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Previous studies have demonstrated an association between HIV infection and coronary artery disease (CAD); little is known about potential associations between HIV infection and extra-coronary calcification (ECC).
We analyzed 621 HIV infected (HIV+) and 384 HIV uninfected (HIV-) men from the Multicenter AIDS Cohort Study who underwent non-contrast computed tomography (CT) from 2010-2013. Agatston scores were calculated for mitral annular calcification (MAC), aortic valve calcification (AVC), aortic valve ring calcification (AVRC), and thoracic aortic calcification (TAC). The associations between HIV infection and the presence of each type of ECC (score > 0) were evaluated by multivariable logistic regression. We also evaluated the association of ECC with inflammatory biomarker levels and coronary plaque morphology.
Among HIV+ and HIV− men, the age-standardized prevalences were 15% for TAC (HIV+ 14%/ HIV− 16%), 10% for AVC (HIV+ 11%/ HIV− 8%), 24% for AVRC (HIV+ 23% HIV− 24%), and 5% for MAC (HIV+ 7%/ HIV− 3%). After adjustment, HIV+ men had 3-fold greater odds of MAC compared to HIV− men (OR=3.2, 95% CI: 1.5-6.7), and almost twice the odds of AVC (1.8, 1.1-2.9). HIV serostatus was not associated with TAC or AVRC. AVRC was associated with higher Il-6 and sCD163 levels. TAC was associated with higher ICAM-1, TNF-α RII, and Il-6 levels. AVC and AVRC calcification were associated with presence of non-calcified plaque in HIV+ but not HIV− men.
HIV infection is an independent predictor of MAC and AVC. Whether these calcifications predict mortality in HIV+ patients deserves further investigation.
Cardiovascular disease (CVD) is a significant cause of morbidity and mortality among the approximately 1.1 million people infected with the human immunodeficiency virus (HIV) in the United States . Due to highly active antiretroviral therapy (HAART), fewer individuals with HIV are dying of opportunistic infections [2,3]. However, more are dying from age-related illnesses such as CVD . Previous studies have shown that HIV-infected (HIV+) individuals have higher rates of myocardial infarction, CVD risk factors, sudden cardiac death, and non-calcified plaque when compared to HIV-uninfected (HIV−) individuals [4-6]. The reason for and etiology of these differences is not clear, but may include several factors including metabolic side effects of HAART, chronic increased systemic inflammation from HIV itself, or a higher prevalence of CVD risk factors, such as smoking [7-9].
In the general population, extra-coronary calcification (ECC) including mitral annular calcification (MAC), aortic valve calcification (AVC), thoracic aortic calcification (TAC), and aortic valve-ring calcification (Figure 1) have been well described and are associated with systemic atherosclerosis [10-12]. In the Multi-Ethnic Study of Atherosclerosis (MESA), Owens et al found AVC to be predictive of future CVD events independent of inflammatory biomarkers and traditional risk factors . A subsequent study also found an association between AVC and CVD events, which was independent of Framingham risk factors . However, ECC has not been well studied in HIV+ populations. To address this issue, we compared the amount and type of ECC between HIV+ and HIV− men in the Multicenter AIDS Cohort Study (MACS).
The MACS is a prospective cohort study, conducted in four U.S. cities, of HIV+ and HIV− men who have sex with men. The MACS has been well characterized previously . We performed cardiac CT scans to characterize the prevalence and predictors of subclinical atherosclerosis. Participants completed non-contrast cardiac computed tomography (CT) scanning for coronary artery calcium (CAC) scoring between January 2010 and August 2013. Eligibility criteria for this MACS CVD ancillary study included being an active MACS participant, age 40 to 70 years, weighing less than 136 kg (300 pounds), and without a history of cardiac surgery or percutaneous coronary intervention as these procedures could interfere with the measurement of coronary atherosclerosis.
Participants were seen during routine MACS semi-annual research visits for standardized interviews, physical examination, and blood and urine collection for concurrent laboratory analyses and storage. Data from the MACS visit prior to the CT measurements were used and included demographic, HIV clinical parameters, and CVD risk factors, including age, race, measured blood pressure, fasting glucose, fasting lipid panel, body mass index (weight/height2), and self-reported medication use. Cumulative pack-years of smoking were calculated from the longitudinal self-report of smoking status and cigarette quantity.
