Findings from the Strategies for Management of AntiRetroviral Therapy (SMART) trial identified key markers of inflammation [eg, high-sensitivity C-reactive protein (hsCRP) and interleukin 6 (IL-6) and coagulation (D-dimer)] that are elevated with HIV infection and associated with risk for cardiovascular disease (CVD) and all-cause mortality.1,2 Furthermore, in SMART, D-dimer changes were correlated with changes in HIV RNA levels resulting from starting or stopping antiretroviral therapy (ART).2,3 Antithrombotic properties of endothelial surfaces are integral to coagulation homeostasis and may be influenced by HIV replication itself and/or exposure to inflammatory cytokines.4–7 Biomarkers of endothelial activation (eg, intercellular and vascular cell adhesion molecules) are consistently elevated in HIV-infected versus uninfected individuals and have been shown to be associated with HIV replication, inflammation, and subclinical atherosclerosis among HIV-infected patients.6–11 The goals of this study were to build on this literature and explore the effect of HIV-related inflammation, resulting from untreated HIV, on nitric oxide (NO)–mediated endothelial dysfunction and endothelial platelet activation.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the NO synthase pathway that reflects endothelial dysfunction12; soluble CD40 ligand (sCD40L) is expressed by lymphocytes, the vascular wall, and activated platelets13; and P-selectin is expressed by both endothelial cells and platelets.14 Plasma levels of ADMA, sCD40L, and P-selectin have been associated with risk for CVD in the general population12–14 and represent interrelated factors that contribute to atherogenesis.15
We hypothesized that these biomarkers would be associated with HIV replication before starting ART and would improve after starting ART. Therefore, using stored samples from the SMART study, we measured ADMA, sCD40L, and P-selectin levels at baseline and 12 months for a subgroup of SMART participants who were not taking ART at study entry that represented a randomized comparison of immediate versus deferred ART.
MATERIALS AND METHODS
The methods and results of the SMART trial, including the subgroup not taking ART at entry, have been reported.3,16,17
Of the 5472 randomized participants in SMART, 477 had never taken ART or had not used ART for at least 6 months before randomization (referred to as the subgroup “off ART”). To reduce the likelihood of recent ART exposure, participants with low HIV RNA levels (<10,000 copies/mL) during the 6 months before randomization were not included in the subgroup off ART. For these participants, the randomized intervention in SMART of a drug conservation (DC) or viral suppression (VS) strategy represented a comparison of immediate versus deferred (until CD4 counts declined to 250 cells/mm3) initiation of ART, respectively.17 Among the 477 participants in the subgroup off ART, 248 participants had specimens from both baseline and month 12 visits available for analysis and, thus, form the basis of this report. A single follow-up time point of 12 months was chosen based on specimen availability and with consideration of time to achieve VS. Among the 229 SMART participants off ART at entry who were excluded, 30 did not consent to have specimens stored, 10 were lost to follow-up, 25 missed the month 12 visit, and 164 did not have sufficient specimen remaining for use at either baseline or month 12 visit.
The SMART study protocol was approved by the institutional review board or ethics committee at each clinical site and at the University of Minnesota, which served as the Statistical and Data Management Center. The institutional review board at the University of Minnesota also approved plans for analysis of stored specimens for consenting participants.
CD4+ cell count and HIV RNA levels were measured at clinical sites. For consenting participants in SMART, plasma specimens were collected using EDTA tubes, processed within 4 hours of collection, frozen at −70°C, and shipped to a central repository where they continued to be stored at −70°C. Samples were not required to be fasting specimens.
