© 1999 American Heart Association, Inc.
Vascular Biology |
Peroxisome Proliferator–Activated Receptor Activators Target Human Endothelial Cells to Inhibit Leukocyte–Endothelial Cell Interaction
From the Department of Medicine (S.M.J., F.P., X.-P.X., W.A.H., R.E.L., L.L.D.), the Division of Cardiology (S.M.J., F.P., L.L.D.), the Division of Endocrinology, Diabetes, and Hypertension (X.-P.X., W.A.H., R.E.L.), and the Department of Pathology (J.A.B.), University of California, Los Angeles, School of Medicine, Los Angeles.
Correspondence to Simon M. Jackson, PhD, Division of Cardiology, UCLA School of Medicine, 47-123 CHS, Box 951679, Los Angeles, CA 90095-1679. E-mail smjack{at}ucla.edu
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Abstract—An early event in acute and chronic inflammation and associated diseases such as atherosclerosis and rheumatoid arthritis is the induced expression of specific adhesion molecules on the surface of endothelial cells (ECs), which subsequently bind leukocytes. Peroxisome proliferator–activated receptors (PPARs), members of the nuclear receptor superfamily of transcription factors, are activated by fatty acid metabolites, peroxisome proliferators, and thiazolidinediones and are now recognized as important mediators in the inflammatory response. Whether PPAR activators influence the inflammatory responses of ECs is unknown. We show that the PPAR activators 15-deoxy-
12,14-prostaglandin J2
(15d-PGJ2), Wyeth 14643, ciglitazone, and troglitazone, but
not BRL 49653, partially inhibit the induced expression of vascular
cell adhesion molecule-1 (VCAM-1), as measured by ELISA, and monocyte
binding to human aortic endothelial cells (HAECs)
activated by phorbol 12-myristate 13-acetate (PMA) or
lipopolysaccharide. The "natural" PPAR
activator 15d-PGJ2 had the greatest potency and
was the only tested molecule capable of partially inhibiting the
induced expression of E-selectin and neutrophil-like HL60 cell binding
to PMA-activated HAECs. Intracellular adhesion molecule-1
induction by PMA was unaffected by any of the molecules tested. Both
PPAR-
and PPAR-
mRNAs were detected in HAECs by using reverse
transcription–polymerase chain reaction and a ribonuclease protection
assay; however, we have yet to determine which, if any, of the PPARs
are mediating this process. These results suggest that certain PPAR
activators may help limit chronic inflammation mediated by
VCAM-1 and monocytes without affecting acute inflammation mediated by
E-selectin and neutrophil binding.
Key Words: peroxisome proliferator–activated receptors • endothelial cells • adhesion molecules • inflammation
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Introduction |
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Akey step in the inflammatory response is adhesion of leukocytes to endothelial cells (ECs) lining the blood vessels, with subsequent extravasation of the leukocytes into the involved tissue.1 ECs are stimulated by inflammatory agents to express selectins, such as endothelial-leukocyte adhesion molecule-1 (E-selectin), which interact with carbohydrate ligands on leukocytes, and to express immunoglobulin superfamily proteins, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which interact with
4ß1 integrin receptors and ß2 integrin
receptors on leukocytes, respectively.1 Acute inflammation is the short-term response to bacterial infections and injuries, whereas chronic inflammation is the longer-term response to sustained infections, chronic injury, or foreign bodies.2 When uncontrolled, chronic inflammation may lead to a variety of common, serious ailments such as atherosclerosis, arthritis, inflammatory bowel disease, and fibrotic lung disease. However, acute inflammation is essential for survival against bacterial infections. Development of new therapeutic agents that preserve the beneficial effects of acute inflammation while controlling the devastating effects of chronic inflammation would have major clinical value. Nonsteroidal anti-inflammatory agents and glucocorticoids achieve this goal to some degree, but with significant side effects. Although both acute and chronic forms of inflammation involve the endothelial-leukocyte interactions described above, E-selectin and neutrophils are associated with acute inflammation, and VCAM-1 and monocytes are associated more closely with chronic inflammation.3
Peroxisome proliferator–activated receptors (PPARs), members
of the nuclear receptor superfamily,4 have a newly
recognized role in inflammation.5 6 All 3 subtypes, ,
, and , are ligand-activated transcription factors.
PPAR- and PPAR- are known to regulate the expression of genes
involved in lipid metabolism. PPARs combine with the
retinoid X receptor- (RXR-) to form a functional heterodimer
bound to specific DNA sequences within the promoters of target genes.
