Skip to main content
  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
  • Impact Factor 13.965
  • Facebook
  • Twitter

  • My alerts
  • Sign In
  • Join

  • Advanced search

Header Publisher Menu

  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

Circulation Research

  • My alerts
  • Sign In
  • Join

  • Impact Factor 13.965
  • Facebook
  • Twitter
  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
Original Contributions

LDLs Impair Vasomotor Function of the Coronary Microcirculation

Role of Superoxide Anions

Travis W. Hein, Lih Kuo
Download PDF
https://doi.org/10.1161/01.RES.83.4.404
Circulation Research. 1998;83:404-414
Originally published August 24, 1998
Travis W. Hein
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lih Kuo
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Tables
  • Info & Metrics

Jump to

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Selected Abbreviations and Acronyms
    • Acknowledgments
    • References
  • Figures & Tables
  • Info & Metrics
  • eLetters
Loading

Abstract

Abstract—Oxidized LDLs (Ox-LDLs) inhibit endothelium-dependent dilation of isolated conduit arteries in a manner comparable to the impairment demonstrated in atherosclerotic vessels. However, it is not known whether the microvessels, which do not develop atherosclerotic lesions, are susceptible to Ox-LDL. Since endothelial release of NO plays an important role in vasodilation and since its dysfunction associated with atherosclerosis has been shown to extend into the coronary microcirculation, we hypothesized that Ox-LDLs impair endothelium-dependent vasodilation of coronary arterioles by reducing the synthesis and/or release of NO. To test this hypothesis, porcine subepicardial vessels (50 to 100 μm) were isolated, cannulated, and pressurized to 60 cm H2O without flow for in vitro study. Isolated vessels developed basal tone and dilated in a dose-dependent manner to the endothelium-dependent vasodilators serotonin, ATP, and ionomycin. These vasodilatory responses were inhibited by the NO synthase inhibitor NG-monomethyl-l-arginine and were subsequently reversed by extraluminal administration of the NO precursor l-arginine (3 mmol/L), suggesting the involvement of NO in these vasomotor responses. Intraluminal incubation of the vessels with native LDL (N-LDL) or Ox-LDL (1 mg protein/mL) significantly attenuated dilations to serotonin, ATP, and ionomycin. Ox-LDL produced more severe inhibition than did N-LDL, and the inhibitory effect was comparable to that of NG-monomethyl-l-arginine. The inhibitory effects of N-LDL and Ox-LDL were reversed by exogenous l-arginine (3 mmol/L) and were prevented by sodium dihydroxybenzene disulfonate (Tiron), a cell-permeable superoxide scavenger. In contrast, administration of the cell-impermeable superoxide scavenger superoxide dismutase prevented the inhibitory effect of N-LDL but not of Ox-LDL. In addition, the inhibitory effects of LDL were not restored by d-arginine or by removal of intraluminal LDL. Neither N-LDL nor Ox-LDL altered endothelium-independent vasodilation to sodium nitroprusside. These results indicate that coronary arterioles are susceptible to LDLs that specifically impair endothelium-dependent vasodilation by reducing NO synthesis. It is suggested that the initiation of superoxide anion production and the subsequent l-arginine deficiency may be responsible for the detrimental effect of LDL.

  • arteriole
  • atherosclerosis
  • endothelium
  • l-arginine
  • nitric oxide
  • superoxide anion

Low density lipoproteins at high plasma concentrations are a major risk factor for the development of atherosclerosis.1 Accumulating evidence suggests that Ox-LDL is closely linked to the atherosclerosis-related pathology.2 Ox-LDL may contribute to atherogenesis by a variety of mechanisms, such as being a chemoattractant for monocytes,3 enhancing lipid accumulation by monocytes,4 impairing metabolic activity of vascular cells,4 and altering endothelial function.5 6 It is generally accepted that the normal endothelium plays an important role in the regulation of vascular function through the release of vasoactive substances in response to various stimuli. One of the most important substances released from the endothelium is endothelium-derived relaxing factor, which has been identified chemically as NO7 or an NO-containing compound.8 In endothelial cells, NO is synthesized from the conversion of l-arginine to l-citrulline by constitutive NO synthase.9 The released NO subsequently activates soluble guanylyl cyclase in underlying vascular smooth muscle cells and thus produces vasodilation. In addition, NO has also been shown to inhibit platelet adherence and aggregation,10 smooth muscle proliferation,11 and endothelial cell–leukocyte interactions,12 all of which are key events in atherogenesis. Interestingly, endothelium-dependent vasodilation is impaired in arteries treated with Ox-LDL in animals5 6 and humans,13 and therefore it is speculated that a reduction in NO synthesis or release may be involved in vascular dysfunction and the development of atherosclerosis.

It is important to note that previous in vitro studies involving Ox-LDL were performed with large-conduit arteries,5 6 13 which have been shown to be the primary site for the formation of atherosclerotic lesions.14 However, it is not clear whether the small arteriolar vessels, which are predominantly responsible for regulation of blood flow to tissues, are also susceptible to Ox-LDL. Although these resistance vessels do not develop atherosclerotic lesions,15 previous studies provide evidence for impaired vascular function in the coronary microcirculation during atherosclerosis.15 16 These results suggest that Ox-LDL may play a pathophysiological role in eliciting microvascular dysfunction by altering vasomotor function. Therefore, the goals of the present study were to (1) quantify the effects of N-LDL and Ox-LDL on endothelium-dependent and -independent vasodilation of coronary microvessels, (2) determine whether NO deficiency is involved in endothelial dysfunction, and (3) elucidate the vascular mechanisms for NO deficiency during exposure to LDL. These goals were accomplished by studying the vasodilatory response of isolated coronary arterioles (50 to 100 μm in diameter) before and after incubation with N-LDL or Ox-LDL, thereby eliminating the confounding influences from blood-borne substances and neurohumoral control mechanisms. Since the majority of coronary resistance (>60%) resides in arterioles <150 μm in diameter,17 it is important to understand the vasomotor regulation of these microvessels during exposure to atherogenic substances, ie, N-LDL and Ox-LDL.

Materials and Methods

General Preparation

Pigs (8 to 12 weeks old of either sex) were sedated with an intramuscular injection of tiletamine and zolazepam (1:1, 4.4 mg/kg) and xylazine (2.2 mg/kg) and then anesthetized and heparinized by intravenous administration of pentobarbital sodium (20 mg/kg) and heparin (1000 U/kg), respectively, via the marginal ear vein. Pigs were intubated and ventilated with room air. After a left thoracotomy was performed, the heart was electrically fibrillated, excised, and immediately placed in cold (5°C) saline solution. The procedures followed were in accordance with guidelines set by the Laboratory Animal Care Committee at Texas A&M University.

