|Home | About | Journals | Submit | Contact Us | Français|
Highly active antiretroviral therapy (HAART) including HIV protease inhibitor ritonavir may be associated with the clinical complications including accelerated atherosclerosis and pulmonary artery hypertension. The objective of this study was to determine whether capsaicin, a major ingredient of hot pepper with antioxidative property, could effectively inhibit ritonavir-induced oxidative injury in porcine pulmonary arteries.
Fresh porcine pulmonary artery rings were treated with ritonavir (15 µM), capsaicin (50 µM) or both for 24 hours and, then, subjected to myograph analysis in response to vasoactive drugs including thromboxane A2 analog U-46619, bradykinin, and sodium nitroprusside (SNP).
In response to U-46619 (3×10−8 M), ritonavir-treated porcine pulmonary artery rings reduced the contraction by 15% compared with control rings. In response to bradykinin (10−6 M), ritonavir-treated rings showed a significant reduction of endothelium-dependent vasorelaxation by 32% compared with untreated control vessels (P < 0.05, n=5, Student t-test). However, ritonavir treatment did not change endothelium-independent vasorelaxation in response to SNP (10−6 M). Capsaicin-treated vessel rings did not show any significant changes in response to U-46619, bradykinin, and SNP compared with untreated control vessels. More importantly, capsaicin co-cultured with ritonavir significantly blocked ritonavir-induced inhibition of endothelium-dependent vasorelaxation and contraction compared with ritonavir alone treatment in porcine pulmonary artery rings (P <0.05, n=5, Student t-test).
Capsaicin effectively inhibits the detrimental effects of HIV protease inhibitor ritonavir on vasomotor functions of porcine pulmonary arteries. These findings may suggest that capsaicin could have clinical applications to prevent vascular and pulmonary complications of HAART drugs in HIV patients.
HIV protease inhibitors were introduced in 1996 for the treatment of HIV and have significantly prolonged the functional immune system for decades. This has resulted in a declining mortality in such treated patients. The proportion of people surviving with AIDS for 2 years or longer increased from 64% in 1993–95 to 85% in 1996–2005 . AIDS death rates declined by 80% in the years 1990–2003 . Given this increased long-term survival, <50% of HIV-infected patients currently die from AIDS defining events and cardiovascular disease is currently the 4th largest cause of death among HIV patients .
Although the purported incidence of pulmonary hypertension in HIV-infected patients is estimated to be 0.5% compared with 0.02% in the general population , there have not been large-scale prospective studies with controls to confirm this. Given the low incidence of pulmonary hypertension, the trials examining the effects of antiretroviral therapy have been mixed. However a retrospective study  found a higher risk of pulmonary artery hypertension in patients treated with a regimen including a protease inhibitor compared to patients treated with only one or two nucleoside reverse transcriptase inhibitors (2.0% versus 0.7%, P = 0.048). In addition case reports have suggested that HIV protease inhibitors had an accelerated course of pulmonary hypertension and a worsening of pulmonary artery systolic pressure .
Initial case reports identified advanced atherosclerotic lesions in patients in their 2nd decade of life treated with HIV protease inhibitors. Antiretroviral therapy has been shown to increase the risk of myocardial infarction (MI) with a 26% relative increase in the annual rate of MI per year of exposure during the first 4–6 years of use from a large, international prospective study . Further studies have demonstrated an association between HIV protease inhibitors (PI) and known cardiovascular risk factors such as hyperlipidemia, lipodystrophy and insulin resistance [8,9]. Increasingly direct effects of PI are noted with a correlation with endothelial dysfunction in a cross sectional study of patients treated with and without PI. , however the mechanism of action remains to be determined.
Capsaicin is the primary active ingredient of red-hot chili pepper and is a member of the capsacinoid family that is extracted from the plant belonging to the genus capsicum. It is extensively used as a cooking ingredient in Asian cuisine. It has been a traditional component of ancient herbal remedies for inflammatory conditions. Recent work has demonstrated beneficial effects in human studies where consumption of chili increased the resistance of serum lipoproteins to oxidation . In addition to its antioxidant effect , capsaicin has been shown to be an inhibitor of tumor proliferation  and possesses anti-inflammatory properties .
Major objective of this study was to examine if capsaicin can protect vascular tissues against ritonavir-induced vascular injury. Porcine pulmonary arteries were used to study the effect of ritonavir and capsaicin on vasomotor function.
U46619 (9,11-Dideoxy-11, 9-epoxymethanoprosta-glandin F2), bradykinin, sodium nitroprusside (SNP), capsaicin and phosphate buffered saline (PBS) solution were obtained from Sigma Chemical Co. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Life Technologies, Inc. Antibiotic-antimycotic solution was obtained from Mediatech Inc. Ritonavir was obtained from AIDS research and Reference Reagent Program (NIH).
Lungs from freshly slaughtered pigs were harvested and placed in cold PBS solution with the main pulmonary arteries flushed with PBS to ensure no clot formation in the arteries. The lungs were transported in ice back to the laboratory. The pulmonary arteries were dissected from the middle and upper lobes and cleared of surrounding connective tissue. The vessels were cut into 5 mm rings and incubated with culture medium for 24 hours at 37°C and 5% CO2 in capsaicin alone (50 µM), 15 µM ritonavir alone and both capsaicin (50 µM) and 15 µM ritonavir together. The culture medium used was DMEM with 1% antibiotic solution. The control used was DMEM with 0.1% dimethylsulfoxide (DMSO) as this was the solvent for ritonovir and capsaicin.
