©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Oxidized Low-density Lipoprotein Decreases the Expression of Endothelial Nitric Oxide Synthase (*)

(Received for publication, July 25, 1994; and in revised form, November 3, 1994)

James K. Liao (§) Wee Soo Shin Wen Yee Lee Stephen L. Clark

From the Cardiovascular Division, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The atherogenic effects of low-density lipoprotein (LDL) may be mediated, in part, by its effect(s) on endothelial-derived nitric oxide (NO). To determine whether LDL can modulate NO production by changing NO synthase expression, we treated human saphenous vein endothelial cells with increasing concentrations of native or oxidized LDL (0-100 µg/ml) for various durations (0-72 h). Oxidized, but not native LDL caused a time-dependent decrease in steady-state NO synthase mRNA levels. This coincided with a maximal 56% decrease in NOS activity as determined by [^3H]arginine to [^3H]citrulline conversion. In the presence of actinomycin D, treatment with oxidized LDL reduced the half-life of NO synthase mRNA from 36 to 10 h. This decrease in NO synthase mRNA correlated with the degree of LDL oxidation and was attenuated by pretreatment with cycloheximide. Nuclear run-off studies showed a biphasic transcriptional pattern of NO synthase gene with an initial 25% decrease during the first 6 h followed by a maximal 2.2-fold increase over baseline during the subsequent 18 h. These results indicate that oxidized LDL regulates endothelial NOS expression through a combination of early transcriptional inhibition and post-transcriptional mRNA destabilization.


INTRODUCTION

Nitric oxide (NO) is produced in endothelial cells by an enzyme known as NO synthase which converts L-arginine to L-citrulline and NO(1, 2) . Endothelial NO synthase is a constitutively expressed enzyme with relatively low basal activity(3, 4) . Its activity, however, is rapidly stimulated by increases in intracellular calcium through specific signal tranduction pathways(5, 6) . Considerable evidence indicates that atherogenic oxidized low-density lipoprotein (LDL) (^1)can cause endothelial-dependent vasomotor abnormalities through inhibition of endothelial-derived NO(7, 8, 9) . Alteration of endothelial NO by oxidized LDL has been shown to occur at the level of membrane signal transduction(10) , NO production(11) , and NO inactivation(12) . These findings are important since nitric oxide can potentially inhibit several components of the atherogenic process such as vascular smooth muscle cell proliferation(13) , platelet aggregation(14) , monocyte adhesion(15) , and even oxidative modification of LDL(16) .

We have previously reported that native LDL can attenuate bradykinin-stimulated endothelial NO production via inhibition of specific guanine nucleotide-binding proteins (G(i)) which couple the bradykinin receptor to NO synthase activation(17) . This is in contrast to oxidized LDL which can inhibit NO production in response to the non-receptor-dependent calcium ionophore A23187(7, 8) . These findings suggest that oxidized LDL differs from native LDL in being able to inhibit NO production at the level of NO synthase. Indeed, recent studies have shown that oxidized, but not native LDL can decrease the activity of the cytokine-inducible NO synthase in murine macrophages without affecting its steady-state mRNA or protein expression(18) . Similar findings have not been reported for the constitutive NO synthase isoform in endothelial cells. Furthermore, the effects of oxidized LDL on the expression of endothelial NO synthase is not known. Since the regulation of endothelial NO production by oxidized LDL may mediate some of its atherogenic effects, we sought to characterize the effects of oxidized LDL on endothelial NO synthase expression at the level of transcriptional and post-transcriptional regulation.


