Human immunodeficiency virus (HIV) protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. Ginsenosides have been shown to have potential benefits on the cardiovascular system through diverse mechanisms, including antioxidative property. The objective of this study was to determine whether ginsenosides could prevent coronary arteries from RTV-induced dysfunction. Porcine coronary artery rings were incubated with RTV and ginsenosides Rb1, Rc, and Re for 24 h. Vasomotor function was recorded by a myograph tension system. In response to the thromboxane A2 analog U-46619, the contraction of the vessel rings was significantly reduced. When cocultured with Rb1, Rc, and Re, the contractility significantly increased. In response to bradykinin at 10−5 M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls (P < 0.05). When cocultured with Rb1, Rc, and Re, the relaxation significantly increased 100%, 90%, and 134%, respectively, compared with the RTV-alone groups (P > 0.05). In response to sodium nitroprusside, RTV significantly reduced vasorelaxation. In addition, the endothelial nitric oxide synthase (eNOS) mRNA levels were significantly reduced by 78% for RTV group (P < 0.05) by real-time PCR analysis. The eNOS protein levels measured by Western blot analysis and nitrite concentrations measured by Griess assay were also decreased, whereas O2− production by lucigenin-enhanced chemiluminescence was significantly increased in the RTV-treated group. These effects of RTV were effectively blocked by ginsenosides. Thus HIV protease inhibitor RTV significantly impaired the vasomotor function of porcine coronary arteries. This effect may be mediated by the downregulation of eNOS and overproduction of O2−. These results suggest that ginsenosides can effectively block RTV-induced vascular dysfunction.
- oxidative stress
- ginseng root
- endothelial nitric oxide
- superoxide anion
- human immunodeficiency virus
human immunodeficiency virus (HIV) infection is characterized by persistent viral replication and progressive immune dysfunction. HIV protease inhibitors (PIs) confer striking immunologic and clinical benefits that have led to their widespread acceptance as key components of highly active antiretroviral therapy (HAART) in patients with HIV infection. Whether HIV itself or HAART leads to cardiovascular diseases is still under investigation. Still, up to 60% of patients receiving HIV PIs develop dyslipidemia, hyperglycemia, and central obesity (4, 10, 11). These metabolic changes adversely affect several risk factors for atherosclerotic vascular disease (1, 21, 30).
Ginseng root is used extensively in traditional Chinese medicine for its alleged tonic effect and possible curative and restorative properties. There are increasing data in the literature on ginseng and its potential role in treating cardiovascular diseases. In the published studies involving cell cultures and animal models, ginseng was shown to have potential benefits on the cardiovascular system through diverse mechanisms such as having antioxidant (13, 19), modifying vasomotor function (15, 26), improving lipid profiles (18), and involving glucose metabolism (22, 31).
Chen's group previously showed that ritonavir (RTV), one of the clinically used PIs, can cause endothelial injury or dysfunction in human endothelial cells (37) and porcine carotid arteries (7). This effect is frequently induced by a remarkable increase of O2− production. In this study, we examined whether ginsenosides Rb1, Rc, and Re could block RTV-induced vascular dysfunction in porcine coronary arteries. Specifically, the vasomotor functions, expression of endothelial nitric oxide synthase (eNOS), and the status of oxidative stress were investigated. The results suggest a new therapeutic strategy to control HIV PIs-associated cardiovascular complications.
MATERIALS AND METHODS
Chemicals and reagents.
