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1.
Figure 8

Figure 8. Schematic representation of the role of MFAP-4 in elastic fiber formation.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

(a) Secreted MFAP-4 and fibrillin-1 interact directly, resulting in the assembly of microfibrils under normal conditions. Tropoelastins assembled with LOX recruited by fibulin-4 are subsequently cross-linked with microfibrils for the formation of complete mature elastic fibers. (b) In the absence of MFAP-4, fibrillin-1 cannot be assembled or matured for the formation of microfibrils. Tropoelastins are never cross-linked due to the failure of microfibril formation.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
2.
Figure 2

Figure 2. More decreased deposition of MFAP4 in photodamaged/photoaged skin than intrinsically aged skin.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

Immunohistological staining with rabbit polyclonal anti-human MFAP-4 and normal rabbit control IgG was conducted on paraffin embedded sections of biopsied skin specimens. They were from the ventral upper arm of the donors in their thirties and sixties, or from the dorsal lower arms of the donors in their sixties. Scale bars = 50 μm.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
3.
Figure 6

Figure 6. MFAP-4 directly interacts with fibrillin-1 to form microfibrils.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

(a) Concentrated supernatants from NHDFs cultured for 8 days were immunoprecipitated with an anti-MFAP-4 antibody or normal rabbit IgG. Immunoprecipitants were analyzed by Western blotting with an anti-fibrillin-1 antibody. (b) NHDFs were transfected with siRNAs for non-specific sequences (Control) or MFAP-4 twice during the culture for 8 days, followed by immunofluorescence staining with an anti-human MFAP-4 antibody (green) and an anti-human fibrillin-1 antibody (red). Nuclear staining (DAPI) and merged images are also shown. Scale bars = 50 μm.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
4.
Figure 4

Figure 4. Effect of MFAP-4 over-expression on the UVB-induced increase in MMP-12 activity.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

(a) Luna staining was performed to evaluate the role of MFAP-4 in the protection of elastic fibers using paraffin-embedded sections from non-exposed skin with a control reporter gene over-expression, a control reporter gene-transduced skin with UVB irradiation for 8 wks and MFAP-4-over-expressed skin with repetitive UVB irradiation for 8 wks. Scale bars = 100 μm. (b) MMP-12 activities in xenografted human skins treated with lentiviral vectors encoding a control reporter gene with or without UVB irradiation for 8 wks and UVB-exposed skin with MFAP-4 over-expression were evaluated using fluorescent-tagged MMP-12 substrate as detailed in the Methods. The values reported represent means ± SD. **p<0.01; vs the value in UVB-exposed skin with a control reporter gene over-expression.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
5.
Figure 1

Figure 1. Expression of MFAP-4 transcripts in photodamaged/photoaged and in intrinsically aged skins.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

Quantitative RT-PCR analysis of MFAP-4 in human skin was performed. (a) The expression of MFAP-4 in xenografted human skin after repeated UVB irradiations with 1 to 2 MED for 6 wks was normalized to the expression of the ribosomal protein, RPLP0, and the relative value compared to the non-irradiated control is shown. (b) MFAP-4 expression in human skin punch biopsies at the ventral area of upper arms from young (twenties) and old (fifties) Caucasian women. The results normalized by RPLP0 expression are presented. Values reported represent means ± SD. *p<0.05, **p<0.01.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
6.
Figure 5

Figure 5. MFAP-4 is essential to the assembly of elastic fibers.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

NHDFs were transfected with siRNAs for non-specific sequences (Control) or MFAP-4 twice during the culture for 8 days. Control siRNA-transfected cells were treated with or without 10 nM human recombinant MFAP-4 during the culture. (a) Quantitative RT-PCR analysis was performed with MFAP-4-specific TaqMan Gene Expression Assays after the reverse transcription of total RNAs from cultured NHDFs. The mRNA expression of the target gene was normalized against the expression of GAPDH mRNA and is relatively presented. The values reported represent means ± SD. ***p<0.001. (b) Western blotting analysis with antibodies specific for MFAP-4 or fibrillin-1 was conducted to confirm the impact of MFAP-4 silencing on their protein abundance using the supernatants from cultured NHDFs. (c) Immunofluorescence staining was performed with an anti-human tropoelastin antibody (green) and an anti-human fibrillin-1 antibody (red). Nuclear staining (DAPI) and merged images are also shown in the diagram. Scale bars = 50 μm. (d) Quantitative RT-PCR analysis of the MMP-12 mRNA expression in cultured NHDFs was performed as described in the legend of Fig. 5a. The values reported represent means ± SD. ***p<0.001, **p<0.01. (e) Western blotting analysis with an anti-human MMP-12 antibody was performed for the supernatants from cultured NHDFs. The obtained bands of the active form of MMP-12 were analyzed using a densitometer and the values in the graph are represented relatively. The values reported represent means ± SD. *p<0.05. (f) NHDFs were cultured for 8 days with or without 20 nM human recombinant MFAP-4 during the culture in FBS-free medium. Immunofluorescence staining was performed as described in the legend of Fig. 5c. Scale bars = 50 μm.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
7.
Figure 7

