Immunohistochemical analysis of TGF-β1-dependent α-SMA expression in aging fibroblasts. Monolayers of patient-matched young (A and C) and aged (B and D) dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (A and B) or serum-free medium containing 10 ng/ml TGF-β1 (C and D) for 72 hours. The cells were then fixed and stained for α-SMA, as described in Materials and Methods. The cells were then mounted in Vectashield fluorescent mountant and viewed under UV light. All results shown are representative of dermal fibroblasts from three patient donors. Original magnification, ×100. E: In a parallel experiment expression of α-SMA mRNA was quantified by QPCR. Confluent monolayers of patient-matched young and aged dermal fibroblasts were growth-arrested in serum-free medium for 48 hours, before addition of either serum-free medium alone (white bar) or serum-free medium containing 10 ng/ml TGF-β1 (black bar) for 72 hours. Total mRNA was extracted, and α-SMA expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control (unstimulated) aged fibroblasts. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of dermal fibroblasts from nine individual experiments with cells isolated from three patient donors. Statistical analysis was performed by the Student’s t-test: *P < 0.05; **P < 0.01, compared with aged cells.

Age-dependent effect on HA assessed by size exclusion chromatography. Confluent monolayers of patient-matched young and aged dermal fibroblasts were growth arrested in serum-free medium for 48 hours. The medium was removed and replaced with either serum-free medium alone or with serum-free medium containing 10 ng/ml of TGF-β1 for 72 hours. Cells were subsequently labeled for 24 hours with 20 μCi/ml [³H]glucosamine. The radiolabeled HA was isolated from the conditioned medium and subjected to size exclusion chromatography on a Sephacryl S500 column. A: Size exclusion chromatography profile of HA purified from resting (nonstimualted) young (closed circles) and aged (open circles) fibroblasts. B and C: Size exclusion chromatography profile of HA purified from young (B) and aged (C) dermal fibroblasts in the presence (open circles) or absence (closed circles) of TGF-β1. All results shown are representative of dermal fibroblasts isolated from at least two separate donors.

Effect of in vitro aging on extracellular HA generation. At the indicated PDL, confluent monolayers of dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for 72 hours. HA concentration in the culture medium was quantified by ELISA. Data represent the mean ± SE from six separate experiments using cells isolated from one donor. Statistical analysis was performed using analysis of variance to show global differences between PDLs for basal (P = 1.1E–09) and TGF-β1 (P = 1.9E–19) conditions, followed by Tukey’s honestly significant difference post hoc test analysis: *P < 0.001 compared with PDL 13.

Effect of age on HAS2 mRNA expression and its involvement in TGF-β1-dependent fibroblast activation. A and B: Confluent monolayers of patient-matched young and aged dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars), and the incubations were continued for 72 hours. HA secreted into the culture medium was quantified by ELISA. After removal of the culture medium, total mRNA was extracted and cDNA prepared as described in Materials and Methods. HAS2 expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control aged fibroblasts. The comparative C_T method was used for relative quantification of gene expression. Data are presented as the mean ± SE of six individual experiments using cells from two patient donors. Statistical analysis was performed by Student’s t-test: ^#not significant; *P < 0.01, compared with aged cells. N/S, not significant. C and D: To further examine the role of HAS2, young dermal fibroblasts were transfected with HAS2 siRNA or scrambled oligonucleotide control (scramble) and incubated in medium supplemented with 10% FBS for 24 hours. The medium was then replaced with serum-free medium for a further 24 hours, before addition of serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for 72 hours. Total mRNA was extracted and HAS2 (C) and α-SMA (D) expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control scramble samples. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of nine individual experiments using cells isolated from three different donors. Statistical analysis was performed by the Student’s t-test: N/S, not significant; *P < 0.01, compared with scramble.

Effect of aging on HA pericellular coat assembly. Subconfluent layers of young (A and C) and aged (B and D) dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (A and B) or with 10 ng/ml TGF-β1 (C and D) for 72 hours. Formalized horse erythrocytes were then added as described in Materials and Methods to visualize the HA pericellular coat. Arrows indicate the cell body; arrowheads show the extent of the pericellular matrix. Results are representative of dermal fibroblasts from three patient donors. Original magnification, ×200.