9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U-46619), bradykinin, sodium nitroprusside (SNP), ginsenosides, β-actin monoclonal antibody, Tri-agent kit, and Tween 20 were obtained from Sigma Chemical (St. Louis, MO). Lucigenin was obtained from Molecular Probes (Eugene, OR). Nitric oxide assay kit was obtained from Calbiochem (San Diego, CA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Life Technologies (Grand Island, NY). Antibiotic-antimycotic solution was obtained from Mediatech (Herndon, VA). iScript cDNA Synthesis Kit, the iQ SYBR Green SuperMix Kit, protein assay kit, and precast polyacrylamide gels were obtained from Bio-Rad Laboratories (Hercules, CA). Antibody against human eNOS was obtained from BD Transduction Laboratories (Lexington, KY). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies and the enhanced chemiluminescence kit were obtained from Amersham Life Sciences (Buckinghamshire, UK). The biotinylated horse anti-mouse IgG and avidin-biotin complex kit were obtained from Vector labs (Burlingame, CA). RTV was obtained through the acquired immunodeficiency syndrome (AIDS) Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. PCR primers were obtained from Sigma Genosys (Woodlands, TX).
Tissue harvest and culture.
Fresh porcine hearts were harvested from young adult farm pigs (6–8 mo old) at a local slaughterhouse, placed in a container filled with cold PBS solution, and immediately transported to the laboratory. The pig right coronary arteries were carefully dissected and cut into multiple 5-mm rings. The rings were then incubated in DMEM with RTV alone (15 μM), or with RTV (15 μM) and Rb1 (1 or 10 μM), or Rc (10 μM), or Re (10 μM) at 37°C and 5% CO2 in a cell culture incubator for 24 h.
The myograph system used in our laboratory has been previously described (5, 12, 23, 28). Briefly, the rings were cultured for 24 h and suspended between the wires of the organ bath myograph chamber (Danish Myo Technology Organ Bath 700 MO, Aarhus, Denmark) in 6 ml of Krebs solution, maintained at 37°C, and oxygenated with pure oxygen gas. Rings were slowly subjected stepwise to a predetermined optimal tension of 30 mN and allowed to equilibrate for at least 30 min. After equilibration, each ring was precontracted with 20 μl of thromboxane A2 analog U-46619 (10−7 M). After 60–90 min of contraction, a relaxation dose-response curve was generated by adding 60 μl of five cumulative additions of the endothelium-dependent vasodilator bradykinin (10−9, 10−8, 10−7, 10−6, and 10−5 M) every 3 min. In addition, 60 μl of SNP (10−6 M) were added into the organ bath, and endothelium-independent vasorelaxation was recorded.
Real time PCR.
After the vessel rings were cultured in the medium for 24 h, they were cut open with Accu-Edge scissors (Sakura Finetek, Torrance, CA). A surgical blade was used to scrape all the endothelial cells off the rings. The cells were then harvested by TriReagent kit, and total RNA was isolated following the manufacturer's instructions (12, 23). Briefly, the RNA was purified from each vessel. The cDNA was generated by reverse transcriptase from mRNA using the iScript cDNA Synthesis Kit according to the manufacturer's instructions. The iQ SYBR Green SuperMix Kit was then used for real-time PCR reaction (36). GAPDH, a housekeeping gene, was used as an internal control for eNOS expression. Porcine eNOS and GAPDH primers were designed using Beacon Designer. The eNOS (GeneBank No. AY266137) primer sequences are the following: forward primer 5′-CCCTACAACGGCTCCCCTC-3′ and reversed primer 5′-GCTGTCTGTGTTACTGGATTCCTT-3′. The GAPDH (GeneBank No. AF017079) primer sequences are the following: forward primer 5′-TGTACCACCAACTGCTTGGC-3′ and reversed primer 5′-GGCATGGACTGTGGTCATGAG-3′. Real-time PCR was performed in an iCycler iQ real-time PCR detection system (Bio-Rad) (36). The thermal cycle conditions used for reverse transcription were as follows: 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C. The thermal cycle conditions used for real-time PCR were as follows: 3 min at 95°C, 40 repeat of 20 s at 95°C, and 1 min at 60°C. Sample cycle threshold (CT) values were determined from plots of relative fluorescence units (RFU) versus PCR cycle number during exponential amplification so that sample measurement comparisons were possible. The eNOS gene expression in each sample was calculated as 2 and further normalized to GAPDH expression as 2.
