Premature atherosclerosis in HIV-infected individuals - focus on protease inhibitor therapy

Objective: Lipid disorders associated with the use of protease inhibitors may contribute to the premature development of atherosclerosis. The purpose of the present study was to determine whether the administration of a protease inhibitor-containing regimen to middle-aged (30-50 years) HIV-infected individuals for 6 months or longer is associated with an increased prevalence of atherosclerosis.

Cited Here...: High-resolution B-mode ultrasound imaging was used to visualize the femoral and carotid arteries of 68 HIV-negative and 168 HIV-infected individuals, including 136 patients who had received protease inhibitors for 26.8 ± 8.9 months (mean ± SD). Atherogenic plaques were defined as a thickening of the intima-media ≥ 1200 mm.

Cited Here...: The proportion of participants with one or more plaques was higher in the HIV-infected group in comparison with the HIV-negative group (55 versus 38%;P = 0.02), and so was the prevalence of cigarette smoking (61 versus 46%;P = 0.03) and hyperlipidaemia (56 versus 24%;P < 0.001). The presence of plaque was independently associated with age, male gender, plasma low-density lipoprotein cholesterol levels and smoking. In univariate logistic regression analysis, an association was also found with HIV infection. Among HIV-infected subjects protease inhibitor therapy was not associated with the presence of plaque.

Conclusions: A large proportion of the middle-aged HIV-infected individuals examined during this study had one or more atherosclerotic plaques within the femoral or carotid arteries. The presence of peripheral atherosclerosis within this population is not associated with the use of protease inhibitors, but rather with 'classic' cardiovascular risk factors such as smoking and hyperlipidaemia, which are amenable to interventions.

Introduction

Administration of antiretroviral therapy containing protease inhibitors (PIs) is accompanied by a variety of metabolic abnormalities ^[1-5] including an elevation in plasma concentrations of low-density lipoproteins (LDL), lipoprotein(a) [Lp(a)] and triglycerides ^[6,7]. Elevation in plasma levels of LDL cholesterol and Lp(a) constitutes a major risk factor for the premature development of atherosclerosis ^[8,9], and hypertriglyceridaemia may also contribute under certain circumstances to the development of heart disease ^[10]. Accordingly, it is a matter of major concern whether long-term administration of PI-containing regimen to HIV-infected patients will lead to cardiovascular complications ^[11-15]. The risk for cardiovascular diseases among PI-treated HIV-infected persons may further be enhanced by the elevated prevalence of lipid abnormalities within this population ^[4,16], which may by itself explain the presence of subclinical atherosclerosis in HIV-infected patients ^[17].

Atherosclerosis is a slowly progressing disease which most commonly culminates in coronary artery disease. As coronary arteries are not immediately amenable to visualization, the presence of ultrasound-detected atherosclerosis within carotid or femoral arteries has been used as a surrogate marker for coronary artery atherosclerosis ^[18,19].

The purpose of the present study was to determine whether prolonged administration of PI-containing regimen to HIV-infected patients is associated with an elevated prevalence of atherosclerotic disease. To do so, high-resolution B-mode ultrasound imaging was used to visualize both carotid and femoral arteries on a group of HIV-infected patients, some of whom had been treated with PIs and a group of HIV-negative individuals. Due to the progressive increase in the prevalence of atherosclerotic plaques with advancing age ^[20,21], only subjects aged 30-50 years were included in the present study.

Methods

HIV-infected individuals were recruited locally among patients registered in the Swiss HIV Cohort Study ^[22], and HIV-negative subjects were identified among visitors to the local anonymous HIV testing centre. Only Caucasian subjects, aged 30 to 50 years, were eligible to participate in the study. Patients who had been treated with PIs were only included if they had received this treatment for at least 6 months. All participants gave their informed consent and the protocol was approved by the local Ethics Committee.

Each participant answered a detailed questionnaire focusing on cardiovascular risk factors and underwent a physical examination. Concentrations of total cholesterol, triglycerides and high-density lipoprotein (HDL) cholesterol were measured on plasma samples collected after the patient had fasted for ≥ 10 h, as described ^[6]. Plasma LDL cholesterol levels were determined using the Friedewald formula. In addition, total cholesterol levels were quantified on plasma samples that had been collected from HIV-infected patients prior to initiation of PI therapy, or stored for an equivalent period of time for non-PI-treated patients. These measurements served as baseline values.

B-mode ultrasound imaging analyses of carotid and femoral arteries were all performed by the same investigator (M.D.) using a colour duplex scanner (Vingmed 5; General Electric Medical System, Milwaukee, Wisconsin, USA) with a high resolution 10 MHz linear array scan head, coupled with the M'ATH software (Metris, Paris, France) which performs semi-automatic measures on frame. The investigator was blinded to the HIV and treatment status of the participants. Carotid arteries were scanned from the lowest part visible in the supraclavicular fossa to the submandibular angle, whereas femoral arteries were scanned from 4 cm above to 4 cm below the inguinal bifurcation. Plaques were defined as a focal echogenic structure which encroached into the lumen and had an intima-media thickness ≥ 1200 μm.

