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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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Cd44 and Has2 were upregulated by 6-h compressive loading, but HA binding proteins and hyaluronidase gene expression were not.Fibroblasts were seeded to collagen sponge and incubated for 24(□) or 200 mmHg (▪) compression for 6 h. Total mRNA was extracted after WST-1 assay, and mRNA expression was assessed using real-time RT-PCR. The expression of the target genes in the 6 h–200 mmHg group relative to the value in the 6 h–0 mmHg group was calculated by the comparative Ct method using the 18S ribosomal RNA gene as an internal control. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Student’s t test between the 0 mmHg group and the 200 mmHg group, and 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. A: Cd44. B: Has2. G: Cox2. J: Vcan and Tnfaip6 are HA binding proteins. Hyal1, 2, and 3 are HA degrading enzyme. C and D: Immunostaining for CD44. Representative sections of (C) the 6 h–0 mmHg group and (D) the 6 h–200 mmHg group. E and F: Immunostaining for HAS2. Representative sections of (E) the 6 h–0 mmHg group and (F) the 6 h–200 mmHg group. H and I: Immunostaining for COX2. Representative sections of (H) the 6 h–0 mmHg group and (I) the 6 h–200 mmHg group. Scale bars = 20 µm for all images.
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pone-0104676-g004: Cd44 and Has2 were upregulated by 6-h compressive loading, but HA binding proteins and hyaluronidase gene expression were not.Fibroblasts were seeded to collagen sponge and incubated for 24(□) or 200 mmHg (▪) compression for 6 h. Total mRNA was extracted after WST-1 assay, and mRNA expression was assessed using real-time RT-PCR. The expression of the target genes in the 6 h–200 mmHg group relative to the value in the 6 h–0 mmHg group was calculated by the comparative Ct method using the 18S ribosomal RNA gene as an internal control. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Student’s t test between the 0 mmHg group and the 200 mmHg group, and 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. A: Cd44. B: Has2. G: Cox2. J: Vcan and Tnfaip6 are HA binding proteins. Hyal1, 2, and 3 are HA degrading enzyme. C and D: Immunostaining for CD44. Representative sections of (C) the 6 h–0 mmHg group and (D) the 6 h–200 mmHg group. E and F: Immunostaining for HAS2. Representative sections of (E) the 6 h–0 mmHg group and (F) the 6 h–200 mmHg group. H and I: Immunostaining for COX2. Representative sections of (H) the 6 h–0 mmHg group and (I) the 6 h–200 mmHg group. Scale bars = 20 µm for all images.

Mentions: A 9.0-fold increase occurred in Cd44, one of the adhesion molecules related to apoptosis via the disruption of adhesion [14], in the 200 mmHg group compared with the 0 mmHg group (p<0.001; Fig. 4A). Subsequently, we investigated Has1, Has2, and Has3, which are HA synthases, known as a primary ligand for CD44 [33]–[35]. A 4.6-fold increase occurred in Has2 in the 200 mmHg group compared with the 0 mmHg group (p<0.001) (Fig. 4B). On the other hand, the levels of Has1 and Has3 were under the detection limit in both the groups. Subsequently, we investigated the expression of CD44 and HAS2 proteins by immunostaining because Cd44 and Has2 were significantly upregulated by compressive loading. Similar to gene expression, CD44 and HAS2 were upregulated by compressive loading in 3D cultured fibroblasts (Fig. 4C, 4D, 4E, and 4F). Based on these results, we decided to quantitatively evaluate HA synthesized by HAS2 in the culture supernatants. Furthermore, we investigated Cox2 gene expression because CD44 and HA interaction upregulates COX2 expression [17]. Cox2 was significantly upregulated by compressive loading (p = 0.007; Fig. 4G). Next, we investigated the expression of the COX2 protein by immunostaining based on the result for Cox2 gene expression. Protein expression, as well as gene expression, for COX 2 was upregulated by compressive loading (Fig. 4H and 4I). Therefore, considering that PGE2 is a secretory substance downstream of COX2 [36], PGE2 would also be a possible molecular marker, that could be quantitatively evaluated in the culture supernatants.