Western blot analysis.
Endothelial cells were harvested from the treated vessel rings with TriReagent kit. Total protein was isolated following the manufacturer's directions (12, 23). Briefly, endothelial proteins (10 μg) were resolved electrophoretically by one-dimensional SDS-PAGE (Ready Gel, 10% polyacrylamide). Subsequently, the proteins were electrophoretically transferred to nitrocellulose filters. Filters were blocked by using 5% nonfat dried milk in PBS with 0.05% Tween 20 (PBS-T). The eNOS protein was detected using a mouse anti-human monoclonal antibody diluted 1:1,000, and β-actin was detected using a mouse anti-human monoclonal antibody diluted 1:10,000. The eNOS and β-actin primary antibodies were detected with HRP-conjugated goat anti-mouse IgG secondary antibodies diluted 1:5,000. Blots were developed by using enhanced chemiluminescence plus and analyzed with AlphaImager gel documentation system and analysis software (Alpha Innotech, San Leandro, CA).
Immunohistochemistry staining procedure was previously described (5, 12, 23, 28). Briefly, the paraffin section of porcine coronary artery rings was incubated with monoclonal antibody against human eNOS antibody (1:1,000) overnight at 4°C. The section was then incubated with biotinylated anti-mouse IgG (1:250) at room temperature for 40 min. For 3,3′-diaminobenzidine (DAB) visualization, the section was incubated in avidin-biotin-peroxidase solution at room temperature for 1 h, followed by 0.1% DAB, 0.003% H2O2 in Tris-buffered saline for 5–10 min. The section was counterstained with hematoxylin-eosin, cover-slipped, and viewed on an Olympus BX41 microscope (Olympus, Melville, NY). Images were captured with an attached SPOT-RT digital camera and software (Diagnostic Instruments, Sterling Heights, MI).
Detection of O2−.
Levels of O2− produced by endothelial cells were detected by using the lucigenin-enhanced chemiluminescence method (28). Briefly, six sets of vessel rings in each group were used. The rings were cut open longitudinally and trimmed into ∼5 × 5-mm pieces. An assay tube (12 × 75 mm) was filled with 500 μl of Krebs-HEPES buffer solution and 25 μl of lucigenin. After being gently vortexed, the vessel segments were placed endothelium-side down in the tubes. Time-based readings of the luminometer were recorded by FB12 software. The data, in relative light units per second (RLU/s) for each sample, were averaged between 5 and 10 min. Values of blank tubes containing the same reagents as the vessel ring samples were subtracted from their corresponding vessel samples. The area of each vessel segment was measured by using a caliper and used to normalize the data for each sample. Final data were represented as means ± SE (in RLU·s−1·mm−2).
Detection of total nitrites.
Nitric oxide (NO) release from vessel rings was determined by measuring the accumulation of its stable degradation products nitrite and nitrate. Nitrate was converted to nitrite by using nitrate reductase. Artery rings were cultured in DMEM medium (without phenol red, Biosource, Rockville, MD) with RTV alone or with combinations of RTV and Rb1, Rc, or Re (10 μM) for 24 h. Supernatant was collected, and total nitrite levels were measured by Griess reaction using a nitric oxide assay kit (Calbiochem, San Diego, CA). The amount of nitrite formed was normalized to the area of each ring. Final data were represented as means ± SE (in μM/mm2).
Maximal contraction and endothelial-dependent and independent vasorelaxation of the rings were compared between the control and treatment groups using one-way ANOVA. Real-time PCR, Western blot, O2−, and total nitrite data from the different groups were compared using the paired Student's t-test (two-tails). Significance was considered if P < 0.05. Data were reported as means ± SE.
Effect of RTV and ginsenosides on vasomotor function in porcine coronary arteries.
