The incorporation of fibrinogen into extracellular matrix is dependent on active assembly of a fibronectin matrix
Marian Pereira1,2,
Brain J. Rybarczyk1,2,
Tatjana M. Odrljin1,
Denise C. Hocking3,
Jane Sottile4 and
Patricia J. Simpson-Haidaris1,2,5
1 Department of Medicine-Vascular Medicine Unit, University of Rochester School
of Medicine and Dentistry, Rochester, NY 14642, USA
2 Department of Pathology and Laboratory Medicine, University of Rochester
School of Medicine and Dentistry, Rochester, NY 14642, USA
3 Department of Pharmacology and Physiology, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642, USA
4 Center for Cardiovascular Research, University of Rochester School of Medicine
and Dentistry, Rochester, NY 14642, USA
5 Department of Microbiology and Immunology, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642, USA
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Fig. 1. Time course of FBG assembly into the ECM. Confluent human foreskin
fibroblasts were incubated with 30 µg/ml Oregon-Green FBG for 1 (A,B), 6
(C,D) or 24 hours (E,F). Cells were washed, fixed and stained with
affinity-purified (to remove contaminating antibodies to FBG) rabbit anti-FN
IgG, followed by incubation with rhodamine-conjugated goat anti-rabbit IgG
(B,D,F). Panels (A,C,E) represent FBG-Oregon-Green fluorescence. Bar in (E)
represents 25 µm.
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Fig. 2. The effect of serum starvation and LPA on FBG assembly in the ECM of
fibroblasts. Confluent monolayers of human foreskin fibroblasts were
serum-starved for 24 hours. Medium containing 10% FBS (A,B), no FBS (C,D) or
500 nM LPA (E,F) supplemented with 40 µg/ml FBG-Oregon Green was added to
cells and incubated for an additional 18 hours. FBG is shown in (A,C,E), and
HSPG staining is shown in (B,D,F). Bar in (F) represents 25 µm.
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Fig. 3. FBG assembly into mature matrix fibrils is Rho dependent. Human foreskin
fibroblasts were serum starved for 24 hours prior to treatment with 1%
FBS-medium with (A) or without (B) PLB (0.1 U/ml) in the presence of
FBG-Oregon Green (40 µg/ml). To inhibit RhoA activation, C3 transferase (2
µg/ml) was delivered to serum-starved HFF via LipofectAMINE (10 µg/ml)
(C); control cells were treated only with LipofectAMINE (10 µg/ml) (D).
FBG-Oregon Green (30 µg/ml) in 1% FBS-medium was added to cells and
incubated for an additional 4 hours. Bar in (B) represents 25 µm.
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Fig. 4. Effect of 9D2 on FBG matrix deposition and recovery. Confluent human
foreskin fibroblasts were treated with 30 µg/ml 9D2 for 18 hours. This was
followed by incubation with 30 µg/ml FBG-Oregon-Green in the continued
presence (A-F) or absence of 9D2 (G-L). MoAb 9D2 was removed (G-L) by washing
the cells after the 18 hours treatment. Cells were washed, fixed and stained
for FN (B,D,F,H,J,L). Bars in (E) and (K) represent 25 µm.
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Fig. 5. FN and FBG incorporation into FN-/- cell matrix. Confluent
FN-/- cells were incubated with 20 µg/ml FBG-Oregon Green in the
presence (A-C) or absence (D-F) of 10 µg/ml FN. FN (A,D), FBG (B,E) and
phase contrast (C,F) images are shown. Bar in (F) represents 25 µm.
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Fig. 6. Binding of 125I-FN to FN-/- cell layers. Confluent
FN-/- cell layers grown in 48 well plates were incubated for 1 hour
at 37°C with 0.2 ml defined medium containing 0.2% BSA and increasing
concentrations of 125I-FN. Nonspecific binding (closed triangles)
was determined by adding excess unlabeled FN (500 µg/ml) to the incubation
medium. Specific binding (closed circles) was assessed by subtracting
nonspecific binding from total binding (open squares). Each point represents
the average of triplicate determinations from a representative experiment.
Bars represent±s.e.m.
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Fig. 7. Effect of MoAb 9D2 on 125I-FBG binding to FN-/- cell
layers. Confluent FN-/- cell layers grown in 48-well plates were
incubated for 1 hour at 37°C with 0.2 ml defined medium containing 0.2%
BSA, FN (25.8 µg/ml) and 125I-FBG (20 µg/ml) in the presence
of increasing concentrations of MoAb 9D2 (closed squares) or nonimmune
IgG1 (open circles). After 1 hour of binding, cell monolayers were
washed, solubilized in 1N NaOH and radioactivity measured. Binding of
125I-FBG is expressed as percentage of binding observed in the
absence of 9D2. These data are representative of two experiments each done in
triplicate at each IgG1 concentration. Bars
represent±s.e.m.
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Fig. 8. Effect of 9D2 on FBG and FN incorporation into ECM. Confluent
FN-/- cells were treated with FN (25.8 µg/ml) and FBG (20
µg/ml) in the absence (A-C) or presence (D-F) of 35 µg/ml MoAb 9D2 or
nonimmune mouse IgG1 (G-I). Cells were washed, fixed and stained
for FBG in green fluorescence (A,D,G) and FN in red fluorescence (B,E,H).
Insets represent background staining for green (D) and red (E) fluorescence.
Composite images shown in (C,F,I) indicate colocalization of FBG and FN in
yellow-orange fluorescence. Bar in panel I represents 25 µm.
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© The Company of Biologists Ltd 2002
