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Cardiovascular disease is increased in HIV patients, but the specific mechanisms are unknown.
To assess arterial wall inflammation in HIV, using 18fluorine-2-deoxy-D-glucose Positron Emission Tomography (18FDG-PET), in relationship to traditional and non-traditional risk markers, including sCD163, a marker of macrophage activation.
81 participants were investigated in a cross-sectional study from November 2009 to July 2011. 27 HIV-infected participants without known cardiac disease underwent cardiac 18FDG-PET and coronary CT imaging for coronary calcium (CAC). Two separate non-HIV control groups were compared. One control group (n=27) was matched to the HIV group for age, gender and Framingham Risk Score (FRS) and had no known atherosclerotic disease (FRS-Matched Controls). The second control group (n=27) was matched on gender and selected based on the presence of known atherosclerotic disease (Atherosclerotic Controls).
Arterial inflammation was prospectively determined as the ratio of FDG uptake in the arterial wall of the ascending aorta/blood background, as the target to background ratio (TBR).
HIV participants demonstrated well-controlled HIV disease (CD4 641±288 cells/mm3, HIV RNA <48 [<48, <48] copies/mL). All were receiving ART (duration 12±4 yrs). The mean Framingham Risk Score (FRS) was low in both HIV and FRS-Matched Controls (6.4 (4.8–8.0) vs. 6.6 (4.9–8.2), P=0.87). Arterial inflammation in the aorta (Aortic TBR) was higher in the HIV vs. FRS-Matched Control Participants (2.23 (2.07–2.40) vs. 1.89 (1.80–1.97), P<0.001), but was similar compared to Atherosclerotic Controls (2.23 (2.07–2.40) vs. 2.13 (2.03–2.23), P=0.29). Aortic TBR remained significantly higher in the HIV group vs. the FRS-Matched Controls after adjusting for traditional cardiovascular risk factors (P=0.002) and in stratified analyses among participants with undetectable viral load, zero calcium, FRS<10, and LDL< 100 mg/dL (2.59 mmol/L) (all P<0.01). Aortic TBR was associated with sCD163 (P=0.04) but not with CRP (P=0.65) or D-Dimer (P=0.08) among HIV-infected patients.
Persons infected with HIV, compared to non-infected controls with similar cardiac risk factors, had signs of increased arterial inflammation which was associated with circulating markers of macrophage activation.
Coronary artery disease is significantly increased in HIV-infected patients, but the specific mechanisms remain unknown1. Immunological modulations may play an important role in the pathogenesis of atherosclerosis in HIV-infected patients. HIV-infected patients demonstrate a high prevalence of noncalcified coronary atherosclerotic lesions that are increased in association with markers of macrophage activation2. This is significant because infiltration of activated monocytes and macrophages into the endothelium contributes to the development of vulnerable atherosclerotic plaque susceptible to rupture3,4.
Positron Emission tomography (PET) with 18fluorine-2-deoxy-D-glucose (18FDG-PET) is widely used for measurement of inflammation in the arterial wall. Accumulation of FDG in human atherosclerotic arteries correlates with the amount of immunohistochemical staining and gene-expression for macrophage-specific markers, including CD685–8 and is based on the fact that activated macrophages have an unusually high metabolic rate9–11.
Accordingly, we employed 18FDG-PET imaging to test the hypothesis that arterial wall inflammation is increased in patients with HIV compared to non HIV-infected patients with similar cardiac risk factors, in association with increased monocyte activation.
Twenty-seven HIV-infected participants without known cardiac disease underwent 18FDG-PET and CT imaging. The HIV-infected participants were prospectively enrolled and had no known coronary disease at the time of imaging. HIV-infected participants were on stable doses of anti-retroviral therapy for at least 3 months prior to the time of the study. Exclusion criteria for the HIV-infected participants consisted of known cardiac disease or symptoms suggestive of cardiovascular or atherosclerotic disease (myocardial infarction, angina, arrhythmias, valvular heart disease, pericarditis, congestive heart failure, stroke and peripheral vascular disease), use of statins or recent acute infection.
