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Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

Kanazawa T, Nakagami G, Minematsu T, Yamane T, Huang L, Mugita Y, Noguchi H, Mori T, Sanada H - PLoS ONE (2014)

Bottom Line: Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased.In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control.These results suggest that compressive loading induces not only apoptosis but also survival activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs), CD44, hyaluronan synthase 2 (HAS2), and cyclooxygenase 2 (COX2) along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

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Sustained compressive loading did not induce apparent cell proliferation and induced apoptosis through disruption of adhesion.Fibroblasts were seeded to collagen sponge and incubated for 24: Collagen sponge samples after loading were transferred to new 12-well plates in 1 ml medium containing 100 µl WST-1 reagent per well, and then incubated for 1.5 h. The absorbance of 450 nm was measured. The cell number was shown relative to base line. The results are represented as the mean ± SEM (error bars) of five experiments. D, E, and F: Collagen sponge samples were fixed, dehydrated, cleared, and processed for embedding in paraffin after loading experiments. Sections were prepared at 4-µm thick. Apoptosis assay were performed by TUNEL stain using tissue slides. The number of TUNEL-positive cells was counted in 5 fields in the central area of the collagen sponge (magnification ×10), and the proportion of positive cells to total cells was calculated. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test: between 2, 4, or 6 h group and 0 h group (A, B, D, and E) or between each of loaded group and nonloaded group (C and F). Statistical significance was taken as p<0.05. A value of p was expressed as: *; p<0.05, **; p<0.01, and ***; p<0.001. G, H, and I: H&E staining for confirming cell morphology. Collagen sponge samples were prepared as aforementioned. Sections were prepared at 5-mm thick. The distinctive cell morphology observed in the 6 h–0 mmHg group was spindle-shaped cells (G), whereas that in the 6 h–200 mmHg group was nonspindle-shaped cells (H) along with apoptotic bodies (I). J and K: Immunostaining for the FA structural protein vinculin (green). Nucleus stained by DAPI (blue). Vinculin expression was observed in the 6 h–0 mmHg group (J), whereas it was scarcely observed in the 6 h–200 mmHg group (K). L and M: Immunostaining for actin stress fibers by phaloidin (red). Nucleus stained by DAPI (blue). Actin stress fibers were observed in the 6 h–0 mmHg group (L), but not in the 6 h–200 mmHg group (M). Scale bars = 20 µm for all images.
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pone-0104676-g002: Sustained compressive loading did not induce apparent cell proliferation and induced apoptosis through disruption of adhesion.Fibroblasts were seeded to collagen sponge and incubated for 24: Collagen sponge samples after loading were transferred to new 12-well plates in 1 ml medium containing 100 µl WST-1 reagent per well, and then incubated for 1.5 h. The absorbance of 450 nm was measured. The cell number was shown relative to base line. The results are represented as the mean ± SEM (error bars) of five experiments. D, E, and F: Collagen sponge samples were fixed, dehydrated, cleared, and processed for embedding in paraffin after loading experiments. Sections were prepared at 4-µm thick. Apoptosis assay were performed by TUNEL stain using tissue slides. The number of TUNEL-positive cells was counted in 5 fields in the central area of the collagen sponge (magnification ×10), and the proportion of positive cells to total cells was calculated. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test: between 2, 4, or 6 h group and 0 h group (A, B, D, and E) or between each of loaded group and nonloaded group (C and F). Statistical significance was taken as p<0.05. A value of p was expressed as: *; p<0.05, **; p<0.01, and ***; p<0.001. G, H, and I: H&E staining for confirming cell morphology. Collagen sponge samples were prepared as aforementioned. Sections were prepared at 5-mm thick. The distinctive cell morphology observed in the 6 h–0 mmHg group was spindle-shaped cells (G), whereas that in the 6 h–200 mmHg group was nonspindle-shaped cells (H) along with apoptotic bodies (I). J and K: Immunostaining for the FA structural protein vinculin (green). Nucleus stained by DAPI (blue). Vinculin expression was observed in the 6 h–0 mmHg group (J), whereas it was scarcely observed in the 6 h–200 mmHg group (K). L and M: Immunostaining for actin stress fibers by phaloidin (red). Nucleus stained by DAPI (blue). Actin stress fibers were observed in the 6 h–0 mmHg group (L), but not in the 6 h–200 mmHg group (M). Scale bars = 20 µm for all images.

Mentions: WST-1 assay and TUNEL staining were used to investigate the proliferative and the apoptotic effects of sustained compressive loading at various loading times and intensities in 3D cultured fibroblasts. While the cell number in nonloaded groups significantly increased compared with the baseline in a time-dependent manner (2, 4, and 6 h groups; p = 0.872,  = 0.147, and  = 0.018, respectively; Fig. 2A), such an increase over the baseline was not observed in 2, 4, and 6 h–200 mmHg groups (each group; p>0.05; Fig. 2B). Moreover, 6-h compressive loading induced a significant reduction in the cell number of the 50, 100, and 200 mmHg groups compared with the 0 mmHg group (each group; p<0.01; Fig. 2C). An increase in apoptosis was not observed in nonloaded groups (each group; p>0.05; Fig. 2D). In contrast, compressive loading significantly induced apoptosis in a time- and load-dependent manner (according to loading time for 2, 4, and 6 h–200 mmHg, p = 0.002, <0.001, and <0.001, respectively; according to loading intensity at 6 h–50, 100, and 200 mmHg, p<0.001, <0.001, and <0.001, respectively; Fig. 2E and 2F). Although these results suggest that proliferation and apoptosis occur simultaneously during compressive loading, apparent cell proliferation did not occur. We thus considered this system as the inhibitory state of granulation.


Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

Kanazawa T, Nakagami G, Minematsu T, Yamane T, Huang L, Mugita Y, Noguchi H, Mori T, Sanada H - PLoS ONE (2014)

Sustained compressive loading did not induce apparent cell proliferation and induced apoptosis through disruption of adhesion.Fibroblasts were seeded to collagen sponge and incubated for 24: Collagen sponge samples after loading were transferred to new 12-well plates in 1 ml medium containing 100 µl WST-1 reagent per well, and then incubated for 1.5 h. The absorbance of 450 nm was measured. The cell number was shown relative to base line. The results are represented as the mean ± SEM (error bars) of five experiments. D, E, and F: Collagen sponge samples were fixed, dehydrated, cleared, and processed for embedding in paraffin after loading experiments. Sections were prepared at 4-µm thick. Apoptosis assay were performed by TUNEL stain using tissue slides. The number of TUNEL-positive cells was counted in 5 fields in the central area of the collagen sponge (magnification ×10), and the proportion of positive cells to total cells was calculated. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test: between 2, 4, or 6 h group and 0 h group (A, B, D, and E) or between each of loaded group and nonloaded group (C and F). Statistical significance was taken as p<0.05. A value of p was expressed as: *; p<0.05, **; p<0.01, and ***; p<0.001. G, H, and I: H&E staining for confirming cell morphology. Collagen sponge samples were prepared as aforementioned. Sections were prepared at 5-mm thick. The distinctive cell morphology observed in the 6 h–0 mmHg group was spindle-shaped cells (G), whereas that in the 6 h–200 mmHg group was nonspindle-shaped cells (H) along with apoptotic bodies (I). J and K: Immunostaining for the FA structural protein vinculin (green). Nucleus stained by DAPI (blue). Vinculin expression was observed in the 6 h–0 mmHg group (J), whereas it was scarcely observed in the 6 h–200 mmHg group (K). L and M: Immunostaining for actin stress fibers by phaloidin (red). Nucleus stained by DAPI (blue). Actin stress fibers were observed in the 6 h–0 mmHg group (L), but not in the 6 h–200 mmHg group (M). Scale bars = 20 µm for all images.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125229&req=5

pone-0104676-g002: Sustained compressive loading did not induce apparent cell proliferation and induced apoptosis through disruption of adhesion.Fibroblasts were seeded to collagen sponge and incubated for 24: Collagen sponge samples after loading were transferred to new 12-well plates in 1 ml medium containing 100 µl WST-1 reagent per well, and then incubated for 1.5 h. The absorbance of 450 nm was measured. The cell number was shown relative to base line. The results are represented as the mean ± SEM (error bars) of five experiments. D, E, and F: Collagen sponge samples were fixed, dehydrated, cleared, and processed for embedding in paraffin after loading experiments. Sections were prepared at 4-µm thick. Apoptosis assay were performed by TUNEL stain using tissue slides. The number of TUNEL-positive cells was counted in 5 fields in the central area of the collagen sponge (magnification ×10), and the proportion of positive cells to total cells was calculated. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test: between 2, 4, or 6 h group and 0 h group (A, B, D, and E) or between each of loaded group and nonloaded group (C and F). Statistical significance was taken as p<0.05. A value of p was expressed as: *; p<0.05, **; p<0.01, and ***; p<0.001. G, H, and I: H&E staining for confirming cell morphology. Collagen sponge samples were prepared as aforementioned. Sections were prepared at 5-mm thick. The distinctive cell morphology observed in the 6 h–0 mmHg group was spindle-shaped cells (G), whereas that in the 6 h–200 mmHg group was nonspindle-shaped cells (H) along with apoptotic bodies (I). J and K: Immunostaining for the FA structural protein vinculin (green). Nucleus stained by DAPI (blue). Vinculin expression was observed in the 6 h–0 mmHg group (J), whereas it was scarcely observed in the 6 h–200 mmHg group (K). L and M: Immunostaining for actin stress fibers by phaloidin (red). Nucleus stained by DAPI (blue). Actin stress fibers were observed in the 6 h–0 mmHg group (L), but not in the 6 h–200 mmHg group (M). Scale bars = 20 µm for all images.
Mentions: WST-1 assay and TUNEL staining were used to investigate the proliferative and the apoptotic effects of sustained compressive loading at various loading times and intensities in 3D cultured fibroblasts. While the cell number in nonloaded groups significantly increased compared with the baseline in a time-dependent manner (2, 4, and 6 h groups; p = 0.872,  = 0.147, and  = 0.018, respectively; Fig. 2A), such an increase over the baseline was not observed in 2, 4, and 6 h–200 mmHg groups (each group; p>0.05; Fig. 2B). Moreover, 6-h compressive loading induced a significant reduction in the cell number of the 50, 100, and 200 mmHg groups compared with the 0 mmHg group (each group; p<0.01; Fig. 2C). An increase in apoptosis was not observed in nonloaded groups (each group; p>0.05; Fig. 2D). In contrast, compressive loading significantly induced apoptosis in a time- and load-dependent manner (according to loading time for 2, 4, and 6 h–200 mmHg, p = 0.002, <0.001, and <0.001, respectively; according to loading intensity at 6 h–50, 100, and 200 mmHg, p<0.001, <0.001, and <0.001, respectively; Fig. 2E and 2F). Although these results suggest that proliferation and apoptosis occur simultaneously during compressive loading, apparent cell proliferation did not occur. We thus considered this system as the inhibitory state of granulation.

Bottom Line: Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased.In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control.These results suggest that compressive loading induces not only apoptosis but also survival activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs), CD44, hyaluronan synthase 2 (HAS2), and cyclooxygenase 2 (COX2) along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

Show MeSH
Related in: MedlinePlus