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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 9, 2001 as doi:10.1096/fj.00-0668fje. |
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* Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, 10043 Orbassano, Torino, Italy;
Department of Molecular Pharmacology and Toxicology, University of South California, School of Pharmacy, Los Angeles, California 90033, USA;
Faculty of Engineering and Natural Sciences, Sabanci University, Karaköy, Istanbul, Turkey; and
CNR Centre of Immunogenetics and Experimental Oncology, 10126 Torino, Italy
2Correspondence: Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, 10043 Orbassano, Torino, Italy. E-mail: guiseppe.poli{at}unito.it
SPECIFIC AIMS
Oxidized low density lipoproteins (oxLDL) may contain variable amounts of oxysterols, i.e., 27-carbon products of cholesterol oxidation, which may become quantitatively important in hypercholesterolemic subjects. Oxysterols have been shown to exert a great many biochemical effects, but their potential action as lipid oxidation products on the macrophage expression of fibrogenic cytokines has not yet been investigated.
PRINCIPAL FINDINGS
1. A biologically compatible mixture of oxysterols is by far less
cytotoxic than comparable amounts of individual oxysterols
The potential cytotoxicity to cultured macrophages of a mixture of
cholesterol oxidation products, whose composition is compatible with
that of oxLDL in hypercholesterolemic patients or of comparable amounts
of single sterols (7-ketocholesterol and unoxidized cholesterol), was
initially evaluated. Cell viability and nuclear morphology were
respectively checked by trypan blue test and fragment end labeling of
DNA (TUNEL). A slight decrease in cell viability, consistently in a
range of 10–15%, was observed only after long-term (24 h) incubation
with the highest (30 µM) concentration used of the oxysterol mixture
or single sterols. On the contrary, a striking difference was observed
as regards the proapoptotic effect of the various treatments. As
reported in Fig. 1
, after 24 h treatment with oxysterol mixture or unoxidized
cholesterol only a few cells showed fragmented and/or condensed nuclei.
Apoptotic cells were, however, numerous in the experimental
samples challenged with comparable amounts of 7-ketocholesterol.
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2. Oxysterols up-regulate transforming growth factor ß1
(TGF-ß1) mRNA level in macrophagic cells
Brief exposure of J774A.1 murine macrophages to the oxysterol
mixture resulted in a marked up-regulation of the steady-state TGF-ß1
mRNA levels (Fig. 2
). The oxysterol mixture was added to the culture medium at a final
concentration ranging from 10 to 30 µM, i.e., in amounts
approximating that found in the plasma of hyperocholesterolemic
subjects. Over this dose range, there was an up-regulation of TGF-ß1
after 30–90 min incubation. The oxysterol mixture at 20 µM
consistently had the strongest effect and none of the components of the
oxysterol mixture was per se responsible for the observed cytokine
up-regulation. Further, contrary to the oxysterol mixture, identical
amounts of 7-ketocholesterol did not significantly modify TGF-ß1 mRNA
steady-state levels in J774A.1 macrophages.
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3. Unlike unoxidized cholesterol or 7-ketocholesterol, oxysterols
up-regulate TGF-ß1 synthesis
To check whether the up-regulation of TGF-ß1 mRNA levels as
induced by the oxysterol mixture was actually followed by increased
TGF-ß1 protein levels, Western blot analysis was performed on samples
from the whole cell suspension after 24 h culture with or without
oxysterol mixture or 7-ketocholesterol. The concentration of the
oxysterol mixture that up-regulated TGF-ß1 mRNA steady-state levels
also produced a significant rise in TGF-ß1 protein levels
(
1.8-fold increase). Consistent with Northern blot data, treatment
of J774A.1 macrophages with 7-ketocholesterol did not modify TGF-ß1
synthesis. The solvent used to suspend oxysterol/s in cell medium had
no effect per se on TGF-ß1 expression.
The treatment of macrophages with the oxysterol mixture also significantly increased the amount of active cytokine as detected by enzyme-linked immunoassay. Again, the 20 µM concentration of the oxysterol mixture appeared to be most effective, increasing steady-state levels of TGF-ß1 by > 30%. A similar finding was obtained when treating human promonocytic U937 cells. In both cell lines, unoxidized cholesterol had no effect on TGF-ß1 synthesis.