Plasma levels of hsCRP and IL-6 were measured from baseline specimens, and levels of D-dimer ADMA, sCD40L, and P-selectin were measured from baseline and month 12 specimens, by the Laboratory for Clinical Biochemistry Research at the University of Vermont. The analytic methods and coefficient of variance for measuring hsCRP, IL-6, and D-dimer levels from SMART specimens have been previously reported.2,3 ADMA levels were measured with a competitive enzyme-linked immunosorbent assay method (Euroimmun, Lübeck, Germany) and sCD40L and P-selectin levels with quantitative sandwich immunoassays (R&D Systems, MN). The lower level of detection for ADMA, sCD40L, and P-selectin were 0.1 μmol/L, 62.5 pg/mL, and 20.2 μg/mL, respectively. The interassay coefficient of variance using these methods was 12.7%–15.6% for ADMA, 6.7%–10.5% for sCD40L, and 6.0%–8.1% for P-selectin, respectively. All samples were analyzed blinded to treatment group.
Unless otherwise stated, comparisons between the VS and DC groups are intent to treat (all randomized participants in the study sample are included in the analysis). Analyses were also carried out in which 2 VS participants who did not initiate ART and 39 DC participants who initiated ART before 12 months are excluded. Simple descriptive statistics are used for baseline cross sectional analyses. Biomarker values were loge transformed before analysis. Analysis of covariance with the baseline level of the biomarker included as a covariate was used to compare treatment groups (VS-DC) for changes in biomarker levels at 12 months. Estimates of the percentage difference between treatment groups at the 12-month visit were estimated by exponentiating the loge-transformed treatment differences. Analysis of covariance models that included interaction terms between treatment group and baseline variables were used for subgroup analyses. Multiple regression analysis was used to study baseline associations and predictors of change after 12 months for VS participants. The following predictors were included: baseline biomarker level of interest; age; sex; race; hepatitis (B or C) coinfection; smoking; blood pressure–lowering medication; lipid-lowering medication; body mass index; diabetes; CD4+ cell count; HIV RNA level (log10 transformed); naive to all ARTs (or not); prior AIDS; and hsCRP, IL-6, and D-dimer levels. Analyses were performed using SAS (version 9.1). All reported P values are 2 sided. A significance level of <0.05 was used for treatment comparisons.
Study Sample and Baseline Associations
When compared with the participants in the SMART, no ART subgroup (n = 477) was included in this report (n = 229), the cohort analyzed in this report (n = 248) had a lower median age (40 versus 42 years), had a higher prevalence of persons previously naive to ART (57% versus 48%), and were less likely to be black (30% versus 43%), be coinfected with hepatitis B or C (9% versus 28%) or be prescribed blood pressure–lowering medication (11% versus 21%). Among the participants in this report, 2 participants deferring ART (DC) and 1 of those randomized to start ART (VS) had a CVD event (ie, myocardial infarction, stroke, coronary artery disease requiring surgery or CVD death) during follow-up.
Baseline characteristics of the DC and VS groups were similar and are given in Table 1. Median CD4+ count was 433 cells per cubic millimeter, 56% of participants had no prior ART exposure, and the percent of patients with a prior AIDS and CVD event were low at 8.5% and 2.8%, respectively. The use of blood pressure and lipid-lowering medication at study entry was 10.9% and 4.4%, respectively.
Median [interquartile range (IQR)] levels of ADMA, sCD40L, and P-selectin were 0.57 (0.49–0.66) μmol/L, 251 (135–696) pg/mL, and 34 (28–44) μg/mL, respectively. ADMA levels, but not sCD40L or P-selectin, were weakly correlated with levels of IL-6 (r = 0.16; P = 0.02) and D-dimer (r = 0.16; P = 0.01). sCD40L and P-selectin levels were significantly correlated with each other (r = 0.63; P < 0.001) but not with ADMA levels (r = 0.02 and r = 0.06, respectively). These 3 biomarkers were not correlated with HIV RNA levels (r = 0.08 for ADMA, r = 0.00 for sCD40L, and r = –0.02 for P-selectin) or CD4+ counts (r = 0.06 for ADMA, r = 0.02 for sCD40L, and r = 0.08 for P-selectin) at baseline (P > 0.20 for all comparisons).
