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Departments of 1 Physiology, 2 Surgery (Division of Pediatric Surgery), 3 Pharmacology and Toxicology, 4 Cardiovascular Center, 5 Free Radical Research Center, and 6 Summer Practicum in Undergraduate Research Studies Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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Low-density
lipoprotein (LDL) and its oxidized derivatives are hypothesized to
impair vascular function by increasing superoxide anion
(O

-nitro-L-arginine methyl ester
(L-NAME)-inhibitable mechanism. In contrast, mmLDL
increased O



native low-density lipoprotein; minimally oxidized low-density lipoprotein; smooth muscle cells; superoxide anion; confocal microscopy; hydroethidine
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INTRODUCTION |
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ONE MECHANISM by
which hypercholesterolemia is believed to induce vascular dysfunction
is by increasing O




Hypercholesterolemia also increases nLDL entry into the vessel wall,
where it becomes trapped and subsequently oxidized (20, 21). Once oxidized it is capable of inducing a wide variety of
potent proinflammatory responses in the vasculature leading to impair
function. Many studies have shown that the effects of LDL on
endothelial and vascular function depend strongly on the degree of
oxidation. For example, one essential function of the endothelium is to
limit the accumulation of lipid hydroperoxides (LOOH) in nLDL
(26). When the content of LOOH in LDL exceeds a critical
threshold, endothelial cells begin to participate in the oxidation
process (26). In addition to endothelial cells, smooth
muscle cells can also oxidize LDL by a
O
Whereas many studies have implicated O

The goal of this study was to test the hypothesis that lipoproteins, in
the absence of other confounding influences, increase O




In this investigation, we incubated isolated carotid arteries with
purified nLDL and minimally oxidized LDL (mmLDL) to determine their
effects on vascular O

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MATERIALS AND METHODS |
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Preparation of nLDL. nLDL was isolated by sequential density ultracentrifugation (1.019-1.063 g/ml) from pooled human plasma (2-5 donors per isolation) as previously described (25). Butylated hydroxytoluene, a lipid-soluble antioxidant, and EDTA were added immediately to the plasma (final concentrations = 20 µM and 0.01%, respectively). The plasma was mixed for 15 min at room temperature before ultracentrifugation. Sterile techniques, reagents, and dialysis solutions were used for all isolation steps. Cholesterol concentrations were determined using the cholesterol oxidase kit from Sigma (catalogue number 352-20). nLDL was used within 1 wk of isolation.
Preparation of mmLDL. nLDL was isolated as outlined above. The LDL was placed in a sterile 50-ml conical tube and stored at 4°C for at least 3-6 mo before use for experiments as described (4, 8). mmLDL had a significantly higher content of LOOH than nLDL as measured by the tri-iodide assay (mmLDL = 73.8 ± 25.0 vs. nLDL = 0.3 ± 5.0 nmol LOOH/mg cholesterol, P < 0.01, n = 5) (26).
Tissue extraction. Canine carotid arteries were obtained from adult mongrel dogs of either gender. Vessels were quickly excised and transferred to physiological saline solution at room temperature, and surrounding advential tissue was quickly cleaned away. The artery was sectioned into segments of at least 2 cm long and placed in RPMI 1640 containing 10% fetal bovine serum (FBS), 20 mM HEPES, and antibiotics-antimycotics (termed RPMI medium hereafter). After being washed twice with RMPI 1640 to remove adherent blood elements, the vessels were placed into organ culture.
Organ culture. Canine carotid artery segments were placed in a 60-mm dish containing either control RPMI medium or RPMI medium containing nLDL (80, 160, or 240 mg/dl) or mmLDL (240 mg/dl). The arterial segments were incubated at 37°C, 5% CO2-95% air and 100% H2O for 24 h.
Superoxide anion detection.
Radical specificity of HEt conversion to Et was examined in phosphate
buffer (250 mM, pH 7.45) containing HEt (10 µM). Fluorescence was
monitored with respect to time on a Perkin Elmer LS50B (excitation wavelength 488 nm, emission wavelength 620 nm, 10-nm slit
widths). Superoxide anion was generated with xanthine/xanthine
oxidase (30, 31). First, xanthine was added (final
concentration = 1 mM) and mixed, and fluorescence was recorded.
Xanthine oxidase was then added (final concentration = 14 U/ml),
the solution mixed, and fluorescence recorded. Finally, superoxide
dismutase (SOD) was added (final concentration = 1,000 U/ml) to
inhibit O





