Skip Navigation

Cardiovascular Research 2004 63(1):168-175; doi:10.1016/j.cardiores.2004.03.020
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Conklin, B. S
Right arrow Articles by Chen, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conklin, B. S
Right arrow Articles by Chen, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

HIV protease inhibitor ritonavir decreases endothelium-dependent vasorelaxation and increases superoxide in porcine arteries

Brian S Conklin1, Weiping Fu, Peter H Lin, Alan B Lumsden, Qizhi Yao and Changyi Chen*

Michael E. DeBakey Department of Surgery, Division of Vascular Surgery and Endovascular Therapy, Molecular Surgeon Research Center, Baylor College of Medicine, One Baylor Plaza, Mail Stop NAB 2010, Houston, TX 77030, USA

*Corresponding author. Tel.: +1-713-798-4401; fax: +1-713-798-6633. Email address: jchen{at}bcm.tmc.edu

Received 15 January 2004; revised 9 March 2004; accepted 23 March 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Although HIV Protease inhibitors significantly reduce the viral load, they are associated with increased risk of cardiovascular disease. The aim of this study was to investigate the effects of HIV protease inhibitor ritonavir on vascular endothelial cell function. Methods: Porcine carotid arteries were perfusion-cultured for 24 h as controls or with 15 µM of ritonavir. Vessels were precontracted with norepinephrine followed by endothelium-dependent vasorelaxation with acetylcholine. Rings of vessels were cultured as controls or with ritonavir for 24 h and basal and NADPH-stimulated superoxide levels were determined using lucigenin-enhanced chemiluminescence. Superoxide levels in situ were also examined using dihydroethidium (DHE) staining, and nitrotyrosine levels were examined using a nitrotyrosine antibody. Results: Endothelium-dependent vasorelaxation was significantly reduced in ritonavir-treated vessels compared to controls. There were significant increases in basal and NADPH-stimulated superoxide production in vessel rings treated with ritonavir compared to control vessels. Dihydroethidium staining and nitrotyrosine staining were also elevated in endothelial cells of ritonavir-treated vessels, indicating increased superoxide production and increased oxidative stress, respectively, in ritonavir-treated vessels compared to controls. Conclusions: These data demonstrate that HIV protease inhibitor ritonavir causes a significant reduction in endothelium-dependent vasorelaxation in cultured porcine carotid arteries. Increased oxidative stress may be a possible mechanism of HIV protease inhibitor ritonavir-induced endothelial dysfunction.

KEYWORDS HIV protease inhibitor ritonavir; Endothelium-dependent vasorelaxation; Porcine arteries


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The use of highly active antiretroviral therapy, including HIV protease inhibitors, has significantly reduced the morbidity and mortality associated with AIDS in HIV-infected patients. HIV protease inhibitors, including saquinavir, ritonavir, indinavir, amprenavir, and nelfinavir, interfere with the ability of the HIV virus to produce mature, infectious virus particles and thus greatly reduce the viral load and increase the CD4+ cell count in HIV patients [1]. Unfortunately, reports dating back to the late 1990s have brought to light serious cardiovascular complications from prolonged use of HIV protease inhibitors [2–5] In these initial reports, patients using HIV protease inhibitors as young as 26 years of age were presenting with advanced atherosclerotic lesions. Further investigations revealed an association between the use of HIV protease inhibitors and hyperlipidemia, lipodystrophy, and insulin resistance [6–8].

While elevated lipid levels may in part account for the increase in atherosclerotic vascular disease associated with the use of HIV protease inhibitors, there may also be effects of these drugs that are not explained by changes in lipid profiles. It has recently been shown, for instance, that HIV protease inhibitors increase CD36-dependent cholesterol ester accumulation in macrophages in both in vitro and in vivo models independent of dyslipidemia [9]. Further, a recent clinical study measured flow-mediated vasodilation (FMD) of the brachial artery as an indicator of endothelial cell function in HIV patients who were either using or not using HIV protease inhibitors [10]. A significant reduction in FMD was found in HIV patients using protease inhibitors versus the control group, indicating a correlation between the use of protease inhibitors and endothelial dysfunction. A linear regression model of brachial FMD demonstrated that most, but not all, of the effects of HIV protease inhibitors on FMD could be accounted for by lipid levels. Thus, there could be a direct effect of HIV protease inhibitors on endothelial cell function, but this possibility has not been thoroughly investigated.