Overexpression of HAS2 in aged dermal fibroblasts. Aged dermal fibroblasts were transfected with either HAS2-pCR3.1 (HAS2 transfected) or pCR3.1 (Mock transfected). HA secreted into the culture medium 48 hours after transfection was quantified by ELISA (A). After removal of the culture medium, total mRNA was extracted. α-SMA expression was subsequently assessed by RT-QPCR (B). Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to mock transfected fibroblasts. The comparative C_T method was used for relative quantification of gene expression, and results represent the mean ± SE of dermal fibroblasts from nine separate experiments using cells isolated from three patient donors. Immunohistochemical analysis was performed to examine α-SMA protein expression on mock transfected (C) and HAS2-overexpressing (D) aged dermal fibroblasts 48 hours after transfection. The cells were then fixed and stained for α-SMA, mounted in Vectashield fluorescent mountant, and viewed under UV light. Original magnification, ×100. In parallel experiments, the HA pericellular coat was visualized. Forty-eight hours after transfection, formalized horse erythrocytes were added to mock-transfected (E) or HAS2-overexpressing (F) aged dermal fibroblasts as described in Materials and Methods to visualize the HA pericellular coat. Arrows indicate the cell body; arrowheads show the extent of the pericellular matrix. Results are representative of dermal fibroblasts from two patient donors. Original magnification, ×200.

Involvement of TSG-6 in TGF-β1-dependent fibroblast activation. A: Confluent monolayers of patient-matched young and aged dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars), and the incubations were continued for 72 hours. Total mRNA was extracted, and cDNA was prepared as described under Materials and Methods. TSG-6 expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control aged fibroblasts. The comparative C_T method was used for relative quantification of gene expression. Data are presented as the mean ± SE of six individual experiments with cells isolated from two patient donors. Statistical analysis was performed by Student’s t-test:*P < 0.05, compared with aged cells. N/S, not significant. B and C: To further examine the role of TSG-6, young dermal fibroblasts were transfected with TSG-6 siRNA or scrambled oligonucleotide control (scramble) and incubated in medium supplemented with 10% FBS for 24 hours. The medium was then replaced with serum-free medium for a further 24 hours, before addition of serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for 72 hours. Total mRNA was extracted and TSG-6 (B), and α-SMA (C) expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control scramble samples. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of nine individual experiments using cells isolated from three different donors. Statistical analysis was performed by Student’s t-test, and statistical significance was taken as P < 0.05. N/S, not significant.

Effect of HAS2 overexpression on pericellular HA coat assembly and phenotype after TGF-β1 in aged dermal fibroblasts. Patient-matched young (A and D) and aged (B, C, E, and F) dermal fibroblasts were transfected either with pCR3.1 alone (A, B, D, and E) or HAS2-pCR3.1 (C and F). Forty-eight hours after transfection, cells were incubated in serum-free medium containing 10 ng/ml TGF-β1 for 72 hours. The pericellular HA coat (A–C) was visualized by addition of formalized horse erythrocytes. Arrows indicate the cell body; arrowheads show the extent of the pericellular matrix. Images are representative of dermal fibroblasts from three patient donors. Original magnification, ×200. After stimulation the cells were fixed, and α-SMA was visualized by immunohistochemistry (D–F). Slides were mounted in Vectashield fluorescent mountant and viewed under UV light. Original magnification, ×100.

Effect of HAS2 overexpression on TGF-β1-dependent responses in aged dermal fibroblasts. Aged dermal fibroblasts were transfected either with HAS2-pCR3.1 or pCR3.1 alone (mock transfected). Parallel mock transfections were performed on patient-matched young dermal fibroblasts. Forty-eight hours after transfection, cells were incubated in either serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for 72 hours. α-SMA (A) and TSG-6 (B) mRNA expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control mock-transfected aged cells. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of nine individual experiments using cells isolated from three different donors. Statistical analysis was performed by Student’s t-test, and statistical significance was taken as P < 0.05. N/S, not significant.