Isolation and Cannulation of Microvessels

The techniques for identification and isolation of porcine coronary microvessels were described previously.18 In brief, a mixture of india ink and gelatin in PSS containing (in mmol/L) NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS 3.0 was perfused into the left anterior descending artery (0.3 mL) and the circumflex artery (0.4 mL) to enable visualization of coronary microvessels. Subepicardial arteriolar branches (50- to 100-μm internal diameter and 0.6 to 1.0 mm long without branches) from the left anterior descending or circumflex arteries were selected and carefully dissected from the surrounding cardiac tissue under cold (5°C) PSS containing BSA (1%, Amersham) at pH 7.4. Each isolated arteriole was then transferred for cannulation to a Lucite vessel chamber containing PSS-albumin equilibrated with room air at ambient temperature. One end of the microvessel was cannulated with a glass micropipette (40 μm in tip diameter) filled with filtered PSS-albumin, and the outside of the microvessel was securely tied to the pipette with an 11-0 ophthalmic suture (Alcon). The ink-gelatin solution inside the vessel was flushed out at low perfusion pressure (<20 cm H2O). Then the other end of the vessel was cannulated with a second micropipette and tied with a suture. We have previously shown that the ink-gelatin solution has no detectable detrimental effect on either endothelial or vascular smooth muscle function.18 19

Instrumentation

After cannulation of a blood vessel, the chamber was transferred to the stage of an inverted microscope (model IM35, Zeiss) coupled to a CCD camera (KP-161, Hitachi) and videomicrometer (Microcirculation Research Institute, Texas A&M University Health Science Center). Internal diameters of the vessel were measured throughout the experiment by using videomicroscopic techniques incorporated with a MacLab (AD Instruments Inc) data acquisition system.20 The micropipettes were connected to independent reservoir systems, and intraluminal pressures were measured through sidearms of the 2 reservoir lines by low-volume-displacement strain-gauge transducers (Statham P23 Db, Gould). The isolated vessels were pressurized without flow by setting both reservoirs at the same hydrostatic level. Leaks were detected by differences between reservoir pressure and intraluminal pressure. Preparations with leaks were excluded from further study.

Preparation of LDLs

Human LDLs (5 mg protein/mL) were obtained from Sigma Chemical Co. LDLs were oxidized by exposure to 10 μmol/L CuCl2 for 8 to 24 hours at room temperature. The degree of LDL oxidation was measured by using a spectrophotometric method21 and TBARS assay.22 One characteristic of LDL oxidation involves the formation of conjugated dienes during the peroxidation of polyunsaturated fatty acids, which was monitored by UV absorption at 234 nm with a spectrophotometer (DU-65, Beckman Instruments Inc). Oxidation was stopped after 90% to 100% of maximal oxidation had been achieved by the addition of 1 mmol/L EDTA to the LDL. For TBARS analysis, LDL samples (10 to 100 μg) were mixed with 1 mL of trichloroacetic acid (20%) and 1 mL of thiobarbituric acid (1%) and heated at 100°C for 30 minutes. After being cooled in a water bath (22°C), the mixture was centrifuged at 12 000g for 15 minutes, and the absorbance was measured at 550 nm with a microplate reader (Molecular Devices Corp). Serial dilutions of 1,1,3,3-tetramethoxypropane, which yields MDA, were used to construct the standard curve. TBARS data were expressed as nanomoles of MDA per milligram of LDL protein. N-LDL and Ox-LDL were dialyzed separately against Dulbecco’s PBS for 24 hours. The 2 forms of LDL were stored at 4°C and used within 2 weeks. Before each experiment, N-LDL and Ox-LDL were filtered with a 0.2-μm filter (Corning) and diluted to their final concentration (1 mg protein/mL) in PSS-albumin. The protein concentration of LDL was determined by using the modified Lowry assay.23 N-LDL used in this study exhibited only negligible oxidation levels (0.50±0.08 nmol MDA/mg LDL protein, n=6), whereas Ox-LDL presented extensive oxidation (13.40±1.90 nmol MDA/mg LDL protein, n=7). These initial levels were not significantly altered after 2 weeks.

Role of NO in Arteriolar Dilations to Serotonin, ATP, and Ionomycin

The following protocol was performed to determine the role of NO in receptor-dependent dilation to serotonin and ATP and in receptor-independent dilation to the calcium ionophore ionomycin.24 The cannulated arterioles were bathed in PSS-albumin and equilibrated with room air; the temperature was maintained at 36°C to 37°C by an external heat exchanger. The vessel was set to its in situ length18 and allowed to develop basal tone at 60 cm H2O intraluminal pressure without flow. This pressure has been demonstrated in coronary arterioles of this size in vivo.17 After the vessels developed basal tone (30 to 40 minutes), the dose-response curves for serotonin (10−10 to 10−6 mol/L), ATP (10−9 to 10−5 mol/L), and ionomycin (10−9 to 3×10−7 mol/L) were examined before and after extraluminal incubation of the NO synthase inhibitor L-NMMA (10 μmol/L, Calbiochem)25 for 40 minutes. Subsequently, the effect of the NO precursor l-arginine (3 mmol/L, 20-minute incubation) on dose-dependent dilations to the aforementioned drugs was examined in the presence of L-NMMA.

Effect of LDL on Endothelium-Dependent, NO-Mediated Vasodilation

To study the effect of LDL (N-LDL and Ox-LDL) on endothelium-dependent vasodilation to serotonin, ATP, and ionomycin, dose-dependent dilations to these agonists were examined before and after replacing the solution inside the vessel with LDL (N-LDL or Ox-LDL, 0.3 or 1 mg protein/mL) and then incubating the fluid-filled vessels for 60 minutes. Finally, l-arginine (3 mmol/L) was administered to the LDL-treated vessels for 20 minutes, and dose-response curves to the various drugs described above were further established.

Effect of Incubation Time on Endothelium-Dependent Vasodilation

To exclude the possibility that the observed vascular dysfunction was a result of nonspecific time-dependent deterioration of vasodilatory function during incubation with N-LDL or Ox-LDL, vessels were subjected to the same experimental interventions as described above except with a vehicle solution. The dose-dependent responses of isolated vessels to serotonin, ATP, and ionomycin were studied after incubating the vessels with vehicle for 60 minutes. In some experiments, 1 mg protein/mL albumin was added to the vehicle solution to examine whether the observed phenomenon was due to a nonspecific effect of increased luminal protein.

Specificity of l-Arginine

To examine whether the vasodilatory function of normal vessels was altered by l-arginine, vasodilations to ATP and serotonin were evaluated before and after treatment of the vessels with l-arginine (3 mmol/L) for 20 minutes. In addition, to determine whether the effect of l-arginine on the impaired vascular function was stereospecific, vasodilations of Ox-LDL–treated vessels to ATP and serotonin (10−7 mol/L) were examined in the presence of d-arginine (3 mmol/L) or l-arginine (3 mmol/L).

Effect of LDL Removal on Endothelium-Dependent Vasodilation

To determine whether the impaired vascular function was a result of extracellular scavenging of NO by LDL, the vasodilation induced by serotonin (10−7 mol/L) was examined in the presence of LDL (N-LDL or Ox-LDL, 60 minute-incubation) and after LDL removal by replacing the intraluminal LDL with vehicle solution. It should be noted that agonist-induced responses were examined within 30 minutes after LDL removal.

Contribution of Superoxide Anions to Vascular Dysfunction

To evaluate whether superoxide anions contributed to the vascular dysfunction elicited by LDL (N-LDL or Ox-LDL), coronary arteriolar dilations to serotonin (10−7 mol/L) and ATP (10−9 to 10−5 mol/L) were established before and after intraluminal administration of LDL (N-LDL or Ox-LDL, 1 mg protein/mL) or of LDL containing the superoxide anion scavengers SOD (100 U/mL, 60-minute incubation) or sodium dihydroxybenzene disulfonate (Tiron, 1 mmol/L, 60-minute incubation). Exogenous SOD enzyme activity is primarily extracellular, whereas Tiron is capable of scavenging superoxide from both the intracellular and extracellular environment.26 27 28 In addition, to examine whether the vasodilatory function of normal vessels was altered by Tiron, vasodilations to ATP, serotonin, and ionomycin were evaluated before and after treatment of vessels with Tiron (1 mmol/L) for 60 minutes.