Inhibitor studies.
To determine the effects of nLDL and mmLDL on
non-O




Effects of nLDL and mmLDL on the association of HSP90 with eNOS. Bovine aortic endothelial cells (BAEC) were incubated with nLDL (240 mg/dl) or mmLDL (240 mg/dl) in RPMI medium for 24 h. After incubation, the cultures were washed three times with HBSS and then incubated in HBSS containing L-arginine (10 µM) with and without A-23187 (5 µM) for 10 min. The HBSS buffer was removed by aspiration, and cell proteins were harvested in modified RIPA buffer containing 1:100 dilution of phosphatase inhibitor cocktail I (P2850, Sigma Chemical; St. Louis, MO) and other inhibitors as described (24). To determine the effects of nLDL and mmLDL on eNOS activation, eNOS was immunoprecipitated from lysates of the cultures using established protocols (24). Coprecipitated proteins were separated by SDS-PAGE (7.5%), transferred to nitrocellulose, and blotted for phospho-eNOS (serine-1177/serine-1179), eNOS, and HSP90 as described (24).
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RESULTS |
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Detection of O

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nLDL increases vascular O









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mLDL increases vascular O




Effects of nLDL and mmLDL on the activation state of eNOS.
Typically, control cultures contain low levels of phospho-eNOS and low
levels of HSP90 associated with eNOS. Upon stimulation, control
cultures display marked increases in phosphorylation of eNOS at
serine-1179 and association of HSP90 with eNOS (24). In
Fig. 5, we see the typical pattern of
phospho-eNOS and HSP90 association with eNOS in the control cells. In
this blot, neither nLDL nor mmLDL appeared to alter basal or
A-23187-stimulated levels of phospho-eNOS. These lipoproteins, however,
did alter the association of HSP90 with eNOS. nLDL increased the
association of HSP90 with eNOS under basal conditions, suggesting that
low levels of oxidative stress activate cultured endothelial cells. In
contrast, in cells incubated with mmLDL, the association of HSP90 with
eNOS remained low. When the lipoprotein-treated endothelial cell
cultures were stimulated with A-23187, the association of HSP90 with
eNOS decreased to baseline levels in cells incubated with nLDL (Fig.
5A, lane 5) but failed to increase over control
levels in cells treated with mmLDL (Fig. 5A, lane
4 vs. lane 6). Figure 5B illustrates the
reproducibility of the effects of nLDL and mmLDL on phospho-eNOS, eNOS,
and association of HSP90 in endothelial cultures exposed to nLDL and
mmLDL for 24 h.
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DISCUSSION |
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In the present study, we used HEt to detect relative changes in
vascular O






In recent years HEt has gained popularity as an O







The cell-free studies using SOD indicate that the conversion of HEt to
Et is relatively specific for O







With appropriate protocols, en face fluorescent confocal microscopy
provided precise and reproducible measurements of Et fluorescence in
vascular tissues. After the endothelium was located by adjusting the
microscope through different focal planes, the surface of the lumen was
quickly assessed for endothelial integrity and fluorescent intensity.
Moistening coverslips with PSS before placing arterial segments
endothelial side down helped maintain the integrity of the endothelium.
Marked increases in Et fluorescence were noted in the blood vessels in
regions of the lumen that were damaged due to cutting carotid arteries
into segments. For our analyses, we cut the arteries into segments 2 cm
in length before incubation. This step increased the surface area of
endothelium available for analysis, making it possible to exclude
damaged regions. En face fluorescence microscopy revealed that the
arterial segments also possessed low levels of nonspecific
fluorescence. Accordingly, background measurements of fluorescence in
regions between nuclei were obtained and subtracted from Et
fluorescence in the nuclei of the vascular endothelium. By avoiding
damaged regions and correcting nuclear pixel intensity for nonspecific
fluorescence, we were able to obtain fluorescent intensity data that
revealed that nLDL increased O