EXPERIMENTAL PROCEDURES

Materials

All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Lipoprotein-deficient serum (Lot 83H9478), calcium ionophore A23187, Me(2)SO, dithiothreitol, L-arginine, heparin sulfate, cupric sulfate (CuSO(4)), polymyxin B, butylated hydroxytoluene, thiobarbituric acid, and 1,1,3,3-tetramethoxypropane were purchased from Sigma. [alpha-P]CTP (3000 Ci/mmol), [alpha-P]UTP (800 Ci/mmol), and [^3H]arginine (40.5 Ci/mmol) were supplied by DuPont NEN. Maloney murine leukemia virus reverse transcriptase was purchased from Life Technologies, Inc. (Gaithersburg, MD). Thermus aquaticus (Taq) DNA polymerase was obtained from Perkin Elmer. PCR primers were synthesized by Michael Berne (Tufts Medical School, Boston, MA). Purified human low-density lipoprotein (LDL, lot 730793), actinomycin D, cycloheximide, and L-N-monomethylarginine were obtained from Calbiochem (San Diego, CA). The Limulus amebocyte lysate kinetic assay was performed by BioWhittaker (Walkersville, MD). Nylon nucleic acid transfer membrane (Hybond) was purchased from Amersham Corp.

Cell Culture

Human endothelial cells were harvested from saphenous veins using Type II collagenase (Worthington Biochemical Corp.) as described previously(19) . Cells of less than three passages were grown to confluence in a culture medium containing Medium 199, 20 mM HEPES, 50 µg/ml endothelial cell growth serum (Collaborative Research Inc., Bedford, MA), 100 µg/ml heparin sulfate, 5 mML-glutamine (Life Technologies, Inc.), 5% fetal calf serum (Hyclone Lot 1112288), and antibiotic mixture of penicillin (100 units/ml), streptomycin (100 µg/ml), Fungizone (1.25 µg/ml). They were characterized by Nomarski optical microscopy (Zeiss ICM 405, 40 times objective) and staining for Factor VIII-related antigens(5) . For all experiments, the endothelial cells were placed in 5% lipoprotein-deficient serum for 48 h prior to treatment with the indicated concentrations of LDL. In some experiments, cells were pretreated with either actinomycin D (5 µg/ml) or cycloheximide (10 µg/ml) for 1 h prior to LDL treatment. Cellular confluence was maintained for all treatment conditions.

Characterization of LDL

Freshly obtained LDL samples were used within 7 days following isolation. Oxidized LDL was prepared by exposing known samples of native LDL to CuSO(4) (5-10 µM) at 37 °C for various durations (6-24 h). The samples were dialyzed with three changes of sterile buffer (150 mM NaCl, 0.01% EDTA, and 100 µg/ml polymyxin B, pH 7.4) prior to filtering through 0.2-µm membrane. Its purity was confirmed by SDS-polyacrylamide and cellulose acetate gel electrophoresis. Cholesterol and triglyceride content were determined as described previously(10) . The protein content was determined by the method of Lowry(20) . The indicated amounts of LDL used in the experiments were based upon the LDL protein concentration.

The degree of LDL oxidation was estimated by measuring the amounts of thiobarbituric acid reactive substances (TBARS) produced using a colorimetric assay for malondialdehyde(21) . LDL samples (50 µg) or supernatant from cell incubation (500 µl) were mixed with 1 ml of trichloroacetic acid (20%) and 1 ml of thiobarbituric acid (1%), and heated at 100 °C for 30 min. After cooling in a water bath (22 °C), the mixture was centrifuged at 12,000 times g for 15 min and the absorbance was measured at 535 nm (Beckman DU-64 Spectophotometer). Serial dilutions of 1,1,3,3-tetramethoxypropane which yields malondialdehyde was used to construct the standard curve. The extent of LDL modification was expressed as nanomole of malondialdehyde per mg of LDL protein.

NO Synthase Activity

NO production was measured by [^3H]arginine to [^3H]citrulline conversion as described previously with some modification(22) . Briefly, confluent endothelial cells treated with the indicated amounts of LDL were washed twice with phosphate-buffered saline and placed in 10 ml of Krebs-Ringer buffer containing NaCl (118 mM), KCl (4.7 mM), CaCl(2) (2.5 mM), MgSO(4) (1.2 mM), KH(2)PO(4) (1.2 mM), NaHCO(3) (25 mM), and glucose (11 mM), pH 7.4. After 5 min, [^3H]arginine (10 µCi) and L-arginine (10 µM) were added to the buffer and the endothelial cells were stimulated with the calcium ionophore A23187 (0.5 µM) for 10 min at 37 °C. In some studies, endothelial cells were pretreated with L-N-monomethylarginine (1 mM) for 15 min prior to A23187 stimulation. The assay was terminated with ice-cold phosphate-buffered saline containing L-arginine (5 mM) and EDTA (5 mM). Cell number (5-7 times 10^6/T-150 cm^2 culture flask) was determined using duplicate sets of confluent endothelial cells in another flask under corresponding lipoprotein treatment conditions followed by trypsinization and counting on a dispersion grid.