Porcine coronary artery rings were cultured for 24 h with a clinically relevant dose of 15 μM of RTV, with or without ginsenosides Rb1 (1 and 10 μM), Rc (10 μM), and Re (10 μM), and subsequently subjected to physiological contraction (U-46619) and endothelium-dependent (bradykinin) and endothelium-independent (SNP) relaxation (n = 8). In response to U-46619, the contraction of the vessel rings was significantly reduced by 76% for RTV compared with controls (P < 0.05, ANOVA, Fig. 1A). When cocultured with Rb1 (1 and 10 μM), Rc, or Re, the contractility increased by 82%, 117%, 222%, and 198%, respectively, compared with the RTV-alone groups (P < 0.01, ANOVA, Fig. 1A). In response to bradykinin at 10−5 M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls (P < 0.05, ANOVA, Fig. 1, B and C). When cocultured with Rb1 (1 and 10 μM), Rc, or Re, the endothelium-dependent relaxation significantly increased by 55%, 100%, 90%, and 134%, respectively, compared with the RTV-alone groups (P < 0.05, ANOVA, Fig. 1, B and C). In response to SNP, RTV treatment significantly reduced vasorelaxation by 50% compared with controls (P < 0.05, ANOVA, Fig. 1D). When cocultured with Rb1 (1 and 10 μM), Rc, or Re, the relaxation increased by 33%, 72%, 92%, and 93%, respectively, compared with the RTV-alone groups (Fig. 1D).
Effect of RTV and ginsenosides on eNOS expression.
To determine whether eNOS expression was correlated to the reduction of endothelium-dependent vasorelaxation in RTV-treated vessels, the eNOS mRNA levels of the endothelial cells isolated from the vessels were determined by real-time PCR analysis (n = 6). The eNOS mRNA levels were significantly reduced by 78% for the RTV-treated group compared with controls (P < 0.05, ANOVA, Fig. 2). When cocultured with 10 μM of Rb1, Rc, or Re, the eNOS mRNA levels were increased to the control level (Fig. 2).
The eNOS protein level was also analyzed by using Western blot analysis (n = 3). RTV treatment significantly decreased the eNOS protein level by 40% compared with controls, whereas Rb1, Rc, or Re coculture totally reversed RTV-induced eNOS reduction (P < 0.05, t-test, Fig. 3, A and B). Immunohistochemistry staining also confirmed that the eNOS protein level in RTV-treated vessel rings was reduced compared with controls (n = 3). When cocultured with Rb1, Rc, or Re, eNOS immunoreactivity was reversed to the control level (Fig. 4).
Effect of RTV and ginsenosides on NO production.
The NO production in rings after RTV and Rb1, Rc, or Re treatment was also analyzed (n = 3). Total nitrite accumulation in the supernatant of cultured vessel rings is shown in Fig. 5. In the control group, the basal nitrite level was 1.12 μM/mm2. In the RTV-treated group, the NO production was decreased to 71% of the control group (P < 0.05, t-test). When cocultured with 10 μM of Rb1, Rc, and Re, the NO production increased to 93%, 84%, and 80% of the control group, respectively. The effects of Rb1 and Rc were significant compared with the RTV-alone groups (P < 0.05, t-test).
Effect of RTV and ginsenosides on O2− production.
Oxidative stress has been shown to cause endothelial dysfunction and vascular injury. To determine whether this is involved in RTV-induced vasomotor dysfunction, O2− production was analyzed by a lucigenin-enhanced chemiluminescence assay (n = 6). The O2− levels of the endothelial layer of vessel rings were significantly increased by 151% for RTV compared with control samples (P < 0.05, t-test, Fig. 6). Cocultured with Rb1, Rc, or Re reversed the O2− level to the control levels (P > 0.1, t-test, compared with controls, Fig. 6).
A principal finding of this study is that a clinically relevant dose of RTV significantly reduced vasocontractility and endothelium-dependent and independent vasorelaxation in porcine coronary artery cultures. RTV also significantly decreased eNOS mRNA and protein levels, as well as NO release, but increased O2− production. Meanwhile, ginsenosides Rb1, Rc, and Re can effectively block these detrimental effects of RTV in porcine coronary arteries. The results from this study reveal several potential mechanisms of RTV-induced vascular dysfunction and possible therapeutic values of ginsenosides in preventing the side effects of HIV PIs in clinical practices.
