© 1999 American Heart Association, Inc.
Vascular Biology |
25-Hydroxycholesterol Increases Eicosanoids and Alters Morphology in Cultured Pulmonary Artery Smooth Muscle and Endothelial Cells
From the Departments of Anesthesiology (E.R.W.) and Pharmacology and Toxicology (W.B.C.), Medical College of Wisconsin, Milwaukee.
Correspondence to Eric R. Wohlfeil, Department of Anesthesiology, Medical College of Wisconsin, MEB 462C, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail wohlfeil{at}mcw.edu
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Abstract |
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Abstract—25-Hydroxycholesterol (25-OHC) is an oxidized derivative of cholesterol that has been implicated in the early development of arteriosclerosis. Changes in arterial smooth muscle cell (SMC) migration and proliferation have also been linked to the pathophysiology of arteriosclerosis. SMCs undergo "activation" in response to vascular injury by changing phenotypically and by increasing prostaglandin G/H synthase-2 (PGHS-2) protein levels and eicosanoid release. Activation is thought to be important in atheroma formation and arteriosclerosis progression. 25-OHC induces SMCs to change morphologically, increase PGHS-2, and increase eicosanoid release. Confluent monolayers were treated with 25-OHC (10 µg/mL) or the PGHS-2 inducer interleukin-1ß (1 ng/mL) for 18 hours at 37°C. The 18-hour treatment resulted in morphological changes. After uptake of [14C]arachidonic acid, released radiolabeled arachidonic acid products were extracted and chromatographed by both normal and reverse-phase high-performance liquid chromatography systems. 25-OHC–treated cells increased their prostaglandin production, with the major component comigrating with a prostaglandin-E2 standard. HETEs and epoxyeicosatrienoic acids were not affected. Immunoprecipitation analysis of treated and control cell lysates using anti–PGHS-1 and -2 and anti–
-actin primary antibodies indicated PGHS-2
induction over control and no change in contractile proteins. These
changes are consistent with SMC activation, which occurs in
vascular injury models. The notion that oxysterols can activate
vascular SMCs may be important in ultimately understanding the
pathophysiology of atheroma formation.
Key Words: prostaglandins • prostaglandin G/H synthase • hydroxyeicosatetraenoic acids • -actin
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Introduction |
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Vascular endothelial cell (EC) injury and atheroma formation are considered early events in the pathophysiology of arteriosclerosis.1 2 Likewise, changes in arterial smooth muscle cell (SMC) migration, proliferation, and phenotype are considered important parts of the disease process.3 4 5 6 7 8 The "activation" of SMCs to secretory, growing phenotypes occurs in response to injury early in the development of arteriosclerosis.7 9
Eicosanoids and oxysterols, the oxidized derivatives of cholesterol, are also thought to play a role in the initial stages of arteriosclerosis.1 2 10 11 25-Hydroxycholesterol (25-OHC) is a much-studied oxysterol that is both formed endogenously and found in the diet.12 13 25-OHC is also present in atheromatous plaques.1 14 We recently reported that 25-OHC enhances eicosanoid production in coronary artery ECs by increasing the amount of prostaglandin (PG) G/H synthase-2 (PGHS-2) protein.15 We postulated that 25-OHC–induced prostanoids could modulate vascular tone either indirectly through endothelial prostanoids or directly by an effect on SMCs.
SMC PGHS-2 mRNA and protein are increased after activation in an experimental model of vessel injury, resulting in marked increases in PG production.9 Here, we show that cultures of SMCs treated with the oxysterol 25-OHC exhibit morphological changes, increases in PGHS-2 protein, and increased eicosanoid release that are consistent with the phenomena of SMC activation described in vascular injury models. Furthermore, the effects of 25-OHC on the pulmonary endothelium mirror those previously described for bovine coronary artery endothelium.15 This is the first time that an oxysterol has been shown to activate SMCs in culture.
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Methods |
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Materials
Cell culture media and materials were purchased from GIBCO. 25-OHC, 22(S)-OHC, 20-OHC, indomethacin, A23187, SDS-PAGE reagents, and buffer salts were from Sigma. Secondary antibodies were from Bio-Rad. PG standards, 6-keto-PGF1
, PGE2,
thromboxane B2,
11-hydroxyeicosatetraenoic acid
(11-HETE), 15-HETE, and 12-hydroxyheptadecatrienoic acid (12-HHT)
standards were purchased from Cayman Chemical.