The objective of this study was therefore to determine the effects of HIV protease inhibitor ritonavir on endothelium-dependent vasorelaxation using an ex vivo artery perfusion culture model. The effect of ritonavir on endothelial superoxide anion production was also investigated as a possible mechanism of HIV protease inhibitors-induced endothelial cell dysfunction. We have demonstrated, for the first time to our knowledge, that HIV protease inhibitor ritonavir causes a decrease in endothelium-dependent vasorelaxation, accompanied by an increase in endothelial superoxide levels. These results may shed light on the mechanisms of HIV protease inhibitor-associated vascular complications.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Tissue harvest and culture
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Carotid arteries from 6–8-month-old male and female domestic swine were harvested within 10 min of death at a local abattoir as described previously [11,12]. Animals were killed by a puncture wound to the aorta after being rendered unconscious via an electrical shock. The carotid arteries were harvested via a ventral midline incision on the neck and quickly placed in ice-cold sterile PBS for transport to the laboratory. Vessels were cultured in either a perfusion culture system or as 6-mm-long rings in static culture.

The perfusion culture methods have been described previously in detail [11,12]. Briefly, segments of carotid arteries 7–10 cm in length were rinsed in sterile ice-cold PBS and mounted on specially designed adjustable cannulae. Side branches were then ligated and vessels were gently stretched to their in vivo lengths. The vessel-cannula assemblies were then placed into the perfusion culture systems which were subsequently filled with 500 ml of Dulbeccos Modified Eagles Medium (DMEM) with 5% dextran (to increase the viscosity to approximately match that of whole blood) and 1% antibiotic–antimycotic (10,000 units of penicillin, 10,000 µg of streptomycin, and 25 µg of amphotericin B per ml) and either 1.25 ml dimethyl sulfoxide DMSO (controls) or 1.25 ml of 6 mM ritonavir in DMSO for a final concentration of 15 µM of ritonavir. The perfusion culture systems were then placed within cell culture incubators maintained at 37 °C and 8.5% CO2 and connected to the pump and pressure transducers. Flow was initiated at approximately 15 ml/min and 10 mm Hg pressure and increased over 2 h to 150 ml/min and 100 mm Hg pressure, which was maintained for the duration of culturing.

Smooth muscle cell (SMC) and endothelial cell function of perfusion-cultured porcine carotid arteries were tested by means of a contraction and relaxation assay as described previously [11,12]. After 24 h of perfusion culture as described above, vessels were precontracted with a 10–4 M dose of norepinephrine. Subsequently, vessels were relaxed with cumulative doses of acetylcholine (Ach, 10–9, 10–7, and 10–5 M concentrations). The vessel diameters were recorded with a video-imaging system and transferred to a personal computer (Dell Computer Corporation, Round Rock, TX). The percent contraction was calculated as (Di–Dc)/Dix100, where Di is the initial diameter and Dc is the contracted diameter. The percent relaxation after each dose of acetylcholine was calculated as (Dr–Dc)/(Di–Dc)x100, where Dr is the relaxed diameter.

Rings of porcine carotid arteries, approximately 6 mm in length, were cut from the arteries harvested as described above. The rings were rinsed several times in sterile ice-cold PBS with 1% antibiotic–antimycotic and placed in the wells of 24 well culture plates. Vessels rings from each artery were randomly divided into either controls with 2 ml DMEM containing 1% antibiotic/antimycotic and 5 µl of DMSO or ritonavir treated with 2 ml DMEM containing 1% antibiotic/antimycotic and 5 µl of 6 mM ritonavir dissolved in DMSO for a final ritonavir concentration of 15 µM. Vessel rings were cultured for 24 h in a cell culture incubator maintained at 37 °C and 5% CO2.

2.2. Detection of superoxide
Levels of superoxide anion produced by endothelial cells were determined using the lucigenin-enhanced chemiluminescence method with a Sirius Luminometer and FB12 software (Berthold Detection Systems, Pforzheim, Germany) [13,14]. After 24 h of culturing, porcine carotid artery rings were rinsed briefly in a modified Krebs HEPES buffer solution (KHBS, 120 mM NaCl, 4.7 mM KCl, 1.18 mM K2HPO4, 20 mM HEPES, 2.5 mM CaCl2, 1.17 mM MgSO4, and 25 mM NaHCO3). The rings were then cut open longitudinally and subsequently into approximately 5x5-mm pieces. Assay tubes (12x75 mm) were filled with 500 µl of KHBS, lucigenin (50 µM for basal readings or 5 µM for NADPH-stimulated readings), and various combinations of NADPH (200 µM final concentration), tiron (5 mM final concentration), or the flavin inhibitor diphenyleneiodonium chloride (DPI, 0.1 mM final concentration). The reagents were gently mixed in the tubes and the vessel segments were placed endothelium-side-down in the tubes. Measurements were taken every 15 s for a total of 12 min. The data in relative light units per second (RLU/s) for each sample was averaged between 7 and 10 min for basal readings and 5 and 10 min for NADPH-stimulated readings. Values of blank tubes containing the same reagents as the vessel ring samples were subtracted from the corresponding vessel samples. The area of each vessel segment was measured using a caliper and used to normalize the data for each sample, the final units thus being RLU/s/mm2.