In univariate associations with traditional CVD risk factors, ADMA levels were 6% higher among current smokers (P = 0.05), 7% lower among males (P = 0.04), and 11% lower among those of black race compared with other ethnic groups, which were similar to one another (P < 0.001); the correlation with age did not reach significance (r = 0.10; P = 0.11). At baseline, P-selectin levels were 14% higher for males (P = 0.02) and were positively correlated with age (r = 0.14; P = 0.03) and inversely correlated with estimated glomerular filtration rate (r = –0.17; P = 0.007). sCD40L levels were also inversely correlated with estimated glomerular filtration rate (r = –0.14; P = 0.03) but with none of the other traditional CVD risk factors. In multivariate regression analyses, associations between biomarker levels with age, sex, and race/ethnicity were similar. Finally, serum platelet counts were available for n = 112 participants (n = 55 from DC and n = 57 from VS group) and were significantly correlated with levels of sCD40L (r = 0.32; P < 0.001) but not P-selectin (r = 0.17; P = 0.07).
Biomarker Changes After 12 Months: Immediate (VS) Versus DC ART
One hundred thirty-two of 134 participants in the VS group initiated ART after randomization, and at 12 months, 116 (88%) remained on ART. Most participants (56%) initiated ART with an nonnucleoside reverse transcriptase inhibitor (efavirenz in 84% of these patients) plus combination nucleoside treatment. The most frequent nucleoside reverse transcriptase inhibitor combination used was zidovudine plus lamivudine. The most common reason given for the 12% of the immediate ART participants (VS group) who stopped ART during follow-up was toxicity (44%). Thirty-nine of 114 deferred ART participants (DC group) started ART at some point before the month 12 visit; 1 (3%) due to disease progression; and 15 (38%) due to a CD4+ count <250 cells per cubic millimeter (the protocol deferral strategy). At 12 months, 25 (23%) participants in the deferred ART group (SMART DC group) and 85 (66%) participants in the immediate ART group (SMART VS group) had an HIV RNA level <400 copies per milliliter (P < 0.001). CD4+ count declines in the DC group and increases in the VS group resulted in a difference between groups of 173 cells per cubic millimeter at 12 months (P < 0.001).
Median biomarker levels over follow-up by treatment group are presented in Table 2, and the difference in biomarker level between groups (VS-DC) at 12 months for intention to treat comparisons is presented in Figure 1. In the immediate ART (VS) group, those randomized to start ART immediately had a −10.3% [95% confidence interval (CI): −16.3 to −3.7; P = 0.003] greater decline in ADMA levels at month 12. The median changes (IQR) in ADMA levels from baseline-to-month 12 were −0.02 μmol/L (−0.15 to 0.13) for the immediate ART (VS) arm (P = 0.12) and 0.02 μmol/L (−0.12 to 0.18) for the deferred ART (DC) arm (P = 0.17). sCD40L and P-selectin did not differ between VS and DC groups at 12 months, and neither marker changed significantly from baseline-to-month 12 when VS and DC groups were examined separately. D-dimer levels declined significantly among VS (P < 0.001) participants, but the differences by treatment groups at 12 months did not reach significance in the randomized intention-to-treat comparison (Fig. 1).
When VS participants who did not initiate ART (n = 2) and DC participants who initiated ART before 6 months (n = 39) are excluded, the corresponding on-treatment comparison for the effect of starting ART on ADMA levels at month 12 was similar (−12.8%; 95% CI: −19.3 to −5.8; P < 0.001) and became more pronounced for D-dimer (−21.4%; 95% CI: −35.8 to −3.8; P = 0.02). When participants with known CVD (n = 7) were excluded, the corresponding difference for ADMA was −11.4% (−17.4 to −5.0; P < 0.001).
