Abstract: This is a 96-week prospective cohort study of antiretroviral therapy (ART)–naive HIV-infected adults and matched healthy controls to assess progression of carotid intima media thickness (CIMT) and its relationship to inflammation. Median common carotid artery (CCA) CIMT increased significantly but similarly in both groups [CCA: 0.02 (interquartile range: 0–0.05); P < 0.01 within HIV-infected adults vs. 0.01 (0–0.05) mm; P < 0.01 within controls; and P = 0.83 between groups]. Change in bulb CIMT yielded similar results. Independent predictors of CCA CIMT progression in HIV-infected adults were higher systolic blood pressure, total cholesterol, and high sensitivity C-reactive protein. Independent predictors of bulb CIMT progression were higher non–high-density lipoprotein cholesterol and high sensitivity C-reactive protein. Other inflammation markers were not associated with CIMT progression.
*Case Western Reserve University School of Medicine, Cleveland, OH;
†Department of Medicine, Division of Infectious Disease, MetroHealth Medical Center, Cleveland, OH;
‡Department of Medicine, Division of Cardiovascular Medicine, University Hospitals Harrington Heart and Vascular Institute, Cleveland, OH; and
§Department of Pediatrics, Division of Infectious Disease, University Hospitals Case Medical Center, Cleveland, OH.
Correspondence to: Grace A. McComsey, MD, Case Western Reserve University, 11100 Euclid Ave, Cleveland, OH 44106 (e-mail: firstname.lastname@example.org).
This study was sponsored by Bristol Myers Squibb.
The results of this study were previously presented at the 20th Conference on Retroviruses and Opportunistic infections, March 3–6, 2013, Atlanta, GA.
C.T.L. has received research grant support from Bristol-Myers Squibb and the Medtronic Foundation. T.L.C. serves on the DSMB of Prairie Education and Research Cooperative. G.A.M. has served as a scientific advisor or speaker for Bristol-Myers Squibb, Merck, and Gilead Sciences; has received research grants from Bristol-Myers Squibb, GlaxoSmithKline, and Gilead Sciences; and is currently serving as the DSMB Chair for a Pfizer-sponsored study.
Received November 03, 2013
Accepted November 16, 2013
With potent antiretroviral therapy (ART), people are living with HIV longer than ever before.1–3 With this longevity, comorbidities including cardiovascular disease (CVD) have become increasingly prevalent.4 Further, HIV-infected individuals are at higher risk for CVD events,5 and this risk seems to accelerate faster with age than in the general population.6 Understanding the pathogenesis behind this accelerated risk is important in determining targets to ameliorate this risk.
Although traditional CVD risk factors have been shown to play an independent role,7 there is still controversy regarding the relative degree that ART plays in the pathogenesis of CVD in HIV. Conducting longitudinal studies in ART-naive patients is challenging; however, it is the only way to dissect the effects of HIV and of ART.
The purpose of this study was not only to compare the progression of CIMT between HIV-infected, ART-naive adults and healthy controls but also to explore the relationship between CIMT progression and markers of inflammation, endothelial cell activation, and coagulation in both groups. Our hypothesis was that CIMT would progress faster in HIV-infected ART-naive adults than in healthy controls, and greater CIMT progression would be associated with higher markers of inflammation.
The Intima Media Thickness Progression Study is a single site prospective cohort study designed to compare changes in CIMT and markers of inflammation in ART-naive HIV-infected adults before and after initiating contemporary ART and healthy HIV-uninfected controls matched by age, sex, and body mass index (BMI). Eligibility criteria have been described previously.8 Briefly, HIV-1–infected adults aged ≥18 years, ART naive, and unlikely to require ART for at least 48 weeks based on ART treatment guidelines at the time (ART initiation at CD4+ T-cell count ≤350 cells/mm3)9 were eligible for the HIV-infected group. Controls were adults aged ≥18 years without known HIV infection or medical conditions requiring prescription medications with the exception of stable hypertension defined as stable antihypertensive medications for ≥3 months. Exclusion criteria for both groups were CVD, diabetes mellitus defined as fasting glucose >126 mg/dL or use of hypoglycemic medication, active infectious or inflammatory condition, pregnancy or breastfeeding. Each participant in the control group was matched by age within 3 years, sex, and BMI within 3 kg/m2 to a previously enrolled HIV-infected participant. For this analysis, all HIV-infected adults who remained ART naive through 96 weeks and all HIV-uninfected participants were included. The primary outcome for this study was change in CIMT over 96 weeks.