Activating ligands for PPARs are semiselective for the subtypes, and
selectivity is ligand-concentration and cell-type
dependent.7 The so-called "natural or
endogenous ligand" prostaglandin
15-deoxy-12,14-prostaglandin
J2 (15d-PGJ2), is
-selective at low concentrations but activates at higher
levels. Many of the eicosanoids, certain nonsteroidal anti-inflammatory
drugs, and the long-chain fatty acids examined are
activators of all PPAR subtypes, whereas peroxisome
proliferators (such as Wyeth 14643) are considered -selective at low
concentrations. The most selective -ligands examined are the
insulin-sensitizing drugs, the thiazolidinediones (such as ciglitazone,
troglitazone, and BRL 49653). Ligands for RXR-, such as
9-cis-retinoic acid, can induce similar responses to PPAR
ligands by activating the PPAR/RXR-
heterodimer.8
Although PPAR- is predominantly localized to the liver where it
orchestrates ß-oxidation of long-chain fatty acids and PPAR- is
expressed predominantly in adipose tissue where it induces
differentiation and triglyceride synthesis,4
PPARs are also now known to be expressed in both the vasculature and in
leukocytes.9 10 11 12 13 14 15 16 Indeed, Ricote et al9 and
Jiang et al10 recently demonstrated that activation of
PPAR- inhibited inflammatory cytokine production by
stimulated monocytes/macrophages. Importantly, Marx et
al11 found that PPAR- activation in
monocytes/macrophages prevented induction of matrix
metalloproteinases, which are associated with atherosclerotic plaque
rupture. Staels et al12 detected PPAR- in human aortic
smooth muscle cells and found that an -selective
activator (Wyeth 14643) inhibited an inflammatory response
in these cells. These authors further showed that patients receiving
fenofibrate (also an -activator) for lipid lowering had
lower plasma C-reactive protein, fibrinogen, and interleukin-6
concentrations, all of which are markers of
inflammation.12 Though not related specifically to the
vasculature, Devchand et al13 showed that the inflammatory
mediator leukotriene B4 is a ligand for PPAR- and that
its activation of PPAR- leads to catabolism of
leukotriene B4, which may ultimately limit the inflammatory
response. These important findings are consistent with a
protective effect of PPAR activators against complications
of atherosclerosis, such as myocardial infarction
caused by plaque rupture.
However, Nagy et al14 and Tontonoz et al15
showed that PPAR- activators promote monocyte
differentiation into macrophages and promote uptake of lipids
by the scavenger receptor, thus leading to increased foam cell
formation. These results suggest the converse, that PPAR-
activators would promote development and growth of
atherosclerotic plaque.
There is preliminary evidence suggesting that ECs express PPARs.17 However, it is not known what affect PPAR activators have on ECs and whether the former can affect even earlier stages of atherogenesis and other chronic inflammatory diseases, specifically leukocyte-EC interaction. This information would be critical in predicting the effects of PPAR activators on chronic inflammatory diseases. In this study, we show that PPAR activators directly affect leukocyte interaction with ECs, partly through preventing upregulation of adhesion molecules by ECs in response to inflammatory stimuli. Most synthetic PPAR activators tested inhibited only the chronic inflammatory mediators—monocyte binding and VCAM-1 expression—and not the acute inflammatory mediators—neutrophil binding and E-selectin expression. These results suggest that PPAR activators may be beneficial in ameliorating chronic inflammatory disease such as atherosclerosis by reducing extravasation of monocytes to the involved tissue without limiting the response to acute infection.
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Methods |
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Cell culture materials were purchased from Fischer Scientific and FBS was from Hyclone Laboratories. Lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical Co. Molecular biology reagents were purchased from Stratagene, and oligonucleotides were synthesized by Gibco BRL. Anti–adhesion molecule monoclonal antibodies were obtained from Biosource. Radiolabel was obtained from Amersham. PPAR activators were purchased from Biomol and Cayman Chemical Co except for BRL 49653 (a kind gift from SmithKline Beecham, King of Prussia, Pa) and troglitazone (kindly provided by Parke Davis, Ann Arbor, Mich).
Cell Culture
Human aortic ECs (HAECs) at passages 4 to 7 were cultured as
previously described.18 Primary human monocytes were
obtained by a modification of the Recalde method.19 The
neutrophil-like cell line, human promyelocytic leukemic cells (HL60),
were obtained from the American Type Culture Collection (Manassas, Va)
and cultured in RPMI medium with 10% FBS.
Leukocyte Adhesion Assay
Leukocytes were added to control or activated HAECs, and
after a 10-minute incubation at 37°C, unbound leukocytes were removed
by washing, the cells were fixed with 4%
paraformaldehyde, and the number of bound leukocytes
was determined by light microscopy as previously
described.20 HAEC activation was achieved by incubation
with 5 to 50 nmol/L PMA (Sigma) or 1 ng/mL LPS (Sigma) for 4 hours.