Arachidonic acid was obtained from Nu Check.
[14C(U)]arachidonic acid (866
mCi/mmol) was from DuPont NEN. Interleukin-1ß (IL-1ß) was purchased
from Boehringer Mannheim. All organic solvents were
high-performance liquid chromatography (HPLC)
grade and were purchased from Burdick and Jackson or Sigma.
Octadecasilyl silica (Bond Elut) extraction columns were obtained from
Varian. Anti–PGHS-1 antibody (PG-20) and purified PGHS-1 and -2
proteins were from Oxford Biomedical Research. Anti–PGHS-2 antibody
used in these experiments was a generous gift from Dr Jacques Maclouf
(Paris, France).
Cell Cultures
Confluent monolayers of rabbit pulmonary artery ECs and
rabbit pulmonary artery SMCs were cultured as previously
described.15 16 17 Cells were maintained in feed medium
consisting of RPMI-1640 supplemented with 15% FCS, 1 mmol/L
glutamine, 25 mmol/HEPES, and 1% (vol/vol) antibiotic-antimycotic
and antibiotics (penicillin G, tylosin, nystatin, and gentamicin).
Cells were isolated and cultured by a modification of methods
previously described.16 17
Pulmonary arteries were dissected to their most distal ends
within the lung, and all branch vessels were ligated at their origin
from the main pulmonary artery. The arteries were then removed
and placed in RPMI medium containing 25 mmol/L HEPES, 1
mmol/L L-glutamine, and antibiotics for 15 minutes. The
rinsed arteries were cleaned of connective tissue and placed on moist
gauze in a culture dish. The vessel lumens were filled with 0.1%
(wt/vol) collagenase in RPMI with a polyethylene
tubing–tipped syringe, and both ends were ligated with nylon sutures.
The arteries were incubated for 30 minutes at 37°C in an atmosphere
of 5% CO2 in air. Detached ECs were collected in
a sterile centrifuge tube by flushing each vessel with RPMI
culture medium containing antibiotics. After
centrifugation at 200g for 10 minutes, the
cells were washed once and resuspended in RPMI supplemented with 20%
FCS. The cells were then plated onto 25-cm2
culture flasks coated with 1% gelatin. Primary cultures remained
undisturbed for 48 hours at 37°C in an atmosphere of 95% air and 5%
CO2. EC feed medium was replaced daily for the
first 3 days and twice weekly thereafter. Cells were subcultured within
3 to 7 days of primary culture by detachment with Pucks-EDTA solution
and trypsin. Generally, the cultures were subcultured 2 to 3 times to
obtain sufficient material for experiments. ECs are characterized by
acetylated LDL (Dil-Ac-LDL, Biomedical Technologies, Inc)
uptake and visualized with fluorescence microscopy. In
addition, they exhibited a typical cobblestone morphology. After
collection of ECs, vessels were cut lengthwise, and strips of denuded
vessel were placed into gelatin-coated flasks containing M199 medium
containing 10% FCS, 1% L-glutamine, 0.1%
tyrosine, and antibiotics (0.15% nystatin and 0.15% gentamycin). SMCs
migrated to the flasks within 3 to 5 days. Once growth was established
on the flasks, the vessels were removed, and the cells were maintained
as described for ECs in M199 supplemented with 10% FCS. The purity of
SMC cultures was confirmed by positive immunohistochemical staining for
-actin with a specific antibody (Sigma).
The effects of different pharmacological agents on eicosanoid production were studied on subconfluent monolayers of SMCs and ECs. 25-OHC, 22(S)-OHC, or 20-OHC (10 µg/mL), indomethacin (10-5 mol/L), IL-1ß (10 ng/mL), or vehicle (95% ethanol) was added directly to the feed medium. Cell monolayers were examined microscopically for morphological changes after treatment or assayed for release of eicosanoids. In all experiments in which comparisons were made between control and treated cells, the control and treated flasks of cells were subcultured identically from the same primary culture.