Our analytic sample included 1005 men, 384 were HIV−, and 621 were HIV+. Details of the CT scanning procedures have been described . Briefly, computed tomography scanning equipment included 64-slice multidetector CT at 3 centers and 320-row multidetector CT at 1 center. Prospective electrocardiography-triggering non-contrast cardiac protocols were used to obtain the CAC scan. The CT images were transferred to the core CT reading center (Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center) and were analyzed by trained, experienced readers who were blinded to participant characteristics and HIV serostatus. The main outcome was the calcification score, calculated as the Agatston Score .
Participants also underwent coronary CT angiography (CTA) to assess plaque morphology unless contraindicated by chronic kidney disease (estimated glomerular filtration rate <60ml/min/m2 using the MDRD equation within 30 days), atrial fibrillation, or IV contrast allergy. Coronary CTA images were analyzed using the modified 15-segment model of the American Heart Association for plaque presence and extent, coronary artery stenosis, and plaque composition . Partially calcified plaque was defined as lesions with less than 50% of plaque area occupied by calcium. Calcified plaque included any structure with attenuation >130 Hounsfield units (HU) visualized separately from the intravascular lumen. Non-calcified atherosclerotic plaque was defined as any structure clearly assignable to the vessel wall, with a CT density less than the contrast-enhanced coronary lumen but greater than the surrounding connective tissue. Plaque types were identified in at least two independent planes. Coronary stenosis was defined as greater than 50% in any coronary segment. The segment involvement score was defined as the number of coronary segments with plaque present. The total plaque score was calculated by adding the plaque size score for each coronary segment that had any plaque (maximum score of 45).
MAC, AVC, AV-ring calcification and TAC scores were computed using the Agatston method from non-contrast cardiac CT scans . According to this method, three contiguous pixels with a density over 130 HUs qualified as an atherosclerotic plaque. The absence of calcium was deemed a score of 0. AVC was defined as any calcified lesion within the aortic valve leaflets, excluding the aortic annulus, proximal aorta, and coronary arteries. AV-ring (root or annulus) calcium was measured at the level of the aortic-ring, and excluded calcium of the valve leaflets as well as the ascending aorta wall. MAC included calcification along the mitral annulus, excluding the mitral leaflets and differentiated from the left circumflex artery. TAC was defined as either ascending or descending thoracic aortic calcium measured from the pulmonary artery bifurcation superiorly to the cardiac apex inferiorly.
HIV− men were tested for HIV at each MACS visit, and HIV serostatus was established by a positive ELISA test confirmed by Western blot. Measures of HIV disease activity, including CD4+ T-cell counts and plasma HIV RNA levels (Roche ultrasensitive assay with limit of detection of 50 copies/mL; Roche Diagnostics, Nutley, NJ), were measured at each visit for HIV+ men. Duration of highly active antiretroviral therapy (HAART), protease inhibitor (PI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and nucleoside reverse transcriptase inhibitor (NRTI) use was calculated based on the longitudinal participant self-report of antiretroviral medication use at each MACS visit. A history of clinically defined AIDS was determined by medical record confirmation of self-reported outcomes.
A panel of biomarkers was measured from blood samples drawn on the day of CT scanning and stored at −70°C until analysis. Biomarkers included high sensitivity C-reactive protein (hsCRP), fibrinogen, monocyte chemotactic protein 1 (MCP-1), interleukin 6 (IL-6), D-dimer, intercellular adhesion molecule 1 (ICAM-1), soluble tumor necrosis factor alpha receptor type I (sTNFαRI), type 2 (sTNFαRII), sCD14, and sCD163. High-sensitivity CRP and fibrinogen were measured using the BNII Nephelometer (Siemens Healthcare Diagnostics, Deerfield, IL). Samples of IL-6, ICAM-1, sCD14, and sCD163 were tested using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). D-dimer was measured using a Stago STA-R analyzer (Parsippany, NJ). A Milliplex (Millipore, Billerica, MA) measured sTNFαRII and sTNFαRII via multiplexing.