PPAR activators were added 18 hours before HAEC activation
and/or during the 4-hour activation period. The activators
were initially prepared as 1000x stock solutions in dimethyl sulfoxide
and then added to an appropriate volume of medium at 37°C. The
samples were mixed and incubated for 30 minutes at 37°C, after which
an appropriate aliquot was placed on the cells in each 48-well plate.
Extensive washing before addition of leukocytes removed both PPAR
activators and the EC activator (PMA or LPS),
thus precluding interaction of these molecules with the leukocytes. As
a validation, we directly treated monocytes with the PPAR
activators over a range of concentrations and found no
effect on binding to activated ECs for the 10-minute
adhesion-time period used in these experiments (data not shown).
Adhesion Molecule Expression
The expression of VCAM-1, E-selectin, and ICAM-1 on control and
on PMA- or LPS-activated HAECs was quantified by ELISA as
previously described.21 In brief, at 95% confluence in
96-well dishes, PPAR activators were added to HAECs 18
hours before activation and/or during the 4-hour activation period. The
cells were washed and incubated with anti–VCAM-1 (1:100),
anti–E-selectin (1:500), or anti–ICAM-1 (1:500) antibody for 2 hours
on ice. Subsequently, the cells were fixed with 4%
paraformaldehyde and incubated for 1 hour with
secondary antibody conjugated to alkaline phosphatase at room
temperature (for VCAM-1, 1:2000; E-selectin, 1:2000; and ICAM-1, 1:5000
dilution) and developed with p-nitrophenyl phosphate
substrate (Sigma). The colorimetric assay was
quantified using a Molecular Devices plate reader. Because the cells
were not permeabilized, this ELISA detected cell
surface–expressed protein.
RNA Analysis
Total RNA was isolated from confluent monolayers of HAECs with
Trizol reagent (Gibco BRL) according to the manufacturer's directions.
RNA (2 µg) was reverse-transcribed (RT) as previously
described.22 Polymerase chain reaction (PCR) was performed
using 3.5 µL of cDNA, 100 ng of each primer (synthesized by Gibco
BRL), 14 µmol/L dNTPs, and 1.75 U of Pfu DNA
polymerase (Stratagene) in a 50-µL reaction mix with a Perkin-Elmer
Gene Amp PCR System 2400. The sense (5'-CCATTCTGGCCCACCAAC-3') and
antisense (5'-CTGAAACCGACAGTACTG-3') primers were capable of amplifying
both human PPAR-1 and -2 isoforms,23 thereby
producing a product of 479 bp. The sense
(5'-CATCACGGACACGCTTTCACC-3') and antisense
(5'-GTTCTTCAAGTAGGCCTCGTAG-3') primers were used for amplification of
human PPAR-24 producing a product of 450 bp. Each
cycle consisted of 45 seconds at 96°C, 45 seconds at 62°C, and 2
minutes at 72°C for 35 cycles, terminating with 5 minutes at 72°C.
A 15-µL aliquot was electrophoresed on a 1.5% agarose gel and
visualized by an ethidium bromide stain. The size of PCR products
was confirmed by comparison with a 1-kb DNA ladder (Stratagene). PPAR
band intensities were normalized to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
levels, amplified as previously described.22
Northern blot analysis was performed for the detection of VCAM-1 mRNA expression by using a VCAM-1 cDNA probe (a kind gift of Dr Myron Cybulsky, Department of Pathology, Brigham and Women's Hospital, Boston, Mass). In brief, total RNA (12 µg) isolated from confluent HAEC monolayers, left untreated or treated with LPS (1 ng/mL) in the presence or absence of Wyeth 14643 (100 µmol/L), was run on a formaldehyde/agarose gel. Fractionated RNA was transferred to a Duralon-UV membrane (Stratagene) and subsequently hybridized with a random primed labeled (Prime-It II, Stratagene) cDNA probe and Rapid-Hyb buffer (Amersham) according to the manufacturers' recommendations at 65°C for 2.5 hours. The blot was subsequently washed once at room temperature in 2x SSC (1x SSC is 0.3 mol/L NaCl, 0.03 mol/L sodium citrate), 0.1% SDS; once at 65°C in 0.5x SSC, 0.1% SDS; and once at 65°C in 0.1x SSC, 0.1% SDS, with each wash requiring 15 minutes. After exposure overnight on film (Hyperfilm MP, Amersham), band intensity was determined by densitometry and normalized to the level of mRNA of the human acidic ribosomal phosphoprotein PO (36B4).25
The solution-hybridization RNase protection assay was performed to
determine PPAR- and RXR- expression in HAECs and human primary
monocytes as previously described.26 In brief, antisense
RNA probes were prepared from pGEM cloning vectors containing partial
cDNA probes for PPAR- (kindly provided by Dr Jeffrey Flier, Harvard
University, Cambridge, Mass)26 27 or RXR- (kindly
provided by Dr Sunil Nagpal, Allergan Inc, Irvine, Calif). Labeled
probes were incubated with RNA (10 µg) for 12 hours at 50°C and
then digested with RNase A (90 µg/mL) and RNase T1 (600 U/mL) for 1
hour at 45°C. Samples were electrophoresed on 6% nondenaturing
polyacrylamide gels. Protected bands of 348 (PPAR-2), 285
(PPAR-1), or 101 (RXR-) bp were detectable. RNA from control or
PMA-activated (5 nmol/L, 4 hours) human monocytes was used as a
positive control for PPAR- expression.9 15 16
Statistical Analysis
Computer-assisted statistical analyses were performed
using the ANOVA program, and probability values were calculated using
Fisher's protected least significant difference test. A value of
P<0.05 was considered significant.
