First published online 17 October 2006
doi: 10.1242/jcs.03171
Journal of Cell Science 119, 4499-4509 (2006)
Published by The Company of Biologists 2006
Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells
Svetlana A. Kuznetsova1,
Philip Issa1,
Elizabeth M. Perruccio1,
Bixi Zeng1,
John M. Sipes1,
Yvona Ward2,
Nicholas T. Seyfried3,
Helen L. Fielder3,
Anthony J. Day3,
Thomas N. Wight4 and
David D. Roberts1,*
1 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
2 Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
3 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
4 The Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101, USA
View larger version (89K):
[in a new window]
|
Fig. 5. Induction of extracellular matrix TSP1 and versican by a TLR3 agonist. (A) HASMC were incubated in control medium or medium containing 5 µg ml–1 poly I:C for 4 hours or 15 hours, followed by fixation and double immunofluorescent staining without permeabilization using antibodies to versican (green) and to TSP1 (red). Nuclei were counterstained with Hoechst 33258 (blue). Note that co-localization of versican and TSP1 appears as yellow close to the cell surface after treatment of HASMC with poly I:C in a time-dependent manner. Bars, 50 µm. Results are representative of at least three independent experiments. (B,C) Confocal images showing extracellular localization of TSP1 and versican after stimulation with poly I:C. HASMC were cultured without treatment (B, left) or treated with 5 µg ml–1 poly I:C for 15 hours (B, right and C) and cells were fixed, permeabilized and double stained for versican (green) and TSP1 (red). Stack of images (C, 1-6) through the Z-plane from inside (1) of the cells to the cell surface was generated using confocal microscopy. Nuclei were counterstained with DAPI (blue). Bar, 10 µm. (D) Confocal 3D reconstruction of 18 hour poly I:C-stimulated HASMC stained without permeabilization for versican (green) and TSP1 (red). Bar, 10 µm.
|
|
View larger version (87K):
[in a new window]
|
Fig. 6. Distinct localization of extracellular hyaluronan and proteins induced by poly I:C. (A) Versican-rich matrix generated by inflammation is mostly distinct from the stress-induced hyaluronan-rich matrix. Image of versican (green, bottom left quarter) and hyaluronan (red, top right) are shown together with phase contrast image (top left) and merge (bottom right). Only limited co-localization of versican and hyaluronan was observed after stimulation with 5 µg ml–1 poly I:C for 17 hours. (B) Inflammation induces increased accumulation and co-localization of TSP1 with elastin. HASMC were cultured with 5 µg ml–1 poly I:C for 4 hours before being processed for immunofluorescence staining. Note that the anti-elastin antibody (green) binds to many of the same structures that are labeled with the anti-TSP1 antibody (red). Nuclei were counterstained with Hoechst 33258 (blue). Bars, 50 µm (A); 20 µm (B).
|
|
View larger version (50K):
[in a new window]
|
Fig. 7. Inflammation-induced binding and turnover of exogenous TSP1. (A) HASMC were cultured for 15 h in either control (left panels) or 5 µg ml–1 poly I:C containing medium (right panels). TSP1 (10 µg ml–1) was added at the indicated time points before completion of the 15 hour incubation period. Immunofluorescent staining of nonpermeabilized cells shows that TSP1 binding to the extracellular matrix is induced by poly I:C in a time dependent manner. However, prolonged incubation with soluble TSP1 in the presence of poly I:C shows reorganization of matrix-associated TSP1 into punctate structures. (B) Confocal microscopy of HASMC, followed by stimulation with 5 µg ml–1 poly I:C in the presence of TSP1 (10 µg ml–1) at 4 hours. Permeabilized or non-permeabilized cells were fixed and stained for TSP1 (green), nuclei (blue) and F-actin (red). Representative confocal images showing that TSP1 containing punctate structures have both an intracellular (bottom, left) and an extracellular distribution (upper right panel). Bars, 50 µm (A); 10 µm (B).
|
|
View larger version (111K):
[in a new window]
|
Fig. 8. High concentrations of exogenous TSP1 inhibit inflammation-induced extracellular deposition of versican and elastin. HASMC were cultured without (control) or with 5 µg ml–1 poly I:C for 4 hours in the presence or absence of 20 µg ml–1 TSP1. Cells were subsequently fixed and double stained as indicated in the Materials and Methods. Exogenous TSP1 diminished versican and elastin accumulation, but not hyaluronan, as seen by double immunofluorescence staining for TSP1 (red), for versican, elastin or hyaluronan (green) and nuclei (blue). Bars, 50 µm.
|
|
View larger version (101K):
[in a new window]
|
Fig. 9. Heparin inhibits TLR3-induced deposition of exogenous TSP1. HASMC were cultured in standard medium (control) or in the presence of 5 µg ml–1 poly I:C for 15 hours, followed by an additional 1 hour incubation in culture medium containing 10 µg ml–1 TSP1 with or without the indicated concentrations of heparin. Staining with antibody against TSP1 showed that binding of exogenous protein to the structures generated by cells following poly I:C treatment was inhibited by heparin in a dose-dependent manner. Bar, 50 µm.
|
|
View larger version (14K):
[in a new window]
|
Fig. 10. Effects of poly I:C on matrix protein synthesis and secretion. (A) Real time PCR. HASMCs were treated with or without 5 µg ml–1 poly I:C for 2 or 24 hours and relative mRNA expression levels using Taqman probes for elastin, versican and TSP1 were quantified using the comparative Ct method with HPRT as the internal standard. Expression data is normalized to that of untreated cells. The graph represents 2 separate experiments, each run in duplicate. All data are mean ± s.d. for quadruplicate determinations. (B) Effect of poly I:C treatment on accumulation of TSP1 in the medium of HASMC at the indicated times following transfer to serum free medium (closed bar) or the same medium containing 5 µg ml–1 poly I:C.
|
|
View larger version (13K):
[in a new window]
|
Fig. 11. Model for inflammation-induced matrix complexes on VSMC.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2006
) or 5 mM EDTA (
) for 3 hours at 37°C. Background values were subtracted from each data point (137.3±13.2 and 87.2±9.6, respectively). All data are mean ± s.e.m. for quadruplicate determinations.
) or 5 mM EDTA (
). The curves represent the best fit displacement curves calculated for a single site model using the program LIGAND. (B) Wells were coated using 0.5 µg per well TSP1, blocked with 3% BSA for 1 hour and incubated with the indicated amounts of biotinylated G1 domains of versican (B) or aggrecan (C) at 37°C for 3 hours in a final volume of 50 µl of DPBS with Ca2+ and Mg2+ containing 0.5% BSA and 0.1 mM phenylmethylsulfonyl fluoride. After washing to remove unbound proteins, the wells were incubated with 1 µg ml–1 [125I]-streptavidin for 1 hour at room temperature, washed and the bound radioactivity quantified. Background values were subtracted from each data point (1150.8±99.2).
), P3E123 (
), or E3CaG (
).
) or 0.3 µg per well of VG1 (
) was determined in the presence of the indicated concentrations of heparin. Binding was normalized to that determined in the absence of heparin (748.8±24.2 and 12658.8±117.4 cpm, respectively). All data are mean ± s.e.m. for quadruplicate determinations.