The supernatant was removed, and the cells were scraped and lysed by a probe sonicator (Model W185F, Ultrasonics, Inc., Plainview, NY). Approximately 4 ml of this cellular extract and supernatant was applied to a column containing 2 ml of Dowex 50WX-8 resin (pre-equilibrated with NaOH) followed by elution of [^3H]citrulline with 2 ml of water. A sample of the eluant (1 ml) was counted for 2 min in a liquid scintillation counter (Beckman LS 1800). Preliminary studies showed that these Dowex columns extracted >95% [^3H]arginine and <8% [^3H]citrulline. Nonspecific activity was determined by [^3H]citrulline production in the absence of A23187 stimulation and represented approximately 5% of total activity. The Me(2)SO (0.1%) used to solubilized A23187 did not produce appreciable changes (<1%) in baseline NO synthase activity.

Generation NO Synthase Oligonucleotide Probe

Total RNA was extracted by guanidine isothiocyanate and isolated by CsCl equilibrium centrifugation(23) . Complimentary DNA template was synthesized using oligo(dT)-primed total RNA and Moloney murine leukemia virus reverse transcriptase in a buffer containing 0.5 mM dNTP (Pharmacia Biotech Inc.), 10 units of RNasin (Promega, Madison, WI), 75 µM KCl, 3 µM MgCl(2), 10 µM dithiothreitol, and 50 mM Tris-HCl, pH 8.3. The NO synthase cDNA probe (JL58) was generated using the following paired primers (sense/antisense: GGCCGCTTIGAIGTGCTGCCTCT/GTCTCCATTICCAAAIGTGCTIGTIACCAC) by the polymerase chain reaction (PCR). The annealing/elongating/denaturing conditions for the PCR reaction was 48/72/95 °C for a total of 40 cycles in a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl(2). The PCR fragment (900 base pairs) was gel-purified, subcloned into PCR II vector (TA Cloning Kit, Invitrogen), and sequenced by the dideoxy chain termination method (Sequenase version 2.0, U. S. Biochemical Corp.).

Northern Blotting

Equal amounts of total RNA (10-20 µg) from approximately 4 times 10^5 endothelial cells were separated by 1% formaldehyde-agarose gel electrophoresis, transferred overnight onto Hybond nylon membranes by capillary action, and baked for 2 h at 80 °C prior to prehybridization. Radiolabeling of JL58 or human beta-actin cDNA probe (ATCC 37997, Rockville, MD) was performed using random hexamer priming, [alpha-P]CTP, and Klenow (Pharmacia). The membranes were hybridized with the probes overnight at 45 °C in a solution containing 50% formamide, 5 times SSC, 2.5 times Denhardt's solution, 25 mM sodium phosphate buffer, pH 6.5, 0.1% SDS, and 250 µg/ml salmon sperm DNA. All Northern blots were subjected to stringent washing conditions (0.2 times SSC, 0.1% SDS at 65 °C) prior to autoradiography with intensifying screen at -80 °C for 24-72 h.

Cloning of Endothelial NO Synthase

Poly(A) mRNA from 10^8 human endothelial cells was isolated from oligo(dT) columns (Collaborative Research Inc.) and used to construct an oligo(dT) and random-primed library (size 5.6 times 10^6) in gt10 phage (Stratagene, La Jolla, CA) according to previously described protocols(24) . Approximately 600,000 plaques were initially screened by hybridization with JL58 probe under similar conditions used for Northern blotting. Upon secondary and tertiary screening, 17 positive clones were eventually identified. Two clones (19A3C1 and 20B4B1) of greater than 4.0 kilobases were sequenced completely using primer extension and both contain open reading frames of 3609 base pairs corresponding to the already published endothelial NO synthase sequence(25) . One of these clones (19A3C1, GenBank/EMBL Data Bank accession number L26914) containing 4219 base pairs was subcloned into pGEM-3z (Promega Inc., Madison, WI) and used as template for transcriptional analysis.

