and PDGFR-β [data normalized to 18S rRNA and presented relative to uaSMCs; *p<0.05 compared to uaSMCs; n = 3]. (E) MPCs were immunostained for antibodies against membrane-bound proteins CD90, NG2, and PDGFR-β, and cytoplasmic protein α-SMA. [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm].

Mentions: Culture of the adult blood cells in EBM-2 media supplemented with SingleQuots induced differentiation of the cells into the endothelial lineage (abEPCs). Culture of blood-derived cells in high glucose media did not significantly alter the number of colonies (Figure 1A). No colonies appeared in either control or high glucose level conditions prior to day 14 (data not shown; plates were screened daily using phase contrast microscopy). abEPCs were then characterized through RT-PCR to confirm expression of endothelial cell-selective genes and through immunocytochemistry to properly localize the cellular markers. cbEPCs and HDMECs were used as controls. RT-PCR confirmed the expression of 5 genes of known significance to endothelial cells: CD31, CD34, VEGFR-2, VE-cadherin, and vWF (Figure 1B). The expression of all endothelial-specific genes tested, except for VEGFR-2, was significantly higher in abEPCs as compared to mature HDMECs (Figure 1B). Immunostaining showed both CD31 and VE-cadherin localized to the cell membrane of abEPCs, as anticipated (Figure 1C). vWF, an intracellular protein stored in Weible Palade bodies, showed intracellular localization.


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

Keats E, Khan ZA - PLoS ONE (2012)

Characterization of human adult blood-derived EPCs (abEPCs) and bone marrow-derived MPCs (bmMPCs).(A) abEPCs (n = 9) and bmMPCs (n = 6) were derived from the mononuclear cell layer of adult peripheral blood samples and bone marrow samples, respectively. No significant change in the number of abEPC and bmMPC colonies was seen with the addition of high glucose (25 mmol/L) to the culture medium. (B) abEPCs were characterized through quantitative RT-PCR analysis for expression of known EC-markers: CD31, CD34, VE-cadherin, vWF, and VEGFR-2 [mRNA data normalized to 18S rRNA and presented relative to HDMECs; *p<0.05 compared to HDMECs; n = 3]. (C) abEPCs were further characterized through immunostaining for antibodies against cell surface markers CD31 and VE-cadherin, and intracellular marker vWF [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm]. (D) Similarly, qRT-PCR analysis of bmMPCs showed expression of mesenchymal markers: calponin, α-SMA, MHC, NG2, and PDGFR-β [data normalized to 18S rRNA and presented relative to uaSMCs; *p<0.05 compared to uaSMCs; n = 3]. (E) MPCs were immunostained for antibodies against membrane-bound proteins CD90, NG2, and PDGFR-β, and cytoplasmic protein α-SMA. [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368917&req=5

pone-0038752-g001: Characterization of human adult blood-derived EPCs (abEPCs) and bone marrow-derived MPCs (bmMPCs).(A) abEPCs (n = 9) and bmMPCs (n = 6) were derived from the mononuclear cell layer of adult peripheral blood samples and bone marrow samples, respectively. No significant change in the number of abEPC and bmMPC colonies was seen with the addition of high glucose (25 mmol/L) to the culture medium. (B) abEPCs were characterized through quantitative RT-PCR analysis for expression of known EC-markers: CD31, CD34, VE-cadherin, vWF, and VEGFR-2 [mRNA data normalized to 18S rRNA and presented relative to HDMECs; *p<0.05 compared to HDMECs; n = 3]. (C) abEPCs were further characterized through immunostaining for antibodies against cell surface markers CD31 and VE-cadherin, and intracellular marker vWF [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm]. (D) Similarly, qRT-PCR analysis of bmMPCs showed expression of mesenchymal markers: calponin, α-SMA, MHC, NG2, and PDGFR-β [data normalized to 18S rRNA and presented relative to uaSMCs; *p<0.05 compared to uaSMCs; n = 3]. (E) MPCs were immunostained for antibodies against membrane-bound proteins CD90, NG2, and PDGFR-β, and cytoplasmic protein α-SMA. [Blue: DAPI for nuclear staining; Green: Alexa Fluorochrome 488; scale bar represents 100 µm].
Mentions: Culture of the adult blood cells in EBM-2 media supplemented with SingleQuots induced differentiation of the cells into the endothelial lineage (abEPCs). Culture of blood-derived cells in high glucose media did not significantly alter the number of colonies (Figure 1A). No colonies appeared in either control or high glucose level conditions prior to day 14 (data not shown; plates were screened daily using phase contrast microscopy). abEPCs were then characterized through RT-PCR to confirm expression of endothelial cell-selective genes and through immunocytochemistry to properly localize the cellular markers. cbEPCs and HDMECs were used as controls. RT-PCR confirmed the expression of 5 genes of known significance to endothelial cells: CD31, CD34, VEGFR-2, VE-cadherin, and vWF (Figure 1B). The expression of all endothelial-specific genes tested, except for VEGFR-2, was significantly higher in abEPCs as compared to mature HDMECs (Figure 1B). Immunostaining showed both CD31 and VE-cadherin localized to the cell membrane of abEPCs, as anticipated (Figure 1C). vWF, an intracellular protein stored in Weible Palade bodies, showed intracellular localization.

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