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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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Stress- and apoptosis-related gene expression was stimulated by 6-h compressive loading.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: The transcription factors of various Hsps. B: various Hsps C: Bcl2 is an antiapoptotic gene, and Bax is a proapoptotic gene. D and E: Immunostaining for HSP90α. Representative sections of (D) the 6 h–0 mmHg group and (E) the 6 h–200 mmHg group. Higher expression and nucleus translocation of HSP90α was observed in the 200 mmHg group (E) when compared with the 0 mmHg group (D). Scale bars = 20 µm for all images.
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pone-0104676-g003: Stress- and apoptosis-related gene expression was stimulated by 6-h compressive loading.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: The transcription factors of various Hsps. B: various Hsps C: Bcl2 is an antiapoptotic gene, and Bax is a proapoptotic gene. D and E: Immunostaining for HSP90α. Representative sections of (D) the 6 h–0 mmHg group and (E) the 6 h–200 mmHg group. Higher expression and nucleus translocation of HSP90α was observed in the 200 mmHg group (E) when compared with the 0 mmHg group (D). Scale bars = 20 µm for all images.

Mentions: A significant increase in the expression of heat shock transcription factor 1 (Hsf1) and Hsf2 was observed in the 200 mmHg group compared with the 0 mmHg group (Hsf1 and Hsf2; p = 0.006 and  = 0.004, respectively; Fig. 3A). Expression of these genes is induced by the disruption of adhesion [26], and HSF1 and HSF2 bind to the regulatory site of various Hsp genes. Following this, we investigated the influence of compressive loading on the gene expression of various HSPs, which are stress-responsive proteins against mechanical stress, elevated temperature, hypoxia, lowered pH, and reactive oxygen species (ROS) [27]. The expression of various Hsps was significantly higher in the 200 mmHg group than in the 0 mmHg group (Hsp32, Hsp40, Hsp47, Hsp60, Hspa5, Hsc70, and Hsp90aa1; p = 0.002,  = 0.019, <0.001,  = 0.024,  = 0.033, <0.001, and <0.001, respectively; Fig. 3B). Upregulation of various Hsps indicates that stress responses by compressive loading occurred in fibroblasts. To examine the condition of nonapoptotic cells, we investigated the expression of antiapoptotic Bcl2[28] and proapoptotic Bax[29]. The results indicated that Bcl2 levels were significantly higher in the 200 mmHg group than in the 0 mmHg group, but Bax levels did not show any significant difference. (p = 0.001 and 0.851, respectively; Fig. 3C). Subsequently, we focused on HSP90α encoded by Hsp90aa1 and investigated the expression of HSP90α by immunocytochemistry, because Hsp32, known as an oxidative stress marker, is upregulated by compressive loading and oxidative stress leads to the release of HSP90α into the extracellular environment [30], [31]. Higher expression and nucleus translocation of HSP90α were observed in the 200 mmHg group when compared with the 0 mmHg group (Fig. 3D and 3E). Nucleus translocation of HSP90 occurs after cellular stress, and HSP90 tightly interacts with histones [32]. We decided to quantitatively evaluate HSP90α in the culture supernatants based on these observations.


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)

Stress- and apoptosis-related gene expression was stimulated by 6-h compressive loading.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: The transcription factors of various Hsps. B: various Hsps C: Bcl2 is an antiapoptotic gene, and Bax is a proapoptotic gene. D and E: Immunostaining for HSP90α. Representative sections of (D) the 6 h–0 mmHg group and (E) the 6 h–200 mmHg group. Higher expression and nucleus translocation of HSP90α was observed in the 200 mmHg group (E) when compared with the 0 mmHg group (D). Scale bars = 20 µm for all images.
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Related In: Results  -  Collection

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pone-0104676-g003: Stress- and apoptosis-related gene expression was stimulated by 6-h compressive loading.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: The transcription factors of various Hsps. B: various Hsps C: Bcl2 is an antiapoptotic gene, and Bax is a proapoptotic gene. D and E: Immunostaining for HSP90α. Representative sections of (D) the 6 h–0 mmHg group and (E) the 6 h–200 mmHg group. Higher expression and nucleus translocation of HSP90α was observed in the 200 mmHg group (E) when compared with the 0 mmHg group (D). Scale bars = 20 µm for all images.
Mentions: A significant increase in the expression of heat shock transcription factor 1 (Hsf1) and Hsf2 was observed in the 200 mmHg group compared with the 0 mmHg group (Hsf1 and Hsf2; p = 0.006 and  = 0.004, respectively; Fig. 3A). Expression of these genes is induced by the disruption of adhesion [26], and HSF1 and HSF2 bind to the regulatory site of various Hsp genes. Following this, we investigated the influence of compressive loading on the gene expression of various HSPs, which are stress-responsive proteins against mechanical stress, elevated temperature, hypoxia, lowered pH, and reactive oxygen species (ROS) [27]. The expression of various Hsps was significantly higher in the 200 mmHg group than in the 0 mmHg group (Hsp32, Hsp40, Hsp47, Hsp60, Hspa5, Hsc70, and Hsp90aa1; p = 0.002,  = 0.019, <0.001,  = 0.024,  = 0.033, <0.001, and <0.001, respectively; Fig. 3B). Upregulation of various Hsps indicates that stress responses by compressive loading occurred in fibroblasts. To examine the condition of nonapoptotic cells, we investigated the expression of antiapoptotic Bcl2[28] and proapoptotic Bax[29]. The results indicated that Bcl2 levels were significantly higher in the 200 mmHg group than in the 0 mmHg group, but Bax levels did not show any significant difference. (p = 0.001 and 0.851, respectively; Fig. 3C). Subsequently, we focused on HSP90α encoded by Hsp90aa1 and investigated the expression of HSP90α by immunocytochemistry, because Hsp32, known as an oxidative stress marker, is upregulated by compressive loading and oxidative stress leads to the release of HSP90α into the extracellular environment [30], [31]. Higher expression and nucleus translocation of HSP90α were observed in the 200 mmHg group when compared with the 0 mmHg group (Fig. 3D and 3E). Nucleus translocation of HSP90 occurs after cellular stress, and HSP90 tightly interacts with histones [32]. We decided to quantitatively evaluate HSP90α in the culture supernatants based on these observations.

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