In Vitro Transcription Studies

NO synthase transcription was measured according to the method previously described by Kavanaugh et al.(26) with some modifications. Confluent endothelial cells (7 times 10^6 cells) were placed in lipoprotein-deficient serum for 48 h to achieve quiescence prior to exposure with indicated amounts of LDL for various durations. Cells were washed twice with phosphate-buffered saline, trypsinized, and centrifuged at 300 times g for 5 min at 4 °C. The cellular pellet was gently resuspended in a buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl(2), and 0.5% Nonidet P-40, allowed to swell on ice for 15 min, and lysed by a Dounce homogenizer (30-35 strokes) with intermittent inspection of nuclei. The lysate was recentrifuged at 300 times g and the resulting nuclear pellet was resuspended in 100 µl of buffer containing 20 mM Tris-HCl, pH 8.1, 75 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 50% glycerol.

In vitro transcription using the nuclear pellet (100 µl) was carried out in a shaking water bath at 30 °C for 30 min in a buffer containing 10 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 300 mM KCl, 50 µM EDTA, 1 mM dithiothreitol, 0.5 units of RNasin, 0.5 mM CTP, ATP, and GTP, and 250 µCi of [alpha-P]UTP. The reaction was terminated by incubating the assay with 40 units of DNase I (Ambion, Austin, TX) for 20 min at 30 °C. Proteins in the mixture were degraded by a solution containing 0.4% SDS, 40 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 400 µg/ml proteinase K (E. Merck, Darmstadt, W. Germany), and extracted by phenol/chloroform. Ammonium acetate (2 M) and an equal volume of EtOH were added to the radiolabeled RNA transcripts. The mixture was kept in dry ice for 20 min prior to precipitation by centrifugation at 12,000 times g for 10 min.

Equal amounts (1 µg) of purified, denatured full-length endothelial NO synthase, human beta-actin, and linearized pGEM-3z cDNA were vacuum-transferred onto nylon membranes using a slot blot apparatus (Schleicher & Schuell). The membranes were baked and prehybridized as described for Northern blots. The precipitated radiolabeled transcripts (5 times 10^7 cpm) were resuspended in 2 ml of hybridization buffer containing 50% formamide, 5 times SSC, 2.5 times Denhardt's solution, 25 mM sodium phosphate buffer, pH 6.5, 0.1% SDS, and 250 µg/ml salmon sperm DNA. Hybridization of radiolabeled transcripts to the nylon membranes was carried out at 45 °C for 48 h. The membranes were then washed with 1 times SSC, 0.1% SDS for 1 h at 65 °C prior to autoradiography for 72 h at -80 °C.

Data Analysis

Band intensities from Northern and nuclear run-off assay blots were analyzed densitometrically by the National Institutes of Health IMAGE program(27) . All values are expressed as mean ± S.E. compared to controls and among separate experiments. Paired and unpaired Student's t tests were employed to determine the significance of changes in NO synthase activity and densitometric measurements. A significant difference was taken for p values less than 0.05.


RESULTS

Cell Culture

Relatively pure (>98%) human saphenous vein endothelial cell cultures were confirmed by their morphological features (i.e. cuboidal, cobble-stone, contact inhibited) using phase-contrast microscopy and immunofluorescent staining with antibodies to Factor VIII (data not shown). There were no observable adverse effects of lipoprotein-deficient serum or LDL on cellular morphology. Oxidized LDL having a higher concentration (>100 µg/ml) and TBARS value (>30 nmol/mg) caused vacuolization and some detachment of endothelial cells after 24 h. Otherwise, cellular confluency (7 times 10^6 cells/T-150 cm^2 flask) and viability as determined by trypan blue exclusion were maintained for all treatment conditions described.