2.3. Oxidative fluorescent microscopy
Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production [15]. DHE is a cell permeable dye that is oxidized by superoxide to ethidium bromide (EB), which subsequently intercalates with DNA and is trapped within the nuclei of cells. EB is excited at 488 nm and emits light at 610 nm. After 24 h of culturing as controls or with ritonavir treatment, rings of porcine carotid arteries were embedded in OCT compound and frozen in a cryostat (Leica Microsystems, Bannockburn, IL). Frozen sections, 10 µm in thickness, were cut and mounted on glass slides. The slides were then rinsed briefly in PBS to remove the OCT compound. DHE was dissolved in DMSO to a concentration of 10 mM which was further diluted in PBS to a working concentration of 2 µM. The tissue sections were covered in DHE, cover-slipped, and incubated in a light-proof humidified container at 37 °C for 30 min. The slides were viewed on an Olympus BX41 fluorescent microscope (Olympus USA, Melville, NY) with a SPOT-RT digital camera and software (Diagnostic Instruments, Sterling Heights, MI).

2.4. Immunohistochemistry
After perfusion culturing as either controls or with ritonavir treatment, vessels were fixed overnight in 10% neutral buffered formalin and subsequently paraffin-processed. Sections, 5 µm thick, were cut, mounted on glass slides, and deparaffinized. Staining was then performed for nitrotyrosine using a polyclonal rabbit anti-nitrotyrosine primary antibody diluted 1:2000 and developed using the avidin–biotin complex immunoperoxidase procedure as described previously [16]. Staining of the tissue without the primary antibody served as a negative control. Sections were counterstained with hematoxylin, cover-slipped, and viewed on an Olympus BX41 microscope and a SPOT-RT digital camera and software.

2.5. Chemicals
All chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise noted. DMEM was purchased from Invitrogen (Grand Island, NY). Antibiotic–antimycotic was purchased from Mediatech (Herndon, VA). Ritonavir was a generous gift from Abbott Laboratories (Abbott Park, IL). Lucigenin, DHE, and the rabbit-anti-nitrotyrosine antibody were obtained from Molecular Probes (Eugene, OR). OCT tissue freezing compound was from Sakura Finetek USA (Torrance, CA). The 10% neutral-buffered formalin was from VWR International (Westchester, PA), and the avidin–biotin complex immunoperoxidase kit was from DAKO (Carpinteria, CA).

2.6. Statistical analysis
Differences between groups were determined using either two-way ANOVA (relaxation curves) or two tail Student's t-test with significance considered to be P<0.05. Results are reported as the mean±the standard error of the mean.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
Porcine carotid arteries were cultured for 24 h with 150 ml/min flow and 100 mm Hg pressure as either controls or with a clinically relevant dose of 15 µM ritonavir, and subsequently subjected to a contraction and endothelial-dependent relaxation assay (Fig. 1). When challenged with norepinephrine (Fig. 1A), control vessels contracted 19.7±3.6% (n=12), while ritonavir-treated vessels contracted 9.9±2.6% (n=11), with the difference being significant (P<0.05). Endothelium-dependent vasorelaxation (normalized to the maximum contraction) was subsequently assessed with three doses of acetylcholine (Fig. 1B). Control vessels relaxed 41.6±8.2%, 62.2±8.3%, and 73.6±6.5% at 10–9, 10–7, and 10–5 M concentrations of acetylcholine, respectively, while ritonavir-treated vessels only relaxed 11.4±12.4%, 23.8±24.3%, and 25.4±24.4% at 10–9, 10–7, and 10–5 M concentrations of acetylcholine, respectively (P=0.033, ANOVA).


