In addition to their critical role in osteoclastogenesis and bone metabolism, receptor activator of nuclear factor-κB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), play important roles in the immune system and vascular biology. RANKL is secreted and expressed on activated T cells1 and endothelial cells,2 and OPG is secreted by dendritic cells and smooth muscle and endothelial cells.3 RANKL and OPG are present in calcified human aortic4 and carotid plaque,5 and dysregulation of the RANKL/OPG axis has been postulated as a potential mediator of vascular calcification.
Recently, there has been significant scientific interest in the relationship between the RANKL/OPG cytokine network and coronary artery disease (CAD). Observational studies in patients with type 2 diabetes and in the general population have shown an association between increased serum OPG levels and severity and progression of coronary artery calcium,6,7 endothelial dysfunction,8,9 and vascular events.6,10 The relationship between RANKL and CAD is less clear with some studies suggesting lower soluble receptor activator of nuclear factor-κB ligand (sRANKL) corresponding with CAD,11,12 whereas another study reported higher sRANKL to be predictive of acute cardiovascular events but not with atherosclerosis as measured by femoral or carotid ultrasound, suggesting that RANKL may be involved in plaque destabilization and rupture.13
HIV infection is associated with an increased prevalence of CAD.14,15 The chronic inflammatory state, direct effects of antiretroviral medications and direct effects of the virus in the endothelium, are thought to contribute to this increased risk of CAD in HIV-infected patients. In work previously described by our group, HIV-infected individuals had a higher prevalence of coronary atherosclerosis and a greater number of coronary segments with plaque compared with HIV-negative controls. Furthermore, a greater proportion of HIV-infected individuals has presence of calcified plaque, as quantified by the Agatston calcium score greater than zero, compared with HIV-negative controls.14
To date, no study has directly explored the relationship between RANKL, OPG, and coronary atherosclerosis in HIV-infected individuals. Studies have shown that HIV infection itself alters RANKL and OPG expression in vitro.16,17 In observational studies investigating the RANKL, OPG, and bone mineral density in HIV-infected individuals, antiretroviral naive HIV-infected individuals had higher levels of RANKL and OPG compared with controls18 and treatment with antiretrovirals decreased serum RANKL and OPG levels.19
In this study, we sought to evaluate the relationship between RANKL and OPG and coronary atherosclerotic indices in HIV-infected patients and controls.
One hundred and ten men were prospectively recruited as previously described.14 Data on sRANKL and OPG have not been previously reported. Seventy-eight HIV-infected men and 32 HIV-negative control men between ages 18–55, without known current or prior cardiac disease or symptoms suggestive of cardiac disease, were studied. The control group was selected to be similar to the HIV group with regards to traditional cardiovascular risk factors. Those HIV-infected subjects on antiretroviral therapy (ART) were required to be on stable therapy for greater than 3 months. Subjects with renal disease or contraindications to administration of contrast agent, beta-blockade, or nitroglycerin, were excluded. All participants provided written informed consent to participate. This study was approved by the institutional review boards of Massachusetts General Hospital and Massachusetts Institute of Technology.
All participants underwent a detailed interview and physical examination by a single investigator. All participants fasted ≥12 hours before blood draws. The presence and characterization of any atherosclerotic plaque was obtained with computed tomography (CT) and coronary CT angiography (CTA) imaging with a 64-slice CT scanner (Sensation 64; Siemens Medical Solutions, Forcheim, Germany). Two cardiac imaging specialists who were blinded to subjects’ HIV status or clinical history analyzed all the scans. The number of coronary segments with evidence of plaque present and plaque volume were assessed as previously described.14 The quantification of atherosclerotic plaque was measured by counting the number of coronary segments with evidence of plaque present (using a modified 17-segment model of the coronary artery tree).20 We also further classified these segments with plaque as having calcified, noncalcified, or mixed plaque.
CD4+ and CD8+ T-cell counts, HIV viral load, and HIV testing were performed as previously described.14 Soluble RANKL was measured via enzyme-linked immunosorbent assay (Biovendor, Candler, NC). OPG was measured via enzyme-linked immunosorbent assay (Kamiya Biomedical, Seattle, WA).
Distributions of sRANKL, OPG, and measures of plaque burden including number of segments with plaque, plaque volume, and Agatston calcium score were non-normal so, therefore, the groups were compared using the Wilcoxon rank sum test for these variables and the Spearman correlation coefficient was used to assess correlations. Multivariate linear regression modeling was performed to adjust for known cardiovascular risk factors and biologically plausible covariates. For all models, Agatston calcium score was log-transformed. Two-tailed probability values are reported and statistical significance was assumed when P < 0.05. All statistical analyses were performed using JMP (SAS Institute Inc).