The distributions of demographic, behavioral, and clinical characteristics between HIV+ and HIV− men were compared using the chi-square or Kruskal-Wallis tests for categorical and continuous variables, respectively. The prevalence of each extra-coronary plaque type was described by HIV serostatus, both crude and age-adjusted, assuming a common age distribution for the HIV+ and HIV− men (we used the age distribution of the total sample) using direct standardization. We used multiple imputation to estimate values for nine CVD risk factor covariates with a small percentage of missing values (<4%). The predictors for the multivariate imputation model included all covariates and outcome variables from the multivariable analyses models. Missing values were imputed five times based on the distribution of covariates using the data augmentation Markov chain Monte Carlo (MCMC) method assuming a multivariate normal distribution . We used logistic regression models, pooled across the multiple imputation datasets, to assess the association between HIV infection and extra-coronary plaque. Multivariable models included age, race, education, study site, enrollment period into the MACS (enrolled pre- vs. post-2001), body mass index (BMI), current smoking status, pack-years of tobacco smoking, use of anti-hypertensive, diabetes or lipid-lowering medications, and systolic blood pressure (SBP), fasting glucose, total and HDL cholesterol for those not on anti-hypertensive, diabetes or lipid-lowering medications. After adjustment for demographic, HIV clinical factors, and CVD risk factors, levels of each of the nine biomarkers were log transformed (base-e) and analyzed for their associations with ECC outcomes in separate multivariable models. Further analyses were conducted among the HIV+ men to evaluate associations between measures of HIV disease and treatment exposures with ECC outcomes. We assessed nadir and current CD4 cell count, HIV viral suppression, history of AIDS, and use of protease inhibitors sequentially, and retained them in the multivariable model if their association with an ECC outcome had a p-value <0.05. Among HIV+ men on HAART, we determined the effects of years of PI, NNRTI, and NRTI use on ECC presence. In both our HIV+ and HIV− groups, we assessed the association between ECC and the presence of coronary plaque morphology after adjusting for age and race. All analyses used Stata 13.1 (StataCorp, College Station, TX).
Our analytic sample included 1005 men who underwent CT scanning [n=621 (61.8%) HIV+/ n=384 (38.2%) HIV−]. TAC was evaluated among 988 men since 17 CT scan images did not include the thoracic aorta. Demographic and clinical characteristics stratified by HIV serostatus are described in Table 1. Compared to the HIV− men, HIV+ men were younger, African-American, had higher fasting glucose and triglyceride levels, were current smokers, and previously injected drugs. In addition, HIV+ men had lower total, HDL and LDL cholesterol levels, and lower BMIs compared to HIV− men.
Thirty-five percent of our sample had extra-coronary calcification in one or more areas (Table 2). Prevalence was highest for AV-ring (24%), followed by TAC (15%), AVC (10%), and MAC (5%). HIV+ men had higher age-standardized prevalences of AVC (HIV+ 11%/ HIV− 8%) and MAC (HIV+ 7%/ HIV− 3%) when compared to HIV− men. Of note, even among the 495 men with no coronary artery calcium (CAC=0), 19% had extra-coronary calcification, with no difference by HIV serostatus (data not shown).
After adjustment for demographic and CVD risk factors, HIV infection was associated with increased odds of AVC (odds ratio, 95% CI: 1.7, 1.1-2.8) and MAC (3.0, 1.4-6.2) (Table 3), but was not associated with AV-ring or TAC. Each decade increase in age was associated with greater odds of all four ECC types. African American men had 40 percent lower odds than white men of AV-ring calcification (0.6, 0.3-0.9). Men on lipid lowering medication had significantly greater odds of TAC (1.6, 1.0-2.4) and AV-ring calcification (1.6, 1.1-2.3). Each additional pack-year of smoking increased the odds of AVC and TAC by 10% (both 1.1, 1.0-1.1). We did not find any statistically significant associations between other demographic or CVD risk factors and any of the four ECC types.
We evaluated the association between each measure of ECC and HIV-specific factors after adjustment for demographics and CVD risk factors. A previous history of AIDS was associated with two-fold higher odds of AV-ring calcification (2.33, 1.26-4.29, p < 0.01), but not with AVC, MAC, or TAC. We did not find any statistically significant associations between other ECC measures and detectable HIV RNA levels, current CD4, nadir CD4, years of HAART, years of NNRTI duration, or NRTI duration. For each year of PI use, however, we found a trend towards increased MAC and AVC (p=0.08 and 0.06, respectively).
To assess inflammation as a potential mechanism for the greater ECC observed in HIV+ men, we evaluated the association between ECC and levels of 9 biomarkers in separate models (Table 4). After full adjustment, TAC was positively associated with levels of ICAM-1 (1.82, 1.07-3.11), TNF-α RII (2.10, 1.18-3.72), and Il-6 (1.32, 1.01-1.72). AV-ring calcifications were positively associated with levels of Il-6 (1.30, 1.04-1.62) and sCD163 (1.77, 1.09-2.88). Only sCD163 was marginally associated (p < 0.10) with AVC. However, its inclusion in multivariate models attenuated the association between HIV infection and AVC [from 1.84 (1.10-3.09) to 1.62 (0.95-2.77)], suggesting it may be a mediating factor. We did not find an association between MAC and any of the biomarkers we tested.