Effect of LDL on Endothelium-Independent Vasodilation

To study the effect of LDL on endothelium-independent vasodilation, the dose-dependent response of isolated vessels to SNP (10−9 to 10−4 mol/L) was studied, and then the vasodilation to this drug was reexamined after incubation of vessels with LDL (N-LDL or Ox-LDL, 1 mg protein/mL) for 60 minutes.

Chemicals

Drugs were obtained from Sigma Chemical Co except where specifically stated otherwise. Serotonin, ATP, L-NMMA, SNP, SOD (bovine), d-arginine, l-arginine, and Tiron were dissolved in PSS. Ionomycin (Calbiochem) was dissolved in DMSO as a stock solution (1 mmol/L), and subsequent concentrations were diluted in PSS. The final concentration of DMSO in the vessel bath was 0.04%. A vehicle control study indicated that this final concentration of DMSO had no effect on arteriolar function.

Data Analysis

At the end of each experiment, the vessel was relaxed with SNP (10−4 mol/L) to obtain its maximal diameter at 60 cm H2O intraluminal pressure. To ensure that this dose of SNP indeed produced maximal dilation, in some studies (control or LDL-treated vessels) an EDTA (1 mmol/L)-calcium–free solution was added to the vessel bath containing SNP (10−4 mol/L) for 30 minutes. It was found that the vessel diameter was not further increased by this treatment, indicating that SNP 10−4 mol/L was sufficient to produce maximal vasodilation. Therefore, all diameter changes were normalized to the diameter in the presence of 10−4 mol/L SNP and expressed as a percentage of maximal dilation. All data are presented as mean±SEM. Statistical comparisons of vasomotor responses under various treatments were performed with 1- or 2-way ANOVA when appropriate and tested with the Fisher protected least significant difference multiple-range test. Differences in resting diameter before and after pharmacological interventions and the vasodilation to serotonin (10−7 mol/L) before and after LDL treatments were compared by the paired Student t test. The extent of vasodilation at the highest concentration of agonists after N-LDL and Ox-LDL treatments was compared by the unpaired Student t test. Significance was accepted at P<0.05.

Results

Role of NO in Vasodilations to Serotonin, ATP, and Ionomycin

All vessels developed a similar level of basal tone (≈68±1% of their maximal diameter) within 40 minutes at a 36°C to 37°C bath temperature with 60 cm H2O intraluminal pressure. The average resting and maximal diameters of all vessels (n=109) studied were 77±2 and 114±2 μm, respectively. Under control conditions, serotonin, ATP, and ionomycin dilated the coronary arterioles in a dose-dependent manner and produced 85%, 90%, and 80% of maximal dilation, respectively, at their highest concentration studied (Figure 1⇓). The NO synthase inhibitor L-NMMA (10 μmol/L) significantly attenuated these vasodilations by increasing the threshold concentration of each drug and by inhibiting the extent of vasodilation (Figure 1⇓). Administration of l-arginine (3 mmol/L) to the L-NMMA–treated vessels subsequently restored the vasodilatory responses (Figure 1⇓), suggesting that these agonists elicited NO-mediated dilation of coronary arterioles. The inhibitory effect of L-NMMA appears to be specific for NO because dilation of isolated vessels to the endothelium-independent vasodilator SNP was not altered by L-NMMA (data not shown), as we have previously demonstrated in the same tissue.19

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

Effect of L-NMMA on arteriolar dilations to serotonin, ATP, and ionomycin. L-NMMA (10 μmol/L) attenuated dilations of vessels to serotonin (A, n=7), ATP (B, n=5), and ionomycin (C, n=6). l-Arginine (3 mmol/L) restored dilations to serotonin, ATP, and ionomycin in L-NMMA–treated vessels. In this set of experiments resting control diameter was 82±5 μm; in the presence of L-NMMA, resting diameter was 73±5 μm; in the presence of l-arginine, resting diameter was 77±6 μm; and maximal diameter was 122±8 μm. *P<0.05 vs control or L-NMMA+l-arginine.

Effect of LDL on Endothelium-Dependent, NO-Mediated Vasodilation

Exposure of coronary arterioles to LDL (N-LDL or Ox-LDL, 1 mg protein/mL for 60 minutes) did not alter resting vascular tone but significantly attenuated dose-dependent dilations to serotonin, ATP, and ionomycin (Figures 2⇓ and 3⇓). The threshold concentration for dilation to each agonist was markedly increased by N-LDL and Ox-LDL. In fact, after Ox-LDL treatment, a slight but significant vasoconstriction was observed at the lower concentration of ATP (10−8 and 10−7 mol/L) (Figure 3B⇓) and ionomycin (10−8 mol/L) (Figure 3C⇓). The dilations of coronary arterioles to the highest concentrations of serotonin, ATP, and ionomycin were diminished to 55%, 60%, and 35%, respectively, after N-LDL treatment. In comparison with N-LDL, arteriolar dilations to the highest dose of the same agonists were reduced to a greater extent, to only 35%, 40%, and 20%, respectively, after Ox-LDL treatment (P<0.05 versus N-LDL). The vasodilatory responses were completely restored by subsequent incubation of LDL-treated vessels with l-arginine (3 mmol/L, 20 minutes) (Figures 2⇓ and 3⇓). Figure 4⇓ shows that the inhibitory effect of LDL on vasodilation to serotonin was dose dependent. A lower concentration of N-LDL (0.3 mg protein/mL) did not have an inhibitory effect on serotonin-induced vasodilation (Figure 4A⇓). However, the lower concentration of Ox-LDL produced a significant attenuation of vasodilation to serotonin. This inhibitory effect was enhanced by increasing the Ox-LDL concentration to 1 mg protein/mL (Figure 4B⇓).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

Effect of N-LDL on arteriolar dilations to serotonin, ATP, and ionomycin. N-LDL (1 mg protein/mL) significantly attenuated dilations of vessels to serotonin (A, n=8), ATP (B, n=8), and ionomycin (C, n=3). l-Arginine (3 mmol/L) completely restored dilations to serotonin, ATP, and ionomycin in N-LDL–treated vessels. In this set of experiments resting control diameter was 72±4 μm; in the presence of N-LDL, resting diameter was 76±4 μm; in the presence of l-arginine, resting diameter was 81±5 μm; and maximal diameter was 113±5 μm. *P<0.05 vs control or N-LDL+l-arginine.

