HSP90 AND ENOS PARTIALLY CO-LOCALIZE AND CHANGE CELLULAR LOCALIZATION IN RELATION TO DIFFERENT ECM COMPONENTS IN 2D AND 3D CULTURES OF ADULT RAT CARDIOMYCYTES

Antonella Marino Gammazza, Giovanni Peri, Valentina Di Felice, Filippo Macaluso, Giovanni Zummo, Angela De Luca, Fabrizio Celestino Minervini, Patrizia Catanese

Risultato della ricerca: Other contribution

Abstract

BACKGROUND INFORMATION: Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). RESULTS: Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. CONCLUSIONS: Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell-ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype
Lingua originaleEnglish
Stato di pubblicazionePublished - 2007

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HSP90 Heat-Shock Proteins
Carbon Monoxide
Cardiac Myocytes
Extracellular Matrix
Nitric Oxide Synthase Type III
Coculture Techniques
Confocal Microscopy
Gels
Regenerative Medicine
Laminin
Mechanics
Transmission Electron Microscopy
Fluorescence Microscopy
Fibronectins
Compliance
Electron Microscopy
Mitochondria
Cell Culture Techniques
Fibroblasts
Cell Count

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title = "HSP90 AND ENOS PARTIALLY CO-LOCALIZE AND CHANGE CELLULAR LOCALIZATION IN RELATION TO DIFFERENT ECM COMPONENTS IN 2D AND 3D CULTURES OF ADULT RAT CARDIOMYCYTES",
abstract = "BACKGROUND INFORMATION: Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). RESULTS: Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. CONCLUSIONS: Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell-ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype",
keywords = "ARTIFICIAL EXTRACELLULAR MATRIX, CO-COLTURES, CONFOCAL MICROSCOPY, ELECTRON MICROSCOPY, PROTEIN CO-LOCALIZATION.",
author = "{Marino Gammazza}, Antonella and Giovanni Peri and {Di Felice}, Valentina and Filippo Macaluso and Giovanni Zummo and {De Luca}, Angela and Minervini, {Fabrizio Celestino} and Patrizia Catanese",
year = "2007",
language = "English",
type = "Other",

}

TY - GEN

T1 - HSP90 AND ENOS PARTIALLY CO-LOCALIZE AND CHANGE CELLULAR LOCALIZATION IN RELATION TO DIFFERENT ECM COMPONENTS IN 2D AND 3D CULTURES OF ADULT RAT CARDIOMYCYTES

AU - Marino Gammazza, Antonella

AU - Peri, Giovanni

AU - Di Felice, Valentina

AU - Macaluso, Filippo

AU - Zummo, Giovanni

AU - De Luca, Angela

AU - Minervini, Fabrizio Celestino

AU - Catanese, Patrizia

PY - 2007

Y1 - 2007

N2 - BACKGROUND INFORMATION: Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). RESULTS: Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. CONCLUSIONS: Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell-ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype

AB - BACKGROUND INFORMATION: Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). RESULTS: Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates. CONCLUSIONS: Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell-ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype

KW - ARTIFICIAL EXTRACELLULAR MATRIX

KW - CO-COLTURES

KW - CONFOCAL MICROSCOPY

KW - ELECTRON MICROSCOPY

KW - PROTEIN CO-LOCALIZATION.

UR - http://hdl.handle.net/10447/39843

M3 - Other contribution

ER -