Figure 1
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Vasomotor function of porcine carotid arteries. Vessels were cultured for 24 h as controls (n=12) or with 15 µM of ritonavir (n=11) and subsequently tested for contraction and endothelium-dependent relaxation. (A) Ritonavir-treated vessels contracted 50% less than control vessels when challenged with norepinephrine (P<0.05). (B) Endothelium-dependent vasorelaxation was also significantly reduced (P=0.033, ANOVA). There was a 72%, 62%, and 65% reduction in relaxation at 10–9, 10–7, and 10–5 M concentrations of acetylcholine (Ach), respectively, in vessels treated with ritonavir relative to controls.

 
In order to investigate a possible mechanism for decreased endothelium-dependent vasorelaxation in vessels cultured with ritonavir, the relative production of superoxide in control and ritonavir-treated vessels was determined using the lucigenin-enhanced chemiluminescence method. Vessel rings were cultured for 24 h as controls or with 15 µM ritonavir and subsequently tested for superoxide production (Fig. 2). The readings were averaged between 7 and 10 min, the blank values were subtracted, and the values were normalized by the area of each sample. Control vessels measured 11.5±1.2 RLU/s/mm2 (n=7) and ritonavir-treated vessels measured 17.9±2.0 RLU/s/mm2 (n=7), with the difference being significant (P<0.02).


Figure 2
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Superoxide detection in cultured vessels. Superoxide levels were detected using the lucigenin-enhanced chemiluminescence method. (A) Typical data from control and ritonavir-treated vessel rings, corrected for blank values and normalized to tissue area, demonstrates an increase in basal superoxide production in ritonavir-treated vessels relative to controls. (B) The readings were averaged between 7 and 10 min where peak values were generally reached. There was a significant increase in superoxide production in ritonavir-treated vessels (n=7) relative to controls (n=7) (P<0.02).

 
Differences in superoxide levels were also investigated using the oxidative fluorescent dye DHE, which becomes ethidium bromide upon interaction with superoxide and produces red fluorescence when excited. Porcine carotid artery ring samples were incubated for 24 h as controls or with 15 µM ritonavir and subsequently frozen sections were cut, incubated with DHE, and viewed on a fluorescent microscope (Fig. 3). There was a marked increase in red fluorescence in ritonavir-treated samples compared to control samples, which agreed well with the results from the lucigenin-enhanced chemiluminescence experiments.


Figure 3
View larger version (107K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 In situ superoxide production. The fluorescent oxidative dye DHE was used to demonstrate superoxide production in frozen, unfixed vessel sections. Red fluorescence indicates positive staining for superoxide while green fluorescence is due to autofluorescence of elastin. Superoxide staining of endothelial cells was faint in control vessels (A) while it was much more pronounced in ritonavir-treated vessels (B). Exposure times and settings were identical in both photomicrographs. Scale bars represent 20 µm.

 
Increased activity of NAD(P)H oxidase was investigated as a possible mechanism for the increased endothelial superoxide production resulting from ritonavir treatment. The lucigenin-enhanced chemiluminescence method was used with NADPH added to the reaction mixture. Readings from vessels were corrected for blank values, averaged between 5 and 10 min, and normalized to the vessel surface areas (Fig. 4). Control vessels with NADPH stimulation measured 39.2±1.8 RLU/s/mm2 (n=8) and ritonavir-treated vessels measured 54.9±4.1 RLU/s/mm2 (n=8), with the difference being significant (P<0.01).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Superoxide detection in vessels with NADPH stimulation. (A) Typical superoxide data from NADPH-stimulated control and ritonavir-treated vessels, with blank values subtracted and normalized by area, showing an increase in NADPH-stimulated superoxide production in ritonavir-treated vessels compared to controls. (B) The data for each sample was averaged between 5 and 10 min where peak values occurred. There was a significant 40% increase in NADPH-stimulated superoxide production in ritonavir-treated vessels (n=8) relative to controls (n=8) (P<0.01).

