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Starting HAART Reduces Endothelial Dysfunction Marker ADMA. ADMA higher levels is associated with CVD & mortality in general population.....
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"ART initiation leads to declines in ADMA levels, a marker of NO2-mediated endothelial dysfunction, with the greatest improvements seen in patients with higher levels of inflammatory and coagulation markers. ADMA levels are associated with risk for CVD and mortality in general population, but it remains unknown whether ART or other treatments that improve NO2-mediated endothelial function, as measured by ADMA, reduces clinical event risk among individuals with HIV infection.....consistent with the thinking that inflammation & coagulation may contribute to HIV-related vascular disease"
Background: HIV infection is associated with premature development of cardiovascular disease (CVD). Understanding the effects of HIV replication on endothelial dysfunction and platelet activation may identify treatment targets to reduce CVD risk.
Methods: A subgroup of HIV+ participants in the Strategies for Management of Antiretroviral Therapy (SMART) study off ART at entry enabled a randomized comparison of immediate versus deferred ART initiation of changes in asymmetric dimethylarginine (ADMA), soluble CD40L and P-selectin levels.
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HIV Replication, Inflammation, and the Effect of Starting Antiretroviral Therapy on Plasma Asymmetric Dimethylarginine, a Novel Marker of Endothelial Dysfunction
JAIDS Journal of Acquired Immune Deficiency Syndromes: POST ACCEPTANCE, 14 March 2012
"Endothelial dysfunction, assessed either by FMD or ADMA levels, has been shown to be impaired among HIV-infected compared to uninfected persons.....In this randomized comparison of immediate versus delayed initiation of ART, we demonstrated improvements in a novel biomarker of NO-mediated endothelial dysfunction (associated with CVD), ADMA.....In the immediate ART (VS) group, those randomized to start ART immediately had a -10.3% (95% CI: -16.3 to -3.7; p=0.003) greater decline in ADMA levels at month 12......Plasma levels of ADMA, sCD40L and P-selectin have been associated with risk for CVD in the general population (12-14), and represent inter-related factors that contribute to atherogenesis.....Among participants in SMART with viral suppression, stopping ART led to significant declines in serum platelet counts that correlated inversely with the rise in HIV RNA and D-dimer levels......ADMA levels were independently associated with risk for all-cause mortality (HR 1.21 per SD higher ADMA level) in the Framingham Offspring Study, and with risk for CVD events (HR 1.29 per SD higher ADMA level) in the Population Study of Women in Gothenburg (23, 24).......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 (e.g., renal), the potential net clinical benefit associated with declines in AMDA 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.......ADMA is an endogenous inhibitor of NO synthase that is inversely associated with flow mediated enthothelium-dependent vasodilation in healthy people (18), and can be used as a biomarker of vascular dysfunction.....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 percent 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).....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.....Compared to those randomized to deferral of ART (n=114), participants randomized to immediate ART (n=134) had 10.3% lower ADMA levels (p=0.003) at 12 months......Plasma levels of ADMA, sCD40L and P-selectin have been associated with risk for CVD in the general population (12-14), and represent inter-related factors that contribute to atherogenesis.....Biomarkers of endothelial activation (e.g., 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....We hypothesized that these biomarkers would be associated with HIV replication prior to 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."
Baker, Jason V.; Neuhaus, Jacqueline; Duprez, Daniel; Freiberg, Matthew; Bernardino, Jose I.; Badley, Andrew D.; Nixon, Daniel E.; Lundgren, Jens D.; Tracy, Russell P.; Neaton, James D.; for the INSIGHT SMART Study Group
1University of Minnesota, Minneapolis, MN, USA
2Hennepin County Medical Center, Minneapolis, MN, USA
3University of Pittsburgh, Pittsburgh, PA, USA
4Hospital La Paz, IdiPAZ, Madrid, Spain
5Mayo Clinic, Rochester, MN, USA
6Virginia Commonwealth University, Richmond, Virginia, USA
7University of Copenhagen & Copenhagen University Hospital, Copenhagen, Denmark
8University of Vermont, Burlington, VT, USA
Abstract
Background: HIV infection is associated with premature development of cardiovascular disease (CVD). Understanding the effects of HIV replication on endothelial dysfunction and platelet activation may identify treatment targets to reduce CVD risk.
Methods: A subgroup of HIV-infected participants in the Strategies for Management of Antiretroviral Therapy (SMART) study off antiretroviral therapy (ART) at entry enabled a randomized comparison of immediate versus deferred ART initiation of changes in asymmetric dimethylarginine (ADMA), soluble CD40L and P-selectin levels.