Measurement of Arachidonic Acid
Metabolites
Eicosanoid production by treated and control cultures
was determined by incubating cells with
[14C]arachidonic acid and
resolving the 14C-metabolites by HPLC. The SMC
and EC cultures (75-cm2 flasks) were washed twice
with 10 mL of 10 mmol/L HEPES buffer containing (in mmol/L)
NaCl 150, CaCl2 2, KCl 5,
MgCl2 1, and glucose 6, pH 7.4, and then
incubated with 10 mL of the same buffer.
[14C]arachidonic acid (0.5
µCi, 866 mCi/mmol) in ethanol and 1.5 µmol/L unlabeled
arachidonate were added to the flasks. The cells were then
incubated at 37°C for 5 minutes, at which time 10 µmol/L
A23187 was added. After 10 minutes at 37°C, the cells were
mechanically disrupted with a rubber spatula, and the mixture
containing the cells and buffer was transferred to a conical
centrifuge tube. The
[14C]arachidonic acid
metabolites were then extracted with octadecylsilica extraction columns
(Varian) as previously described.15
High-Performance Liquid Chromatography
Extracted
[14C]arachidonic acid
metabolites were identified and quantified by both reverse-phase (RP)
and normal-phase (NP) HPLC.15 17 RP-HPLC was used to
isolate the PGs, and NP-HPLC was used to isolate the HETEs.
To separate the PGs from the other major eicosanoids and unmetabolized arachidonic acid, an RP-HPLC system with a Nucleosil-C18 (5-µm, 4.6x250-mm) column (method 1) was used. For method 1, solvent A was water and solvent B was 0.01% glacial acetic acid in acetonitrile. The products were eluted by a linear gradient of 50% solvent A in solvent B to 100% solvent B over 40 minutes. Flow rate was 1 mL/min, and absorbance was monitored at 235 nm. Column effluent was collected in fractions, and radioactivity in the fractions was determined by liquid scintillation spectrometry. The retention times of the radioactive peaks were compared in all cases with known eicosanoid standards separated under identical chromatographic conditions after coinjection.
Fractions corresponding to the peaks containing [14C]PGs (retention time of 3 to 10 minutes, method 1) and [14C]HETEs (22 to 25 minutes) were collected separately. After evaporation of the acetonitrile under a nitrogen stream and then acidification of the pooled fractions, the radioactive metabolites were extracted into cyclohexane/ethyl acetate (50:50). The organic layers containing the separated metabolites were dried under a nitrogen stream, and the residues were stored at -40°C for further HPLC analysis.
The PG fraction (3 to 10 minutes, method 1) was resolved into its component PGs with the same Nucleosil-C18 column with RP-HPLC, method 2. Method 2 used solvent C (0.025% phosphoric acid in water) and solvent D (acetonitrile). Elution was carried out isocratically over 40 minutes with 31% solvent D in solvent C followed by a 20-minute linear gradient to 100% solvent D and a 10-minute isocratic elution at 100% solvent D. The flow rate was 1 mL/min, and absorbance was monitored at 207 nm. The column effluent was collected in 0.5-mL fractions and analyzed for radioactivity by liquid scintillation spectroscopy.
Method 3 is used to separate the HETE fraction (22 to 25 minutes, method 1). A Nucleosil silica (5-mm, 4.6x250-mm) NP-HPLC column was used. For method 3, solvent E was hexane containing 0.1% acetic acid, and solvent F was hexane containing 2% isopropanol and 0.1% acetic acid. A linear gradient of 25% solvent F in solvent E to 100% solvent F over 45 minutes was used. The flow rate was 3 mL/min, and 0.6-mL fractions of the column effluent were collected. Absorbance was monitored for method 3 at 235 nm, and fractions were analyzed for radioactivity.
Electrophoresis and Immunoprecipitation
To measure changes in PGHS-1, PGHS-2, and -actin protein in
SMC and EC lysates, SDS-PAGE was performed by the method of
Laemmli18 with 12% resolving gels and 4% stacking gels.