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Results |
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PPAR Activators Inhibit Monocyte Binding and VCAM-1 Expression
We chose PMA and LPS to activate HAECs, and under our experimental conditions, PMA (1 and 5 nmol/L) and LPS (1 ng/mL) induced a 3- to 4-fold increase in the binding of monocytes to HAECs. This induction was partially blocked by the synthetic PPAR activators ciglitazone, troglitazone, Wyeth 14643, and the natural PPAR activator 15d-PGJ2 (Figures 1A
and 2A). The synthetic PPAR
activator BRL 49653 had no effect on monocyte binding
(Figure 2A). These findings were mirrored by the effects on
expression of VCAM-1 protein, an adhesion molecule implicated in EC
binding of monocytes.1 3 The induction of VCAM-1
expression on activated HAECs was partially blocked by the PPAR
activators with the exception of BRL 49653 (Figures 1B and 2B).
The approximate relative effectiveness was
similar to that for monocyte binding, such that
15d-PGJ2 (10 µmol/L) > ciglitazone
(60 µmol/L) > Wyeth 14643 (100 µmol/L)=troglitazone
(15 µmol/L)=ciglitazone (30 µmol/L). The PPAR
activators alone did not significantly increase or decrease
the protein expression levels of the adhesion molecules relative to
control untreated cells (data not shown). To examine whether PPAR
activation could affect VCAM-1 mRNA levels, we performed Northern blot
analysis, which showed that the induction in VCAM-1 mRNA levels
on activation by LPS could be partially inhibited by treatment with
100 µmol/L Wyeth 14643 (39.4±4.9% inhibition versus LPS alone,
Figure 3). This inhibition was comparable
to the reduced induction in VCAM-1 protein expression after LPS
activation in the presence of Wyeth 14643 (53.3±11.5% inhibition
versus LPS alone, Figure 2B). Thus, the PPAR
activators tested were capable of reducing monocyte binding
to activated HAECs by partially inhibiting the induction of
VCAM-1 expression.
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PPAR Activators Fail to Inhibit Neutrophil Binding and
E-Selectin Expression
To determine whether the synthetic PPAR activators
also affect neutrophil binding, we performed the same experiments as
above after replacing the monocytes with the neutrophil-like cell line
HL60. In contrast to their effects on monocyte binding, the synthetic
PPAR activators failed to inhibit, and in some cases
further increased, the induced binding of HL60 cells to
PMA-activated HAECs (ciglitazone 30 µmol/L, 45.8±15.4%
increased binding; ciglitazone 60 µmol/L, 58.7±14.1% increased
binding over PMA alone; Wyeth 14643, 34.6±16.3% increase; and
troglitazone, 11.4±13.2% decreased binding versus PMA alone; Figure 4A). A similar trend was observed for
protein levels of E-selectin (Figure 4B), an adhesion molecule
implicated in the binding of neutrophils by ECs.1 3 The
synthetic PPAR activators tested therefore block the
induction in monocyte binding and VCAM-1 expression but not neutrophil
binding and E-selectin expression.
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Greater Potency of 15d-PGJ2
The prostaglandin-like PPAR ligand
15d-PGJ2, a metabolite of
PGD2, blocked the induction of E-selectin protein
expression (56.6±2.0% inhibition versus PMA alone), unlike the
synthetic PPAR activators tested (Figure 4B
). To
investigate the activity of 15d-PGJ2 further, we
applied more stringent assay conditions. Instead of preincubating HAECs
with PPAR activators, cells were exposed to PPAR
activators only during the 4-hour PMA activation period.
Moreover, a higher dose of PMA (50 nmol/L) was utilized. Under these
conditions, 15d-PGJ2 partially blocked the
induction of monocyte binding (75.2±7.7% inhibition versus PMA alone,
Figure 5A) and VCAM-1 protein expression
(72.7±2.2% inhibition versus PMA alone, Figure 5B), whereas
ciglitazone, Wyeth 14643, troglitazone, and BRL 49653 were ineffective.
