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Keratocyte phenotype is enhanced in the absence of attachment to the substratum.

Funderburgh ML, Mann MM, Funderburgh JL - Mol. Vis. (2008)

Bottom Line: Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling.The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin.These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion.

View Article: PubMed Central - PubMed

Affiliation: UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Sciences Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213-2588, USA.

ABSTRACT

Purpose: Keratocytes, mesenchymal cells populating the corneal stroma, secrete the unique transparent connective tissue of the cornea as well as opaque scar tissue after injury. Previous studies identified factors mediating keratocyte phenotype in vitro, particularly the expression of the keratan sulfate proteoglycans, which are essential for vision. Whereas earlier work emphasized effects of cytokines, the current study examines the effects of substratum attachment on keratocyte phenotype.

Methods: Primary keratocytes from collagenase digestion of bovine corneas were cultured on tissue-culture plastic or on poly (2-hydroxyethylmethacrylate)(polyHEMA)-coated, non-adhesive surfaces. Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling. Gene expression was characterized with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Secreted matrix was examined with immunostaining.

Results: We observed that virtually all primary keratocytes participate in the formation of spheroidal aggregates, remaining viable for at least four weeks in vitro. Spheroid keratocytes secrete more keratan sulfate and keratocan than attached cells in the same culture medium. In spheroids, keratocytes accumulate substantial matrix in intercellular spaces, including keratan sulfate, lumican, keratocan, and collagens V and VI. The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin. Similarly, the platelet derived growth factor, a cytokine initiating the fibroblastic phenotype in attached keratocytes, had a limited effect on the spheroid-associated keratocytes. Ascorbate-2-phosphate was the only agent stimulating keratan sulfate secretion in the spheroid keratocytes.

Conclusions: These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion. For primary keratocytes, the alteration of the cellular environment in terms of cell-cell and cell-matrix interactions mediates and can override signals from soluble cytokines in influencing matrix expression and also in adopting other aspects of the fibroblastic and myofibroblastic phenotypes found in healing wounds.

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All keratocytes participate in sphere formation. A: Freshly isolated primary keratocytes were cultured in polyHEMA-coated dishes either in Advanced-DMEM (ADV, dark solid bars) or in Advanced-DMEM containing transforming growth factor beta-1 (TGFβ-1), fibroblast growth factor 2 (FGF2), and platelet derived growth factor BB (PDGF; light-stippled bars). Viable cells were labeled with Calcein AM, and the number labeled cells was determined by direct counting after trypsinization. Cell number is shown as a percentage of viable cells added to the original culture. B: Primary stromal cells were plated in Advanced-DMEM containing 10 ng/ml FGF2 under attachment conditions (solid lines) or as spheroids in polyHEMA-coated dishes (broken lines). Cells numbers were estimated by conversion of the dye Alamar Blue to a fluorescent form and normalized to the number of cells added to the culture. Error bars show the SD of triplicate analyses.
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f2: All keratocytes participate in sphere formation. A: Freshly isolated primary keratocytes were cultured in polyHEMA-coated dishes either in Advanced-DMEM (ADV, dark solid bars) or in Advanced-DMEM containing transforming growth factor beta-1 (TGFβ-1), fibroblast growth factor 2 (FGF2), and platelet derived growth factor BB (PDGF; light-stippled bars). Viable cells were labeled with Calcein AM, and the number labeled cells was determined by direct counting after trypsinization. Cell number is shown as a percentage of viable cells added to the original culture. B: Primary stromal cells were plated in Advanced-DMEM containing 10 ng/ml FGF2 under attachment conditions (solid lines) or as spheroids in polyHEMA-coated dishes (broken lines). Cells numbers were estimated by conversion of the dye Alamar Blue to a fluorescent form and normalized to the number of cells added to the culture. Error bars show the SD of triplicate analyses.

Mentions: To determine what proportion of primary cells were incorporated into spheroids, a time course was performed documenting the total number of cells and their viability after freshly isolated primary keratocytes were cultured in substratum-independent conditions. An ADV serum-free medium (as in Figure 1) was compared to the medium supplemented with fibroblast growth factor 2 (FGF2), TGFβ1, and PDGF, factors that induce cell division in attached keratocytes. As shown in Figure 2A, the number of viable cells in the culture remained constant over a two-day period compared to the number of cells in the inoculum. The presence of cytokines had no effect on the cell number. This experiment suggests that all keratocytes participate in the formation of these substratum-independent spheroids and not just the cells with progenitor character.


Keratocyte phenotype is enhanced in the absence of attachment to the substratum.