Since the introduction of the HAART regimen in 1996, the mortality and morbidity of HIV-infected patients have declined sharply. These patients showed a longer lifespan and improved quality of life (24). However, despite these benefits, all antiretroviral agents, including HIV PIs, nucleoside reverse transcriptase inhibitor (NRTI), and nonnucleoside reverse transcriptase inhibitor (NNRTI), have been associated with cardiovascular complications (3, 8). Although the mechanisms of these complications are not fully understood, metabolic abnormalities as risk factors may play crucial roles in HIV PI-associated cardiovascular lesion formation. In the current investigation, a clinically relevant dose of RTV (15 μM) significantly induced vasomotor dysfunction, including decreasing vessel contractility and endothelium-dependent vasorelaxation as well as independent vasorelaxation. These results are consistent with several clinical studies (8, 29). HIV-infected individuals receiving HAART showed impaired vasodilation by using a flow-mediated brachial artery vasodilation assay, a noninvasive technique that relies on high-resolution ultrasound of the brachial artery. In this study, we used only one concentration of U-46619 for constrictions of these arterial rings. This could be a limitation. A full dose response to U-46619 may demonstrate whether this finding is a shift in sensitivity to the agonist or whether maximal constrictions are altered. It could be possible that the different levels of preconstriction may contribute to the altered relaxation responses observed. These issues warrant further investigations.
NO produced from eNOS is a key regulator of vascular homeostasis, including basal vascular tone (blood flow) and blood pressure (34), as well as acting as an anti-thrombogenic agent (32). An impairment of endothelium-dependent relaxation is present in atherosclerotic vessels even before vascular structural changes occur and represents the reduced eNOS-derived NO activity (16). Bradykinin induces vasodilation via endothelial bradykinin type 2 (B2) receptors. This effect can be blocked partly by inhibitors of NO synthase, suggesting a role for de novo synthesis of NO from l-arginine by NO synthase (2, 25). It has been reported that bradykinin-induced relaxations of porcine coronary artery rings precontracted with U-46619 were attenuated by the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) (33). Our real-time PCR, Western blot, and immunohistochemistry data indicated that eNOS expression in endothelial cells was significantly reduced in RTV-treated pig coronary artery rings. RTV can also decrease bioavailability of NO, which may be caused by reaction with O2− or thiol groups. It is well known that an increase of reactive oxygen species can result in uncoupling of mitochondrial oxidative phosphorylation and eNOS activity, reducing NO availability and generating more reactive oxygen species. Notably, when coronary artery rings were cocultured with RTV and ginsenosides (Rb1, Rc, and Re), these damaging effects of RTV on vasomotor functions were significantly reversed, indicating protective effects of these ginsenosides on vessel walls. Furthermore, ginsenosides can also reverse the effects of RTV on eNOS mRNA and protein expression, as well as NO and O2− production. These data are consistent with a study that showed another ginsenoside Rg1 can enhance NO production, and the expression of eNOS mRNA in tumor necrosis factor-α stimulated human umbilical vein endothelial cells (20). Ginsenoside-induced NO release in cultured endothelial cells has also been well documented. Chen and his colleagues (6) reviewed relaxation effects of ginsenoside Rb1 and Rg1 on pulmonary vessels and discovered that it was eliminated by nitro-l-arginine, an inhibitor of NO synthase. Scott et al. (26) examined the effects of ginsenosides Rb1 and Re on rat cardiac myocytes and discovered that both ginsenosides decreased cardiac contraction in adult rat ventricular myocytes. Additionally, they showed that pretreatment with NO synthase inhibitor attenuated the effects of Rb1 and Re, which further indicated that NO production may be a mediator (26). These data are particularly important because eNOS has been reported to be a target molecule of RTV at the clinically relevant plasma concentration (15 μM) (7). Future studies investigating the phosphorylation of eNOS are warranted to further understand this issue.