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

Effect of Ox-LDL on arteriolar dilations to serotonin, ATP, and ionomycin. Ox-LDL (1 mg protein/mL) significantly attenuated dilations of vessels to serotonin (A, n=7), ATP (B, n=6), and ionomycin (C, n=6). l-Arginine (3 mmol/L) completely restored dilations to serotonin, ATP, and ionomycin in Ox-LDL–treated vessels. In this set of experiments resting control diameter was 73±4 μm; in the presence of Ox-LDL, resting diameter was 71±4 μm; in the presence of l-arginine, resting diameter was 68±3 μm; and maximal diameter was 105±5 μm. *P<0.05 vs control or Ox-LDL+l-arginine.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

Concentration-dependent effect of LDL on arteriolar dilation to serotonin. A, Serotonin-induced vasodilation was not altered by 0.3 mg protein/mL N-LDL (resting diameter, 101±12 μm; maximal diameter, 136±12 μm; n=3) but was significantly inhibited by 1 mg protein/mL N-LDL (resting diameter, 70±6 μm; maximal diameter, 106±7 μm; n=8). B, Ox-LDL at 0.3 mg protein/mL (resting diameter, 73±8 μm; maximal diameter, 105±8 μm; n=4) significantly inhibited vasodilation to serotonin, and this inhibitory effect was enhanced by increasing the concentration to 1 mg protein/mL (resting diameter, 65±7 μm; maximal diameter, 96±8 μm; n=7). *P<0.05 vs control; †P<0.05 between Ox-LDL groups.

Time-Dependent Effect on Arteriolar Function

Since impaired vasodilation was observed after LDL (N-LDL or Ox-LDL) treatment for 60 minutes, it is possible that the altered vascular response was a result of time-dependent deterioration of endothelial function rather than the specific action of LDL. To address this issue, another set of experiments was performed in isolated coronary arterioles treated intraluminally with a vehicle solution for 60 minutes. As shown in the Table⇓, dose-dependent dilations of coronary arterioles in response to serotonin, ATP, and ionomycin were not altered after this treatment. It should be noted that these vasodilatory responses were also not altered by excess albumin in the vehicle solution.

View this table:
  • View inline
  • View popup
Table 1.

Endothelium-Dependent Vasodilations to Pharmacological Agonists Before and After Administration of Vehicle

Specificity of l-Arginine

Pretreatment of coronary arterioles with l-arginine (3 mmol/L) for 20 minutes did not alter the vasodilatory response to ATP (Figure 5A⇓) and serotonin (data not shown). To determine whether l-arginine was stereospecific for the restoration of vascular function impaired by LDL, dilation of isolated vessels to serotonin (10−7 mol/L) was examined in the presence of d-arginine (3 mmol/L) or l-arginine (3 mmol/L). Figure 5B⇓ shows that the impaired vasodilation to serotonin by Ox-LDL was not affected by d-arginine but was completely reversed by l-arginine.

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5.

Specificity of l-arginine. A, l-Arginine (3 mmol/L) did not affect dose-dependent dilation to ATP, demonstrating that vascular function of normal vessels is not influenced by exogenous l-arginine (resting diameter, 54±5 μm; maximal diameter, 82±6 μm; n=3). B, In the presence of l-arginine but not of d-arginine (3 mmol/L), impaired dilation to serotonin in Ox-LDL–treated vessels was completely restored (resting diameter, 81±10 μm; maximal diameter, 117±8 μm; n=3). *P<0.05 vs control.

Effect of LDL Removal on Serotonin-Induced Vasodilation

Coronary arteriolar dilation to serotonin (10−7 mol/L) was examined in the LDL-treated vessels after the intraluminal LDL (N-LDL or Ox-LDL) had been replaced with vehicle solution. Both N-LDL and Ox-LDL impaired vasodilation to serotonin, which is in agreement with the results shown in Figures 2A⇑ and 3A⇑. After removal of intraluminal LDL (N-LDL or Ox-LDL), vasodilation to serotonin was still attenuated (Figure 6⇓), and this inhibitory effect was not different from that observed in the presence of LDL.

Figure 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 6.

Effect of LDL removal on endothelium-dependent vasodilation. Bar graphs show that vasodilation to serotonin was inhibited by N-LDL (A; resting diameter, 82±2 μm; maximal diameter, 131±13 μm; n=3) and Ox-LDL (B; resting diameter, 78±9 μm; maximal diameter, 112±9 μm; n=3). Inhibited vasodilations to serotonin were still present after removal of intraluminal LDL from the vessels. *P<0.05 vs control.

Contribution of Superoxide Anions to Vascular Dysfunction

Under control conditions, serotonin (10−7 mol/L) produced ≈80% of maximal dilation of coronary arterioles. This dilation was significantly attenuated by intraluminal LDL (N-LDL or Ox-LDL, Figure 7⇓). On administration of N-LDL with SOD (100 U/mL), impairment of vasodilation to serotonin (10−7 mol/L) was not observed (Figure 7A⇓). However, coadministration of Ox-LDL and SOD did not influence the inhibitory effect of Ox-LDL on serotonin-induced dilation (Figure 7B⇓). l-Arginine (3 mmol/L) administered extraluminally to these vessels completely restored the vasodilation in response to serotonin as shown in Figure 7B⇓. In another series of experiments, intraluminal administration of LDL (N-LDL or Ox-LDL) with Tiron (1 mmol/L), a cell-permeable superoxide scavenger, eliminated the inhibitory action of N-LDL and Ox-LDL on vasodilations to ATP (Figure 8A⇓ and 8B⇓), serotonin, and ionomycin (data not shown). Treatment of coronary arterioles with Tiron (1 mmol/L) for 60 minutes did not alter the resting diameter (76±10 μm before Tiron versus 81±11 μm after Tiron) or the vasodilatory response to ATP (Figure 8C⇓), serotonin, and ionomycin (data not shown).

Figure 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 7.

Effects of SOD and l-arginine on vascular dysfunction caused by LDL. A, Serotonin-induced dilation was inhibited by N-LDL. Coadministration of N-LDL with SOD (100 U/mL) prevented the inhibitory effect of N-LDL (resting diameter, 67±12 μm; maximal diameter, 101±16 μm; n=3). B, Serotonin-induced dilation was inhibited by Ox-LDL (resting diameter, 74±4 μm; maximal diameter, 107±8 μm; n=4). Inhibitory effect of Ox-LDL was not altered by adding SOD (100 U/mL) to the vessels. Administration of l-arginine (3 mmol/L) to Ox-LDL–treated vessels with SOD completely restored vasodilation to serotonin. *P<0.05 vs control.

Figure 8.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 8.

Effect of Tiron on vascular dysfunction caused by LDL. N-LDL (A; resting diameter, 104±9 μm; maximal diameter, 140±11 μm; n=5) and Ox-LDL (B; resting diameter, 71±7 μm; maximal diameter, 99±8 μm; n=5) significantly attenuated dilation of vessels to ATP. In the presence of Tiron (1 mmol/L) intraluminally, inhibitory effects of N-LDL and Ox-LDL were prevented. Treatment of normal coronary arterioles with Tiron (1 mmol/L) did not affect dose-dependent dilation to ATP, demonstrating that vasodilatory function of these vessels was not influenced by Tiron (C; resting diameter, 76±10 μm; maximal diameter, 117±13 μm; n=5). *P<0.05 between groups.

Effect of LDL on Endothelium-Independent Vasodilation

Vascular smooth muscle function of isolated coronary arterioles was assessed by examining endothelium-independent vasodilation to SNP (10−9 to 10−4 mol/L) before and after incubation of vessels with LDL for 60 minutes. The dose-response curve for SNP after N-LDL or Ox-LDL treatment was identical to that before LDL incubation (Figure 9⇓), suggesting that the vasodilatory function of vascular smooth muscle was not affected by LDL.