Seventy-eight HIV-infected men (age 46.5 ± 6.5 years) and 32 healthy controls (age 45.4 ± 7.2 years) with similar traditional cardiovascular risk factors and without any known current or prior cardiovascular disease participated in the study as previously described.14 Amongst those individuals with HIV infection, the duration of HIV diagnosis was 13.5 ± 6.1 years. About 95% of these patients were receiving ART and 53% (n = 41) were on a regimen that included protease inhibitors (PIs). The average duration on ART was 7.1 ± 4.6 years and the average duration on a PI was 3.8 ± 4.2 years. The average CD4 T-lymphocyte count was 523 ± 282 cells per cubic millimeter, and 81% of HIV-infected individuals had undetectable HIV viral load (Table 1). To provide context, traditional cardiovascular risk factors, and detailed measures of CT angiography are reported in Supplemental Table 1, consistent with previous data of CTA results in these patients (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A347). Traditional cardiovascular risks factors such as age, body mass index, hemoglobin A1c, total cholesterol, LDL and HDL levels were similar between the groups (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A347).
RANKL and OPG Levels in HIV-Infected Individuals and Controls
sRANKL was lower in HIV-infected individuals compared with controls [2.52 (1.08–3.98) vs. 3.33 (2.44–4.64), P = 0.01, median (IQR), respectively] (Table 1). There were no significant differences in levels of OPG (P = 0.11) or the sRANKL/OPG ratio (P = 0.65) between the 2 groups.
Amongst HIV-infected individuals, sRANKL was positively associated with CD4 counts (Spearman ρ = 0.29, P < 0.01) and negatively associated with duration since HIV diagnosis (ρ = −0.32, P < 0.005). Exclusion of the 4 HIV-positive ART-naive subjects from analysis had no significant impact on the results. Those HIV-infected individuals on PIs (n = 41) had lower sRANKL levels compared with those on regimens without PIs (n = 37) [1.92 (0.92–3.45) vs. 3.20 (1.72–4.77) pg/mL, P = 0.02]. Those HIV-infected individuals on non–PI-based regimens had sRANKL levels that were similar to healthy controls [3.20 (1.72–4.77) vs. 3.33 (2.44–4.64) pg/mL, P = 0.38].
Associations Between sRANKL and Cardiac CT and CT Angiography Measurements
sRANKL negatively correlated with measurements of plaque burden in the HIV-infected individuals (Table 2). In particular, sRANKL was observed to have a strong negative correlation with the number of coronary segments with plaque present (ρ = −0.41, P < 0.001), the Agatston calcium score (ρ = −0.30, P < 0.01), the number of segments with mixed calcified and noncalcified plaque (ρ = −0.31, P = 0.007), and the number of segments with calcified plaque (ρ = −0.26, P = 0.03). These relationships were not observed in healthy controls. In contrast, a marker of generalized inflammation such as C-reactive protein had no relationship with sRANKL (ρ = −0.07, P = 0.46) or any measurements of plaque burden amongst HIV-infected and non–HIV-infected individuals. Importantly, the relationship between sRANKL and coronary calcium score remained significant even after controlling for traditional cardiovascular risk factors as estimated by the 10-year Framingham Risk (P = 0.03), which has been previously shown to relate highly to coronary artery calcium in HIV.14 The relationship between sRANKL and total number of segments with plaque also remained significant after adjusting for Framingham risk (P = 0.04). Furthermore, the relationship between sRANKL and calcium score and sRANKL and total number of plaque segments remained significant after controlling for other factors associated with HIV infection including ART use, duration of ART use, and CD4 T-lymphocyte count (P = 0.02 and P = 0.008, respectively).
The relationship between sRANKL and calcium score and sRANKL and total number of plaque segments also remained significant after controlling for PI use (P = 0.02 and P = 0.01, respectively). Furthermore, in sensitivity analyses examining the subgroup of HIV-infected patients not on PI therapy (n = 37), sRANKL remained significantly inversely associated with total number of segments with plaque (ρ = −0.51, P = 0.002) and with calcium score (ρ = −0.35, P = 0.03).
Lower OPG levels were seen in individuals with plaque compared with no plaque [6.3 (3.1–18.1) vs. 11.2 (4.5–42.8) ng/mL, P < 0.05]. However, there was no significant correlation between OPG and coronary calcium scores.