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)

Cd44 and Has2 were upregulated by 6-h compressive loading, but HA binding proteins and hyaluronidase gene expression were not.Fibroblasts were seeded to collagen sponge and incubated for 24(□) or 200 mmHg (▪) compression for 6 h. Total mRNA was extracted after WST-1 assay, and mRNA expression was assessed using real-time RT-PCR. The expression of the target genes in the 6 h–200 mmHg group relative to the value in the 6 h–0 mmHg group was calculated by the comparative Ct method using the 18S ribosomal RNA gene as an internal control. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Student’s t test between the 0 mmHg group and the 200 mmHg group, and 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. A: Cd44. B: Has2. G: Cox2. J: Vcan and Tnfaip6 are HA binding proteins. Hyal1, 2, and 3 are HA degrading enzyme. C and D: Immunostaining for CD44. Representative sections of (C) the 6 h–0 mmHg group and (D) the 6 h–200 mmHg group. E and F: Immunostaining for HAS2. Representative sections of (E) the 6 h–0 mmHg group and (F) the 6 h–200 mmHg group. H and I: Immunostaining for COX2. Representative sections of (H) the 6 h–0 mmHg group and (I) the 6 h–200 mmHg group. Scale bars = 20 µm for all images.
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pone-0104676-g004: Cd44 and Has2 were upregulated by 6-h compressive loading, but HA binding proteins and hyaluronidase gene expression were not.Fibroblasts were seeded to collagen sponge and incubated for 24(□) or 200 mmHg (▪) compression for 6 h. Total mRNA was extracted after WST-1 assay, and mRNA expression was assessed using real-time RT-PCR. The expression of the target genes in the 6 h–200 mmHg group relative to the value in the 6 h–0 mmHg group was calculated by the comparative Ct method using the 18S ribosomal RNA gene as an internal control. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Student’s t test between the 0 mmHg group and the 200 mmHg group, and 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. A: Cd44. B: Has2. G: Cox2. J: Vcan and Tnfaip6 are HA binding proteins. Hyal1, 2, and 3 are HA degrading enzyme. C and D: Immunostaining for CD44. Representative sections of (C) the 6 h–0 mmHg group and (D) the 6 h–200 mmHg group. E and F: Immunostaining for HAS2. Representative sections of (E) the 6 h–0 mmHg group and (F) the 6 h–200 mmHg group. H and I: Immunostaining for COX2. Representative sections of (H) the 6 h–0 mmHg group and (I) the 6 h–200 mmHg group. Scale bars = 20 µm for all images.
Mentions: A 9.0-fold increase occurred in Cd44, one of the adhesion molecules related to apoptosis via the disruption of adhesion [14], in the 200 mmHg group compared with the 0 mmHg group (p<0.001; Fig. 4A). Subsequently, we investigated Has1, Has2, and Has3, which are HA synthases, known as a primary ligand for CD44 [33]–[35]. A 4.6-fold increase occurred in Has2 in the 200 mmHg group compared with the 0 mmHg group (p<0.001) (Fig. 4B). On the other hand, the levels of Has1 and Has3 were under the detection limit in both the groups. Subsequently, we investigated the expression of CD44 and HAS2 proteins by immunostaining because Cd44 and Has2 were significantly upregulated by compressive loading. Similar to gene expression, CD44 and HAS2 were upregulated by compressive loading in 3D cultured fibroblasts (Fig. 4C, 4D, 4E, and 4F). Based on these results, we decided to quantitatively evaluate HA synthesized by HAS2 in the culture supernatants. Furthermore, we investigated Cox2 gene expression because CD44 and HA interaction upregulates COX2 expression [17]. Cox2 was significantly upregulated by compressive loading (p = 0.007; Fig. 4G). Next, we investigated the expression of the COX2 protein by immunostaining based on the result for Cox2 gene expression. Protein expression, as well as gene expression, for COX 2 was upregulated by compressive loading (Fig. 4H and 4I). Therefore, considering that PGE2 is a secretory substance downstream of COX2 [36], PGE2 would also be a possible molecular marker, that could be quantitatively evaluated in the culture supernatants.

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