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Olivier Le Saux, Severa Bunda, Christopher M

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1 Serum Factors from Pseudoxanthoma Elasticum Patients Alter Elastic Fiber Formation In Vitro 
Olivier Le Saux, Severa Bunda, Christopher M. VanWart, Vanessa Douet, Laurence Got, Ludovic Martin, Aleksander Hinek  Journal of Investigative Dermatology  Volume 126, Issue 7, Pages (July 2006) DOI: /sj.jid Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Extracellular matrix components deposited by control and PXE fibroblasts. Fibroblasts were derived from normal and PXE patients and were cultured in medium supplemented with 10% FBS. Fibroblasts were grown for 10 days and the deposited matrix network was revealed by immunofluorescence using antibodies specific to (a, b) elastin, (c, d) fibrillin-1, (e, f) fibronectin, and (g, h) chondroitin sulfate-containing glycoaminoglycan. Representative photomicrographs are presented. (b) No structural difference in the matrix network was visible between normal and PXE cells, except for the overabundance of elastin. Nuclei were counterstained with propidium iodide to reveal cell density. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Extracellular matrix components detected by immunohistochemistry. 10-day-old cultures of normal and PXE fibroblasts supplemented with 10% FBS were used. The morphometric analysis of extracellular matrix components was performed using Image-Pro Plus software from Media Cybernetics. MAGP: microfibril-associated glycoprotein-1, CS-GAG: chondroitin sulfate-containing glycoaminoglycan. Each ECM group was analyzed from three separate cultures of three normal and three PXE fibroblast cell lines, and the area occupied by the particular immunodetectable component was quantified. The abundance of each component was then expressed as a percentage of the entire analyzed field (mean±SD (*P<0.002; **P<0.05). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Quantitative analysis of insoluble elastin. The total insoluble elastin labeled with [3H]valine and produced by three normal skin fibroblasts (□) and three PXE skin fibroblasts (▪) after 10 days of cultures was quantified. PXE cells deposited on average 41% more insoluble elastin than their unaffected counterparts in the presence of 10% FBS in the culture medium. Values of mean±SD from four different experiments were collected for statistical evaluation (*P<0.01). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Extracellular matrix gene expression in normal and PXE fibroblasts. The fibroblasts were cultured in medium supplemented with 10% FBS. Total RNA was extracted after 10 days of culture and was reversed transcribed. Real-time PCRs were performed using TaqMan probes (Applied Biosystems) specific for tropoelastin (ELN), fibrillin-1 (FBN-1), microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), lysyl oxidase-like (LOXL), collagen type I (COL1), and the β-actin cDNAs. Units are ratio of the relative gene expression to β-actin and are shown normalized to expression in normal fibroblasts. Standard errors are indicated (n=3, *P<0.01). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Immunofluorescent labeling of elastin deposited by normal and PXE fibroblasts. The dermal fibroblasts derived from normal and PXE patients were cultured in medium supplemented with 10% serum samples from normal individuals or PXE patients. Fibroblasts were grown for 10 days and the network was revealed by immunofluorescence using an anti-elastin antibody. Representative photomicrographs of normal and PXE fibroblasts grown in normal serum are shown in panels a and b, respectively, while panels c–h represent three different PXE serum samples. Arrows point to large abnormal deposits of immunodetectable elastin. Nuclei were counterstained with propidium iodide to reveal cell density. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Representative electron micrographs of cultures of skin fibroblasts maintained in 10% human sera. Upper panel: 5-day-old cultures of normal skin fibroblasts grown with (a) normal sera and (b) from a PXE patient. While cells maintained in the presence of normal human serum produce normal microfibrills, fibroblasts treated with PXE-patient serum deposited irregular and fragmented microfibrils. Lower panel: electron micrographs showing 10-day-old cultures of normal skin fibroblasts maintained in sera from (c) unaffected individuals and (d) from a PXE subject. Cells maintained with normal serum deposit well-assembled elastic fibers, but cultures treated with PXE serum contain abnormal and fragmented fibers on an irregularly assembled microfibrillar scaffold. MF: microfibrils, EF: elastic fiber, F: fibroblasts. Image scale is indicated on panel d. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Extracellular matrix gene expressions in normal and PXE fibroblasts. The fibroblasts were cultured in medium supplemented with 10% normal or PXE serum. Total RNA was extracted after 10 days of culture and was reversed transcribed. Real-time PCRs were performed using TaqMan probes (Applied Biosystems) specific for tropoelastin (ELN), fibrillin-1 (FBN-1), microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), lysyl oxidase-like (LOXL), collagen type I (COL1), and the β-actin cDNAs. Units are ratio of the relative gene expression to β-actin and are shown normalized to expression in normal fibroblasts. Standard errors are indicated (n=3, **P<0.05). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Immunofluorescent labeling of elastin deposited by aortic smooth muscle cells. The smooth muscle cells were derived from a normal 6-year-old individual in culture medium supplemented with 10% serum samples from normal individuals or PXE patients. The cells were grown for 5 days and the network was revealed by immunofluorescence using an anti-elastin antibody. Representative pictures of smooth muscle cells grown in normal serum are shown in panels a and c, respectively, while panels b and d represent cells cultured with PXE serum samples. Arrows point to large abnormal deposits of immunodetectable elastin. Nuclei were counterstained with propidium iodide. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

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