Furthermore, 15d-PGJ2 partially blocked the
induction of HL60 binding (84.2±5.1% inhibition versus PMA alone,
Figure 6A) and E-selectin protein
expression (46.1±6.7% inhibition versus PMA alone, Figure 6B),
whereas ciglitazone, Wyeth 14643, troglitazone, and BRL 49653 were
ineffective. Therefore, 15d-PGJ2 appears to have
a much greater potency than any of the other molecules tested.
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PPAR Activators Fail to Inhibit ICAM-1
Expression
The level of expression of ICAM-1 protein on unactivated
control HAECs was greater than that for the other adhesion molecules
examined, but the levels were induced 2- to 3-fold by PMA activation
(data not shown). The synthetic PPAR activators ciglitazone
(30 µmol/L) and troglitazone (15 µmol/L) and the natural
activator 15d-PGJ2 (5 µmol/L)
were incapable of blocking the induction in ICAM-1 protein expression
in HAECs activated by 5 to 50 nmol/L PMA, even under
preincubation conditions (data not shown). The PPAR
activator effects were therefore rather specific among
adhesion molecules.
Expression of PPARs in ECs
The ribonuclease protection assay detected low levels of PPAR-1
mRNA but no PPAR-2 mRNA in HAECs (Figure 7A). Human monocytes expressed relatively
high levels of PPAR-1 mRNA and detectable PPAR-2 mRNA after
stimulation by PMA (Figure 7A), in agreement with previous
studies.15 16 The level of RXR- was approximately
equivalent in HAECs, monocytes, and PMA-activated monocytes
(Figure 7A). The mRNA level of PPAR- (both 1 and 2
isoforms together) was compared with PPAR- in HAECs by
semiquantitative RT-PCR. The expression of PPAR- was greater than
that of PPAR- in HAECs (Figure 7B), and PPAR- expression
was lower in HAECs than in human monocytes (as shown by the
ribonuclease protection assay). GAPDH expression was approximately
equivalent in HAECs and monocytes (Figure 7B). No PCR
product was detected when cDNA derived from mouse 3T3-L1 adipocytes
was used as a template for the human PPAR- and PPAR- primers,
thus demonstrating the specificity of this system (Figure 7B).
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Discussion |
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These results indicate that some synthetic PPAR activators (structures shown in Figure 8
), such as a peroxisome proliferator and
certain thiazolidinediones, inhibit monocyte binding by
activated HAECs and their expression of VCAM-1, an adhesion
molecule relatively specific for monocyte and lymphocyte adhesion and
chronic inflammation. These activators showed no effect on
neutrophil binding by activated HAECs or their expression of
E-selectin, an adhesion molecule associated with acute inflammation. A
natural PPAR activator, 15d-PGJ2, a
prostaglandin metabolite of PGD2,
inhibited both monocyte and neutrophil binding concomitant with the
inhibition of both induced VCAM-1 and E-selectin expression. These
results further fuel the discussion on the positive versus negative
aspects of PPAR activation on atherosclerosis.
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Induction of adhesion molecules by stimuli such as LPS and various
cytokines is mediated by signal transduction pathways that
subsequently regulate the activity of transcription
factors.28 29 There are various binding sites for
oxidation, proliferation, and inflammation-related transcription
factors in the promoter elements of the adhesion molecule
genes.30 31 32 The transcription factor nuclear factor-
B
(NF-B) is critical for the induction of adhesion molecules; however,
it works in concert with other transcription factors that are specific
for each adhesion molecule.30 Ricote et al9
postulated that PPAR- activation inhibited the activity of
transcription factors such as NF-B, activating protein-1, and STAT,
subsequently inhibiting the inducible form of NO synthase, gelatinase
B, and scavenger receptor A expression in activated monocytes.
Such a molecular mechanism may apply here; however, NF-B is required
for induction of all of the adhesion molecules examined in this study.
So why do we observe the specific inhibition of VCAM-l? We can only
speculate that because NF-B is a dimeric complex that can comprise a
range of distinct subunits, such as p50 (NF-B1), p52 (NF-B2), p65
(Rel A), c-Rel, and Rel B,33 34 the mechanism for the
specificity of the synthetic PPAR activators may be due to
an inhibitory interaction with a specifically composed
NF-B complex or specific auxiliary transcription factors on the
VCAM-1 promoter. A similar specificity of inhibition of adhesion
molecule expression was previously observed after EC treatment with
all-trans-retinoic acid35 or certain
antioxidant molecules.36 Troglitazone contains a vitamin E
moiety that endows it with some antioxidant properties,37
perhaps partially accounting for its activity in this study. The other
PPAR activators used in our study have no reported
antioxidant activity. In addition, using an assay that detects lipid
hydroperoxide formation38 (in LDL, in this case), we found
that although troglitazone prevented lipid hydroperoxide formation in
LDL, the activators Wyeth 14643, ciglitazone, and
15d-PGJ2 at the concentrations used in the
adhesion experiments did not (data not shown). This result suggests
that an antioxidant effect is not a major factor accounting for the
observed effects of PPAR activators. The mechanism by which
certain PPAR activators induce E-selectin expression and
HL60 binding to ECs in our study may also pertain to specific
interactions with the transcription factor complexes on the promoter or
with the promoter itself. Further studies are required to define the
mechanism(s) involved.
