From the Department of Geriatric Medicine (R.M., J.H., N.T., I.K., M.A.,
A.M., S.H., T.O.) and the Institute for Molecular and Cellular Biology (Y.K.),
Osaka University Medical School, Suita; Shinotest Co, Tokyo (S.Y.); and the
Ohmiya Medical Center, Jichi Medical School, Omiya (I.S.), Japan.
Correspondence to Toshio Ogihara, MD, PhD, Department of Geriatric Medicine, Osaka University Medical School, Suita 565, Japan.
Abstract—Lipoprotein(a) [Lp(a)] is
well known to stimulate growth of vascular smooth muscle cells (VSMCs),
resulting in atherosclerosis. Its mechanism is
postulated to be decreased in active transforming growth factor
(TGF)-ß. However, the exact mechanisms and cellular processing from
apolipoprotein(a) [apo(a)] to Lp(a) have not yet been clarified
because no cultured cells producing apo(a) are available. Therefore, it
is necessary to establish apo(a)-producing cells to study the role of
apo(a). We evaluated the effects of overexpression of human apo(a) gene
on human aortic VSMC growth. First, we tested whether transfection of
apo(a) gene into human hepatoma cells, HepG2 cells, producing human
apoB resulted in the formation of Lp(a). Transfection of apo(a) gene
into HepG2 cells resulted in detectable levels of Lp(a) in the medium,
as assessed by ELISA and Western blot, whereas no Lp(a) was detected in
the medium of HepG2 cells transfected with control vector and
untransfected HepG2 cells. Expression of apo(a) mRNA was also confirmed
by reverse transcription–polymerase chain reaction. In contrast,
Western blotting showed a single band detected by specific anti-apo(a)
antibody, but not anti-apoB antibody, in the medium of
apo(a)-transfected VSMCs. These results demonstrate that Lp(a) can be
formed from apo(a) on HepG2 cells, whereas transfection of apo(a) gene
into VSMCs resulted in the production of apo(a) alone but not
Lp(a). Next, we examined the biological effects of overexpression of
apo(a) gene on growth of VSMCs and endothelial cells.
Incubation of cultured medium of HepG2 cells transfected with apo(a)
gene with human VSMCs or endothelial cells resulted in
a significant increase in cell number compared with the conditioned
medium of HepG2 cells transfected with control vector. In contrast,
transfection of apo(a) gene directly into VSMCs caused no significant
effect on VSMC growth. Therefore, we measured TGF-ß concentration in
the conditioned medium of VSMCs. However, using ELISA, only latent but
not active TGF-ß was detected in the medium of VSMCs. Moreover,
addition of neutralizing anti–TGF-ß antibody did not alter VSMC
growth. These results suggest that Lp(a) could stimulate growth of
VSMCs via the independent mechanisms from the inhibition of TGF-ß
activation. Overall, these data demonstrate that overexpression of
apo(a) gene in cells producing apoB results in formation of Lp(a),
resulting in a mitogenic action on human
endothelial cells and VSMCs. These results provide new
information to understand the mechanisms of the mitogenic
action of Lp(a) and suggest the role of Lp(a) in the pathogenesis
of atherosclerosis.
© 1998 American Heart Association, Inc.
Scientific Contributions
Conditioned Medium From HepG2 Cells Transfected With Human Apolipoprotein(a) Gene Stimulates Growth of Human Vascular Smooth Muscle Cells
Effects of Overexpression of Human Apolipoprotein(a) Gene
Key Words: atherosclerosis • transforming growth factors • hyperplasia • liposomes • gene transfer
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