Two separate non HIV-infected control groups were selected prospectively for comparison to the HIV group from a large data base of 15,587 participants, and had undergone whole body PET/CT between 2005–10, but in whom neither aortic FDG uptake nor coronary calcium had been previously assessed. The primary indication for PET imaging for those patients was evaluation for neoplastic process. However, in both control groups we excluded any patients with active malignancy, presence of active systemic inflammatory disease or use of systemic steroid therapy at the time of PET imaging, or known HIV.
The first control group, consisting of 27 non HIV-infected participants, was selected based on the absence of known atherosclerotic disease and matching to the HIV group for age, gender and Framingham Risk Score (FRS) (FRS-Matched Controls) using a 1:1 case matching technique. In this regard, the first HIV patient was considered, with respect to age, gender and FRS. The database was examined to find the first patient of the same age and gender, with a FRS within ± 4 to the index subject. Matches were found for all participants within this range except for 1. Only participants in whom all data to establish FRS risk were available were eligible for selection. Participants with known cardiac or atherosclerotic disease were not eligible for selection. This process was repeated to obtain a match for each HIV patient.
The second group of 27 control participants was selected based on the presence of known atherosclerotic disease and matching to the HIV group by gender (Atherosclerotic Controls). Known atherosclerotic disease was defined as the presence of either: 1) documented atherothrombotic events (such as prior stroke, myocardial infarction or limb ischemia), or 2) documented presence of occlusive atherosclerotic disease (such as carotid stenosis or peripheral artery disease) using a 1:1 case matching technique. The gender of the first HIV patient was considered and the database was examined to find the first non-HIV control subject of this gender with known atherosclerotic disease for selection. This process was repeated to obtain a match for each HIV patient.
The selection of control participants was performed based on clinical and demographic matching criteria above, prior to and independent of determination of the aortic FDG uptake. After matching and subject selection were completed, arterial inflammation and coronary calcium were prospectively determined from April to July 2011. Written informed consent was obtained from HIV-infected participants. Non HIV-infected controls underwent scanning for clinical purposes and were not consented for the study, but permission was received from the Partners Healthcare IRB to use the data from participants in the clinical database for comparison in this study.
Patients underwent PET imaging after an overnight fast to reduce myocardial FDG uptake. The PET imaging was performed 3 h after administration of 13 mCi of 18F-FDG (Siemens ECAT Exact HR+ PET or biograph 64 system, Knoxville, Tennessee)12. The system provides 63 planes, a 15.5-cm field of view, and a maximum 4.2-mm intrinsic resolution at the center of the field of view. Images were acquired in 3 dimensional mode over 20 minutes with an effective resolution of 5mm.
All image analysis was performed while blinded to clinical data. Co-registration of the PET and CT images was performed using anatomical landmarks (such as ascending aorta, left atrium, spine). The ascending aorta was chosen for measurement. The target-to-background ratio (TBR) was calculated (see Figure 1 for Methods).
For the HIV patients, coronary calcification was evaluated using standardized methods as previously described13,14. Non-HIV participants underwent hybrid PET CT, and coronary calcium score was determined using similar parameters. Low attenuation CT scanning was performed using a pitch 1.0, tube voltage 120 kVp, tube current of 40 mAs, and slice thickness of 3mm. Prior studies have demonstrated that coronary calcium scores measured on CT images obtained from a hybrid PET-CT scanner are comparable to those obtained on a dedicated CT scanner15. For the purpose of this study, after coronary calcium score was recorded, the status of coronary calcium was dichotomously characterized as present or absent. Evaluation of coronary calcium score was performed while investigators were blinded to clinical information or PET data, by a separate group of investigators (EC and SA) from those who performed the PET analyses.