Figure 7. MFAP-4 over-expression prevents UVB-induced deterioration of collagen I.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

(a) Immunohistochemical analysis with a rabbit anti-human collagen I antibody or a non-specific control rabbit IgG was carried out using paraffin embedded sections from the xenografted skin treated with a lentiviral vector encoding a control reporter gene with or without continuous UVB irradiation for 8 wks and using xenografted skin over-expressing MFAP-4 with UVB exposure for 8 wks. Scale bars = 100 μm. (b) Western blotting analysis with an anti-human collagen I or an anti-human β-actin antibody to assess the protein levels of collagen I in xenografted skin treated with lentiviral vectors encoding a control reporter gene or MFAP-4 before and after UVB exposure for 4 wks. (c) NHDFs were transfected with siRNAs for non-specific sequences (Control) or MFAP-4 twice during the culture for 8 days. Control siRNA-transfected cells were treated with or without 10 nM human recombinant MFAP-4 during the culture. Quantitative RT-PCR analysis of the MMP-1 mRNA expression in cultured NHDFs was performed as described in the legend of Fig. 5a. The values reported represent means ± SD. ***p<0.001. (d) Western blotting analysis with an anti-human MMP-1 antibody was conducted for supernatants from cultured NHDFs treated as described in Fig. 7c. The obtained bands of the active form of MMP-1 were analyzed using a densitometer and the values in the graph are represented relatively. The values reported represent means ±SD. **p<0.01, *p<0.05.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.
8.
Figure 3

Figure 3. Protection of skin from UVB-induced photodamage by enhancement of MFAP-4 expression.. From: Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

(a) Lentiviral vectors to over-express MFAP-4 or a control reporter gene were intradermally administered into human xenografted skin twice with a week interval, subjected to continuous UVB irradiations at 1 to 2 MED for 8 wks as indicated. (b) Immunohistological staining with rabbit polyclonal anti-human MFAP-4 and normal rabbit control IgG was performed on paraffin embedded sections from non-exposed skin with a control reporter gene over-expression, a control reporter gene-transduced skin with UVB irradiation for 8 wks and MFAP-4-over-expressed skin with repetitive UVB irradiation for 8 wks. Scale bars = 100 μm. (c) En face representative images of xenografted skin were captured at 0 (before the lentiviral administration), 6 (before the UVB irradiation), 10, 14 (just after the completion of continuous UVB exposures) and 20 wks (6 wks after the final UVB irradiation) using a hand-held microscope. The non-UVB irradiated control with a control reporter gene over-expression, the long-term UVB-irradiated skin with a control reporter gene over-expression and MFAP-4-over-expressed skin with repetitive UVB exposure are shown at the top, middle and bottom, respectively. Scale bars = 3 mm. (d) Skin profilometry of replicas was utilized to evaluate surface roughness using arithmetic mean roughness (Sa) and mean peak to valley height (Sz) at the indicated time points. The values reported represent means ± SD. *p<0.05, **p<0.01. 1; vs the values of the UVB-irradiated control with a control reporter gene over-expression. 2; vs the values of the non-irradiated control with a control reporter gene over-expression. (e) Skin elasticity was evaluated using a Cutometer SEM575 at the same time points as the assessments of skin surface roughness. Ur/Ue represents pure elasticity ignoring creep. The values reported represent means ± SD. *p<0.05. 1; vs the values of the UVB-irradiated control with a control reporter gene over-expression.

Shinya Kasamatsu, et al. Sci Rep. 2011;1:164.

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