Results: At study entry, median (IQR) levels of ADMA, sCD40L, and P-selectin were 0.57 (0.49-0.66) [mu]g/mL, 251 (135-696) [mu]mol/L, and 34 (28-44) pg/mL. Compared to those randomized to deferral of ART (n=114), participants randomized to immediate ART (n=134) had 10.3% lower ADMA levels (p=0.003) at 12 months; treatment differences in sCD40L (95% CI: -17 to 44%; p=0.53) and P-selectin (95% CI: -10 to 10%; p=0.95) were not significant. The difference in ADMA for those assigned immediate ART compared to those assigned ART deferral was greater among younger patients and those with higher levels of hsCRP and D-dimer (p<=0.05 for interaction for both), but not HIV RNA level at baseline (p=0.51).
Discussion: ART initiation leads to declines in ADMA levels, a marker of nitric-oxide-mediated endothelial dysfunction. Improvement in ADMA levels was related to the degree of inflammation and coagulation, suggesting that up-regulation of these pathways contributes to premature vascular disease among individuals with HIV infection. Whether declines in ADMA levels impact risk of disease requires further research.
Introduction
Findings from the Strategies for Management of AntiRetroviral Therapy (SMART) trial identified key markers of inflammation (e.g. 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 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). Anti-thrombotic 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 (e.g., 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 dysfunction (12); soluble CD40 ligand (sCD40L) is expressed by lymphocytes, the vascular wall, and activated platelets (13); and Pselectin 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 population (12-14), and represent inter-related factors that contribute to atherogenesis (15).
We hypothesized that these biomarkers would be associated with HIV replication prior to 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.
DISCUSSION
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 AMDA 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 up-regulation 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 people (18), and can be used as a biomarker of vascular dysfunction. Reference ranges for ADMA have been obtained using Framingham participants that 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 gender) 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 percent 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), while our finding that women had higher ADMA levels than men has not been consistently reported (19-22). However, one study of 500 participants found 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 gender 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 (HR 1.21 per SD higher ADMA level) in the Framingham Offspring Study, and with risk for CVD events (HR 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 (e.g., renal), the potential net clinical benefit associated with declines in AMDA 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 (FMD), 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 biological relevance of these findings by describing ART-related improvements in ADMA levels that varies by the degree of inflammation and coagulation activity present prior to starting HIV treatment.
Endothelial dysfunction, assessed either by FMD or ADMA levels, has been shown to be impaired among HIV-infected compared to uninfected persons (26, 27). ADMA levels were associated with levels of the macrophage inflammatory marker neopterin, but not CRP, 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 (81-1300mg) (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 that 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 a greater burden of vascular disease among older persons may, to some extent, limit the capacity for improvement in endothelial dysfunction (e.g., 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 viral suppression, 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 anti-viral response and CD40L expression may be down-regulated 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 (i.e., 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.
Results
Study Sample and Baseline Associations
When compared with the participants in the SMART no ART subgroup (n=477) not included in this report (n=229), the cohort analyzed in this report (n=248) had a lower median age (40 vs. 42 years), had a higher prevalence of persons previously naïve to ART (57 vs. 48%), and were less likely to be black (30 vs. 43%), be co-infected with hepatitis B or C (9 vs. 28%), or be prescribed BP lowering medication (11 vs. 21%).
Among the participants in this report, 2 of participants deferring ART (DC) and 1 of those randomized to start ART (VS) had a CVD event (i.e., 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/mm3, 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 (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). Soluble CD40L 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, r=-0.02 for P-selectin) or CD4+ counts (r=0.06 for ADMA, r=0.02 for sCD40L, 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 to 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 eGFR (r=-0.17; p=0.007). Soluble CD40L levels were also inversely correlated with eGFR (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, gender 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 Deferred (DC) ART
One hundred and thirty-two of 134 participants in the VS group initiated ART following randomization, and at 12 months 116 (88%) remained on ART. Most participants (56%) initiated ART with an NNRTI (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 followup was toxicity (44%). Thirty-nine of 114 deferred ART participants (DC group) started ART at some point prior to the month 12 visit; 1 (3%) due to disease progression and 15 (38%) due to a CD4+ count <250 cells/mm3 (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/mL (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/mm3 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 are presented in Figure 1. In the immediate ART (VS) group, those randomized to start ART immediately had a -10.3% (95% 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 (-0.15-0.13) for the immediate ART (VS) arm (p = 0.12) and 0.02 μmol/L (-0.12-0.18) for the deferred ART (DC) arm (p = 0.17). Soluble CD40L 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 (figure 1).
When VS participants that did not initiate ART (n=2) and DC participants that 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 (i.e., 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 following 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/mL 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/mL at 12 months and 6% for participants with viral load ≥400 copies/mL at 12 months.
Subgroup Findings
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 (Figure 2). Significant treatment x 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.
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