Characterization of Lipoproteins

SDS-polyacrylamide gel electrophoresis of native LDL revealed a single protein band migrating at 510 kDa corresponding to apoB-100. This band became progressively degraded following oxidative modification (data not shown). Lipoprotein-deficient serum on gel electrophoresis was devoid of apoB-100 protein staining with Coomassie Blue and lipid staining with fat red 7B. The native LDL had a protein, cholesterol, and triglyceride concentration of 6.13 ± 0.24, 23.1 ± 1.11, and 1.55 ± 0.17 mg/ml, respectively. Neither the lipoprotein-deficient serum nor the LDL samples had detectable levels of endotoxin (<0.10 units/ml).

Native LDL had an initial TBARS value of 0.1 ± 0.1 nmol/mg which increased to 0.6 ± 0.3, 2.3 ± 0.8, and 4.5 ± 1.2 nmol/mg after 24, 48, and 72 h of incubation with endothelial cells in Medium 199 and lipoprotein-deficient serum, respectively. LDL samples which have been oxidized by exposure to CuSO(4) exhibited much higher levels of oxidative modification with TBARS values ranging from 7.3 ± 1.4 nmol/mg after a 6-h exposure with 5 µM CuSO4 to 48.8 ± 4.3 nmol/mg after a 24-h exposure with 10 µM CuSO4.

Effects of LDL on NO Synthase mRNA

The sequence of the JL58 probe (882 base pairs) corresponding to amino acids 284-578 is identical to the human endothelial NO synthase sequence previously reported(25) . Northern blotting using this probe showed that native LDL (100 µg/ml) produced a slight increase in the steady-state NO synthase mRNA levels after 24 h with respect to beta-actin mRNA (Fig. 1A). Lower concentrations of native LDL (10-50 µg/ml), however, did not produce any significant change in NO synthase mRNA levels after 72 h (data not shown). In contrast, oxidized LDL (50 µg/ml, TBARS 24.4 nmol/mg) caused a time-dependent progressive decrease in NO synthase mRNA levels with a steady-state half-life of 15 ± 3 h (Fig. 1B). Overall, the NO synthase steady-state mRNA level was reduced by 3.2-fold after 72 h with respect to beta-actin. In addition, changes in NO synthase mRNA levels was also observed in human aortic endothelial cells (Fig. 2) and correlated with the degree of LDL oxidation as assessed by TBARS (Fig. 3). For oxidized LDL (50 µg/ml) with TBARS value of greater than 30 nmol/mg, there was a marked decrease in NO synthase mRNA after 24 h. The calculated TBARS IC value was 15.7 ± 4.3 nmol/mg.


Figure 1: Northern blots (20 µg total RNA/lane) showing the time course of NO synthase mRNA expression in response to treatment with (A) native LDL (100 µg/ml) and (B) oxidized LDL (50 µg/ml, TBARS 24.4 nmol/mg). RNA loading was determined by hybridization to human beta-actin. This is representative of three separate experiments.




Figure 2: Northern blots (10 µg total RNA/lane) showing dose-dependent effects of oxidized LDL (TBARS 24.4 nmol/mg) at 24 h on NO synthase mRNA levels from primary human aortic endothelial cells. Equal RNA loading for each experiment was verified by hybridization to beta-actin. Experiments were performed two times.




Figure 3: NO synthase mRNA expression in response to increasing oxidative modification of LDL (50 µg/ml) as assessed by TBARS. Equal RNA loading for each experiment was verified by hybridization to beta-actin. Experiments were performed two times.



The half-life of NO synthase mRNA was determined in the presence of actinomycin D (5 µg/ml). Oxidized LDL (50 µg/ml) shortened the half-life of NO synthase mRNA from 36 ± 6 to 10 ± 3 h (Fig. 4, A and B). This reduction in NO synthase half-life suggests that oxidized LDL caused post-transcriptional decreases in NO synthase mRNA stability. Pretreatment with cycloheximide (10 µg/ml), however, prevented the decrease in NO synthase mRNA prior to 18 h (Fig. 5, A and B). These findings indicate that the effect(s) of oxidized LDL on NO synthase mRNA destabilization requires new protein synthesis.