To our knowledge, this is the first study to demonstrate a significant relationship between RANKL and coronary artery calcification and plaque in HIV-infected individuals. We show that amongst HIV-infected individuals, lower sRANKL levels correlate with increased calcified and noncalcified coronary plaque. Moreover, the relationship between sRANKL and plaque indices remained significant even after controlling for traditional cardiovascular risk factors. Those patients with lower CD4 T-lymphocyte counts, longer duration of disease, and PI use had lower sRANKL levels. These data suggest that RANKL may play a role in the development of CAD in HIV-infected patients. One possible explanation is that HIV infection is a unique state of chronic inflammation with dysregulation of T-lymphocyte cells, which are an important source of RANKL expression. In this regard, it is notable that RANKL was related to CD4 count among the HIV-infected patients in our study. Further studies are needed to investigate the contribution of T-cell dysfunction to RANKL activation in HIV-infected patients.
We show that PI use is associated with lower sRANKL levels, and therefore our findings are consistent with a study exploring the effects of ART initiation on bone markers which found that sRANKL levels decreased after initiation of ART.19 In vitro studies have shown that different PIs exert different dose dependent effects on RANKL signaling.16,21 Interestingly, the relationship between coronary artery calcium and sRANKL remained significant even after controlling for duration of ART and PI and other ART use in our study. In addition, the relationship between sRANKL and coronary calcification indices remained significant in a sensitivity analysis amongst those not receiving a PI, suggesting that the relationship is mediated by other factors in addition to PI use.
There has been some evidence to suggest that serum levels of RANKL may be very different from local tissue expression and activity. Interestingly, in human atherosclerotic plaques, RANKL mRNA levels have been found to be higher than in normal vessels22; however, many observational studies report an inverse relationship between serum RANKL levels and measures of CAD.11,12 One explanation for this finding is that with increased local RANKL activity there is less release of cell surface RANKL to soluble RANKL. In fact, Findlay et al23 reported that circulating RANKL levels were inversely associated with local bone RANKL mRNA levels. In HIV infection, in vitro data suggests that RANKL activity is increased at the tissue level possibly via HIV infection itself16,17 or medication effects.16 This raises an intriguing possibility that these factors increase local RANKL activity, and thus mediate the vascular calcification processes seen in HIV-infected patients. Our findings highlight the need for further studies to evaluate local RANKL activity within the arterial wall or within the calcified and noncalcified plaques of HIV-infected individuals.
Prior studies examining OPG levels in the general population showed associations between OPG levels and measures of CAD. In contrast, although our data showed a significant relationship between lower OPG levels and presence of plaque, we found no consistent relationships between OPG and coronary calcification.
We took advantage of a prior study with data available from over 100 well-phenotyped HIV patients and matched control subjects undergoing CTA to look for the first time at the relationship of RANKL, detailed measures of coronary atherosclerosis and T-cell indices in HIV patients and to compare these results to a well-matched non–HIV-infected group. Limitations of this study include its cross-sectional design. In addition, our study population consisted of men only, so our findings cannot be generalized to women in whom estrogen is known to have significant effects on the RANKL/OPG axis.24 Our population also had relatively well-controlled and stable HIV disease so our findings cannot be generalized toward individuals with more active and uncontrolled HIV disease. Dysregulation of the RANKL/OPG cytokine network may play a role in plaque stability, and it remains unclear whether those HIV-infected individuals with acute cardiovascular events or with worse cardiovascular risk factors would have similar alterations in their sRANKL levels.
In conclusion, this study found a novel and interesting inverse relationship between serum sRANKL levels and coronary artery calcification amongst HIV-infected individuals, which persisted after controlling for traditional cardiac risk factors. Further studies are necessary to understand the physiological regulation and potential effects of RANKL on coronary atherosclerosis development in the HIV population.
The authors thank the subjects for their participation and the nurses and staff of the Massachusetts General Hospital Clinical Research Center for their dedicated patient care.
1. Anderson DM, Maraskovsky E, Billingsley WL, et al.. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature. 1997;390:175–179.
2. Collin-Osdoby P, Rothe L, Anderson F, et al.. Receptor activator of NF-kappa B and osteoprotegerin expression by human microvascular endothelial cells, regulation by inflammatory cytokines, and role in human osteoclastogenesis. J Biol Chem. 2001;276:20659–20672.
3. Towler DA, Demer LL. Thematic series on the pathobiology of vascular calcification: an introduction. Circ Res. 2011;108:1378–1380.