High-resolution ultrasound scans of the carotid arteries were performed at entry and week 96 using a Philips iU22 ultrasound system with a L9-3 MHz linear array transducer according to American Society of Echocardiography guidelines.10 R-wave gated still frame images of the distal 1 cm of the common carotid artery (CCA) far wall were obtained at 3 separate angles bilaterally (anterior, lateral, and posterior) and of the carotid bulb bilaterally. CIMT was measured offline by 2 readers (T.L.C. and C.T.L.) who were blinded to the participant’s HIV status using semiautomated edge detection software (Medical Imaging Applications LLC, Coralville, IA). The mean–mean CIMT of the CCA (6 segments) and bulb (2 segments) were examined in separate analyses. Interreader variability was excellent (concordance correlation coefficient 0.943, 95% confidence interval: 0.918 to 0.960).
Evaluation of Glucose Metabolism, Lipoproteins, and Biomarkers
Participants had blood drawn fasting at entry and week 96 with real-time measurements of glucose, insulin, and lipoproteins. Plasma samples from each participant stored at −80°C were batched and tested for inflammation markers including high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), soluble tumor necrosis factor α receptors (sTNFR-l and sTNFR-II), endothelial activation markers including soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1), and coagulation markers including D-Dimer and fibrinogen as previously described.8,11–14
All demographic, HIV and CVD risk factors, and endpoints were compared between groups using unpaired t tests or Wilcoxon rank sum tests for continuous variables, and y using χ2 tests, Fisher exact tests, or Pearson exact χ2 tests for categorical variables. Within-group changes were tested using paired t tests or Wilcoxon signed rank tests. All statistical tests were 2-sided and considered significant with P < 0.05. Analysis of covariance was used to adjust mean changes in log-transformed CCA and bulb CIMT for clinically important variables known to effect CVD risk—age, sex, race, smoking, BMI, family history of myocardial infarction, systolic blood pressure, and baseline CIMT.
For the HIV-infected and control groups separately, multivariable linear regression was used to explore relationships between change in log-transformed CCA and bulb CIMT and log-transformed markers of inflammation, endothelial activation and coagulation, and CVD risk factors in 2 separate models. In the HIV-infected group, nadir CD4+, HIV-1 RNA level, and known duration of HIV (years since HIV diagnosis) were also considered. Stepwise selection was used to generate final models. All analyses were performed using SAS v. 9.2 (The SAS Institute, Carey, NC).
From July 2, 2008, to April 30, 2010, 130 participants (85 HIV-infected adults and 45 matched controls) were enrolled. For this analysis, 83 participants were included (42 ART-naive HIV-infected adults and 41 controls). Baseline and 48-week results have been published previously.8
Overall, the 2 groups were similar with regard to baseline demographic and cardiovascular factors, except that the HIV-infected group had more African Americans (69% vs. 27%), smokers (67% vs. 17%), and lower total cholesterol (162 vs. 194 mg/dL), non–high-density lipoprotein (HDL) cholesterol (115 vs. 142 mg/dL), and HDL cholesterol levels (42 vs. 48 mg/dL). Overall, 69% were men, median (interquartile range) age was 39.6 (31.2–47.7) years, median BMI was 27.3 (25–30.7) kg/m2 and waist to hip ratio was 0.93 (0.87–0.98). Sixteen percent of all participants were on antihypertensive medication. One control participant was on a statin at entry. The median Framingham risk score was 1% (1%–4%).
In the HIV-infected group, the median baseline and nadir CD4+ T cells were 630 (507–812) and 502 (444–590) cells per cubic millimeter, respectively. The median HIV-1 RNA level was 4900 (430–14400) copies per milliliter, and time since HIV diagnosis was 4.8 (1.9–9.7) years. The median change in CD4+ T cells and HIV-1 RNA level over 96 weeks was −82 (−162 to −13; P = 0.0004) cells per cubic millimeter and 340 (−1773 to 14,223; P = 0.14) copies per milliliter in this group. Compared with the ART-naive HIV-infected individuals included in this analysis, the HIV-infected participants in the Intima Media Thickness Progression Study who initiated ART before week 96 (32 of 85 HIV-infected participants enrolled) had lower baseline and nadir CD4+ T cells (509 and 400 cells/mm3, respectively).
There were no differences in baseline CCA or bulb CIMT between ART-naive HIV-infected and control participants. Over 96 weeks, CIMT at both the CCA and bulb sites changed significantly, but similarly in both groups (Table 1). Adjustment for CVD risk factors did not change the between-group results.
Of the eight biomarkers tested, IL-6, sTNFR-II, sVCAM-1, and sICAM-1 were higher in the HIV-infected group at both time points and D-Dimer was higher at week 96 (P < 0.01 for all, except week 96 IL-6, P = 0.02). sTNFR-I and sTNFR-II increased over 96 weeks in the HIV-infected group (P < 0.05 for both), whereas sVCAM-1 and sICAM-1 both decreased (P < 0.05 for both). There were no between-group differences with regard to the changes in the biomarkers over 96 weeks (Table 1).