Predictors of Change in ADMA Levels After Starting ART in the VS Group
Baseline predictors of change in ADMA levels at 12 months for VS participants were also explored. In univariate models, smaller improvements (ie, less decline) in ADMA levels were seen for those of older age (6% less decline per 10 years older; P = 0.04), whereas greater declines were seen for those with higher baseline hsCRP (6% greater decline per log-e unit higher; P = 0.003), IL-6 (7%; P = 0.06), and D-dimer (6%; P = 0.04) levels. Baseline CD4+ count (P = 0.36) and HIV RNA level (P = 0.64) did not predict changes in ADMA levels. In mulitivariate models, only older age (P < 0.001) and higher hsCRP levels (P = 0.02) at baseline remained independently associated with the degree of change in ADMA levels after ART initiation for the VS group. None of the other HIV-related or traditional CVD risk factors examined at baseline predicted change in ADMA levels in the VS group.
We compared biomarker declines for those who achieved an HIV viral load <400 copies per milliliter at 12 months versus those who did not, and no differences were observed except for D-dimer (P < 0.001). Among the VS participants, the percentage decline in ADMA was 3% for those with HIV viral load <400 copies per milliliter at 12 months and 6% for participants with viral load ≥400 copies per milliliter at 12 months.
To further explore the above findings on the influence of age, inflammation, coagulation, and HIV replication on ART-related improvements in ADMA, we examined treatment differences in ADMA according to these baseline factors (Fig. 2). Significant treatment × baseline subgroup interactions were found for age, hsCRP, and D-dimer but not HIV RNA or IL-6. For younger participants and those with higher levels of hsCRP or D-dimer at entry, the effects of immediate versus deferred ART treatment on ADMA levels are greater.
In this randomized comparison of immediate versus delayed initiation of ART, we demonstrated improvements in a novel biomarker of NO-mediated endothelial dysfunction, ADMA, but not in plasma markers of endothelial platelet activation (sCD40L and P-selectin). Improvements in ADMA were greater in younger patients and in patients with higher levels of baseline hsCRP and D-dimer. These findings expand our understanding of HIV-related endothelial injury and dysfunction and suggest that upregulation of inflammatory and coagulation pathways contribute to premature vascular dysfunction and disease among individuals with HIV infection.
ADMA is an endogenous inhibitor of NO synthase that is inversely associated with flow-mediated enthothelium-dependent vasodilation in healthy people18 and can be used as a biomarker of vascular dysfunction. Reference ranges for ADMA have been obtained using Framingham participants who were free of clinical CVD, hypertension, diabetes or obesity, and did not smoke.19 Median (IQR) ADMA levels were 0.51 (0.45–0.59) μmol/L (results similar by sex), and the 97.5 percentile was 0.732 μmol/L.19 Median (IQR) ADMA levels at baseline in our study were 0.57 (0.49–0.66) μmol/L; 11.3% had levels >0.732 μmol/L. We describe modest improvements in ADMA levels after 12 months of ART, and the percentage of patients with ADMA >0.732 μmol/L was 25.4% for the deferred group (DC) and 16.4% for the immediate ART group (VS). Our finding that ADMA levels were lower for black participants is consistent with reports from general population studies,20,21 whereas our finding that women had higher ADMA levels than men has not been consistently reported.19–22 However, one study of 500 participants found that women had lower ADMA levels than men at ages <50 years but higher ADMA levels at ages >50 years.22 Whether HIV infection has an effect similar to advancing age on sex differences in ADMA levels is not clear and should be explored in larger epidemiologic studies.
ADMA levels were independently associated with risk for all-cause mortality (hazard ratio 1.21 per SD higher ADMA level) in the Framingham Offspring Study and with risk for CVD events (hazard ratio 1.29 per SD higher ADMA level) in the Population Study of Women in Gothenburg.23,24 Although SMART participants differ from those in Framingham and the Population Study of Women both by HIV-specific and traditional CVD risk factors, the degree of ART-related change in ADMA we observed corresponds to approximately a 5% lower risk of death and 7% lower risk of CVD, respectively, in these studies.23,24 If morbidity from other non–AIDS-related end organ diseases were considered (eg, renal), then the potential net clinical benefit associated with declines in ADMA levels of this degree could be greater. Currently, these assertions require validation in larger clinical event studies where the longer-term consequences of ART exposure are also assessed.