Samples were prepared for electrophoresis by removing medium from
75-cm2 tissue culture flasks and washing cell
monolayers 3 times with 10 mL HEPES buffer, pH 7.4, at 37°C. Cells
were then scraped and pelleted. Cold lysis buffer (1 mL, buffer A)
consisting of 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl,
1 mmol/L EDTA, 1% (vol/vol) Triton X-100, 1% sodium
deoxycholate, 0.1% sodium dodecylsulfate, and 0.25 mmol/L
PMSF was added to the pelleted SMCs or ECs, and the mixture was
incubated at 4°C for 5 minutes. The crude cell lysate was briefly
sonicated, then centrifuged at 15 000g for 15
minutes at 4°C. The supernatant was stored at -80°C until further
use. Protein was determined on all samples by the method of
Bradford,19 and the amount of sample in each lane was
normalized to protein content of crude cell lysate. Cell lysates were
then precleared with protein A Sepharose (Pierce) for 45 minutes at
4°C, incubated with a 1:100 dilution of primary antibody for 2 hours
at 4°C, and then precipitated with protein A Sepharose. The pelleted
beads were washed extensively with buffer A, resuspended in 25 µL of
sample buffer, boiled 3 minutes, and subjected to SDS-PAGE as above.
Immunoprecipitated proteins were visualized with Coomassie blue R-250.
Kaleidoscope prestained standards (7000 to 208 000 kDa; Bio Rad) were
used for molecular-weight determinations. Coomassie-stained gels were
quantified, where indicated, by densitometry with an AMBIS gel
documentation system.
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Results |
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Morphological changes were observed in both SMCs and ECs treated with 25-OHC. Photomicrographs of control and IL-1ß– and 25-OHC–treated SMC cultures are shown in Figure 1
. Disorganized orientation, cytoplasmic
swelling, and increased vacuolization were noted in the 25-OHC–treated
SMC cultures after 18 hours of incubation. No morphological changes
were noted in control or IL-1ß–treated SMC cultures. Morphological
changes of ECs are shown in Figure 2. The
25-OHC–treated ECs show cytoplasmic swelling, increased vacuolization,
and membrane changes. These effects were noted after 48 hours of
treatment. As in SMC cultures, no morphological changes were noted in
the control or IL-1ß–treated EC monolayers. In both SMC and EC
cultures, the chronic addition of indomethacin
(10-5 mol/L) during 25-OHC treatment did not
block the morphological changes noted (data not shown).
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[14C]arachidonic acid
metabolism was studied in EC cultures treated with vehicle
and 25-OHC and in SMC cultures treated with vehicle, 25-OHC, or
IL-1ß. Figure 3 shows the separation of
the major EC [14C]eicosanoids by RP-HPLC
(method 1). Endothelial 25-OHC treatment results in a
large increase in eicosanoid production compared with control
cultures. PGs, HHT, and HETEs are all increased, as evidenced by the
increase in radioactivity in the PG (3 to 10 minutes’ retention time),
HHT (15 to 18 minutes), and HETE (22 to 25 minutes) fractions. No
change in epoxyeicosatrienoic acid fractions were noted between
25-OHC–treated and control EC cultures.
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The 14C-labeled PG and HETE fractions from Figure 3
were pooled and rechromatographed by RP and
straight-phase HPLC, respectively (Figure 4). The PG fraction from the
25-OHC–treated cells contained 2 eicosanoids that comigrated with
PGE2 and 6-keto-PGF1.
The HETE fraction from the 25-OHC–treated EC cultures contained equal
quantities of 11- and 15-HETE.
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The RP separations of the
[14C]arachidonic acid
metabolites from SMCs are shown in Figure 5. Both 25-OHC and IL-1ß treatment
resulted in increased PG production compared with vehicle
control. No significant amounts of HHT or HETEs were noted in any SMC
culture. The PG-containing fractions from Figure 5 were pooled
and rechromatographed with RP-HPLC (Figure 6). [14C]
metabolites that comigrated with PGE2 and
6-keto-PGF1 were the predominant PGs observed
in control, 25-OHC–treated, and IL-1ß–treated cultures. SMC and EC
cultures were also treated in an identical fashion with
22(S)-OHC and 20-OHC (10 µg/mL for 18 and 48 hours,
respectively), but no morphological changes were noted, and eicosanoid
production was not affected by these oxysterols. The chronic
addition of indomethacin during 25-OHC treatment
completely blocked eicosanoid production in SMC and EC cultures
(data not shown).