Funderburgh ML, Mann MM, Funderburgh JL - Mol. Vis. (2008)

All keratocytes participate in sphere formation. A: Freshly isolated primary keratocytes were cultured in polyHEMA-coated dishes either in Advanced-DMEM (ADV, dark solid bars) or in Advanced-DMEM containing transforming growth factor beta-1 (TGFβ-1), fibroblast growth factor 2 (FGF2), and platelet derived growth factor BB (PDGF; light-stippled bars). Viable cells were labeled with Calcein AM, and the number labeled cells was determined by direct counting after trypsinization. Cell number is shown as a percentage of viable cells added to the original culture. B: Primary stromal cells were plated in Advanced-DMEM containing 10 ng/ml FGF2 under attachment conditions (solid lines) or as spheroids in polyHEMA-coated dishes (broken lines). Cells numbers were estimated by conversion of the dye Alamar Blue to a fluorescent form and normalized to the number of cells added to the culture. Error bars show the SD of triplicate analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2255023&req=5

f2: All keratocytes participate in sphere formation. A: Freshly isolated primary keratocytes were cultured in polyHEMA-coated dishes either in Advanced-DMEM (ADV, dark solid bars) or in Advanced-DMEM containing transforming growth factor beta-1 (TGFβ-1), fibroblast growth factor 2 (FGF2), and platelet derived growth factor BB (PDGF; light-stippled bars). Viable cells were labeled with Calcein AM, and the number labeled cells was determined by direct counting after trypsinization. Cell number is shown as a percentage of viable cells added to the original culture. B: Primary stromal cells were plated in Advanced-DMEM containing 10 ng/ml FGF2 under attachment conditions (solid lines) or as spheroids in polyHEMA-coated dishes (broken lines). Cells numbers were estimated by conversion of the dye Alamar Blue to a fluorescent form and normalized to the number of cells added to the culture. Error bars show the SD of triplicate analyses.
Mentions: To determine what proportion of primary cells were incorporated into spheroids, a time course was performed documenting the total number of cells and their viability after freshly isolated primary keratocytes were cultured in substratum-independent conditions. An ADV serum-free medium (as in Figure 1) was compared to the medium supplemented with fibroblast growth factor 2 (FGF2), TGFβ1, and PDGF, factors that induce cell division in attached keratocytes. As shown in Figure 2A, the number of viable cells in the culture remained constant over a two-day period compared to the number of cells in the inoculum. The presence of cytokines had no effect on the cell number. This experiment suggests that all keratocytes participate in the formation of these substratum-independent spheroids and not just the cells with progenitor character.

Bottom Line: Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling.The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin.These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion.

View Article: PubMed Central - PubMed

Affiliation: UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Sciences Research Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213-2588, USA.

ABSTRACT

Purpose: Keratocytes, mesenchymal cells populating the corneal stroma, secrete the unique transparent connective tissue of the cornea as well as opaque scar tissue after injury. Previous studies identified factors mediating keratocyte phenotype in vitro, particularly the expression of the keratan sulfate proteoglycans, which are essential for vision. Whereas earlier work emphasized effects of cytokines, the current study examines the effects of substratum attachment on keratocyte phenotype.

Methods: Primary keratocytes from collagenase digestion of bovine corneas were cultured on tissue-culture plastic or on poly (2-hydroxyethylmethacrylate)(polyHEMA)-coated, non-adhesive surfaces. Secreted proteoglycans from culture media and cell-associated proteins were characterized using western blotting or isotopic labeling. Gene expression was characterized with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Secreted matrix was examined with immunostaining.

Results: We observed that virtually all primary keratocytes participate in the formation of spheroidal aggregates, remaining viable for at least four weeks in vitro. Spheroid keratocytes secrete more keratan sulfate and keratocan than attached cells in the same culture medium. In spheroids, keratocytes accumulate substantial matrix in intercellular spaces, including keratan sulfate, lumican, keratocan, and collagens V and VI. The unattached cells undergo limited cell division and do not differentiate into myofibroblasts in response to transforming growth factor beta (TGFbeta), which is based on the expression of extra domain A (EDA) fibronectin and alpha-smooth muscle actin. Similarly, the platelet derived growth factor, a cytokine initiating the fibroblastic phenotype in attached keratocytes, had a limited effect on the spheroid-associated keratocytes. Ascorbate-2-phosphate was the only agent stimulating keratan sulfate secretion in the spheroid keratocytes.

Conclusions: These results provide a new paradigm for understanding signals that regulate extracellular matrix secretion. For primary keratocytes, the alteration of the cellular environment in terms of cell-cell and cell-matrix interactions mediates and can override signals from soluble cytokines in influencing matrix expression and also in adopting other aspects of the fibroblastic and myofibroblastic phenotypes found in healing wounds.

Show MeSH
Related in: MedlinePlus