Downregulation of adhesion molecule expression for therapeutic purposes has followed a number of strategies. Monoclonal antibodies to adhesion molecules were shown to significantly reduce leukocyte binding,39 and oligonucleotides were utilized as transcription factor decoys.40 Most pertinent to the present study, De Caterina et al41 showed that certain lipids could block the induction of VCAM-1 expression on activated ECs These authors suggested that this may partially explain the purported protective effect of various unsaturated dietary lipids in atherogenesis. Such lipids, for instance, docosahexaenoic acid, are now understood to be activators of PPARs.42 43
In contrast to the synthetic PPAR activators tested, the
natural PPAR activator 15d-PGJ2
blocked both E-selectin as well as VCAM-1 expression and acted without
the need for pretreatment of the cells. This difference may be related
to the prostaglandin nature of
15d-PGJ2, which, by analogy with other
prostaglandins such as
PGE2,44 may interact with a cell
surface receptor to generate an increase in intracellular cAMP levels.
Such an increase in cAMP in this manner has been shown previously to
block induction of VCAM-1 and E-selectin but not ICAM-1 expression on
activated ECs.45 46 However, no known specific
cell surface receptor for 15d-PGJ2 has been
identified. It is also possible that 15d-PGJ2 has
other pharmacological properties, such as conjugation with glutathione,
as occurs with prostaglandin metabolites containing an
,ß-unsaturated carbonyl group resulting in varied biological
activity.47 48 Alternatively, one might argue that these
effects are solely mediated by PPAR and that
15d-PGJ2 may be a very effective PPAR
activator in ECs. Even though
15d-PGJ2 is less effective than most
thiazolidinediones at inducing adipocyte differentiation or
transactivation of a PPAR- promoter-reporter construct in
fibroblast-type cells,5 7 49 50 it has often been shown to
have the greatest efficacy in other processes in various cell types,
such as inhibiting cytokine production by
activated monocytes,10 inducing the
differentiation of monocytes into macrophages,15
and inhibiting growth of breast cancer cells.51 The reason
for this paradox is currently unknown.
We are unable to state, as yet, which PPAR subtype might be mediating
this process in ECs. The ability of the peroxisome proliferator Wyeth
14643 to have an effect suggests that PPAR- is involved, whereas the
effects of the thiazolidinediones indicate that PPAR- is involved.
The potency of 15d-PGJ2 also predicts a role for
PPAR- in this process; however, the ineffectiveness of BRL 49653,
the most potent -activator in most systems, is puzzling.
Analysis of the mRNA levels indicates that PPAR- is more
abundant and perhaps more active than PPAR- in ECs; however, this is
not definitive, since we have as yet to determine the functional
protein levels of the PPARs in ECs. Given that there is no truly
specific activator of a particular PPAR, we can only
conclude that the process described in this study may be mediated by
any of the PPARs expressed in ECs.
Our results do not exclude a role for the effects of PPAR activators on the expression of other factors involved in leukocyte-EC interaction, such as inhibition of endothelium-derived chemokines and other adhesion molecules. Nor do they rule out the possibility that the molecules tested may act through unrelated mechanisms in addition to PPAR activation. Regardless of the exact mechanism(s), it is hoped that such molecules will have the same effects in vivo.
Because inflammatory lipid factors such as prostaglandins, eicosanoids, and leukotrienes are well-recognized activators of PPARs,13 42 43 49 these transcription factors are poised to mediate inflammatory responses in general and in specific diseases. The present results suggest that, even though certain PPAR activators may promote foam cell formation in the atherosclerotic lesion, they may also block the initial entry of monocytes into the artery wall, thus preventing the onset of atherosclerotic lesion formation but aggravating preexisting lesions. These findings carry enormous clinical significance, as increasing numbers of diabetic patients are receiving PPAR agonists as routine therapy for insulin resistance.
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Acknowledgments |
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This work was supported in part by National Institutes of Health (Bethesda, Md) grant HL30568 (to J.A.B. and L.L.D.) and the Laubisch Fund (to S.M.J. and F.P.). We thank Susan Hama, Linda Jin, and Leslie Ingram-Drake for assistance with HAECs and monocytes.
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Footnotes |
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S.M.J. and F.P. contributed equally to this work.