Brachial artery flow-mediated dilation, a functional measure of NO-mediated endothelial responsiveness, was shown to improve with ART initiation in a nonrandomized comparison conducted by the AIDS Clinical Trial Group.25 Our randomized comparison expands on the biologic relevance of these findings by describing ART-related improvements in ADMA levels that varies by the degree of inflammation and coagulation activity present before starting HIV treatment. Endothelial dysfunction, assessed either by flow-mediated dilation or ADMA levels, has been shown to be impaired among HIV-infected compared with HIV-uninfected persons.26,27 ADMA levels were associated with levels of the macrophage inflammatory marker neopterin, but not C-reactive protein, in a study of 112 HIV-infected participants.28 In a proof of concept study of 70 HIV-negative patients with the metabolic syndrome, aspirin therapy led to declines in ADMA levels across a wide range of doses (81–1300 mg).29 Thus, it may be possible to modify biomarkers such as ADMA with simple adjunctive therapies. Whether any resulting changes influence risk of disease will require larger trials.
The mechanisms underlying the interaction between ART-related improvements in ADMA levels and advancing age described in our subgroup analyses are not entirely clear. At baseline, the positive correlation between ADMA levels and advancing age did not reach significance. Our study had limited power to detect modest associations, and the correlation between ADMA levels and advancing age was of similar magnitude between our study (r = 0.10; P = 0.11) and a subset of 1126 Framingham participants who were free of clinical CVD, hypertension, diabetes, or obesity and did not smoke (r = 0.14; P < 0.001).19 We describe smaller ART-related declines in ADMA levels with advancing age that suggests that a greater burden of vascular disease among older persons may, to some extent, limit the capacity for improvement in endothelial dysfunction (eg, ADMA levels). Understanding the interaction between HIV infection, advancing age, and vascular disease should be a focus of future research.
Several studies have demonstrated greater platelet activation or higher levels of platelet microparticles among HIV-infected versus uninfected participants.30–32 Among participants in SMART with VS, stopping ART led to significant declines in serum platelet counts that correlated inversely with the rise in HIV RNA and D-dimer levels.33 In our study, sCD40L and P-selectin levels at baseline were tightly correlated with one another and inversely with platelet counts, but neither marker changed significantly after starting ART. Prior data are inconsistent with respect to changes in P-selectin levels after starting or stopping ART,7,34 and sCD40L has not been extensively studied in this context. In normal physiology, platelets account for the main source of circulating sCD40L and P-selectin levels.14,35–37 However, in the context of HIV infection, the CD40 receptor–ligand system is also integral to the host antiviral response and CD40L expression may be downregulated directly by HIV proteins.38 Thus, interpreting these markers may be difficult in the context of suppressing HIV replication due to competing forces, including the potential contribution of ART-related toxicity.39 Cumulatively, these data support the need for additional research on platelet activation and platelet function among HIV-infected patients.
Strengths of this study include use of a randomized design to estimate ART-related changes in endothelial biomarkers. A limitation is that specific ART regimens were chosen by patients and providers (ie, not randomized in SMART) and several different regimens were used, limiting any comparisons of specific antiretroviral combinations. The lack of platelet-free plasma or cell-based specimens in SMART also prevented a more comprehensive assessment of platelet activation and function. Finally, the sample size may lack power to detect more modest associations between ADMA levels and traditional CVD risk factors.
In summary, ART reduces ADMA levels. Reductions with ART are greater for those with higher levels of inflammatory and coagulation markers at entry. Futures studies are warranted to determine whether treatments that improve NO-mediated endothelial function, as measured by ADMA, leads to a reduced risk for morbidity and mortality among individuals with HIV infection.
The authors would like to sincerely thank the SMART study participants, the SMART study team (see N Engl J Med. 2006;355:2294–2295 for list of investigators), and the INSIGHT Executive Committee.
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Keywords:© 2012 Lippincott Williams & Wilkins, Inc.
HIV infection; antiretroviral therapy; inflammation; endothelial dysfunction; asymmetric dimethylarginine (ADMA); CD40 ligand; P-selectin