CONCLUSIONS AND SIGNIFICANCE
The results reported here indicate that the incubation of murine and human macrophages with a biologically representative mixture of oxysterols is able to promote markedly both expression and synthesis of one of the most potent proinflammatory and fibrogenic cytokines, TGF-ß1. On the contrary, no effect on TGF-ß1 synthesis was found with unoxidized cholesterol.
As appropriately stressed in a couple of recent reviews on origin and biological relevance of oxysterols, only a few reports address the effects of these cholesterol oxidation products on inflammatory cytokine expression. Individual oxysterols were shown to up-regulate the secretion of interleukin 1ß (IL-1ß) by U937 cells and human endothelial cells and the production of IL-8 in human macrophages. In all these studies, the effect of unoxidized cholesterol was not evaluated. An increasing amount of data supports the possibility that oxidized LDL modulates the expression of a large number of genes, including some coding for inflammatory cytokines. It appears that one specific effect related to the fibrogenic action of oxidized LDL may be associated with oxysterol content.
Oxysterols may account for 1–2% of total cholesterol in
atherosclerotic lesions, whereas plasma oxysterol content has been
reported to be around 0.3 µM. They have been reported to comprise up
to 30–40% of total cholesterol in atherosclerotic lesions or in
plasma lipoproteins of hypercholesterolemic subjects, however, these
high levels may be due to oxidation artifacts during isolation and
analysis. Conclusive evidence that all the oxysterols studied are
present in human plasma is not available at this time, but several
reports suggest that some, if not all, can be found in plasma to a
variable extent whereas others studies indicate that they are nil or
absent. Indeed, analysis of samples derived from human clinical trials
(270 samples) indicates that levels range from 0 to 7% of plasma
cholesterol. We selected a mixture of oxysterol representative of
plasma samples analyzed in our lab but similar in proportion as
reported by others. These oxysterols are likely derived from
cholesterol peroxidation in various lipid matrices.
Another lipid oxidation product, the aldehyde 4-hydroxynonenal (HNE),
was previously shown to induce a profibrogenic response through
up-regulation of TGF-ß1 gene in a manner comparable to oxysterols.
HNE has been shown to derive from oxidative breakdown of 
-6
polyunsaturated fatty acids (PUFA), which are abundant in LDL. It is
plausible that the formation of cholesterol oxidation products is an
intrinsic component of the free radical chain initiation and
propagation reactions involved in PUFA oxidation, and both HNE and
oxysterols are consistently detectable in the fibrotic plaques.
Together, these findings point to the primary role for oxidation of LDL
lipids in stimulating fibrogenic cytokine formation as an integral step
in the atherogenic process (see Fig. 3
). In particular, the results described here indicate a possible
mechanistic link between increased cholesterol in plasma and arteries
and the stimulated fibrogenesis, a process that is characteristic of
atherosclerotic lesion formation. Uptake of cholesterol by macrophages
can give rise to foam cells that characterize the formation of
atheroma, but these phagocytes appear to require cholesterol oxidation
products in order to adequately generate fibrogenic stimuli.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0668fje ; to cite this article, use FASEB J. (May 9, 2001) 10.1096/fj.00-0668fje 
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) and blood monocytes (M) become attracted and
activated by this way driving an inflammatory reaction. The phagocyte
uptake of LDL polyunsaturated fatty acids and cholesterol, already
oxidized or undergoing oxidation in the vasculature, gives rise to foam
cells (FoC) and up-regulates expression of inflammatory and fibrogenic
cytokines. TGF-ß overexpression due to oxysterols and other lipid
oxidation products like 4-hydroxynonenal (HNE) likely plays a pivotal
role in smooth muscle cell (SMC) chemoattraction and differentiation
into myofibroblast-like cells (MyoF), with consequent excessive
production of extracellular matrix (ECM). A further contribution to
overexpression of fibrogenic cytokines may derive from platelet
(Ptls) aggregation.