 
The specificity of the lucigenin-enhanced chemiluminescence assay for superoxide was tested using the cell permeable superoxide scavenger tiron. Tiron was added to the reaction mixtures and basal superoxide levels in vessels were determined (Fig. 5A). The addition of tiron reduced basal superoxide readings from 9.8±2.4 RLU/s/mm2 (n=2) to –0.7±0.4 RLU/s/mm2 (n=3) for control vessels and from 15.6±4.8 RLU/s/mm2 (n=2) to 1.3±1.9 RLU/s/mm2 (n=3) for ritonavir-treated vessels. To determine the potential source of the increased superoxide production in ritonavir-treated vessels, the flavin inhibitor DPI was used (Fig. 5A). When DPI was added to the reaction mixtures, the readings from control vessels remained relatively unchanged at 10.4±1.9 RLU/s/mm2 (n=3) while the reading of ritonavir-treated vessels was reduced to a level similar to controls of 7.5±1.7 RLU/s/mm2 (n=3). Likewise, for NADPH-stimulated vessels, tiron reduced control vessels from 44.2±0.7 RLU/s/mm2 (n=2) to 4.6±1.9 RLU/s/mm2 (n=3) and ritonavir-treated vessels from 57.7±2.0 RLU/s/mm2 (n=2) to 3.7±0.1 RLU/s/mm2 (n=3) (Fig. 5B). Inhibition of the flavin-containing enzymes NAD(P)H oxidase, nitric oxide synthase, and xanthine oxidase with DPI also dramatically reduced the superoxide levels in NADPH-stimulated vessels. Control vessel readings were reduced to 2.8±0.5 RLU/s/mm2 (n=3) with the addition of DPI and ritonavir-treated vessels were reduced to 3.4±0.4 RLU/s/mm2 (n=3) when DPI was included in the reaction mixture (Fig. 5B).


Figure 5
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Superoxide measurements with tiron and DPI. The specificity of the assay and possible mechanism of increased superoxide were tested using tiron and diphenyleneiodonium chloride (DPI), respectively. (A) For basal superoxide levels, adding tiron to the reaction mixture completely abolished the superoxide reading, indicating the assay was specific to superoxide. Adding DPI to the reaction mixture decreased the ritonavir-treated reading (n=3) to the level of the controls (n=3). (B) Likewise, for NADPH-stimulated readings, adding tiron to the reaction mixtures reduced the readings to nearly zero, indicating the specificity of the assay for superoxide, and the addition of DPI to the reaction mixtures reduced the readings to nearly zero for both controls (n=3) and ritonavir-treated vessels (n=3).

 
Nitrotyrosine immunostaining was performed on vessel segments after 24 h of culturing as controls or with 15 µM ritonavir to asses possible oxidation of amino acids by free radicals. The nitrotyrosine immunoreactivity in ritonavir-treated vessels was markedly increased, particularly in endothelial cells, compared to control vessels (Fig. 6). Negative controls (no primary antibody) showed no immunoreactivity.


Figure 6
View larger version (105K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Nitrotyrosine staining of vessels. Nitrotyrosine immunohistochemical staining of vessels after 24 h of culture as controls or with ritonavir treatment was used to demonstrate the effects of ritonavir on oxidative stress. Control vessels (A) showed much lighter staining than ritonavir-treated vessels (B), particularly in the endothelium. Scale bars represent 20 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The use of HIV protease inhibitors has greatly reduced the morbidity and mortality of HIV-infected individuals. Unfortunately, there is a high incidence of severe side effects resulting from the prolonged use of these drugs including dyslipidemia, hyperglycemia, central obesity, coronary artery disease, and carotid artery disease [2–4,6,17,18]. Our studies demonstrate that HIV protease inhibitor ritonavir directly causes endothelial cell dysfunction by reducing endothelium-dependent vasorelaxation in arteries. The recommended clinical dose of ritonavir of 600 mg every 12 h results in a maximum plasma concentration of 8–15 µM [19,20]. In the present study, the dose used (15 µM) was therefore representative of the maximal clinical concentration, but the exposure to ritonavir was only 24 h, whereas patients may continually use ritonavir for >year. Smooth muscle mediated vasoconstriction was also reduced by ritonavir treatment, suggesting a possible effect on smooth muscle as well. The mechanism of SMC dysfunction remains unknown, however, and warrants further investigation. Furthermore, an increase in endothelial cell superoxide anion levels was demonstrated in vessels treated with ritonavir. The increase in superoxide production in ritonavir-treated vessels may have been due to increased NAD(P)H oxidase activity, as evidenced by elevated NADPH-stimulated superoxide production in treated vessels, which was abolished by the inhibitor DPI. Nitrotyrosine immunostaining also demonstrated increased oxidative stress in ritonavir-treated vessels relative to controls. Taken together, the results may explain in part the increased incidence of vascular complications in HIV patients using HIV protease inhibitors.