HIV-infected subjects were characterized in terms of immune function, inflammatory, and monocyte activation. HIV viral load was determined by ultrasensitive RT PCR (Roche Amplicor Monitor) (lower limit of detection=48 copies/mL). HIV testing was performed by ELISA (Abbott) and confirmed by Western blot. CD4 count was assessed by flow cytometry. High sensitivity C-reactive protein (hs-CRP) was measured by Immunochemiluminometric assay (LabCorp). sCD163 was measured from serum by ELISA (Trillium Diagnostics)2. D-Dimer was measured using particle enhanced immunoturbidimetric assay (Roche Cobas).
Comparison of TBR and other variables in the three groups 1) HIV, 2) non-HIV FRS-Matched Controls, and 3) non-HIV Atherosclerotic Controls was performed using ANOVA for normally distributed variables and the Kruskal-Wallis test for non-normally distributed variables. If the P value for the overall ANOVA/Kruskal-Wallis test between groups was significant (P<0.05), comparisons of variables between two groups were performed using Student’s t-test for normally distributed continuous variables or the Wilcoxon rank sum test for non-normally distributed variables. For comparison of means, 95% CI are provided. Additional comparisons of TBR between the groups were performed in stratified analyses among those without coronary calcium (calcium score of zero), low FRS (0–10), LDL < 100 mg/dL (2.59 mmol/L), not receiving a statin, and limiting the HIV group for comparison to those with undetectable viral load. In addition, least squares multivariate regression modelling was performed to assess the differences in TBR between the HIV and non-HIV FRS-Matched Control groups simultaneously controlling for FRS, calcium score, smoking, statin use and LDL in a single model among all patients in these two groups.
Pearson correlation coefficients were used to assess correlations for normally distributed data. For non-normally distributed variables, Spearman rho (ρ) was used to assess correlations and P value was determined by the Hoeffdings D Test. Two-tailed probability values are reported and statistical significance was assumed when P<0.05. With 54 patients in each two group comparison, the study was powered at 0.85, with a two-sided significance level of 0.05, to detect a 0.83 SD difference between the groups. All statistical analyses were performed using SAS JMP (SAS Institute). Complete data on all subjects were available for PET-CT, and CVD risk markers, as well as for CRP, and sCD163 in the HIV patients. D-Dimer was available in a subset of HIV patients (n=14). No imputation was made for missing data.
The clinical characteristics of the HIV-infected and control participants are shown in Table 1. Age was similar in the HIV and FRS-Matched Control groups and increased in the Atherosclerotic Control group (Table 1). All of the HIV-infected participants and all the FRS-Matched Controls demonstrated either low or intermediate FRS. Based on the matching, mean FRS was not significantly different between the HIV group and FRS-Matched Controls (6.4 (95% CI 4.8–8.0) vs. 6.6 (4.9–8.2), P=0.87, respectively). As expected, cardiovascular risk parameters were markedly increased and statin use more prevalent in the Atherosclerotic Control participants (Table 1). The majority of HIV-infected participants demonstrated well-controlled HIV disease with mean CD4 count 641±288 cells/mm3 and virologic suppression [median HIV viral RNA <48 [<48, <48] copies/mL] (Table 1). The minimum duration of HIV infection was 5 years and mean duration of infection 16±6 years. The average duration of treatment with antiretroviral treatment (ART) was 12±4 years.
Arterial inflammation (TBR) in the aorta was higher in the HIV vs. FRS-Matched Controls (2.23 (2.07–2.40) vs. 1.89 (1.80–1.97), P<0.001) (Table 1). In comparison, the arterial inflammation in the aorta was not significantly different between the HIV and the Atherosclerotic Control group (2.23 (2.07–2.40) vs. 2.13 (2.03–2.23), P=0.29). The analysis was also repeated, limiting the comparison to those HIV-infected patients with undetectable viremia. TBR of the aorta remained increased among the HIV-infected, patients with undetectable viremia (n=21, 81%) compared to the FRS-Matched Control participants (2.24 (2.03–2.45) vs. 1.89 (1.80–1.97), P<0.001, Table 2 and Supplemental Table 6) and similar to that in the Atherosclerotic Control group (2.24 (2.03–2.45) vs. 2.13 (2.03–2.23), P=0.31). TBR of the aorta did not differ by use of ART class (2.21 (2.03–2.39) vs. 2.25 (1.98–2.52), P=0.81 [protease inhibitor (PI) vs. no PI]; 2.25 (1.94–2.56) vs. 2.22 (2.07–2.37), P=0.84 [nonnucleoside reverse transcriptase inhibitor (NNRTI) vs. no NNRTI)]). TBR remained similar (P=0.82) between the HIV and the group with established CAD, controlling for age and statin use.