Received November 13, 1998; accepted February 8, 1999.
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References |
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H. J. Patel, M. G. Belvisi, D. Bishop-Bailey, M. H. Yacoub, and J. A. Mitchell Activation of Peroxisome Proliferator-Activated Receptors in Human Airway Smooth Muscle Cells Has a Superior Anti-inflammatory Profile to Corticosteroids: Relevance for Chronic Obstructive Pulmonary Disease Therapy J. Immunol., March 1, 2003; 170(5): 2663 - 2669. [Abstract] [Full Text] [PDF]
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D. S. Calnek, L. Mazzella, S. Roser, J. Roman, and C. M. Hart Peroxisome Proliferator-Activated Receptor {gamma} Ligands Increase Release of Nitric Oxide From Endothelial Cells Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 52 - 57. [Abstract] [Full Text] [PDF]
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 C. Kluft, R. Kleemann, and M.P.M. de Maat How best to counteract the enemies? By controlling inflammation in the coronary circulation Eur. Heart J. Suppl., November 1, 2002; 4(suppl_G): G53 - G65. [Abstract] [PDF]
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N. Wang, L. Verna, N.-G. Chen, J. Chen, H. Li, B. M. Forman, and M. B. Stemerman Constitutive Activation of Peroxisome Proliferator-activated Receptor-gamma Suppresses Pro-inflammatory Adhesion Molecules in Human Vascular Endothelial Cells J. Biol. Chem., September 6, 2002; 277(37): 34176 - 34181. [Abstract] [Full Text] [PDF]
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 S. Sethi, O. Ziouzenkova, H. Ni, D. D. Wagner, J. Plutzky, and T. N. Mayadas Oxidized omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPARalpha Blood, July 30, 2002; 100(4): 1340 - 1346. [Abstract] [Full Text] [PDF]
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Q. N. Diep, M. El Mabrouk, J. S. Cohn, D. Endemann, F. Amiri, A. Virdis, M. F. Neves, and E. L. Schiffrin Structure, Endothelial Function, Cell Growth, and Inflammation in Blood Vessels of Angiotensin II-Infused Rats: Role of Peroxisome Proliferator-Activated Receptor-{gamma} Circulation, May 14, 2002; 105(19): 2296 - 2302. [Abstract] [Full Text] [PDF]
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O. Barbier, I. P. Torra, Y. Duguay, C. Blanquart, J.-C. Fruchart, C. Glineur, and B. Staels Pleiotropic Actions of Peroxisome Proliferator-Activated Receptors in Lipid Metabolism and Atherosclerosis Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 717 - 726. [Abstract] [Full Text] [PDF]
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G. J. Etgen, B. A. Oldham, W. T. Johnson, C. L. Broderick, C. R. Montrose, J. T. Brozinick, E. A. Misener, J. S. Bean, W. R. Bensch, D. A. Brooks, et al. A Tailored Therapy for the Metabolic Syndrome : The Dual Peroxisome Proliferator-Activated Receptor-{alpha}/{gamma} Agonist LY465608 Ameliorates Insulin Resistance and Diabetic Hyperglycemia While Improving Cardiovascular Risk Factors in Preclinical Models Diabetes, April 1, 2002; 51(4): 1083 - 1087. [Abstract] [Full Text] [PDF]
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 B. Molavi, N. Rasouli, and J. L. Mehta Peroxisome Proliferator-Activated Receptor Ligands as Antiatherogenic Agents: Panacea or Another Pandora's Box? Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2002; 7(1): 1 - 8. [Abstract] [PDF]
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R. B. Clark The role of PPARs in inflammation and immunity J. Leukoc. Biol., March 1, 2002; 71(3): 388 - 400. [Abstract] [Full Text] [PDF]
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L. L. Demer Adipose Rex: Fat and Fats That Rule Differentiation Circ. Res., February 22, 2002; 90(3): 241 - 243. [Full Text] [PDF]
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Y. Oyama, N. Akuzawa, R. Nagai, and M. Kurabayashi PPAR{gamma} Ligand Inhibits Osteopontin Gene Expression Through Interference With Binding of Nuclear Factors to A/T-Rich Sequence in THP-1 Cells Circ. Res., February 22, 2002; 90(3): 348 - 355. [Abstract] [Full Text] [PDF]
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A.-L. Levonen, D. A. Dickinson, D. R. Moellering, R. T. Mulcahy, H. J. Forman, and V. M. Darley-Usmar Biphasic Effects of 15-Deoxy-{Delta}12,14-Prostaglandin J2 on Glutathione Induction and Apoptosis in Human Endothelial Cells Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1846 - 1851. [Abstract] [Full Text] [PDF]
