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O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells.

Zafir A, Bradley JA, Long BW, Muthusamy S, Li Q, Hill BG, Wysoczynski M, Prabhu SD, Bhatnagar A, Bolli R, Jones SP - PLoS ONE (2015)

Bottom Line: Differentiation significantly decreased the abundance of O-GlcNAcylated proteins.Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited.We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology; Diabetes and Obesity Center, Department of Medicine, Division of Cardiovascular Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase--the protein responsible for adding O-GlcNAc to proteins--is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be considered carefully in the context of cardiac cell therapy.

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Related in: MedlinePlus

Flow cytometric characterization of Sca-1+/lin- murine cardiac cells.A, B: Gating strategy, and C-K: flow cytometric analyses for C: CD105, D: CD29, E: Sca-1, F: CD90.2, G: CD73, H: CD45, I: CD31, J: CD34, and K: c-kit.
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pone.0142939.g001: Flow cytometric characterization of Sca-1+/lin- murine cardiac cells.A, B: Gating strategy, and C-K: flow cytometric analyses for C: CD105, D: CD29, E: Sca-1, F: CD90.2, G: CD73, H: CD45, I: CD31, J: CD34, and K: c-kit.

Mentions: We used flow cytometry to immunophenotype the cells (Fig 1A–1K), and found them to be positive for CD105 (endoglin, 97%, Fig 1C), CD29 (integrinβ1, Fig 1D), and Sca-1 (95%, Fig 1E). The cells expressed minimally other mesenchymal markers CD90.2 (3%, Fig 1F) and CD73 (2%, Fig 1G), and, other relevant markers CD45 (0%, Fig 1H), CD31 (0%, Fig 1I), CD34 (1%, Fig 1J), and c-kit (2%, Fig 1K). We previously documented the loss of c-kit expression during passage[2, 19].


O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells.

Zafir A, Bradley JA, Long BW, Muthusamy S, Li Q, Hill BG, Wysoczynski M, Prabhu SD, Bhatnagar A, Bolli R, Jones SP - PLoS ONE (2015)

Flow cytometric characterization of Sca-1+/lin- murine cardiac cells.A, B: Gating strategy, and C-K: flow cytometric analyses for C: CD105, D: CD29, E: Sca-1, F: CD90.2, G: CD73, H: CD45, I: CD31, J: CD34, and K: c-kit.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643874&req=5

pone.0142939.g001: Flow cytometric characterization of Sca-1+/lin- murine cardiac cells.A, B: Gating strategy, and C-K: flow cytometric analyses for C: CD105, D: CD29, E: Sca-1, F: CD90.2, G: CD73, H: CD45, I: CD31, J: CD34, and K: c-kit.
Mentions: We used flow cytometry to immunophenotype the cells (Fig 1A–1K), and found them to be positive for CD105 (endoglin, 97%, Fig 1C), CD29 (integrinβ1, Fig 1D), and Sca-1 (95%, Fig 1E). The cells expressed minimally other mesenchymal markers CD90.2 (3%, Fig 1F) and CD73 (2%, Fig 1G), and, other relevant markers CD45 (0%, Fig 1H), CD31 (0%, Fig 1I), CD34 (1%, Fig 1J), and c-kit (2%, Fig 1K). We previously documented the loss of c-kit expression during passage[2, 19].

Bottom Line: Differentiation significantly decreased the abundance of O-GlcNAcylated proteins.Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited.We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology; Diabetes and Obesity Center, Department of Medicine, Division of Cardiovascular Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase--the protein responsible for adding O-GlcNAc to proteins--is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be considered carefully in the context of cardiac cell therapy.

Show MeSH
Related in: MedlinePlus