Following 24 hours of incubation in culture medium and respective compound, the vessel rings were suspended between the wires of the organ bath myograph chamber (Danish Myo Technology Organ Bath 700 MO, Aarhus Denmark) in 6 ml Kreb’s solution at 37°C and oxygenated with pure oxygen. The rings were subjected to slow, stepwise increase in tension till 30 mN and allowed to equilibrate for a minimum of 30 minutes. Each ring was then precontracted with 20 µl thrombaxane A2 analogue U46619 (3×10−8M). After a minimum of 60 minutes contraction to equilibrium, relaxation was generated by adding 60 µl of bradykinin at concentrations of 10−10 10−9, 10−8, 10−7, and 10−6 M every 3 minutes. Finally 60 µl SNP (10−6 M) was added and endothelium-independent vasodilatation was recorded.
Statistical analysis was performed on the Data Analysis tool of the Microsoft Excel program (Microsoft Office 2003, Microsoft, Seattle, WA). Data were expressed as means ± SE. Significant difference of data between the control and treated groups was determined by the paired Student’s t-test (two-tail). The final data points of all contractions and relaxations among different groups were also analyzed by ANOVA test. P < 0.05 was considered statistically significant.
Porcine pulmonary artery rings were cultured for 24 hours with a clinically relevant concentration of 15 µM ritonavir alone or together with 50 µM capsaicin, and subsequently subjected to contraction (U46619), and endothelium-dependent (bradykinin) and endothelium-independent (SNP) relaxation assays. Endothelium-dependent vasorelaxation was achieved with cumulative additions of bradykinin.
In response to U46619 (3×10−8 M), the control vessels contracted at 15.29 mN, while ritonavir-treated vessels contracted at 12.95 mN, showing a 15% reduction as compared with controls (Figure 1, P < 0.05, n =5).
In response to bradykinin at 10−6 M, the control vessels relaxed by 62.43±4.72%, while ritonavir-treated vessels only relaxed by 42.30±13.86%, showing a 32% reduction. In contrast, the ritonavir in combination with the capsaicin-treated vessels showed a considerable improvement of vasomotor functions as compared with the ritonavir-treated vessels with relaxation reversed to 63.98±3.40% (Figure 2 and Figure 3, P < 0.05, n=5).
In response to SNP, endothelium-independent relaxation showed no difference for the 4 groups (Figure 4).
A principal finding of this study is that a clinically relevant concentration of ritonavir significantly reduced vasocontractility and endothelium-dependent vasorelaxation in porcine pulmonary artery cultures. Meanwhile capsaicin can effectively block these detrimental effects of ritonavir in porcine pulmonary arteries.
Since the introduction of the anti-retroviral regimens in 1996, the mortality and morbidity of HIV-infected patients have declined sharply. These patients showed a longer lifespan and improved quality of life . However, despite these benefits, anti-retroviral agents have been associated with cardiovascular complications [9, 16]. Although the mechanisms of these complications are not fully understood, metabolic abnormalities as risk factors may play crucial roles in HIV protease inhibitor-associated cardiovascular lesion formation. The detrimental effect of anti-retroviral agents also extends to the pulmonary bed with increased incidence of pulmonary artery hypertension. The mechanism of action has recently been shown to be via impaired vasomotor function of porcine arteries and mediated by endothelial nitric oxide synthase (eNOS) downregulation, oxidative stress, and ERK1/2 activation .
In the current study, ritonavir at the concentration near clinical plasma levels (15 µM) notably induced vasomotor dysfunction, including decreased vessel contractility and endothelium-dependent vasorelaxation. These changes indicate ritonavir can have direct effects on the vascular endothelium. These results are consistent with several clinical studies . HIV-infected individuals receiving HIV protease inhibitors showed impaired vasodilatation by using a flow-mediated brachial artery vasodilatation assay, a noninvasive technique that relies on high-resolution ultrasound of the brachial artery .
In this study, capsaicin effectively blocked the impaired vasoreactivity induced by ritonavir. Further experiments elucidating the mechanism of this action are in progress although the postulated action is likely via loss of nitric oxide bioavailability in the vessel wall [18,19]. Decline in NO bioavailability could be caused by decreased expression of eNOS , lack of substrate or co-factor for eNOS [21,22], and altered cell signaling pathway, which leads to inappropriate eNOS activation , and accelerated NO degradation by reactive oxygen species .
It would not be unreasonable to postulate that capsaicin may mediate its effect via the above pathways given its known actions on other tissues. The antioxidant activity of capsaicin has been demonstrated in human, animal and cell cultures. Ahuja et al. demonstrated that 4-week chili supplementation in men caused a decrease in their resting heart rate and an increase in the effective myocardial perfusion pressure  with an increase in the resistance of serum lipoproteins to oxidation . In rats, capsaicin was noted to have a protective effect against lipid peroxidation . In melanoma cell lines, capsaicin was noted to retard growth of the tumor cell line with a reduction in superoxide anion generation .
Capsaicin is a compound found in natural food sources that could potentially have a cardio protective role against ritonavir-induced vascular dysfunction. Capsaicin effectively inhibits the detrimental effects of HIV protease inhibitor ritonavir on the functions of endothelial and smooth muscle cells in porcine pulmonary arteries. These findings may suggest that capsaicin could have clinical applications to prevent vascular and pulmonary complications of HAART drugs in HIV patients.
Ritonavir was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
Source of support: This work is partially supported by research grants from the National Institutes of Health (Yao: AI 49116 and DE15543; Lin: HL076345; and Chen: HL65916, HL72716, and EB-002436). This study is also supported by the Michael E. DeBakey Department of Surgery, Baylor College of Medicine, and Michael E. DeBakey VA Medical Center, Houston, Texas, USA.