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D. Hornung, L. L. Waite, E. A. Ricke, F. Bentzien, D. Wallwiener, and R. N. Taylor Nuclear Peroxisome Proliferator-Activated Receptors {{alpha}} and {{gamma}} Have Opposing Effects on Monocyte Chemotaxis in Endometriosis J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3108 - 3114. [Abstract] [Full Text] [PDF]
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V. Pasceri, J. Chang, J. T. Willerson, and E. T. H. Yeh Modulation of C-Reactive Protein-Mediated Monocyte Chemoattractant Protein-1 Induction in Human Endothelial Cells by Anti-Atherosclerosis Drugs Circulation, May 29, 2001; 103(21): 2531 - 2534. [Abstract] [Full Text] [PDF]
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 S. X. Cheng and T. Kupper A New Rexinoid for Cutaneous T-Cell Lymphoma Arch Dermatol, May 1, 2001; 137(5): 649 - 652. [Full Text] [PDF]
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A. R. Collins, W. P. Meehan, U. Kintscher, S. Jackson, S. Wakino, G. Noh, W. Palinski, W. A. Hsueh, and R. E. Law Troglitazone Inhibits Formation of Early Atherosclerotic Lesions in Diabetic and Nondiabetic Low Density Lipoprotein Receptor-Deficient Mice Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 365 - 371. [Abstract] [Full Text] [PDF]
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Z. Chen, S. Ishibashi, S. Perrey, J.-i. Osuga, T. Gotoda, T. Kitamine, Y. Tamura, H. Okazaki, N. Yahagi, Y. Iizuka, et al. Troglitazone Inhibits Atherosclerosis in Apolipoprotein E-Knockout Mice : Pleiotropic Effects on CD36 Expression and HDL Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 372 - 377. [Abstract] [Full Text] [PDF]
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 J. Calles-Escandon and M. Cipolla Diabetes and Endothelial Dysfunction: A Clinical Perspective Endocr. Rev., February 1, 2001; 22(1): 36 - 52. [Abstract] [Full Text]
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M. Otsuki, H. Saito, X. Xu, S. Sumitani, H. Kouhara, T. Kishimoto, and S. Kasayama Progesterone, but Not Medroxyprogesterone, Inhibits Vascular Cell Adhesion Molecule-1 Expression in Human Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 2001; 21(2): 243 - 248. [Abstract] [Full Text] [PDF]
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W. A. Hsueh, S. Jackson, and R. E. Law Control of Vascular Cell Proliferation and Migration by PPAR-{gamma}: A new approach to the macrovascular complications of diabetes Diabetes Care, February 1, 2001; 24(2): 392 - 397. [Abstract] [Full Text]
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 Y. Miwa, T. Sasaguri, H. Inoue, Y. Taba, A. Ishida, and T. Abumiya 15-Deoxy-Delta 12,14-prostaglandin J2 Induces G1 Arrest and Differentiation Marker Expression in Vascular Smooth Muscle Cells Mol. Pharmacol., October 1, 2000; 58(4): 837 - 844. [Abstract] [Full Text]
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H. Lee, W. Shi, P. Tontonoz, S. Wang, G. Subbanagounder, C. C. Hedrick, S. Hama, C. Borromeo, R. M. Evans, J. A. Berliner, et al. Role for Peroxisome Proliferator-Activated Receptor {alpha} in Oxidized Phospholipid-Induced Synthesis of Monocyte Chemotactic Protein-1 and Interleukin-8 by Endothelial Cells Circ. Res., September 15, 2000; 87(6): 516 - 521. [Abstract] [Full Text] [PDF]
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J. G. Hunter, M. F. van Delft, R. A. Rachubinski, and J. P. Capone Peroxisome Proliferator-activated Receptor gamma Ligands Differentially Modulate Muscle Cell Differentiation and MyoD Gene Expression via Peroxisome Proliferator-activated Receptor gamma -dependent and -independent Pathways J. Biol. Chem., October 5, 2001; 276(41): 38297 - 38306. [Abstract] [Full Text] [PDF]
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Y. Oyama, N. Akuzawa, R. Nagai, and M. Kurabayashi PPAR{gamma} Ligand Inhibits Osteopontin Gene Expression Through Interference With Binding of Nuclear Factors to A/T-Rich Sequence in THP-1 Cells Circ. Res., February 22, 2002; 90(3): 348 - 355. [Abstract] [Full Text] [PDF]
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D. M. Flavell, Y. Jamshidi, E. Hawe, I. Pineda Torra, M.-R. Taskinen, M. H. Frick, M. S. Nieminen, Y. A. Kesaniemi, A. Pasternack, B. Staels, et al. Peroxisome Proliferator-Activated Receptor {alpha} Gene Variants Influence Progression of Coronary Atherosclerosis and Risk of Coronary Artery Disease Circulation, March 26, 2002; 105(12): 1440 - 1445. [Abstract] [Full Text] [PDF]
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