Figure 9.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 9.

Effect of LDL on arteriolar dilation to SNP. Neither N-LDL (A; resting diameter, 77±6 μm; maximal diameter, 116±9 μm; n=7) nor Ox-LDL (B; resting diameter, 73±4 μm; maximal diameter, 105±5 μm; n=13) altered dose-dependent dilation to SNP.

Discussion

The major findings of the present study are that LDLs (N-LDL or Ox-LDL) inhibit endothelium-dependent dilations of isolated coronary arterioles to serotonin, ATP, and ionomycin in a manner similar to that under NO synthase inhibition. The inhibitory effects of LDL on endothelium-dependent vasodilation were reversed by subsequent administration of l-arginine and were prevented by the cell-permeable superoxide scavenger Tiron. In contrast, administration of the cell-impermeable superoxide scavenger SOD only prevented the inhibitory effect elicited by N-LDL. This is the first study to report the direct impairment of endothelial function by LDL in the coronary microcirculation. Since N-LDL and Ox-LDL impair both receptor- and non–receptor-mediated vasodilations and since application of l-arginine or Tiron preserved vascular function, it is suggested that the impairment of endothelium-dependent vasodilation results from the deficiency of NO release associated with the cellular production of superoxide anions by LDL. To provide a perspective for our observations and conclusions, methodological considerations such as isolated-vessel preparations and the treatment of LDL will be discussed. In addition, the effect of LDL on vascular function and the possible mechanism involved will be addressed.

Methodological Considerations

In the present study, use of the isolated-vessel technique allowed us to directly examine the effect of LDL on microvascular function without confounding influences from the interaction of vascular cells with either blood-borne substances or circulating cells. Since the effect of LDL was examined after incubation of arterioles in the bath solution without l-arginine for 60 minutes, it is possible that the observed vascular dysfunction resulted from nonspecific depletion of vascular l-arginine or from the time-dependent deterioration of vasomotor function. However, in the time-control study without l-arginine, vascular function remained intact after a 60-minute incubation with vehicle solution (the Table⇑). This finding argues against the idea that the observed vascular dysfunction was a result of time-dependent deterioration of endothelial function or nonspecific depletion of l-arginine during the course of LDL incubation. Furthermore, vasodilatory function was not altered by an increase of inert protein in the lumen, suggesting the specific inhibitory effect of LDL. Therefore, the impaired vasodilations appear to be a direct effect of LDL rather than a nonspecific effect from experimental interventions.

Recent studies have demonstrated that oxidation of LDL occurs in vivo,29 30 and oxidatively modified LDLs have been detected in both plasma30 and atherosclerotic lesions of various species, including humans.31 Although the plasma concentration of Ox-LDL in vivo is not known, it has been predicted to be 0.5 to 2 mg protein/mL in human atherosclerotic lesions.5 These concentrations of Ox-LDL have been shown to inhibit vascular relaxation of large-conduit arteries in vitro.5 13 32 A recent clinical study has demonstrated increased plasma levels of autoantibodies against Ox-LDL in hypercholesterolemic patients.33 Interestingly, these patients also exhibited impaired endothelial function of forearm resistance vessels. In the present study, the microvascular dilations to endothelium-dependent agonists were impaired by 0.3 and 1 mg protein/mL of Ox-LDL, concentrations that have been reported to be within the pathophysiological range.32 34 It is worth noting that the detrimental effect of Ox-LDL observed in the present study might have been underestimated because of the possible absorption of lysophosphatidylcholine (a lysophospholipid contained in Ox-LDL) by albumin in the incubation solution. This consideration is based on the evidence that lysophosphatidylcholine-induced impairment of endothelium-dependent relaxation was attenuated by albumin in rabbit aortic ring preparations.32

The inhibitory effect of N-LDL found in our present study is surprising, since other investigators have demonstrated little, if any, effect of N-LDL on vascular responses.6 35 It is worth noting that these studies with negative results were primarily performed in large-conduit vascular rings or strips. These vascular tissues were preconstricted with various constrictors for vasodilatory study. It is likely that these constrictors may initially mask the effect of N-LDL, since its detrimental effect is moderate compared with that of Ox-LDL at the same concentration and incubation time (Figures 2⇑ and 3⇑). Our microvessels developed spontaneous basal tone and thus may have been more sensitive or susceptible to LDL insult. On the other hand, a recent study showed that the inhibitory effect of N-LDL was evident in the large-coronary-vessel preparation when a longer incubation time (4 hours) was allowed.36 This result indicates that N-LDL has the potential to elicit a detrimental effect on vascular function even in large-conduit vessels.

Effect of LDL on NO-Dependent Vasodilation in the Coronary Microcirculation

Although coronary vascular dilations to serotonin, ATP, and ionomycin require an intact endothelium,24 37 38 it is not clear whether these dilations are mediated by the release of NO, especially at the microcirculatory level. In the present study, vasodilations to these agonists were significantly attenuated by the NO synthase inhibitor L-NMMA. Subsequent administration of excess NO precursor l-arginine restored vasodilation in the presence of L-NMMA (Figure 1⇑), indicating that vasodilations to these endothelium-dependent agonists in coronary arterioles are primarily mediated by NO. The role of prostanoids in the present preparation was not apparent, since indomethacin (10−5 mol/L), which has previously been shown to inhibit the cyclooxygenase pathway of the same vessel,19 did not affect the vasodilatory response to these agonists (authors’ unpublished data, 1998). Interestingly, the inhibitory effects of LDL on vasodilations to serotonin, ATP, and ionomycin were comparable to those of L-NMMA, and these inhibitions were also effectively reversed by l-arginine (Figures 2⇑ and 3⇑). Since l-arginine had no effect on the vasodilation of control vessels (Figure 5A⇑) and impaired vascular function was specifically restored by l-arginine and not by d-arginine (Figure 5B⇑), it is suggested that a deficiency of NO is likely responsible for the LDL-associated vascular dysfunction. This contention is supported by our preliminary studies showing that NO-mediated, flow-induced coronary arteriolar dilation was specifically compromised by Ox-LDL39 but that hyperosmolarity-induced, endothelium-dependent vasodilation40 via an NO-independent mechanism was not altered.39 Although these studies are preliminary, they suggest selective impairment of NO-mediated vasodilation by Ox-LDL.

Mechanism of LDL-Induced Vascular Dysfunction

There are several proposed mechanisms that may explain the observed vascular dysfunction elicited by LDL. First, a selective loss of receptor-mediated, endothelium-dependent vasodilation has been described in various animal models of atherosclerosis, including human.41 42 In addition, Flavahan43 suggested that endothelium-dependent vasodilation mediated by receptor-incorporated pertussis toxin–sensitive Gi proteins may be selectively affected by an early stage of atherosclerosis or by a low concentration of Ox-LDL (≤50 μg protein/mL). However, the present study indicated that impaired vasodilation to both receptor-dependent and receptor-independent agonists occurred after a 60-minute exposure of the vessel to LDL. It is likely that the high concentration of LDL (1 mg protein/mL) used in our study may have produced a general inhibitory effect on vascular function beyond the receptor level.