The results presented here agree well with a recent clinical study that demonstrated impaired flow-mediated vasorelaxation of brachial arteries in HIV patients being treated with protease inhibitors compared to HIV patients that were not being treated with protease inhibitors [10]. It is important to note that there is a strong correlation between endothelial dysfunction of the brachial artery and the coronary arteries and future adverse coronary events [21,22]. Their analysis showed that most, but not all, of the differences observed in brachial flow-mediated dilation could be explained by differences in lipid profiles resulting from the use of HIV protease inhibitors. Our results demonstrate a direct effect of ritonavir on endothelial cell nitric oxide (NO)-mediated vasorelaxation that is independent of changes in lipid concentrations, thereby offering a possible explanation for part of the endothelial dysfunction noted in patients. While flow-mediated dilation was not directly measured in our study, Ach-induced relaxation and flow-mediated vasorelaxation both rely on nitric oxide, and therefore, the results presented here may shed some light on endothelial dysfunction in HIV patients using ritonavir. Indeed, we previously demonstrated a direct cytotoxic effect of ritonavir on endothelial cells and an increase in endothelial cell necrosis [23]. Likewise, a direct effect of HIV protease inhibitors amprenavir, indinavir, and ritonavir on macrophage cholesterol ester accumulation has also been recently demonstrated, with ritonavir effecting the largest changes [9]. The increase in cholesterol accumulation was dependent on an upregulation of CD36, as demonstrated with CD-36 blocking antibodies and CD-36 null mice. Taken together, these recent findings demonstrate that HIV protease inhibitors may cause dysfunction in vascular cells beyond that caused by dyslipidemia.

The changes in endothelium-dependent relaxation demonstrated in the present study may have been due to the increase in endothelial cell superoxide production. A 56% increase in basal superoxide production was shown in vessel rings treated with 15 µM of ritonavir compared to vehicle controls. Likewise, an increase in endothelial cell DHE staining was observed in vessels treated with ritonavir compared to vehicle controls, also indicating an increase in superoxide production. Even before endothelial-derived relaxation factor (EDRF) was determined to be nitric oxide, it was demonstrated that superoxide anion decreased its bioavailability [24]. Nitric oxide reacts with superoxide to form the peroxynitrite anion (ONOO–), which subsequently decomposes to form the highly reactive hydroxyl radical (OH) [25,26]. The interaction between NO and O2– occurs approximately three times faster than the rate for O2– with superoxide dismutase (SOD), and therefore, some NO may always be reacting with superoxide and thus be unavailable for other biological functions [27,28]. It is also possible that the decrease in vasorelaxation with ritonavir treatment resulted from decreased NO availability from decreased expression of endothelial nitric oxide synthase (eNOS). Our preliminary studies, however, showed no change in eNOS expression on either an mRNA or protein level (data not shown). Other than decreased vasorelaxation, the consequences of decreased NO bioavailability and increased superoxide levels may include increased platelet aggregation and monocyte adhesion, increased smooth muscle cell proliferation, activation of matrix metalloproteinases, and endothelial cell apatosis [29–32]. The increase in superoxide production demonstrated here may also help explain in part the mitochondrial DNA damage we demonstrated previously, as the product of superoxide and nitric oxide interaction, peroxynitrite, has been shown to induce such damage in endothelial cells [23,33].

The increase in superoxide production from ritonavir treatment may have been due to increased NAD(P)H oxidase activity. The addition of the flavin inhibitor DPI to the reaction mixture abolished the increase in basal superoxide levels in ritonavir-treated vessels compared to controls, indicating NAD(P)H oxidase, nitric oxide synthase, or xanthine oxidase as possible sources of the increased superoxide production. The cell permeable superoxide scavenger tiron demonstrated the specificity of the assay for superoxide anion. When vessel rings were stimulated with NADPH, ritonavir-treated vessels showed a significant 40% increase in superoxide levels as compared to NADPH-stimulated controls, also demonstrating a possible increase in the activity of NAD(P)H oxidase activity resulting from ritonavir treatment. Again tiron demonstrated the specificity of the assay. The addition of DPI to the reaction mixtures of NADPH-stimulated vessel rings reduced both control and ritonavir-treated vessels to approximately zero, indicating that NAD(P)H oxidase activity was most likely responsible for the superoxide production resulting from the addition of NADPH.

The effects of increased free radical stress resulting from ritonavir treatment were also demonstrated by anti-nitrotyrosine staining. Although only superoxide levels were measured, it is likely that other radical species were also increased due to ritonavir treatment. Peroxynitrite, the product formed by the interaction between superoxide and nitric oxide can modify tyrosine residues to form 3-nitrotyrosine [34]. Increased anti-nitrotyrosine immunoreactivity in ritonavir-treated vessels suggests that reactive nitrogen species were also increased, particularly in the endothelium.