Statistical analyses were performed using STATA (Stata Corp., College Station, Texas, USA) software. Univariate and multivariate logistic regression analyses were used to identify factors associated with the presence of one or more plaques at any of the four sites examined.

Results

A total of 168 HIV-infected individuals aged 39.0 ± 5.5 years (mean ± SD) were compared with a group of 68 significantly younger HIV-negative persons (37.5 ± 5.7 years, P = 0.04). Among HIV-infected participants, 32 were not receiving any PI (16 subjects did not receive any antiretroviral agents, nine patients were receiving zidovudine and lamivudine, three patients stavudine and didanosine, two patients zidovudine and didanosine, one patient stavudine and lamivudine, and one patient stavudine, lamivudine and efavirenz), whereas the remaining 136 patients were currently receiving a PI-containing regimen [seven (5%) were receiving amprenavir, 18 (13%) indinavir, 33 (25%) nelfinavir, 10 (7%) ritonavir, 44 (32%) and 24 (18%) saquinavir in combination with ritonavir or nelfinavir, respectively, at doses recommended by the manufacturers]. The mean duration of exposure to PIs was 26.8 ± 8.9 months (range, 6-45 months).

In comparison with HIV-negative individuals, HIV-infected subjects had a lower body mass index, a higher prevalence of cigarette smoking, higher current plasma levels of total cholesterol and triglycerides, lower HDL cholesterol levels and a higher total cholesterol : HDL cholesterol ratio (Table 1). The proportion of subjects with hyperlipidaemia (defined as an elevation in plasma levels of total cholesterol > 6.5 mmol/l or triglycerides > 2.0 mmol/l) or mixed hyperlipidaemia (defined as a simultaneous elevation in plasma concentrations of both cholesterol and triglycerides) was higher among HIV-infected than HIV-negative individuals.

Table 1

Imaging of both femoral and carotid arteries detected the presence of one or more plaques in 93 of 168 (55.4%) HIV-infected subjects compared with 26 of 68 (38.2%) HIV-negative subjects, with this difference being significant (P = 0.02). In univariate analysis (crude odds ratio, Table 2), male gender, more advanced age, elevated plasma levels of LDL cholesterol, cigarette smoking and HIV infection were associated with the presence of plaque. In multivariate analysis (adjusted odds ratio, Table 2), the presence of plaque was independently associated with age, male gender, elevated plasma LDL cholesterol levels and cigarette smoking, whereas the association was lost with HIV infection and total cholesterol levels (P = 0.09).

Table 2

To gain insight into the contribution of PIs to the presence of peripheral atherosclerosis among HIV-infected patients, we next compared the group of 136 individuals receiving a PI-containing regimen with the group of 32 patients who did not receive any PI. The PI-treated and non-PI-treated patients were well matched for age, cigarette smoking, body mass index, mode of acquisition of HIV infection and actual number of CD4 cells (Table 3). The PI-treated subjects had a lower viraemia and a lower nadir of CD4 cell count. As expected ^[1,3,4,6], PI therapy was associated with higher plasma levels of total and LDL cholesterol and triglycerides, a higher total cholesterol : HDL cholesterol ratio and a higher proportion of subjects with hyperlipidaemia or mixed hyperlipidaemia, despite similar baseline plasma cholesterol levels. The proportion of HIV-infected patients with plaque was slightly, but not significantly, higher among PI-treated than non-PI-treated subjects (57 versus 50%, P = 0.50). Univariate logistic regression analysis revealed that, among HIV-infected participants, age and LDL cholesterol levels were significantly associated with the presence of plaque, whereas cigarette smoking tended to be associated with the presence of plaque (Table 4). In contrast, PI therapy , duration of exposure to PI therapy, plasma HDL cholesterol levels and the total cholesterol : HDL cholesterol ratio, body mass index, mode of contamination, viraemia, actual CD4 cell count, nadir CD4 cell count and pre-treatment plasma levels of total cholesterol were not associated with the presence of plaque. Multivariate logistic regression analysis indicated that the presence of plaque was independently associated with increasing age and plasma LDL cholesterol levels and smoking, but not with PI therapy. Due to possible colinearity between PI therapy and plasma LDL cholesterol levels, we also repeated the analysis without this latter variable. In this model PI therapy was not independently associated with the presence of plaque either (odds ratio, 1.4; 95% confidence interval, 0.6-3.4;P = 0.45).