4. Dhore CR, Cleutjens JP, Lutgens E, et al.. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2001;21:1998–2003.
5. Golledge J, McCann M, Mangan S, et al.. Osteoprotegerin and osteopontin are expressed at high concentrations within symptomatic carotid atherosclerosis. Stroke. 2004;35:1636–1641.
6. Anand DV, Lahiri A, Lim E, et al.. The relationship between plasma osteoprotegerin levels and coronary artery calcification in uncomplicated type 2 diabetic subjects. J Am Coll Cardiol. 2006;47:1850–1857.
7. Abedin M, Omland T, Ueland T, et al.. Relation of osteoprotegerin to coronary calcium and aortic plaque (from the Dallas Heart Study). Am J Cardiol. 2007;99:513–518.
8. Shin JY, Shin YG, Chung CH. Elevated serum osteoprotegerin levels are associated with vascular endothelial dysfunction in type 2 diabetes. Diabetes Care. 2006;29:1664–1666.
9. Xiang GD, Sun HL, Zhao LS. Changes of osteoprotegerin before and after insulin therapy in type 1 diabetic patients. Diabetes Res Clin Pract. 2007;76:199–206.
10. Omland T, Ueland T, Jansson AM, et al.. Circulating osteoprotegerin levels and long-term prognosis in patients with acute coronary syndromes. J Am Coll Cardiol. 2008;51:627–633.
11. Crisafulli A, Micari A, Altavilla D, et al.. Serum levels of osteoprotegerin and RANKL in patients with ST elevation acute myocardial infarction. Clin Sci (Lond). 2005;109:389–395.
12. Schoppet M, Schaefer JR, Hofbauer LC. Low serum levels of soluble RANK ligand are associated with the presence of coronary artery disease in men. Circulation. 2003;107:e76; Author reply: e76.
13. Kiechl S, Schett G, Schwaiger J, et al.. Soluble receptor activator of nuclear factor-kappa B ligand and risk for cardiovascular disease. Circulation. 2007;116:385–391.
14. Lo J, Abbara S, Shturman L, et al.. Increased prevalence of subclinical coronary atherosclerosis detected by coronary computed tomography angiography in HIV-infected men. AIDS. 2010;24:243–253.
15. Triant VA, Lee H, Hadigan C, Grinspoon SK. Increased acute myocardial infarction rates and cardiovascular risk factors among patients with human immunodeficiency virus disease. J Clin Endocrinol Metab. 2007;92:2506–2512.
16. Fakruddin JM, Laurence J. HIV envelope gp120-mediated regulation of osteoclastogenesis via receptor activator of nuclear factor kappa B ligand (RANKL) secretion and its modulation by certain HIV protease inhibitors through interferon-gamma/RANKL cross-talk. J Biol Chem. 2003;278:48251–48258.
17. Chakravarti A, Marceau AA, Flamand L, et al.. Normal human primary CD4+ T lymphocytes synthesize and release functional osteoprotegerin in vitro. Lab Invest. 2008;88:171–184.
18. Gibellini D, Borderi M, De Crignis E, et al.. RANKL/OPG/TRAIL plasma levels and bone mass loss evaluation in antiretroviral naive HIV-1-positive men. J Med Virol. 2007;79:1446–1454.
19. Brown TT, Ross AC, Storer N, et al.. Bone turnover, osteoprotegerin/RANKL and inflammation with antiretroviral initiation: tenofovir versus non-tenofovir regimens. Antivir Ther. 2011;16:1063–1072.
20. Austen WG, Edwards JE, Frye RL, et al.. A reporting system on patients evaluated for coronary artery disease. Report of the Ad Hoc Committee for Grading of Coronary Artery Disease, Council on Cardiovascular Surgery, American Heart Association. Circulation. 1975;51(4 suppl):5–40.
21. Wang MW, Wei S, Faccio R, et al.. The HIV protease inhibitor ritonavir blocks osteoclastogenesis and function by impairing RANKL-induced signaling. J Clin Invest. 2004;114:206–213.
22. Sandberg WJ, Yndestad A, Oie E, et al.. Enhanced T-cell expression of RANK ligand in acute coronary syndrome: possible role in plaque destabilization. Arterioscler Thromb Vasc Biol. 2006;26:857–863.
23. Findlay D, Chehade M, Tsangari H, et al.. Circulating RANKL is inversely related to RANKL mRNA levels in bone in osteoarthritic males. Arthritis Res Ther. 2008;10:R2.
24. Bord S, Ireland DC, Beavan SR, et al.. The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts. Bone. 2003;32:136–141.