Calcium score was significantly higher in the Atherosclerotic Control Group than in either the HIV or FRS-Matched Control group (Table 1), however, TBR of the aorta was not related to calcium score in univariate regression analysis among all participants (P=0.60) or within the individual groups (data not shown). Arterial inflammation in the aorta remained higher in HIV vs. FRS-Matched Controls in stratified analyses limited to those participants with no coronary calcium (P=0.009, Table 2 and Supplemental Table 1), and separately among those with coronary calcium score > 0 (P=0.02). Smoking rates did not differ between HIV and FRS-Matched Controls. Moreover, aortic TBR was increased among the HIV compared to FRS-Matched Control participants in an analysis limited to non-smokers (2.23 (2.04–2.43) vs. 1.90 (1.81–1.99), P=0.001, Table 2) (for further smoking analysis, see Supplemental Text and Supplemental Table 5). Similarly, arterial inflammation in the aorta remained higher in HIV vs. FRS-Matched Controls in stratified analyses limited to patients with low FRS (FRS 0–10), LDL < 100 mg/dL, and those not receiving statins (all P≤0.01, Table 2 and Supplemental Tables 2–4).
Adjusting simultaneously for traditional cardiovascular risk factors in a multivariate regression model including Framingham risk score, statin use, calcium score, smoking and LDL, aortic TBR remained higher in the HIV-infected group compared to the FRS-Matched Control group (P=0.002). In contrast, traditional risk factors were not significant in the model (Supplemental Table 7).
The mean sCD163 level was 1200±988 ng/mL and higher than that seen in a group of previously published comparable non HIV controls (883±561 ng/mL)2. The aortic arterial inflammation (TBR) significantly correlated with sCD163 levels (P = 0.04) (Table 3). In contrast to sCD163, hsCRP and D-dimer were not significantly associated with aortic arterial inflammation (TBR) (Table 3). Limiting the analysis to HIV patients with undetectable viral load (n=21, 81%), sCD163 remained similarly and significantly associated with aortic TBR (P=0.03) (Figure 2).
FDG accumulates within metabolically active macrophages infiltrating affected vessels such that increased FDG uptake reflects heightened vascular inflammation5–8. Indeed, through pathologic/histologic analyses of plaque specimens from participants with occlusive carotid disease who went on to carotid endarterectomy, we and others have previously shown that arterial FDG uptake correlates closely with plaque macrophage infiltration characterized by increased CD68 staining5–8. Increased aortic FDG uptake is known to correlate with increased FDG uptake in the left main coronary artery16 Moreover, increased arterial PET-FDG uptake is associated with subsequent progression of atherosclerotic plaques17 and identifies patients at risk for subsequent atherothrombotic events12,18. Hence, the signal that we see likely reflects atherosclerotic inflammation with macrophage infiltration into arterial atheroma. The results from the current study using the PET-FDG technique suggest that macrophage infiltration and resulting arterial inflammation, measured here in the aorta, are increased among HIV-infected patients.