Second, it has been demonstrated that endothelium-derived relaxing factor released from cultured endothelial cells is inactivated by both N-LDL and Ox-LDL in a bioassay system, suggesting that LDL may directly contribute to the degradation of NO and thus attenuate vasodilation to agonists.44 In this cultured cell study, the investigators used the acyltransferase inhibitor thimerosal to stimulate endothelium-derived relaxing factor release and assumed that the released factor was NO. However, this assumption is weak, since thimerosal has recently been shown to stimulate the release of endothelium-derived hyperpolarizing factor rather than NO in both cultured endothelial cell and intact-vessel preparations.45 46 Therefore, these investigators might have studied the effect of LDL on endothelium-derived hyperpolarizing factor instead of NO. Furthermore, in our intact-microvessel study, we found that the impaired vasodilation was still present within 30 minutes of LDL removal (Figure 6⇑). Therefore, this result does not favor the idea of degradation of NO by LDL. Nevertheless, it remains to be elucidated whether the impaired vascular function is reversible beyond 30 minutes of LDL removal, since endothelium-dependent function has been shown to be partially restored after correction of plasma lipid concentrations in hypercholesterolemic animals47 and humans.48

Third, the deficiency of NO production or release associated with hypercholesterolemia and atherosclerosis has generally been proposed as a primary mechanism for vascular dysfunction in various animal models49 and in humans.50 51 This contention is based on the fact that impaired endothelium-dependent vasodilation can be normalized by administration of the NO precursor l-arginine.49 50 51 It is believed that the increased NO production from exogenous l-arginine reverses this aberrant response. This idea may hold true only under conditions with unsaturated NO synthase. Normally, intracellular levels of arginine (≈0.1 mmol/L)52 are high enough to saturate NO synthase, whose Km has been determined to be in the micromolar range (≈2.9 μmol/L).53 In this regard, it is expected that excess l-arginine would not enhance NO-dependent relaxation of normal vessels, as evident in the present study (Figure 5A⇑) and in other studies.15 54 However, if l-arginine availability were reduced to a level where NO synthase was no longer saturated, this effect could limit the stimulated production of NO. This may be the case in the presence of LDL, since exogenous l-arginine could then restore NO-dependent vasodilation (Figures 2⇑ and 3⇑). Interestingly, reduced levels of l-arginine have been shown to enhance the generation of superoxide anions from constitutive NO synthase by uncoupling the l-arginine/NO pathway.55 56 This perturbation could further decrease functional levels of NO through direct inactivation of the synthesized NO by superoxide.

Superoxide Anions and Microvascular Dysfunction

Several in vitro models have demonstrated an increase in superoxide production by endothelial cells during hypercholesterolemia.57 58 Similarly, stimulation of superoxide production from endothelial cells and neutrophils by N-LDL and Ox-LDL was also reported.57 59 Since the superoxide anion inactivates NO60 and has been implicated in the alteration of endothelium-dependent relaxation in hypercholesterolemia61 and atherosclerosis,62 its contribution to LDL-induced vascular dysfunction should be considered in the present study. Our results show that administration of Tiron, an antioxidant that is capable of scavenging superoxide from both the intracellular and extracellular environment,26 27 28 prevented the inhibitory action of both N-LDL (Figure 8A⇑) and Ox-LDL (Figure 8B⇑) on vasodilation. However, the salutary effect of Tiron was not evident in control vessels (Figure 8C⇑). These results indicated that LDL-induced vascular dysfunction is associated with the production of superoxide anions.

In contrast to the Tiron study, treatment of the vessels with SOD for 60 minutes prevented the inhibitory action of N-LDL but not of Ox-LDL (Figure 7⇑). It is possible that the oxidation of N-LDL during this incubation period is responsible for the observed vascular dysfunction, since SOD has been shown to prevent oxidation of N-LDL in vitro.35 At this time, it is unclear where the LDL is oxidized, in terms of either the intracellular space or at the cell membrane. However, our SOD data suggest that oxidation is likely to take place at the cell membrane, since SOD is rather impermeable. A recent study on isolated coronary arteries indicated that endothelial dysfunction induced by N-LDL (0.2 mg protein/mL) was time dependent, since the inhibitory effect was observed only after a longer period (4 hours versus 20 minutes) of incubation.36 In a similar manner, we noted that vessels treated with N-LDL for 2 hours exhibited impairment of vasodilation in a manner comparable to that of Ox-LDL–treated vessels for 60 minutes (n=3, data not shown). It appears that time-dependent oxidation of N-LDL is likely involved in the initiation of vascular impairment.

Taken together, the ability of superoxide scavengers to prevent LDL-induced vascular dysfunction and of excess l-arginine to restore impaired vascular function suggests that the initiation of superoxide production and the subsequent reduced intracellular l-arginine for NO synthesis are responsible for the inhibitory effect of LDL. However, the intracellular pathway involved in the l-arginine deficiency remains unclear. Endogenous levels of l-arginine in endothelial cells have been proposed to be maintained in part by the recycling of l-citrulline to l-arginine.63 It is possible that the initial production of superoxide by LDL inhibits this pathway and thus reduces the availability of cellular l-arginine for NO synthase. A decrease in l-arginine levels may also enhance superoxide anion production55 56 and consequently further aggravate this detrimental process. In this respect, it is conceivable that excess l-arginine would not only overcome the reduction in l-arginine and replenish NO for normal vasodilation but also restore vascular function by reducing superoxide generation. The results of the present study are consistent with recent studies suggesting that supplementation of hypercholesterolemic animals and humans with l-arginine or antioxidants decreases the vascular release of superoxide anion and partly restores NO production.64 65 66

In summary, the findings of the present study indicate that isolated coronary arterioles are susceptible to an oxidized form of LDL that specifically impairs endothelium-dependent vasodilation by reducing NO synthesis. This deleterious effect may result from a reduction in the cellular level of l-arginine after the enhanced production of superoxide anions. We speculate that the impaired coronary flow regulation observed in patients and animals with hypercholesterolemia or atherosclerosis16 51 may be due in part to LDL-induced microvascular dysfunction that is associated with superoxide anion–mediated NO deficiency. In this regard, antioxidants and l-arginine may be beneficial not only in the prevention of LDL oxidation and oxygen-derived free-radical formation but also in amelioration of vasomotor function in the microcirculation.

Selected Abbreviations and Acronyms

L-NMMA=NG-monomethyl-l-arginine
MDA=malondialdehyde
N-LDL=native LDL
Ox-LDL=oxidized LDL
PSS=physiological salt solution
SNP=sodium nitroprusside
SOD=superoxide dismutase
TBARS=thiobarbituric acid–reactive substances

Acknowledgments

This study was supported by National Heart, Lung, and Blood Institute (Bethesda, Md) grants HL-55524 and K02 HL-03693 (Research Career Award) to Dr Kuo and by American Heart Association (National Center) Grant 95009970 to Dr Kuo.

  • Received December 16, 1997.
  • Accepted April 17, 1998.
  • © 1998 American Heart Association, Inc.

