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Protease Inhibitors Adversely Affect Coronary Artery Function  
  NEW YORK (Reuters Health) Sept 27 - HIV protease inhibitors significantly impair the in vitro function of coronary arteries, according to a report in the September 1st issue of the Journal of Acquired Immune Deficiency Syndromes.
Previous studies in HIV-infected patients have identified correlations between the use of protease inhibitors (PIs) and endothelial dysfunction, the authors explain, but it remains unclear whether different PIs differ in their effects.
Dr. Changyi Chen and associates from Baylor College of Medicine, Houston, Texas evaluated the effects of five PIs (ritonavir, amprenavir, saquinavir, indinavir, and nelfinavir) on vasomotor functions in porcine coronary arteries in vitro.
Porcine coronary arteries showed significantly reduced contraction after incubation in solutions containing ritonavir (63% reduction) or saquinavir (32% reduction), the authors report. Insignificant reductions in contractility were seen with amprenavir and nelfinavir, but indinavir did not affect contractility.
Endothelium-dependent relaxation was also significantly impaired after exposure to ritonavir, amprenavir, and saquinavir, the report indicates. Smaller, nonsignificant reductions were seen after exposure to indinavir and nelfinavir.
Other significant changes included reduction of endothelial nitric oxide synthase (eNOS) mRNA (ritonavir, amprenavir, and saquinavir), reduced production of nitrite (ritonavir and amprenavir), and increased production of superoxide anion (ritonavir and amprenavir).
The effects of PI exposure were largely reversed by coculture with the antioxidant SeMet, the researchers note.
"Antioxidant SeMet can successfully block ritonavir-induced effects, indicating a major molecular mechanism and a potential therapeutic strategy in HIV patients with antiviral therapy," the researchers write.
"Further investigations including in vivo experiments are warranted to gain a better understanding of this important issue," the investigators conclude.
J Acquir Immune Defic Syndr 2005;40:12-19
Effects of 5 HIV Protease Inhibitors on Vasomotor Function and Superoxide Anion Production in Porcine Coronary Arteries
JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 40(1) 1 September 2005 pp 12-19
Chai, Hong MD, PhD; Yang, Hui MD, PhD; Yan, Shaoyu PhD; Li, Min PhD; Lin, Peter H MD; Lumsden, Alan B MD; Yao, Qizhi MD, PhD; Chen, Changyi MD, PhD
From the Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX.
HIV protease inhibitors (PIs) have been implicated to cause cardiovascular complications. Previous studies demonstrated that the PI ritonavir (RTV) caused endothelial dysfunction in porcine arteries.
This study investigated and compared the effects of 5 commonly used PIs on vasomotor function, endothelial nitric oxide synthase (eNOS) expression, and oxidative stress in porcine coronary arteries. Vessel rings were incubated with 15 mM of RTV, amprenavir (APV), saquinavir (SQV), indinavir (IDV), or nelfinavir (NFV) for 24 hours. Vasomotor function was studied using a myograph system.
The contractility of the rings was significantly reduced for RTV and SQV. In response to bradykinin at 10-5 M, the endothelium-dependent relaxation was significantly reduced for RTV, APV, and SQV. The eNOS mRNA levels were significantly reduced for RTV, APV, and SQV. Furthermore, the superoxide anion (O2 -) levels of the vessels were significantly increased for RTV and APV. It was found that nitric oxide production was decreased, whereas the level of nitrotyrosine proteins was increased in RTV-treated vessels. Furthermore, antioxidant seleno-L-methionine (SeMet) reversed RTV-induced O2 - production and vasomotor dysfunction.
Thus, the HIV PIs RTV, APV, and SQV at 15 mM have more potent in vitro effects on vasomotor dysfunction, eNOS downregulation, and O2 - production than IDV and NFV. The antioxidant SeMet can block these adverse effects of RTV. The results suggest that antioxidant therapy may have applications for controlling PI-associated cardiovascular complications.