Our observation that HIV-infection is associated with increased arterial inflammation, even among relatively young HIV-infected patients with low FRS and undetectable viremia, is concordant with the epidemiological observations that patients with HIV have a higher risk of stroke and MI than non-HIV patients1,19 and demonstrates that this risk may not be measured adequately by traditional risk assessment tools, such as the FRS. Indeed, recent studies among non HIV-infected patients demonstrate that consideration of TBR can improve net reclassification index compared to use of FRS and traditional risk factors20. Moreover, these studies demonstrate that a TBR > 1.7 is associated with an approximate 40% reduction in CVD event free survival over 3 years12, whereas a TBR > 2.25 (vs. < 1.84) is associated with a markedly increased risk of CVD events over 5 years20. These data suggest a clinically relevant degree of added CVD risk due to increased arterial inflammation in the HIV population we studied. One potential mechanistic link to this observation is suggested by our demonstration that a marker of monocyte/macrophage activation, sCD163, was significantly associated with this inflammatory signal. CD163 is expressed specifically on the surface of monocytes and macrophages and has a known role as a scavenger receptor involved in the uptake of hemoglobin-haptoglobin complexes21. Soluble CD163 (sCD163) is shed via proteolytic cleavage at the cell surface and can be found in the circulation. sCD163 has been previously shown as a circulating marker of atherosclerosis in non HIV-infected patients22,23. Macrophages expressing CD163 have been found in human atherosclerotic plaques of non HIV-infected patients24 as well as within plaque lesions in SIV-infected monkey models25. In chronically HIV-infected patients, we have previously demonstrated sCD163 to be independently associated with increased noncalcified plaque among young, asymptomatic men2. Here we extend the observations further, by observing a significant correlation between sCD163 and the extent of arterial inflammation. In contrast, markers of generalized inflammation (hsCRP) and thrombosis (D-dimer) were not statistically significant in terms of their relationships to vascular inflammation in the current study. Hence, in HIV, macrophage activation markers correlate with non-calcified plaques and arterial wall inflammation, two separate predictors of subsequent atherothrombosis. These observations suggest that sCD163 may be able to uniquely provide an index of risk of atherosclerotic disease in HIV. For example, in this study we show that among HIV-infected patients, a sCD163 level of > 800 ng/ml, identifies a group with a markedly elevated TBR, >2.35. Further studies are needed to determine if the demonstration of an elevated sCD163 level in clinical practice will predict events and provide unique information to that of traditional risk indices.
One hundred percent of the HIV patients studied were receiving antiretroviral therapy, and had been receiving such therapy for a long duration of approximately 12 years. A significant majority had undetectable HIV viral load. Viral load was not related to TBR and the observation of markedly increased TBR in HIV was confirmed in the subset with undetectable viremia. Thus, the observation of increased vascular inflammation by PET occurred in well–treated patients in whom significant detectable viremia was neither present nor likely to be a contributing factor. In contrast, increasing degrees of monocyte activation even within this well controlled group were associated with increased arterial inflammation. The patients we studied are similar to the majority of patients undergoing treatment with ART today, with well-controlled virus and absent history of cardiovascular disease. Such patients, particularly with low FRS, are not considered to be at high risk for cardiovascular disease, yet we now show that such patients have increased arterial wall inflammation, equal to that of non-HIV patients with established CAD.
CAC was higher in non-HIV patients with established CAD than in the HIV group. This difference may be due to the increased rate of traditional CVD risks in the atherosclerotic controls compared to the HIV group, and persisted controlling for age. It is notable that the degree of inflammation is similar between the HIV group with very little CAC and low FRS and the established CAD group with significant CAC and traditional risks, suggesting that inflamed noncalcified plaque related more to nontraditional risk factors is likely to be present in the HIV group. Over time, the increased inflammation seen in the HIV group might itself induce an increase in CAC.
The design of this study limits definitive conclusions regarding causality of increased inflammation, but our data suggest monocyte/macrophage activation may be contributing. We cannot completely rule out an effect of ART directly on arterial inflammation, but evidence from SMART26 and STACCATO27 showing that ART decreases inflammation and endothelial activation, the lack of any ART class effect in our data and the low traditional risk factors in our group on ART (ruling out an indirect effect) make this unlikely. We included a relatively small proportion of women, and thus our findings may not be fully generalizable for women. Additionally, while the HIV population was prospectively identified, the control groups were subsequently selected from a database of imaged individuals. However, the analysis of aortic TBR was identical for all participants in the study, was performed only after matching and subject selection and was performed blinded to clinical history. The study was adequately powered to detect a clinically relevant 0.83 SD difference between the study groups.