References

  1. ↵
    Kannel WB. Contribution of the Framingham Study to preventive cardiology. J Am Coll Cardiol. 1990;15:206–211.
    OpenUrlCrossRefPubMed
  2. ↵
    Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
  3. ↵
    Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A.. 1987;84:2995–2998.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.
    OpenUrlCrossRefPubMed
  5. ↵
    Jacobs M, Plane F, Bruckdorfer KR. Native and oxidized low-density lipoproteins have different inhibitory effects on endothelium-derived relaxing factor in the rabbit aorta. Br J Pharmacol. 1990;100:21–26.
    OpenUrlCrossRefPubMed
  6. ↵
    Tanner FC, Noll G, Boulanger CM, Lüscher TF. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries: role of scavenger receptor and endothelium-derived nitric oxide. Circulation. 1991;83:2012–2020.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524–526.
    OpenUrlCrossRefPubMed
  8. ↵
    Myers PR, Minor RL, Guerra R Jr, Bates JN, Harrison DG. Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature. 1990;345:161–163.
    OpenUrlCrossRefPubMed
  9. ↵
    Palmer RMJ, Rees DD, Ashton DS, Moncada S. l-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun. 1988;153:1251–1256.
    OpenUrlCrossRefPubMed
  10. ↵
    Radomski MW, Palmer RMJ, Moncada S. An l-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A.. 1990;87:5193–5197.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774–1777.
  12. ↵
    Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A.. 1991;88:4651–4655.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Galle J, Bauersachs J, Busse R, Bassenge E. Inhibition of cyclic AMP– and cyclic GMP–mediated dilations in isolated arteries by oxidized low density lipoproteins. Arterioscler Thromb. 1992;12:180–186.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.
    OpenUrlCrossRefPubMed
  15. ↵
    Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation: restoration of endothelium-dependent responses by l-arginine. Circ Res. 1992;70:465–476.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Chilian WM, Dellsperger KC, Layne SM, Eastham CL, Armstrong MA, Marcus ML, Heistad DD. Effects of atherosclerosis on the coronary microcirculation. Am J Physiol. 1990;258:H529–H539.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Chilian WM, Eastham CL, Marcus ML. Microvascular distribution of coronary vascular resistance in beating left ventricle. Am J Physiol. 1986;251:H779–H788.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Kuo L, Davis MJ, Chilian WM. Myogenic activity in isolated subepicardial and subendocardial coronary arterioles. Am J Physiol. 1988;255:H1558–H1562.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Kuo L, Chilian WM, Davis MJ. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J Physiol. 1991;261:H1706–H1715.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Kuo L, Davis MJ, Chilian WM. Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am J Physiol. 1990;259:H1063–H1070.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. 1989;6:67–75.
    OpenUrlCrossRefPubMed
  22. ↵
    Chait A. Methods for assessing lipid and lipoprotein oxidation. Curr Opin Lipidol. 1992;3:389–394.
    OpenUrlCrossRef
  23. ↵
    Markwell MAK, Haas SM, Tolbert NE, Bieber LL. Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol. 1981;72:296–303.
    OpenUrlCrossRefPubMed
  24. ↵
    Stork AP, Cocks TM. Pharmacological reactivity of human epicardial coronary arteries: characterization of relaxation responses to endothelium-derived relaxing factor. Br J Pharmacol. 1994;113:1099–1104.
    OpenUrlPubMed
  25. ↵
    Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res. 1989;65:1–21.
    OpenUrlFREE Full Text
  26. ↵
    Krishna CM, Liebmann JE, Kaufman D, DeGraff W, Hahn SM, McMurry T, Mitchell JB, Russo A. The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage. Arch Biochem Biophys. 1992;294:98–106.
    OpenUrlCrossRefPubMed
  27. ↵
    Münzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance: a novel mechanism underlying tolerance and cross-tolerance. J Clin Invest. 1995;95:187–194.
  28. ↵
    Kaminski PM, Wolin MS. Hypoxia increases superoxide anion production from bovine coronary microvessels, but not cardiac myocytes, via increased xanthine oxidase. Microcirculation. 1996;1:231–236.
    OpenUrl
  29. ↵
    Palinski W, Rosenfeld ME, Ylä-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A.. 1989;86:1372–1376.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Hodis HN, Kramsch DM, Avogaro P, Bittolo-Bon G, Cazzolato G, Hwang J, Peterson H, Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-). J Lipid Res. 1994;35:669–677.
    OpenUrlAbstract
  31. ↵
    Hammer A, Kager G, Dohr G, Rabl H, Ghassempur I, Jürgens G. Generation, characterization, and histochemical application of monoclonal antibodies selectively recognizing oxidatively modified apoB-containing serum lipoproteins. Arterioscler Thromb Vasc Biol. 1995;15:704–713.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Plane F, Bruckdorfer KR, Kerr P, Steuer A, Jacobs M. Oxidative modification of low-density lipoproteins and the inhibition of relaxations mediated by endothelium-derived nitric oxide in rabbit aorta. Br J Pharmacol. 1992;105:216–222.
    OpenUrlCrossRefPubMed
  33. ↵
    Heitzer T, Ylä-Herttuala S, Luoma J, Kurz S, Münzel T, Just H, Olschewski M, Drexler H. Cigarette smoking potentiates endothelial dysfunction of forearm resistance vessels in patients with hypercholesterolemia: role of oxidized LDL. Circulation. 1996;93:1346–1353.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Rangaswamy S, Penn MS, Saidel GM, Chisolm GM. Exogenous oxidized low-density lipoprotein injures and alters the barrier function of endothelium in rats in vivo. Circ Res. 1997;80:37–44.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Simon BC, Cunningham LD, Cohen RA. Oxidized low density lipoproteins cause contraction and inhibit endothelium-dependent relaxation in the pig coronary artery. J Clin Invest. 1990;86:75–79.
  36. ↵
    Abebe W, Mustafa SJ. Effect of low density lipoprotein on adenosine receptor-mediated coronary vasorelaxation in vitro. J Pharmacol Exp Ther. 1997;282:851–857.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Richard V, Tanner FC, Tschudi M, Lüscher TF. Different activation of l-arginine pathway by bradykinin, serotonin, and clonidine in coronary arteries. Am J Physiol. 1990;259:H1433–H1439.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    White TD, Angus JA. Relaxant effects of ATP and adenosine on canine large and small coronary arteries in vitro. Eur J Pharmacol. 1987;143:119–126.
    OpenUrlCrossRefPubMed
  39. ↵
    Hein TW, Kuo L. Oxidized LDL specifically impairs nitric oxide-mediated dilation of coronary arterioles. Circulation. 1997;96(suppl II):I-114. Abstract.
  40. ↵
    Ishizaka H, Kuo L. Endothelial ATP-sensitive potassium channels mediate coronary microvascular dilation to hyperosmolarity. Am J Physiol. 1997;273:H104–H112.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Shimokawa H, Flavahan NA, Vanhoutte PM. Loss of endothelial pertussis toxin–sensitive G protein function in atherosclerotic porcine coronary arteries. Circulation. 1991;83:652–660.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5′-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest. 1987;79:170–174.
  43. ↵
    Flavahan NA. Atherosclerosis or lipoprotein-induced endothelial dysfunction: potential mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation. 1992;85:1927–1938.
    OpenUrlFREE Full Text
  44. ↵
    Galle J, Mülsch A, Busse R, Bassenge E. Effects of native and oxidized low density lipoproteins on formation and inactivation of endothelium-derived relaxing factor. Arterioscler Thromb. 1991;11:198–203.
    OpenUrl
  45. ↵
    Mombouli J-V, Bissiriou I, Agboton V, Vanhoutte PM. Endothelium-derived hyperpolarizing factor: a key mediator of the vasodilator action of bradykinin. Immunopharmacology. 1996;33:46–50.
    OpenUrlCrossRefPubMed
  46. ↵
    Mombouli J-V, Bissiriou I, Agboton VD, Vanhoutte PM. Bioassay of endothelium-derived hyperpolarizing factor. Biochem Biophys Res Commun. 1996;221:484–488.
    OpenUrlCrossRefPubMed
  47. ↵
    Osborne JA, Lento PH, Siegfried MR, Stahl GL, Fusman B, Lefer AM. Cardiovascular effects of acute hypercholesterolemia in rabbits: reversal with lovastatin treatment. J Clin Invest. 1989;83:465–473.
  48. ↵
    Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med. 1995;332:488–493.
    OpenUrlCrossRefPubMed
  49. ↵
    Rossitch E Jr, Alexander E III, Black PM, Cooke JP. l-Arginine normalizes endothelial function in cerebral vessels from hypercholesterolemic rabbits. J Clin Invest. 1991;87:1295–1299.
  50. ↵
    Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ, Cooke JP. l-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248–1253.
  51. ↵
    Drexler H, Zeiher AM, Meinzer K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolaemic patients by l-arginine. Lancet. 1991;338:1546–1550.
    OpenUrlCrossRefPubMed
  52. ↵
    Nakaki T, Kata R. Beneficial circulatory effect of l-arginine. Jpn J Pharmacol. 1994;66:167–171.
    OpenUrlPubMed
  53. ↵
    Pollock JS, Förstermann U, Mitchell JA, Warner TD, Schmidt HHHW, Nakane M, Murad F. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A.. 1991;88:10480–10484.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Amezcua JL, Palmer RMJ, de Souza BM, Moncada S. Nitric oxide synthesized from l-arginine regulates vascular tone in the coronary circulation of the rabbit. Br J Pharmacol. 1989;97:1119–1124.
    OpenUrlCrossRefPubMed
  55. ↵
    Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem. 1992;267:24173–24176.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Heinzel B, John M, Klatt P, Böhme E, Mayer B. Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase. Biochem J. 1992;281:627–630.
  57. ↵
    Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995;77:510–518.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546–2551.
  59. ↵
    Maeba R, Maruyama A, Tarutani O, Ueta N, Shimasaki H. Oxidized low-density lipoprotein induces the production of superoxide by neutrophils. FEBS Lett. 1995;377:309–312.
    OpenUrlCrossRefPubMed
  60. ↵
    Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986;320:454–456.
    OpenUrlCrossRefPubMed
  61. ↵
    Ohara Y, Peterson TE, Sayegh HS, Subramanian RR, Wilcox JN, Harrison DG. Dietary correction of hypercholesterolemia in the rabbit normalizes endothelial superoxide anion production. Circulation. 1995;92:898–903.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    White CR, Brock TA, Chang L-Y, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM. Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A.. 1994;91:1044–1048.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Hecker M, Sessa WC, Harris HJ, Änggård EE, Vane JR. The metabolism of l-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle l-citrulline to l-arginine. Proc Natl Acad Sci U S A.. 1990;87:8612–8616.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    Böger RH, Bode-Böger SM, Mügge A, Kienke S, Brandes R, Dwenger A, Frölich JC. Supplementation of hypercholesterolaemic rabbits with l-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis. 1995;117:273–284.
    OpenUrlCrossRefPubMed
  65. ↵
    Keaney JF Jr, Xu A, Cunningham D, Jackson T, Frei B, Vita JA. Dietary probucol preserves endothelial function in cholesterol-fed rabbits by limiting vascular oxidative stress and superoxide generation. J Clin Invest. 1995;95:2520–2529.
  66. ↵
    Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF Jr, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996;93:1107–1113.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top
Previous ArticleNext Article