Highly active antiretroviral therapy (HAART) has greatly improved clinical outcomes for patients with HIV infection.1 Among all the treatments, HIV protease inhibitors (PIs) have been considered one of the most significant advances in the past 2 decades in controlling HIV infection.1,2 With marked improvement in the long-term survival, however, concern is growing about serious cardiovascular complications from the prolonged use of PIs. Some investigations have revealed a possible association between HIV PIs and metabolic abnormality including hyperlipidemia, lipodystrophy, and insulin resistance.3,4
Previous studies have shown that HIV PIs increased CD36-dependent cholesterol ester accumulation in macrophages in both in vitro and in vivo models independent of dyslipidemia.5 In addition, a clinical cross-sectional study of HIV patients revealed a correlation between the use of PIs and endothelial dysfunction independent of lipid levels.6 Furthermore, we have recently demonstrated that the HIV PI ritonavir (RTV) significantly impaired vasomotor activities through the increase of oxidative stress and the decrease of endothelial nitric oxide synthase (eNOS).7-9 Thus, there could be a direct effect of HIV PIs on endothelial cell functions. Direct comparison of 5 HIV PIs for their adverse effects is not available. It is possible that some HIV PIs have more potent effects than others under the same conditions such as drug concentrations, cell types, and culture environments.
The objective of this study was to determine and compare the effects of HIV PIs including RTV, amprenavir (APV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV) on vasomotor functions as well as underlying molecular mechanisms in porcine coronary arteries. Specifically, the expression and activity of eNOS and the status of oxidative stress were investigated. This study may provide a better understanding of the mechanisms of HIV PI-associated vascular complications and suggest a new therapeutic strategy to control this clinical problem.
This study has compared the direct effects of 5 HIV PIs on coronary artery injury as well as eNOS expression and superoxide anion production. Our results demonstrated that in the model of porcine coronary artery cultures, the HIV PIs RTV, APV, and SQV have more detrimental effects than IDV and NFV, resulting in vasomotor dysfunction, eNOS downregulation, and O2 - overproduction. Furthermore, antioxidant therapy could completely block these adverse effects of HIV PIs.
The pharmacokinetic properties of RTV, APV, SQV, IDV, and NFV have been evaluated in both healthy volunteers and AIDS patients. The maximal plasma concentrations of clinical doses of RTV, APV, and IDV are about 15 mM, whereas those for SQV and NFV are 3.7 and 6.0 mM, respectively (Table 1).14 In a previous study, we determined a dose-response curve of RTV on endothelial function.9 RTV at 7.5 mM showed no significant effect, whereas RTV at 15 and 30 mM showed a significant decrease in both vasomotor function and eNOS expression. In the current study, we have carefully chosen the drug concentration of 15 mM for all 5 PIs to simulate a clinical situation and make a meaningful comparison. At this identical condition, RTV significantly impairs vessel contractility, endothelium-dependent vasorelaxation, and endothelium-independent vasorelaxation, indicating RTV-induced smooth muscle cell and endothelial cell injury. APV and SQV had similar but less potent effects on vasomotor function as compared with RTV. IDV and NFV had very limited effects on vasomotor function in porcine coronary arteries. These data are consistent with clinical observations showing impaired flow-mediated vasorelaxation of brachial arteries in HIV patients using PIs.6 Direct effects shown in this study and indirect adverse effects of HIV PIs by clinical dyslipidemia could contribute to vascular complications. Our study provides evidence of these direct effects of HIV PIs. Indeed, we previously demonstrated a direct cytotoxic effect of RTV on endothelial cell culture.15 Recently, a direct effect of HIV PIs (ie, RTV and APV) on macrophage cholesterol ester accumulation has been reported, and the most potent effects have been seen with RTV.5
A key process in the early pathogenesis of atherosclerosis is diminished bioavailability of the endothelium-derived signaling molecule NO, which is generated by eNOS and is a potent vasodilator with multiple additional cardiovascular functions.16 Both basic and clinical studies have shown that eNOS plays a very important role in cardiovascular disease.17 In this study, HIV PI-induced vessel injury may be due to the decrease in eNOS expression and NO production. Indeed, the real-time PCR data showed a 34% to 43% decrease in eNOS mRNA expression in RTV- and APV-treated vessel rings, and immunohistochemistry data also showed a decrease in eNOS protein levels. In addition, APV- and RTV-treated groups showed a marked decrease of NO, which can be reversed by co-culturing with an antioxidant in the porcine coronary artery culture model.