Since ECC has been associated with CVD events [13,14], we used coronary CTA to investigate the associations between the presence of each ECC and coronary artery plaque presence, extent and composition, stratified by HIV serostatus (Table 5A and andB).B). The presence of AVC was associated with the presence of CAC, calcified plaque, partially calcified plaque, coronary stenosis > 50%, a greater segment involvement score, and total plaque score among both HIV+ and HIV− participants. Among men with AVC, there was a greater prevalence of non-calcified plaque in HIV+ men compared with HIV− men (81.3% vs. 52.9%), p-value for HIV serstatus interaction =0.001. After adjustment for age and race, HIV+ participants with AVC were 2.2 times more likely to have non-calcified plaque than men without AVC. Similar associations were seen with AV-ring calcification. TAC presence was associated with all coronary plaque types except non-calcified plaque among both HIV+ and HIV− men. After adjustment for age and race, there were no statistically significant associations between MAC and plaque morphologies.
In the MACS, we found that HIV+ men were 70% more likely than HIV− men to have AVC and 3 times more likely to have MAC. Using coronary CTA, the presence of ECC was strongly correlated with the presence and extent of coronary artery plaque and stenosis. Furthermore, the presence of AVC and AV-ring calcification in HIV+ but not HIV− men was associated with the presence non-calcified plaque, a plaque type previously demonstrated to exist in excess among HIV+ compared to HIV− men in this cohort . In contrast, the presence of MAC was not independently associated with coronary plaque type or extent overall or by HIV serostatus.
Although the presence and extent of CAC has a well-established link with future CVD events [20,21] in the general population, growing evidence suggests that further prognostic information can be gleaned from calcifications in other-than-coronary vascular beds [22-24]. ECC has the added benefit of high reproducibility and visualization with several imaging modalities, such as CT and echocardiography [25-27]. Additionally, ECC has been associated with angiographic coronary artery disease (CAD) extent and severity. Yamamoto et al found that AVC was the strongest predictor of obstructive coronary disease. Further, descending aorta calcifications were associated with multivessel coronary disease . Our findings build upon these earlier reports and go one step further by comparing ECC types and their associations with coronary plaque morphology. These findings could potentially offer clinicians valuable information regarding the likelihood of coronary disease if ECC is found. Moreover, they could trigger further testing and potentially early intervention of CAD.
In our cohort, HIV+ men with AVC were more likely to have non-calcified coronary plaque, which is associated with a higher risk of coronary events [28,29] than calcified plaque. If AVC is found on imaging with CT or other modalities, it could alert clinicians to the increased likelihood of high-risk plaque, which may not be represented by CAC scoring. Our group has previously described the increased prevalence of non-calcified coronary plaque in HIV+ men . These analyses suggest that there may be a shared mechanism whereby HIV infection predisposes to a greater prevalence of non-calcified plaque as well as to AVC.
Two studies from different cohorts (MESA and The Heinz Nixdorf Recall Study) have previously described the predictive ability of AVC for future CVD events [13,14]. A subsequent study, also from MESA, included all four ECC types and showed their predictive value for CVD and all-cause mortality risk beyond traditional CVD risk factors . Furthermore, they found that the odds of CVD events, CVD mortality, and all-cause mortality increased with incremental sites of ECC involvement. Risk prediction was further improved with the addition of traditional CVD risk factors and CAC. Their study built upon findings from both the Rotterdam Study and Cardiovascular Health Study, each of which reported upon the prognostic ability of ECC as assessed by echocardiography [27,30]. However, the applicability and use of ECC in CVD risk prediction for other populations such as persons with HIV infection is unclear.
The development of ECC appears to share similar cardiovascular risk factors and mechanisms with atherosclerosis [31-33]. All four ECC types included in these analyses are areas of altered shear stress and turbulent blood flow, both of which predispose to endothelial dysfunction, lipid accumulation, and calcium deposition [33-36]. Furthermore, inflammation and inflammatory cells, including monocyte activation, play a large role in vascular calcifications [37-39]. We also know that inflammation plays a significant role in HIV disease and related complications [9,40], and that HIV leads to advanced vascular aging .