Numerous reports have shown that oxidative stress plays a pivotal role in endothelial dysfunction, cardiovascular diseases, and other pathogenic conditions (9, 14). Free radicals have been well documented to play a key role in atherosclerotic plague formation and endothelial dysfunction (17). Among them, superoxide anions (O2−) could react with endothelium-derived NO rapidly and inactivate its effect (25), resulting in reduction or loss of endothelium-dependent vasorelaxation and increase of other atherogenic processes. In this study, we showed a significant increase of O2− production in the endothelial layer of the RTV-treated porcine coronary artery rings. Thus RTV-induced oxidative stress may elucidate its effects on vascular dysfunction, especially on endothelium-dependent vasorelaxation where NO is an important mediator. We also report herein that ginsenosides can reverse RTV-induced overproduction of O2−, which in turn leads to the normal vasomotor function of the coronary artery rings. Extensive studies have been conducted on the protective effects of ginseng against free radical damage on the vascular endothelium. Zhong et al. (38) discovered that certain ginsenosides (Rb1, Rb2, Rb3, Rc, Rg1, Rg2, Re, and Rh1) can counteract the action of free radicals induced by xanthine. Using an animal model, Chen and his colleagues (6) showed that ginsenoside protected against myocardial reperfusion injury with a concomitant increase in 6-keto-PGF1α and a decrease in lipid peroxidation. In addition, Gillis (13) demonstrated protective effects of ginsenoside on injured rabbit pulmonary endothelium induced by a variant of reactive oxygen species. He further reviewed other studies and concluded that ginseng was associated with vasorelaxation and prevented manifestations of oxygen-derived free radical injury by promoting the release of NO. It appears that overproduction of O2− plays a very crucial role in the RTV-induced vascular dysfunction, and ginsenosides with the antioxidative property may be useful in preventing the cardiovascular side effects of HIV PIs.
It is interesting that RTV decreased SNP relaxation to a similar extent as the bradykinin relaxations. This suggests that RTV may cause an inability of the arteries to relax to NO as well as cause a decreased production of NO. RTV induced an increase of O2−, which may readily interact with NO released by SNP, thereby reducing the action of SNP. In addition, RTV may cause a cytotoxic/necrotic process (37), resulting in the global decrease in cellular function, which is demonstrated by decreased smooth muscle contraction, inability to respond to dilator factors, and decreased release/production of endothelial cell relaxing factors. Thus the effect of ginsenosides may also alter the necrotic process by antioxidant activity.
The mechanism by which ginsenosides reverse RTV-induced eNOS downregulation is still unclear. Both direct and indirect effects may be possible. Besides, different ginsenosides may have very different chemical and biological characteristics. A recent study (27) explained the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active compounds in the extract. It showed that ginsenoside Rg1 can promote angiogenesis, whereas Rb1 exerts an opposing effect (27). Future studies are warranted to fully understand this issue.
In summary, a clinically relevant dose of RTV (15 μM) can cause vasomotor dysfunction, decrease eNOS expression, and increase O2− production. Ginsenosides Rb1, Rc, and Re can effectively block all these detrimental effects of RTV. With the increasing use of HAART regimen in HIV-infected patients, more vascular complications resulting from HIV infection or side effects of antiviral drugs are expected. The data from this study raise the possibility of a new therapeutic strategy for this significant clinical problem.
This work was supported by NIH Grants R01HL-61943, R01HL-65916, R01HL-60135 and R01HL-72716 (to C. Chen); R21AI-49116 and R01DE-015543 (to Q. Yao); R01HL-75824 (to A. Lumsden); and K08 HL-076345 (to P. Lin).
RTV was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH).
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- Copyright © 2005 by the American Physiological Society