Oxidative stress contributes to mechanisms of vascular dysfunction.18 Endothelium can generate a substantial amount of O2 -. Nitric oxide reacts with O2 - to form peroxynitrite anion (ONOO-), which subsequently decomposes to form the highly reactive hydroxyl radical (OH). This interaction occurs approximately 3 times faster than the rate for O2 -with superoxide dismutase, and therefore, a certain portion of NO may always be reacting with O2 -and thus become unavailable for other biologic functions.19 Evidence from cell culture and animal studies suggests that overproduction of O2 - and subsequent oxidative inactivation of NO may both be important in the pathogenesis of atherosclerosis.20 In our study, about a 50% increase in O2 - production was shown in RTV- and APV-treated samples as compared with controls. Likewise, an increase in endothelial cell DHE staining was observed in vessels treated with RTV and APV, indicating an increase in O2 - production. Because RTV showed the most potent effect on vasomotor functions as compared with other HIV PIs, it was chosen as a representative of HIV PIs to further investigate protein nitration and the role of the antioxidant SeMet. Western blot and immunohistochemistry showed an elevated level of nitrotyrosine in RTV-treated rings. This increase can be reversed by co-culturing with the antioxidant SeMet. Furthermore, when rings were co-cultured with RTV and antioxidant SeMet, endothelium-dependent vasorelaxation as well as vessel contractility and endothelium-independent vasorelaxation were returned to the normal levels of control vessels. SeMet is a commonly used antioxidant. We have used a single concentration of 100 mM of SeMet based on our previous experience and publications in this porcine artery culture model.21,22 SeMet (100 mM) alone did not affect endothelial function including vasomotor activity and eNOS expression in our studies. Other studies also used this concentration of SeMet.23-25 In a previous study, we have tested other antioxidants including curcumin26 and ginsenosides,8 which also significantly blocked RTV-induced endothelial dysfunction in the porcine coronary arteries. These data are well correlated with O2 - production in these vessels, indicating that oxidative stress may be the major mechanism in HIV PI-induced vascular injury.
Five HIV PIs have some effects in common and some differences. For example, RTV and APV showed more potent effects on vasomotor function relative to others. Both drugs significantly reduced endothelium-dependent relaxation, but APV had no effects on contraction or endothelium-independent relaxation. In contrast, both drugs significantly reduced the levels of eNOS mRNA and nitrites as well as enhanced generation of superoxide radicals. The underlying reasons for these differences between RTV and APV are not clear. Besides oxidative stress and NO availability affecting HIV PI-induced vascular injury, other mechanisms may exist. The data regenerated from RTV may be applicable to other HIV PIs including APV. This issue must be directly verified by direct experiments with these specific HIV PIs in future investigations. In addition, cytotoxicity of RTV could play a role in vascular injury. The viability of RTV-treated vessels was not directly measured, however. Instead, the potent mechanism of RTV-induced vessel injury was studied, and it was related to oxidative stress and eNOS downregulation as compared with the untreated vessels as controls. Antioxidant SeMet could effectively block these effects of RTV on porcine coronary arteries.
HIV PIs tested in vitro may have different biologic activities in vivo because of plasma protein-binding properties of these drugs. In fact, all of the HIV PIs tested except IDV are highly bound to serum proteins, and RTV, SQV, and NFV are >97% bound.14 For example, using a reasonable Cmax concentration of 2500 ng/mL for SQV and 97% plasma protein binding,14 the free plasma drug concentration is calculated to be 0.11 mM. This in vitro concentration of 15 mM of SQV represents 136-fold the calculated free plasma Cmax concentration. Similarly, the concentration of NFV tested in vitro represents approximately 160-fold the calculated free plasma concentration. Thus, the 15-mM concentration of tested HIV PIs in vitro may represent the excessive (supratherapeutic) concentrations for some tested HIV PIs after consideration of free plasma concentrations in vivo. This concern could be a limitation of current investigation in vitro. Kinetics between plasma-bound and free PIs in vivo are not clear. The impact of protein-bound PIs on endothelial functions is also not clear. Further investigations including a dose range of HIV PIs and in vivo models as well as human cells or tissues are warranted. Whereas the potency of the PIs on vasomotor function differed, we may also consider the therapeutic concentrations of the PIs to define the therapeutic window in regards to potential effects on vasomotor function.
In summary, we have demonstrated that several HIV PIs can significantly cause vasomotor dysfunction, decrease eNOS expression, and increase O2 - production in porcine coronary arteries. RTV and APV showed more potent effects than SQV. However, IDV and NFV had very limited effects in the same condition. Antioxidant SeMet can successfully block RTV-induced effects, indicating a major molecular mechanism and a potential therapeutic strategy in HIV patients with antiviral therapy. Further investigations including in vivo experiments are warranted to gain a better understanding of this important issue.
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