In the Strategies for Management of Antiretroviral Therapy (SMART) study, investigators interrupted HAART therapy, which resulted in higher levels of inflammatory markers, such as IL-6, and greater CVD events as well as mortality compared to persons in whom HAART was not interrupted . We found that both AV-ring calcifications and TAC were associated with elevated levels of IL-6. Furthermore, higher levels of sCD163, a marker of monocyte activation , were positively associated with AV-ring calcification and borderline associated with TAC. Elevation of these and other inflammatory markers could partially explain the higher prevalence of ECC in HIV+ men in our cohort. Also supporting inflammation as a potential mechanism included our findings that a history of AIDS, which has been associated with higher levels of HIV-related inflammation, was associated with a two times greater risk of AV-ring calcifications.
Another potential mechanism for the increased CVD morbidity and mortality of HIV+ individuals are ART medications, especially protease inhibitors, and resulting metabolic disorders [7,8]. However, we did not find that overall HAART duration was associated with any of the ECC types. Of the specific HAART classes, years of NNRTI and NRTI were not associated with ECC. However, the presence of MAC and AVC was more likely with increasing years of protease inhibitor use, but these relationships did not meet statistical significance. It may be that cumulative use of specific protease inhibitors increases the risk of ECC, but this sample is too small to parse out these associations.
Racial differences in ECC have also been well described . Similar to previous studies from MESA on CAC , they found that whites had the highest prevalence of all four ECC types compared to African-Americans, Hispanics, and Chinese participants. In our cohort, we did not find an association between race and AVC, MAC, or TAC prevalence. However, African-American men had lower odds of AV-ring calcification compared to whites, which persisted after adjustment for other demographic and CVD risk factors.
Our study has several important strengths, including a large sample size with an HIV− group from a similar, ethnically diverse population. More importantly, to our knowledge, this is the first study addressing the association of ECC with HIV infection. It also includes novel results comparing the presence of ECC with coronary plaque composition. However, our study needs to be interpreted in light of its limitations. Although we statistically adjusted for demographic and CVD risk factors differences between the two populations (HIV+ and HIV−), it is possible there may be residual or unmeasured confounding. Furthermore, we measured prevalent extracoronary calcification at a single time point, and therefore the associations that we found may differ from the factors associated with its development or progression. Finally, given sex-based differences in the manifestations of subclinical cardiovascular disease, our findings may have limited generalizability to HIV+ women.
We found that HIV+ men were more likely than HIV− men to have AVC and MAC. Furthermore, AVC was associated with the presence of non-calcified plaque in HIV+ but not HIV− men. Levels of several inflammatory and immune activation biomarkers were positively associated with AV-ring calcification and TAC. Given the higher rates of CVD morbidity among HIV+ compared HIV− persons, further studies are needed to evaluate whether the increased prevalence of AVC and MAC is predictive of CVD clinical events among HIV+ persons.
Acknowledgements: Dr. Russell Tracy's laboratory at the University of Vermont Laboratory for Clinical Biochemistry Research (Burlington, VT).
Funding Sources: The MACS CVD study is funded by NHLBI: RO1 HL095129 (Post). Data in this manuscript were collected by the MACS with centers (Principal Investigators) at: Johns Hopkins University Bloomberg School of Public Health (Joseph Margolick), U01-AI35042; Northwestern University (Steven Wolinsky), U01-AI35039; University of California, Los Angeles (Roger Detels), U01-AI35040; University of Pittsburgh (Charles Rinaldo), U01-AI35041; the Center for Analysis and Management of MACS, Johns Hopkins University Bloomberg School of Public Health (Lisa Jacobson), UM1-AI35043. LA Biomed CTSI funding source UL1TR000124 (Mallory Witt). The MACS is funded primarily by the National Institute of Allergy and Infectious Diseases (NIAID), with additional co-funding from the National Cancer Institute (NCI). MACS data collection is also supported by UL1 TR 001079 (JHU CTSA). Website located at http://www.statepi.jhsph.edu/macs/macs.html. The contents of this publication are solely the responsibility of the authors and do not represent the official views of the National Institutes of Health
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Disclosures: Dr. Jacobson is a consultant for Bristol-Myers Squibb. Dr. Palella is on the speaker’s bureau for Gilead Sciences, Janssen Pharmaceuticals, Merck & Co., Inc., and Bristol- Myers Squibb. Dr. Matthew Budoff has received grants from General Electric Company and National Institute of Health (NIH).
The other authors have no conflicts of interest to disclose.