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All of the eicosanoids that were stimulated by treatment with 25-OHC
and IL-1ß in both SMCs and ECs are PGHS metabolites. To determine
whether 25-OHC affects PGHS, Western immunoprecipitation
analysis of control, 25-OHC–treated, and IL-1ß–treated EC
and SMC lysates were performed with anti–PGHS-1 and -2 specific
antibodies. Cellular -actin, a major SMC-specific protein, was
determined by immunoblot analysis to determine
whether 25-OHC or IL-1ß treatment induced the contractile protein.
Figure 7 shows
representative immunoblots of the 3
proteins obtained from EC and SMC lysates after treatment with vehicle,
25-OHC, or IL-1ß.
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PGHS-1 protein content did not change after 25-OHC or IL-1ß treatment
in either SMCs or ECs. PGHS-2 protein was increased by 25-OHC in both
SMC and EC lysates compared with control cultures. IL-1ß effects on
SMCs and ECs were different. Although IL-1ß increased PGHS-2 in ECs,
it had no significant effect on PGHS-2 in SMC lysates. -Actin was
determined in SMC cultures only. It increased 39% after IL-1ß
treatment and 10% after 25-OHC treatment.
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Discussion |
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The migration and proliferation of vascular SMCs is thought to play an important role in the pathogenesis of arteriosclerosis. SMCs are the predominant cell type in atherosclerotic plaques, and their proliferation leads to vascular occlusion.4 The response of SMCs to vascular injury is considered important in the initial pathophysiological sequence of events leading to atheroma formation.2 Vascular SMCs respond to injury by changing from a contractile, nongrowing phenotype to a secretory, growing phenotype by a process called activation.7 9 PGHS-2 induction and increased eicosanoid release are characteristic of activation. In this article, we report that the oxysterol 25-OHC enhances differential eicosanoid release in rabbit pulmonary artery SMCs and ECs. Furthermore, gross morphological changes are noted, eicosanoid release is enhanced, and PGHS-2 protein contents are increased after oxysterol treatment. All of these effects of 25-OHC treatment on SMCs are consistent with SMC activation. This is the first time that oxysterol-mediated SMC activation has been described.
Eicosanoid production in experimental arteriosclerosis has been investigated in a variety of vascular preparations. The majority of observations suggest that biosynthesis of eicosanoids is reduced in arteriosclerotic vessels. Initially, Dembinska-Kiec et al20 reported that experimental arteriosclerosis was associated with a suppression of prostacyclin generation in mesenteric rings. Other decreases in PG production were subsequently shown in human umbilical vein endothelium,21 whole-vessel aorta,22 aortic microvessels,23 renal microvessels,24 human hepatocellular carcinoma cells,25 rabbit platelets,26 and macrophages.27 Whole-vessel studies have found that prostacyclin responses to experimental arteriosclerosis models appear to be time-dependent.23 28 Initial decreases in PG production were followed by increases in production.
Fewer studies have examined the effects of
arteriosclerosis on SMC eicosanoid modulation. The
release of PGI2 (as
6-keto-PGF1), PGE2,
PGF2, and 15-HETE have been reported in
various SMC preparations.29 30 31 Nonoxidized
cholesterol treatment was shown to decrease PGHS-2 pathway
eicosanoids 8-fold in arterial SMCs.31
Eicosanoid release in SMCs after oxidized cholesterol
derivative (oxysterol) treatment has not been described previously.
Oxysterols have been shown to decrease PG production in
hepatocellular carcinoma cells and in vascular
ECs.25 32
We have previously described eicosanoid stimulation after 25-OHC treatment secondary to elevated PGHS-2 in bovine coronary artery ECs. Gross morphological changes were also noted.15 Morphological changes after 25-OHC treatment have also been described in aortic SMCs.33 These cells exhibit phenotypic features of bone-forming osteoblasts in vitro, like those seen in advanced atherosclerotic plaque.