Figure 4: A, Northern blots (20 µg total RNA/lane) showing the effects of actinomycin D (ACT, 5 µg/ml) on NO synthase (NOS) mRNA in the presence and absence (control) of oxidized (ox) LDL (50 µg/ml; TBARS 24.4 nmol/mg). Corresponding ethidium bromide-stained blots did not reveal any significant differences in ribosomal 28 S band intensities. B, densitometric analysis of Northern blots showing NO synthase mRNA levels (relative intensity) as a semi-log function of time in the presence of actinomycin D alone or in combination with oxidized LDL.




Figure 5: A, Northern blots (10 µg total RNA/lane) showing the effects of oxidized (ox) LDL (50 µg/ml; TBARS 24.4 nmol/mg) on NO synthase (NOS) mRNA expression in the presence and absence of cycloheximide (CHX; 10 µg/ml). Equal RNA loading was verified by equal ribosomal 28 S band intensities. B, densitometric analysis of Northern blots showing NO synthase mRNA levels (relative intensity) as a semi-log function of time.



Effects of LDL on NO Synthase Activity

Using the conversion of [^3H]arginine to [^3H]citrulline as a measure of NO synthase activity, stimulation of untreated endothelial cells by the calcium ionophore A23187 produced an activity of 1.33 ± 0.10 pmol/min/million cells. In the presence of L-N-monomethylarginine, NO synthase activity was reduced to 0.20 ± 0.06 pmol/min/million cells. Native LDL (100 µg/ml) caused a small, but significant decrease in A23187-stimulated NO synthase activity after 24 h (1.11 ± 0.06 pmol/min/million cells, p < 0.05) (Fig. 6A). In contrast, oxidized LDL (50 µg/ml, TBARS 24.4 nmol/mg) substantially reduced A23187-stimulated citrulline production in a time-dependent manner beginning after 6 h of treatment (1.12 ± 0.08 pmol/min/million cells, p < 0.05) and achieving maximal inhibition by 24 h of treatment (0.74 ± 0.05 pmol/min/million cells).


Figure 6: NO synthase activity as determined by calcium ionophore A23187 (0.1 µM)-stimulated [^3H]arginine to [^3H]citrulline conversion. A, oxidized LDL (50 µg/ml; TBARS 24.4 nmol/mg) time course; B, oxidized LDL (TBARS 24.4 nmol/mg) dose-response after 24 h. Each experiment was performed three times in duplicate.



Similar decreases in NO synthase activity were also observed with native and oxidized LDL (TBARS 24.4 nmol/mg) in a concentration-dependent manner with maximal effect occurring at a concentration of 50 µg/ml (Fig. 6B). After 24 h of treatment, native LDL decreased NO synthase activity to 1.07 ± 0.05 pmol/min/million cells; whereas, oxidized LDL had a greater effect in decreasing NO synthase activity to 0.65 ± 0.06 pmol/min/million cells. The calculated IC values for native and oxidized LDL were 14.4 ± 0.18 and 11.2 ± 0.12 nmol/mg, respectively. Furthermore, decrease in NO synthase activity also correlated with the degree of LDL oxidation. After exposure to LDL (50 µg/ml) for 24 h, the calculated TBARS IC value was 21.4 ± 3.8 nmol/mg (data not shown).

Effects of LDL on NO Synthase Transcription

Nuclear run-off studies using oxidized LDL (50 µg/ml, TBARS 24.4 nmol/mg) showed a biphasic pattern of transcriptional regulation of NO synthase gene (Fig. 7). Compared to baseline levels, treatment with oxidized LDL caused a 25% decrease in NO synthase mRNA transcription during the first 6 h followed by a 1.8- and 2.2-fold increase over baseline at 12 and 24 h, respectively.


Figure 7: Blots from a representative nuclear run-off experiment showing the effects of oxidized LDL (50 µg/ml; TBARS 24.4 nmol/mg) on time-dependent transcriptional activity of beta-actin and NO synthase (NOS) gene. Band intensities of NO synthase compared to beta-actin (relative index) at the indicated time point for three separate experiments are shown. Nonspecific activity was determined by hybridization to pGEM vector.