Our study demonstrates that HIV is associated with a high degree of inflammation within the arterial wall, even in patients with low FRS and well-controlled viremia. These findings advance our understanding of the unique pathophysiology and predilection to early increased CVD among HIV-infected patients and suggest that monocyte/macrophage activation could play a critical role in the early expression of subclinical atherosclerosis in HIV-infected patients. These data have clinical relevance and suggest that HIV patients with chronic infection have significant vascular inflammation, and thus added CVD risk, beyond that estimated by traditional risk factors. This information should now be considered in determining optimal monitoring and CVD prevention strategies for this group. Future studies will be useful to further investigate unique immune-based mechanisms of arterial inflammation and potential agents to reduce the pro-atherogenic activation of monocytes/macrophages with hopes of reducing risk of atherothrombosis in HIV-infected patients.
Funding Sources: The project was supported by NIH R01 HL 095123 to Dr. Grinspoon. Relevant grants supporting investigators were NIH K23 HL092792 to Dr. Lo, NIH-NS37654 and NIH-NS40237 to Dr. Williams, and NIH K24 DK064545 to Dr. Grinspoon. The GCRC grant number is M01 RR01066-25S1. Funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.
Disclosures: The authors have no conflicts of interest to declare relevant to this manuscript. Dr. Grinspoon has received research support from Amgen, Bristol-Myers-Squibb and Theratechnologies, consulted for Theratechnologies, Alize Pharmaceuticals, Hoffmann-LaRoche and Serono and received lecture fees from Ferrer and Sanofi Aventis all unrelated to the manuscript. Dr. Tawakol has consulted for Roche, Bristol-Myers Squibb, and Novartis and has received grant support from Merck, Glaxo Smith Kline, Genetech/Roche, Vascular Biogenics Ltd. and Bristol-Myers-Squibb all unrelated to the manuscript. Dr. Abbara has consulted for Perceptive Informatics and Partners Imaging, received grant support from Bracco and Becton, Dickenson and Company and received royalties from Elsevier and Amirsys all unrelated to the manuscript. Dr. Hoffmann has received research support from Siemens Healthcare, GE Healthcare, Bracco Diagnostics, the American College of Radiology Imaging Network, and NIH all unrelated to the manuscript.
Author’s Contributions:Study Concept and Design: Subramanian, Tawakol, Lo, Grinspoon
Acquisition of Data: Subramanian, Tawakol, Burdo, Abbara, Wei, Vijayakumar, Corsini, Hoffmann, Williams, Lo, Grinspoon
Analysis and Interpretation of Data: Subramanian, Tawakol, Burdo, Abbara, Wei, Vijayakumar, Corsini, Abdelbaky, Zanni, Hoffmann, Williams, Lo, Grinspoon
Drafting of the Manuscript: Subramanian, Tawakol, Abbara, Lo, Grinspoon
Critical Revision of the Manuscript for Important Intellectual Content: Subramanian, Tawakol, Burdo, Abbara, Wei, Vijayakumar, Corsini, Abdelbaky, Zanni, Hoffmann, Williams, Lo, Grinspoon
Statistical Analysis: Tawakol, Wei, Lo, Grinspoon
Obtained Funding: Tawakol, Williams, Grinspoon
Administrative, Technical or Material Support: Wei, Corsini, Zanni, Hoffmann
Study Supervision: Tawakol, Grinspoon.
Trial Registration: clintrials.gov Identifier: NCT00965185. Data from HIV-infected patients were obtained as part of the screening process for NCT00965185. Control data were obtained from clinical scanning and database review with prospective assessment of aortic PET/CT data after case matching to HIV-infected patients.
S.K.G. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Presentation at Scientific Meeting: These data were presented at the 2012 Conference on Retroviruses and Opportunistic Infections on March 7th, 2012 in Seattle, Washington.