In summary, we have demonstrated for the first time to our knowledge that HIV protease inhibitor ritonavir, in doses similar to clinical plasma levels, decreases endothelium-dependent vasorelaxation and increases superoxide anion production in porcine arteries. The increase in superoxide production may have reduced the bioavailability of nitric oxide, thus offering a possible explanation for the decreased endothelium-dependent vasorelaxation. The exact mechanisms by which ritonavir upregulates superoxide in endothelial cells is unknown at this time and thus further investigation is warranted. However, it seems clear that ritonavir has a variety of direct effects on vascular biology that may shift the balance toward a pro-atherogenic state. These direct effects, when coupled with the indirect effects of dyslipidemia, may help explain the high incidence of premature atherosclerosis in HIV-infected patients being treated with HIV protease inhibitors. While the results of this study are only indicative of endothelial dysfunction related to ritonavir use, it is possible that other HIV protease inhibitors may have similar effects. These studies are currently underway in our laboratory.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by NIH/NHLBI Grants HL61943-01, HL65916, HL72716, and HL60135 (C. Chen), as well as NIH/NIAID AI49116 (Q. Yao). The authors would like to thank Dr. E. O'Brian Smith for his help with the statistical analysis.


   Notes
 
1 Current Address: Section of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. Back

Time for primary review 13 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Hammer S.M, Squires K.E, Hughs M.D. A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. N. Engl. J. Med. (1997) 337:725–733.[Abstract/Free Full Text]
  2. Behrens G, Schmidt H, Meyer D, Stoll M, Schmidt R.E. Vascular complications associated with use of HIV protease inhibitors. Lancet (1998) 351:1958.[ISI][Medline]
  3. Gallet B, Pulik M, Genet P, Chedin P, Hiltgen M. Vascular complications associated with use of HIV protease inhibitors. Lancet (1998) 351:1958–1959.[ISI][Medline]
  4. Laurence J. Vascular complications associated with use of HIV protease inhibitors. Lancet (1998) 351:1960.[ISI][Medline]
  5. Vittecoq D, Escaut L, Monsuez J.J. Vascular complications associated with use of HIV protease inhibitors. Lancet (1988) 351:1959.[CrossRef]
  6. Periard D, Telenti A, Sudre P, et al. Atherogenic dyslipidemia in HIV-infected individuals treated with protease inhibitors. The Swiss HIV Cohort Study. Circulation (1999) 100:700–705.[Abstract/Free Full Text]
  7. Sullivan A.K, Nelson M.R. Marked hyperlipidaemia on ritonavir therapy. AIDS (1997) 11:938–939.[ISI][Medline]
  8. Carr A, Samaras K, Chisholm D.J, Cooper D.A. Pathogenesis of HIV-1-protease inhibitor-associated preipheral lipodystrophy, hyperlipidaemia, and insulin resistance. Lancet (1998) 351:1881–1883.[CrossRef][ISI][Medline]
  9. Dressman J, Kincer J, Matveev S.V, et al. HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages. J. Clin. Invest. (2003) 111:389–397.[CrossRef][ISI][Medline]
  10. Stein J.H, Klein M.A, Bellehumeur J.L, et al. Use of human immunodeficiency virus-1 protease inhibitors is associated with atherogenic lipoprotein changes and endothelial dysfunction. Circulation (2001) 104:257–262.[Abstract/Free Full Text]
  11. Conklin B.S, Surowiec S.M, Lin P.H, Chen C. A simple physiologic pulsatile perfusion system for the study of intact vascular tissue. Med. Eng. Phys. (2000) 22:441–449.[CrossRef][ISI][Medline]
  12. Conklin B.S, Surowiec S.M, Ren Z, et al. The effects of nicotine and cotinine on porcine arterial endothelial cell function. J. Surg. Res. (2001) 95:23–31.[CrossRef][ISI][Medline]
  13. Miller F.J.J, Sharp W.J, Fang X, Oberley L.W, Oberley T.D, Weintraub N.L. Oxidative stress in human abdominal aortic aneurysms: a potential mediator of aneurysmal remodeling. Arterioscler. Thromb. Vasc. Biol. (2002) 22:560–565.[Abstract/Free Full Text]
  14. Hathaway C.A, Heistad D.D, Piegors D.J, Miller F.J Jr. Regression of atherosclerosis in monkeys reduces vascular superoxide levels. Circ. Res. (2002) 90:277–283.[Abstract/Free Full Text]