Preliminary studies using different amounts of radiolabeled RNA transcripts demonstrate that under our experimental conditions, hybridization was linear and nonsaturable. The density of each NO synthase band was standardized to the density of its corresponding beta-actin band at each time point (relative index). To exclude the possibility that changes in NO synthase relative index is due to changes in beta-actin transcription caused by oxidized LDL, another marker, beta-tubulin, was included on each of the nuclear run-off blots. Similar relative indices were obtained when NO synthase transcription was standardized to beta-tubulin transcription (data not shown). The specificity of each band was determined by the lack of hybridization to the nonspecific pGEM cDNA vector.


DISCUSSION

In this study, we have characterized the effects of LDL on endothelial NO synthase mRNA expression and correlated these effects with decreases in NO production as assessed by L-arginine to L-citrulline conversion. We found that exposure to noncytotoxic concentrations of oxidized, but not native LDL caused a progressive time-dependent decrease in steady-state NO synthase mRNA levels. Furthermore, additional studies using human aortic endothelial cells yielded similar results. The mechanism(s) responsible for this decline is dependent upon oxidative modification of LDL and involves a combination of early transcriptional inhibition and predominantly, post-transcriptional mRNA degradation. This latter effect of oxidized LDL on NO synthase mRNA apparently requires the participation of newly synthesized protein(s) as demonstrated by studies using the protein synthesis inhibitor, cycloheximide. It is not known whether these transcriptional and post-transcriptional processes are regulated separately by oxidized LDL, and are therefore, perhaps differentially sensitive to specific characteristics of the LDL particle such as the concentration or the degree of modification of its constituents.

Increases in NO synthase mRNA degradation have been observed with exposure to certain proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1beta(28, 29) . Both tumor necrosis factor-alpha and interleukin-1beta are associated with atherogenesis and their presence has been demonstrated in atherosclerotic lesions(30) . Endothelial cells can produce interleukin-1beta which in turn, can regulate diverse vascular functions in an autocrine and paracrine fashion(31) . Analysis of our endothelial NO synthase cDNA reveals a sequence (AUUUA) in the 3`-untranslated region which is known to be involved in mRNA destabilization(32) . It remains to be determined whether this sequence is utilized as the putative site of regulation by oxidized lipids and cytokines. Since atherosclerotic vessels contain oxidized LDL (33) and respond abnormally with respect to altered endothelial NO activity(34) , it is interesting to speculate whether translational induction of these cytokines by oxidized LDL is the mechanism by which oxidized LDL destabilizes NO synthase mRNA.

The regulation of NO synthase gene transcription by oxidized LDL is complex and consists of at least two separate components. Early transcriptional inhibition occurring at 6 h is most probably due to direct effects of oxidized LDL since small decreases in transcriptional activity can be observed within 30-120 min of exposure to oxidized LDL. (^2)The 5`-flanking promoter region of the human constitutive endothelial NO synthase has recently been characterized and found to contain a DNA binding sequence (GCGGGGCG) for an inhibitory transcription factor(35) . It is not known whether oxidized LDL represses NO synthase transcription through activation of this DNA binding protein or through other yet unidentified silencer elements. Increases in NO synthase transcriptional activity occurring after 12 h is probably the result of indirect effects of oxidized LDL or secondary to some feedback counter-regulatory mechanism induced by declining NO synthase activity and steady-state mRNA levels. This delayed increase in NO synthase transcription suggests translational induction of new protein(s) as the most likely mechanism for enhanced transcription although direct effects of oxidized LDL components with delayed access into endothelial subcellular compartments cannot be excluded. Nevertheless, despite a greater than 2-fold increase in transcription rate, steady-state NO synthase mRNA levels continue to fall after 12 h as a result of an even higher rate of NO synthase mRNA degradation. This marked effect on mRNA stability is evident by the more than 3-fold reduction in NO synthase half-life as determined in the presence of the transcriptional inhibitor, actinomycin D.