This Issue

Circulation Research
August 24, 1998, Volume 83, Issue 4
  • Table of Contents
Previous ArticleNext Article

Jump to

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Selected Abbreviations and Acronyms
    • Acknowledgments
    • References
  • Figures & Tables
  • Info & Metrics

Article Tools

  • Print
  • Citation Tools
    LDLs Impair Vasomotor Function of the Coronary Microcirculation
    Travis W. Hein and Lih Kuo
    Circulation Research. 1998;83:404-414, originally published August 24, 1998
    https://doi.org/10.1161/01.RES.83.4.404

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
  •  Download Powerpoint
  • Article Alerts
    Log in to Email Alerts with your email address.
  • Save to my folders

Share this Article

  • Email

    Thank you for your interest in spreading the word on Circulation Research.

    NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

    Enter multiple addresses on separate lines or separate them with commas.
    LDLs Impair Vasomotor Function of the Coronary Microcirculation
    (Your Name) has sent you a message from Circulation Research
    (Your Name) thought you would like to see the Circulation Research web site.
  • Share on Social Media
    LDLs Impair Vasomotor Function of the Coronary Microcirculation
    Travis W. Hein and Lih Kuo
    Circulation Research. 1998;83:404-414, originally published August 24, 1998
    https://doi.org/10.1161/01.RES.83.4.404
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Circulation Research

  • About Circulation Research
  • Editorial Board
  • Instructions for Authors
  • Abstract Supplements
  • AHA Statements and Guidelines
  • Permissions
  • Reprints
  • Email Alerts
  • Open Access Information
  • AHA Journals RSS
  • AHA Newsroom

Editorial Office Address:
3355 Keswick Rd
Main Bldg 103
Baltimore, MD 21211
CircRes@circresearch.org

Information for:
  • Advertisers
  • Subscribers
  • Subscriber Help
  • Institutions / Librarians
  • Institutional Subscriptions FAQ
  • International Users
American Heart Association Learn and Live
National Center
7272 Greenville Ave.
Dallas, TX 75231

Customer Service

  • 1-800-AHA-USA-1
  • 1-800-242-8721
  • Local Info
  • Contact Us

About Us

Our mission is to build healthier lives, free of cardiovascular diseases and stroke. That single purpose drives all we do. The need for our work is beyond question. Find Out More about the American Heart Association

  • Careers
  • SHOP
  • Latest Heart and Stroke News
  • AHA/ASA Media Newsroom

Our Sites

  • American Heart Association
  • American Stroke Association
  • For Professionals
  • More Sites

Take Action

  • Advocate
  • Donate
  • Planned Giving
  • Volunteer

Online Communities

  • AFib Support
  • Garden Community
  • Patient Support Network
  • Professional Online Network

Follow Us:

  • Follow Circulation on Twitter
  • Visit Circulation on Facebook
  • Follow Circulation on Google Plus
  • Follow Circulation on Instagram
  • Follow Circulation on Pinterest
  • Follow Circulation on YouTube
  • Rss Feeds
  • Privacy Policy
  • Copyright
  • Ethics Policy
  • Conflict of Interest Policy
  • Linking Policy
  • Diversity
  • Careers

©2018 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. The American Heart Association is a qualified 501(c)(3) tax-exempt organization.
*Red Dress™ DHHS, Go Red™ AHA; National Wear Red Day ® is a registered trademark.

  • PUTTING PATIENTS FIRST National Health Council Standards of Excellence Certification Program
  • BBB Accredited Charity
  • Comodo Secured