Here, in rabbit pulmonary artery SMCs and ECs cultured individually, similar morphological changes were noted. 25-OHC–treated SMCs showed cytoplasmic swelling and increased vacuolization as well as disorganized growth and orientation. These SMC morphological changes are similar to those described for activated SMCs after injury,7 34 which were noted to develop an epithelioid shape, resembling newborn rat SMCs, with cellular rounding up and cytoplasmic swelling.
Rabbit pulmonary artery ECs also exhibited gross morphological changes after 25-OHC treatment. These changes paralleled those previously described for bovine coronary ECs. EC cytoplasmic swelling and increased vacuolization were noted. 25-OHC effects on endothelial morphology seem to be consistent between species and tissue types. It should be noted that as previously reported, EC cultures were treated for 48 hours with 25-OHC and SMCs were treated for 18 hours. Confluent EC cultures, in our hands, required longer treatment than SMC cultures to elicit the morphological changes described.
25-OHC dramatically increased eicosanoid release in both ECs and SMCs.
Eicosanoid production by SMCs increased 4-fold after 25-OHC
treatment, primarily reflecting increases in the PG fraction.
PGE2 was the predominant PG produced by SMCs,
followed by 6-keto-PGF1. HHT and HETE
production were not noted to be increased above control levels
in 25-OHC– or IL-1ß–treated SMC cultures. IL-1ß, a known inducer
of PGHS-2, showed an identical eicosanoid pattern of release to 25-OHC,
with a 2-fold increase in total eicosanoids. Both the morphological and
eicosanoid-stimulating effects of 25-OHC treatment were found to be
dose-dependent (data not shown). 25-OHC was used at 10 µg/mL because
this concentration yielded maximal morphological, enzyme, and
eicosanoid changes without causing cell death.
Interestingly, chronic addition of indomethacin during 25-OHC treatment had no effect on the phenotypic changes observed but completely blocked the eicosanoid increases noted. This suggests that morphological changes and eicosanoid enhancement mediated by 25-OHC may be independent and perhaps unrelated events.
Because increases in PGHS-2 mRNA and protein are characteristic of SMC activation after injury, we studied PGHS-2 protein in SMC and EC lysates before and after 25-OHC treatment. These results were compared with identical cultures treated with the known PGHS-2 inducer IL-1ß. Treatment with 25-OHC results in an increase in PGHS-2 in SMCs and ECs. No change was noted in PGHS-1 protein, the constitutive form of the enzyme. Interestingly, IL-1ß treatment increased PGHS-2 only in ECs and not in SMC cultures. This may suggest that the mechanism by which IL-1ß increases PGHS-2 in ECs is not present in SMCs.
Because PGs, 11-HETE, 15-HETE, and HHT may be PGHS-derived,17 we believe that the eicosanoid increases with 25-OHC are due predominantly to PGHS-2 induction. Along these lines, the production of 11- and 15-HETE is greater with PGHS-2 than PGHS-1.35
Because activated SMCs are also characterized by a loss of
contractile function and decreases in levels of contractile
proteins,7 experiments comparing the content of -actin
in control, IL-1ß–treated, and 25-OHC–treated SMCs were performed.
IL-1ß increased -actin content in SMCs by 40% over control in our
experiments. However, 25-OHC also increased -actin 10% compared
with control SMCs. Thus, in our hands, contractile proteins are not
inhibited by 25-OHC but rather are slightly elevated.
In summary, the characteristics of SMC activation secondary to intravascular injury that have been described are observed with 25-OHC treatment: morphological changes, enhanced eicosanoid release, and increases in PGHS-2. This study indicates that oxysterols may activate SMCs in vitro in a manner similar to that associated with early arteriosclerotic development demonstrated in vivo. We must remember, however, that arteriosclerosis is a chronic-progressive disease. Cell culture experiments, as described here, reflect predominantly acute responses of cell phenotypes that may differ from those occurring in vivo. These findings may be important in understanding the role of oxysterols and related oxidized cholesterol derivatives in the pathophysiology of arteriosclerosis.
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Acknowledgments |
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This study was supported by grants from the National Heart, Lung, and Blood Institute (HL-37981), the American Heart Association, Wisconsin Affiliate (95-GB-59), and NIH Anesthesiology Research Training Grants (GM-36144 and GM-08377).
Received December 7, 1998; accepted July 8, 1999.
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