Despite increases in NO synthase mRNA after exposure to native LDL, NO synthase activity remains mildly reduced. This suggests that native LDL can also inhibit NO production by either decreasing NO synthase protein expression or attenuating its enzymatic activity. This is in agreement with previous studies showing that short-term exposure of rabbit aorta to native LDL can diminish its endothelial-dependent response to the calcium ionophore A23187(36) . In contrast, our results showed that oxidized LDL produced a more profound inhibitory effect on NO synthase activity with most of the reduction occurring within 24 h. The reduction in NO synthase mRNA expression most likely contributes to this exaggerated effect although a direct effect on protein expression and/or activity cannot be ruled out. This finding is consistent with animal studies showing that the inhibitory effects of native and oxidized LDL operate through different mechanisms in attenuating the release of endothelial-derived NO(37) .

Interestingly, the IC values of NO synthase activity for native and moderately oxidized LDL were fairly comparable indicating that oxidative modification of the LDL particle rather than its concentration is more important in determining its inhibitory effects. Indeed, we found that NO synthase mRNA expression is negatively regulated by the degree of LDL oxidation as measured by the formation of malondialdehyde. Changes in the LDL particle associated with oxidative modification include lipid peroxidation, formation of conjugated dienes, carbonyl modification of apoB-100, and enzymatic conversion of phosphatidylcholine to lysophosphatidylcholine by phospholipase A(2) in apoB-100(38, 39) . Recent reports suggest that attenuation of bradykinin-stimulated calcium transients and inhibition of endothelial-dependent relaxation by oxidized LDL is mediated by its lysophosphatidylcholine component(40, 41) . However, studies using different preparations of human oxidized LDL with similar lysophosphatidylcholine content demonstrate varying inhibitory effects on endothelium-dependent relaxations of rabbit aorta(8) . These findings suggest that components in oxidized LDL other than lysophosphatidylcholine may also be influencing NO synthase expression and activity. It remains to be determined what the roles of each of these components in oxidized LDL are in regulating NO synthase mRNA expression and enzymatic activity.

The effect of oxidized LDL on NO synthase expression in atherosclerosis, however, remains controversial. Studies of human atherosclerotic plaques with in vitro hybridization techniques demonstrate normal expression of NO synthase in the overlying endothelium(42) . An increase in NO production was reportedly found in aorta from cholesterol-fed rabbits although NO activity was greatly reduced as a result of enhanced inactivation(11) . Yet, considerable evidence including the findings in this study suggest that oxidized LDL reduces endothelial NO production at the level of NO synthase(43) . Indeed, we have found a strong correlation between decreases in NO synthase mRNA expression and reduction in NO synthase activity.

Several possible explanations could account for this discrepancy. First, oxidized lipids may affect other cell types such as macrophages and vascular smooth muscle cells which could indirectly influence NO synthase expression in vivo. Second, the regulation of NO synthase protein by oxidized LDL is not known and may differ from that of NO synthase mRNA. Third, oxidized LDL preparations used in various studies or found in atherosclerotic arteries may differ with respect to the concentrations encountered by endothelial cells or the degree of oxidative modification. Perhaps exposure of endothelial cells to lower concentrations of native or mildly-modified LDL would not alter NO synthase mRNA expression while higher levels of oxidative modification would inhibit expression. Finally, NO synthase mRNA transcription and its degradation may be regulated independently by different components of oxidized LDL. For example, certain factors in oxidized LDL which destabilize NO synthase mRNA may either be absent or counteracted by other mediators in the atherosclerotic lesion. Consequently, increases in NO synthase mRNA expression would result from the delayed transcriptional induction by oxidized LDL.

In summary, regulation of endothelial NO synthase expression involves complex interactions between transcriptional and post-transcriptional effects of oxidized LDL. It remains to be determined which component(s) of oxidized LDL and which newly synthesized protein(s) mediate these effects.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL02508 and the American Heart Association Grant-in-Aid Award (to J. K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cardiovascular Div., Department of Medicine, 221 Longwood Ave., LMRC-307, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-732-6961.

(^1)
The abbreviations used are: LDL, low density lipoprotein; PCR, polymerase chain reaction; TBARS, thiobarbituric acid reactive substances; NO, nitric oxide.

(^2)
J. K. Liao, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Drs. Peter Libby and Maria Muszynski for generously providing human saphenous vein and aortic endothelial cells.


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