The mechanisms by which LDL induces vascular dysfunction are complex. Previously, our laboratory focused on mechanisms of endothelial dysfunction induced by nLDL. When LDL crosses the endothelium and becomes trapped in the vessel wall, it can be oxidized to a proinflammatory LDL particle by endothelial cells, smooth muscle cells and monocytes (4, 6, 13). In this way, multiple mechanisms can be induced in the vessel wall by the initial insult of increased plasma concentrations of nLDL. Our findings suggest that the association of HSP90 with eNOS, which is required for ·NO production and activity (9, 10), might also be regulated by oxidative stress. The evidence supporting this statement comes from the fact that mmLDL contained significantly higher levels of LOOH than nLDL before incubation. Such changes in oxidation are similar to the levels in mmLDL reported earlier (8).
The finding that the amount of Et fluorescence inhibited by L-NAME in mmLDL-treated arterial segments was essentially equal to that inhibited in the nLDL-treated segments suggests that these atherogenic lipoproteins uncoupled eNOS to the same extent. Western blot analysis revealed that the activation state of eNOS was altered with respect to its association with HSP90. However, neither lipoprotein significantly altered phospho-eNOS levels under basal or A-23187-stimulated conditions. nLDL increased the association of HSP90 with eNOS under basal conditions, whereas mmLDL maintained this association at low levels. In A-23187-stimulated cells, nLDL decreased the level of HSP90 associated with eNOS compared with the levels in nLDL cells under basal conditions and the levels in A-23187-stimulated control cells. In mmLDL-treated cells, the association of HSP90 with eNOS failed to increase when stimulated with A-23187. Taken together, these data indicate that one mechanism may be a decrease in or failure of HSP90 to associate with eNOS when eNOS is activated. It should be noted that differences in the regulation of the association of HSP90 with eNOS appeared to be related to the levels of oxidative stress induced by nLDL and mmLDL. Furthermore, it is important to note that the lipoprotein with the highest level of oxidative change, mmLDL, impaired HSP90 interactions with eNOS under both basal and A-23187-stimulated conditions. Such differences in regulation of this protein-protein interaction suggest a role for oxidative stress.
Recent findings by Go et al. (11) also indicate that
oxidized LDL uncouples eNOS activity. They report that oxidized LDL induced a nearly threefold increase in phospho-eNOS (serine-1179) levels in BAEC over a period of 30 min. In the initial incubations, citrulline conversion kept pace with phospho-eNOS. However, at a later
time point (30 min), citrulline conversion decreased even though
phospho-eNOS levels remained elevated (11). Our findings here are consistent with those of Go et al. (11) in that a
decrease in citrulline production in the presence of increased levels
of phospho-eNOS also suggests that eNOS generates
O
In separate studies using diverse redox cycling agents (dichromate VI, adriamycin, and bis-N-methylacridinium nitrate) to induce oxidative stress, we found that low levels of oxidative stress appeared to increase, whereas high levels appeared to decrease HSP90 interactions with eNOS (data not shown). The changes in the association of HSP90 with eNOS in cells acutely treated with the redox cycling agents (15 min to 4 h) were similar to the changes induced by 24 h incubation with nLDL representing low levels, and mmLDL, representing high levels of oxidative stress. Taken together, such changes indicate that the effects of oxidative stress on the association of HSP90 with eNOS are both concentration and time dependent.
In summary, nLDL and mmLDL increase vascular endothelial
O





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ACKNOWLEDGEMENTS |
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The authors thank Jennifer S. Pollock (Vascular Biology Center, Medical College of Georgia, Augusta, GA) for providing anti-eNOS antibodies and William M. Chilian and Neil Hogg (Medical College of Wisconsin) for helpful discussions.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-61414 (to K. A. Pritchard, Jr.). D. W. Stepp is the recipient of an American Heart Association Scientist Development award. S. Welak was a participant in the Summer Program for Undergraduate Research at the Medical College of Wisconsin working in A. Pritchard's laboratory.
Address for reprint requests and other correspondence: K. A. Pritchard, Jr., Medical College of Wisconsin, Division of Pediatric Surgery, Cardiovascular Center, M4066 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: kpritch{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 28, 2002;10.1152/ajpheart.00029.2002
Received 15 January 2002; accepted in final form 18 March 2002.
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J. Ou, Z. Ou, D. W. Jones, S. Holzhauer, O. A. Hatoum, A. W. Ackerman, D. W. Weihrauch, D. D. Gutterman, K. Guice, K. T. Oldham, et al. L-4F, an Apolipoprotein A-1 Mimetic, Dramatically Improves Vasodilation in Hypercholesterolemia and Sickle Cell Disease Circulation, May 13, 2003; 107(18): 2337 - 2341. [Abstract] [Full Text] [PDF] |
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J. Ou, Z. Ou, D. G. McCarver, R. N. Hines, K. T. Oldham, A. W. Ackerman, and K. A. Pritchard Jr. Trichloroethylene Decreases Heat Shock Protein 90 Interactions with Endothelial Nitric Oxide Synthase: Implications for Endothelial Cell Proliferation Toxicol. Sci., May 1, 2003; 73(1): 90 - 97. [Abstract] [Full Text] [PDF] |
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Z. Ou, J. Ou, A. W. Ackerman, K. T. Oldham, and K. A. Pritchard Jr L-4F, an Apolipoprotein A-1 Mimetic, Restores Nitric Oxide and Superoxide Anion Balance in Low-Density Lipoprotein-Treated Endothelial Cells Circulation, March 25, 2003; 107(11): 1520 - 1524. [Abstract] [Full Text] [PDF] |
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