HAS2 responsiveness is retained in aged cells after addition of IL-1β. Confluent monolayers of patient-matched young and aged dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (white bars), serum-free medium containing 10 ng/ml TGF-β1 (gray bars), or serum-free medium containing 1 ng/ml IL-1β (black bars) for a further 72 hours. mRNA was extracted, and cDNA prepared as described in Materials and Methods. HAS2 (A), TSG-6 (C), and α-SMA (D) expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control aged cells. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of six individual experiments using cells isolated from two different donors. Statistical analysis was performed by Student’s t-test and statistical significance was taken as P < 0.05. N/S, not Significant. B: HA secreted into the culture medium of aged cells was quantified by ELISA. Data are the mean ± SE of six individual experiments using cells isolated from two different donors. Statistical analysis was performed by Student’s t-test: *P < 0.05; **P < 0.01, compared with control cells.

Effect of IL-1β on HA pericellular coat assembly. Subconfluent layers of patient-matched young (A and C) and aged (B and D) dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (A and B) or 1 ng/ml IL-1β (C and D) for 72 hours. Formalized horse erythrocytes were then added as described to visualize the HA pericellular coat. Zones of exclusion were visualized using a Zeiss Axiovert 135 inverted microscope. Arrows indicate the cell body; arrowheads show the extent of the pericellular matrix. Images are representative of dermal fibroblasts from two patient donors. Original magnification, ×200.

Consequences of inhibiting HA coat assembly on TGF-β1-dependent phenotypic activation of fibroblasts. Confluent monolayers of young dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. Bovine testicular hyaluronidase (200 μg/ml) was then added in serum-free medium at 37°C for 1 hour before the addition (to the hyaluronidase) of either serum-free medium alone (white bars) or 10 ng/ml TGF-β1 (black bars) for a further 72 hours. In control experiments hyaluronidase treatment was replaced by adding serum-free medium alone. HAS2 (A) and α-SMA (B) mRNA expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to the control treatment in nonstimulated cells. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of six individual experiments using cells isolated from two different donors. Statistical analysis was performed by the Student’s t-test: *P < 0.05, compared with control treatment; N/S, not significant. In parallel experiments, serum-free medium alone (C) or bovine testicular hyaluronidase (200 μg/ml) (D) were added to growth-arrested cells for 1 hour before the addition of 10 ng/ml TGF-β1, and the incubations were continued for 72 hours. Formalized horse erythrocytes were then added to visualize the HA pericellular coat. Zones of exclusion were visualized using a Zeiss Axiovert 135 inverted microscope. Arrows indicate the cell body; arrowheads show the extent of the pericellular matrix. Images are representative of dermal fibroblasts from two patient donors. Original magnification, ×200. Immunohistochemical analysis was also performed to assess dermal fibroblast phenotype after treatment with hyaluronidase. Serum-free medium alone (E) or bovine testicular hyaluronidase (200 μg/ml) (F) was added to growth arrested cells for 1 hour before the addition of 10 ng/ml TGF-β1, and the incubations were continued for 72 hours. The cells were fixed and stained for α-SMA, mounted in Vectashield fluorescent mountant, and viewed under UV light. Original magnification, ×100. The relationship between the pericellular coat and regulation of the cell phenotype was also examined by CD44 gene silencing (G and H). Total mRNA was extracted from young dermal fibroblasts after transfection with CD44 siRNA or scrambled oligonucleotide control (scramble) and incubated in medium supplemented with 10% FBS for 24 hours. The medium was then replaced with serum-free medium for a further 24 hours, before addition of serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for 72 hours. CD44 (G) and α-SMA (H) expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control and gene expression was assessed relative to control-scrambled samples. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of six individual experiments using cells isolated from two different donors. Statistical analysis was performed by Student’s t-test: *P < 0.05; **P < 0.01, compared with scramble.