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J. Biol. Chem., Vol. 266, Issue 26, 17686-17694, Sep, 1991
I Takasaki, AV Chobanian and P Brecher
The in vitro interactions between vascular cells and fibronectin have been
shown to influence phenotypic expression of both cultured endothelial and
smooth muscle cells. To more effectively assess the potential functional
role of fibronectin in vivo in modulating vascular phenotypes, we have
established methodology for studying fibronectin biosynthesis in the rabbit
aorta using aortic rings that are morphologically and functionally intact
and metabolically active. Aortic rings were incubated with 35S-labeled
methionine in a supplemented physiological salt solution. The tissue was
fractionated, and quantitative immunoprecipitation was performed using a
polyclonal antibody directed against human plasma fibronectin. Newly
synthesized fibronectin was most abundant in the fraction solubilized using
4% sodium dodecyl sulfate and in the incubation medium. In all fractions
studied, fibronectin was present predominantly as a dimer with no
detectable aggregates of fibronectin. Pulse-chase experiments showed that a
substantial amount of newly synthesized fibronectin was found in the 4%
sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin
was rapidly incorporated into the extracellular matrix. The more soluble
forms of newly synthesized fibronectin appeared to be the precursors for
secreted fibronectin, and no precursor-product relationship between soluble
and insoluble fibronectin was found. Dissection of aortic rings following
incubation with labeled methionine showed that newly synthesized
fibronectin was uniformally distributed in both intima-media and
media-adventitia segments. Endothelial cell denudation caused only a 20%
decrease of fibronectin biosynthesis concomitant with similar changes in
total protein biosynthesis, consistent with the medial smooth muscle cell
as the major source of newly synthesized fibronectin. Biosynthesis of
fibronectin was increased following a 24-h preincubation of the aortic
rings, and concomitant increases in steady state mRNA for fibronectin were
found. These in vitro studies documented the utility of aortic rings for
the general purpose of studying protein synthesis in vascular cells and
provide new information on the characteristics of fibronectin biosynthesis
by aortic tissue.
Biosynthesis of fibronectin by rabbit aorta
Department of Medicine, Boston University School of Medicine, Massachusetts 02118.
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