  15. Miller F.J, Gutterman D.D, Rios C.D, Heistad D.D, Davidson B.L. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ. Res. (1998) 82:1298–1305.[Abstract/Free Full Text]
  16. Chen C, Li J, Mattar S.G, et al. Boundary layer infusion of basic fibroblast growth factor accelerates intimal hyperplasia in endarterectomized canine artery. J. Surg. Res. (1997) 69:300–306.[CrossRef][ISI][Medline]
  17. Carr A, Samaras K, Burton S, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS (1998) 12:F51–F58.[CrossRef][ISI][Medline]
  18. Henry K, Melroe H, Huebsch J, et al. Severe premature artery disease with protease inhibitors. Lancet (1998) 351:1328.[ISI][Medline]
  19. Arky R, Allen C.B, Ehardt T.W, et al. Physicians Desk Reference, 51st ed. (1997) Oradell (NJ): Medical Economics—Thompson Healthcare. 447–451.
  20. Deeks S.G, Smith M, Holodniy M, Kahn J.O. HIV-1 protease inhibitors: a review for clinicians. JAMA (1997) 277:145–153.[Abstract]
  21. Hikita H, Takase B, Satomura K, Kurita A, Nakamura H. Differences in heart rate variability in the acute phase of first and second attacks of myocardial infarction. Scand. Cardiovasc. J. (1998) 82:1535–1539.
  22. Suwaidi J.A, Hamasaki S, Higano S.T, Nishimura R.A, Holmes D.R Jr., Lerman A. Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction. Circulation (2000) 101:948–954.[Abstract/Free Full Text]
  23. Zhong D.S, Lu X.H, Conklin B.S, et al. HIV protease inhibitor ritonavir induces cytotoxicity of human endothelial cells. Arterioscler. Thromb. Vasc. Biol. (2002) 22:1560–1566.[Abstract/Free Full Text]
  24. Rubanyi G.M, Vanhoutte P.M. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J. Physiol. (1986) 250:H822–H827.[ISI][Medline]
  25. Beckman J.S, Beckman T.W, Chen J, Marshall P.A. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U. S. A. (1990) 87:1620–1624.[Abstract/Free Full Text]
  26. Lum H, Roebuck K.A. Oxidant stress and endothelial cell dysfunction. Am. J. Physiol. (2001) 280:C719–C741.[ISI]
  27. Thompson L, Trujillo M, Telleri R, Radi R. Kinetics of cytochrome c+2 oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems. Arch. Biochem. Biophys. (1995) 319:491–497.[CrossRef][ISI][Medline]
  28. Cai H, Harrison D.G. Endothelial dysfunction in cardiovascular disease: the role of oxidant stress. Circ. Res. (2000) 87:840–844.[Abstract/Free Full Text]
  29. Ohara Y, Peterson T.E, Harrison D.G. Hypercholesterolemia increases endothelial superoxide anion production. J. Clin. Invest. (1993) 91:2546–2551.[ISI][Medline]
  30. Rajagopalan S, Meng X.P, Ramasamy S, Harrison D.G, Galis Z.S. Reactive oxygen species produced by macrophage-derrived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. J. Clin. Invest. (1996) 98:2572–2579.[ISI][Medline]
  31. Griendling K.K, Ushio-Fukai M. Redox control of vascular smooth muscle proliferation. J. Lab. Clin. Med. (1998) 132:9–15.[CrossRef][ISI][Medline]
  32. Li P.-F, Dietz R, von Harsdorf R. Differential effect of hydrogen peroxide and superoxide anion on apotosis and proliferation of vascular smooth muscle cells. Circulation (1997) 96:3602–3609.[Abstract/Free Full Text]
  33. Ballinger S.W, Patterson C, Yan C.N, et al. Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ. Res. (2000) 86:960–966.[Abstract/Free Full Text]
  34. MacMillan-Crow L.A, Crow J.P, Kerby J.D, Beckman J.S, Thompson J.A. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc. Natl. Acad. Sci. U. S. A. (1996) 93:11853–11858.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 CirculationHome page
M. P. Dube, S. E. Lipshultz, C. J. Fichtenbaum, R. Greenberg, A. D. Schecter, S. D. Fisher, and for Working Group 3
Effects of HIV Infection and Antiretroviral Therapy on the Heart and Vasculature
Circulation, July 8, 2008; 118(2): e36 - e40.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Conklin, B. S
Right arrow Articles by Chen, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conklin, B. S
Right arrow Articles by Chen, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?