Table 3

Table 4

Discussion

In this study, we used an ultrasound-assisted imaging technology to examine whether PI-containing antiretroviral regimens were associated with premature atherosclerosis in peripheral arteries. Our data reveals that the proportion of middle-aged subjects with atherosclerotic disease within femoral or carotid arteries was higher among HIV-infected than HIV-negative individuals. The elevated prevalence of premature atherosclerosis in the HIV-infected patients examined here was partly accounted for by 'classic' risk factors for cardiovascular diseases, including age, male gender, plasma LDL cholesterol levels and cigarette smoking. In contrast, HIV infection or PI therapy per se did not seem to contribute significantly and independently to the presence of peripheral atherosclerosis in this population.

The association we observed here between the presence of plaque within peripheral arteries and 'classic' risk factors for coronary artery disease is consistent with atherosclerosis being a diffuse disease, as the same factors appear to promote the development of both peripheral and coronary disease. Next, the fact that the same risk factors appeared to be associated with peripheral atherosclerosis in HIV-infected patients and with coronary artery disease in HIV-negative populations indicates that the mechanism responsible for atherogenesis is similar in both HIV-infected and HIV-negative populations. As such, our data provides indirect evidence for a higher risk of coronary artery disease among HIV-infected subjects. Conversely, HIV-infected patients may benefit as much as HIV-negative subjects from interventions aimed at reducing the progression of atherosclerosis such as cessation of cigarette smoking or administration of lipid-lowering agents.

In the present study, the presence of plaques within peripheral arteries was not independently associated with HIV infection nor with the use of PIs. As such, the data indicates that HIV infection or PI therapy only contribute minimally, if anything, to the development of atherosclerosis in this population. We cannot rule out, however, that our study may have underestimated the association between HIV infection or PI therapy and atherosclerosis. One limitation of our study relates to the relatively small HIV-negative sample size and the lack of matching for cardiovascular risk factors between HIV-negative controls and HIV-infected patients. It should be pointed out, however, that matching HIV-negative individuals and HIV-infected patients for cardiovascular risk factors is hardly feasible given the metabolic effects of PIs and the high prevalence of cigarette smoking in these patients, which render comparison with healthy volunteers inappropriate. In our study, we attempted to find an HIV-negative control group as closely matched as possible with HIV-infected patients by recruiting our HIV-negative participants among visitors to the HIV testing clinic. This approach did not suffice, however, to have two groups perfectly matched for cardiovascular risk factors. Next, the size of the non-PI-treated group was also limited, given the widespread use of PI-containing regimens. In addition, comparison of PI-treated subjects was made with patients receiving two non-nucleosidic reverse transcriptase inhibitors, not with patients on triple therapy. Moreover, mean duration of exposure to PI was limited to 26.8 ± 8.9 months, and the possibility remains open that a more prolonged exposure to PIs may be harmful. In our study, we only visualized peripheral arteries, and a detailed imaging of the coronary vessels (for instance using intravascular ultrasound imaging) would be of greater value. Finally, it is now well established that not only the size, but also the composition of atherosclerotic plaques plays a crucial role in the development of cardiovascular events ^[23].

Despite these limitations, our study is perfectly in agreement with retrospective analyses by Klein et al from Kaiser Permanente in California. In these analyses, the incidence of cardiovascular events was higher among HIV-infected subjects, compared to HIV-negative subjects, but was similar for PI-treated and non-PI-treated HIV-infected persons ^[24]. This analysis, coupled to our present observation, allows to draw some suggestions regarding the risk of heart disease in HIV-infected patients and the use of PIs in this population. First, clinicians shall be aware that HIV-infected patients may be at higher risk for cardiovascular events than HIV-negative individuals. Next, from a cardiovascular standpoint, there is no pressing need, at the present time, to stop or limit the administration of PIs, even when administered to older patients with cardiovascular risk factors such as smoking. As importantly, our study provides evidence that atherosclerosis in HIV-infected patients is partly accounted for by modifiable risk factors such as smoking and hyperlipidaemia. Interventions on these risk factors has led to a major decline in the incidence of heart disease in the general population ^[25,26]. As there is no evidence that HIV-infected individuals may benefit less from these interventions than HIV-negative populations, vigorous interventions on these factors, similar to those recommended for patients suffering from other chronic diseases such as obesity or diabetes, would probably be equally beneficial to HIV-infected subjects. Caution shall be paid, however, to the potential toxic interactions between lipid-lowering agents and antiretroviral therapy.

Acknowledgements

The authors are very grateful to the participants in this study, and wish to thank Patrick Yerly for his contribution, Vincent Lenain, Gilda Crespell and her colleagues for their excellent technical assistance, the medical and nurse staff of the HIV outpatient clinic (Médecine-2) of CHUV University Hospital and the HIV testing centre of Lausanne University Medical Policlinic for their support and interest, the Geneva HIV group and Bernard Hirschel for providing a subset of non-PI-treated HIV-infected individuals, Philippe Burgisser for storing and retrieving plasma samples for the Swiss HIV Cohort Study, Nicole Aebischer, Giuseppe Pantaleo and Gérard Waeber for helpful discussions.