Table 2 shows the results of multivariable linear regression for change in CIMT over 96 weeks at the CCA and bulb sites in the HIV-infected group. In the HIV-infected group, higher systolic blood pressure, total cholesterol, and hsCRP were independently associated with greater CCA CIMT progression; higher non–HDL cholesterol and hsCRP were independently associated with greater bulb CIMT progression. In the HIV-uninfected participants, hsCRP was not associated with CCA or bulb CIMT progression.
In this 96-week prospective cohort study, we report for the first time that there is no difference in CIMT progression at the CCA or bulb between HIV-infected ART-naive adults and matched healthy controls. Importantly, hsCRP was the only measured marker independently associated with CCA and bulb CIMT progression in the HIV-infected group.
We had hypothesized that CIMT progression would be greater in ART-naive HIV-infected adults versus controls; however, in this study, the first to our knowledge involving solely ART-naive adults and HIV-uninfected controls matched by age, sex, and BMI, we found the contrary. Several explanations for the lack of difference in CIMT progression are possible. The duration of this study is relatively short, although prior studies showing faster progression in ART-treated HIV-infected adults were as short as 48 weeks.15 Also, our study participants had a relatively short duration of known HIV infection, a commonly encountered observation in studies involving ART-naive individuals.7,16 We have shown that baseline inflammatory (IL-6 and sTNFR-II) and endothelial activation markers (sVCAM-1 and sICAM-1) were higher in the ART-naive HIV-infected group. Therefore, perhaps over a longer duration of untreated HIV infection, a difference in CIMT progression would be seen. Additionally, our study participants, similarly to other ART-naive studies,7 were relatively young, had low CVD risk with a Framingham risk score of median (interquartile range) 1 (1–4) and low baseline CIMT (Table 1). This is in contrast with studies of ART-experienced participants such as that by Hsue et al,15 where baseline mean CIMT in the ART-experienced cohort (n = 121) was 0.93 mm, considerably higher than in our study, which may be explained by either longer duration of HIV and consequent heightened inflammation, or by the effects of ART and consequent negative effects on lipids, visceral adiposity, and insulin resistance. In a study by Currier et al,17 participants with baseline median CIMTs, comparable with those in our population, had a similarly slower rate of CIMT progression (0.0096, 0.0058, and 0.0085 mm per year) with no difference between groups.18 This, along with our study, suggests that CIMT progression may be dependent on CVD risk factors and baseline CIMT values.
An important finding of our study is that in addition to usual CVD risk factors, that is, higher systolic blood pressure and higher total and non–HDL cholesterol, higher baseline hsCRP was associated with greater CIMT progression at the CCA and bulb sites in the ART-naive HIV-infected group. The finding that hsCRP predicts CIMT progression has been reported in ART-experienced individuals,19 but we extend these observations to ART-naive HIV-infected adults as well. In HIV, higher CRP levels have been associated with progression to AIDS and mortality in a wide variety of settings20–25 and have been predictive of clinical cardiovascular events.26,27 In the general population, hsCRP is a well-established predictor for cardiovascular outcomes28,29 with practice guidelines suggesting routine screening for patients at intermediate risk for CVD determined by global risk assessment.30,31 Whether hsCRP is merely a marker of increased CVD risk or in the causal pathway is a subject of ongoing debate.32–34 There is biologic plausibility for why CRP would be higher in those who have more rapid progression of atherosclerosis. Deposits of CRP have been demonstrated in foam cells and colocalized with complement within the intima layer of early atherosclerotic lesions in human coronary arteries.35 Further, although CRP is primarily synthesized by hepatocytes, arterial tissue itself has been shown to produce CRP.36 Also, CRP has been shown to modulate the inflammatory response of vascular cells through the NF-κB pathway37 and decrease vasoreactivity in hypercholesterolemic patients.38 It stands to reason that chronic inflammation and immune activation in HIV would lead to faster progression of atherosclerosis.
Limitations of this study include a sample size that precluded adjustment for all clinically important CVD risk factors in the multivariable analyses. Additionally, although the results of this study may be generalizable to those adults with high CD4+ counts at diagnosis, these results may not be applicable to those individuals with CD4+ counts ≤350 cells per cubic millimeter who may have had a longer duration of uncontrolled systemic inflammation. For this study we did not collect peripheral blood mononuclear cells, thus could not determine expression of activated T cells or monocytes which may shed further light into the association of hsCRP with CIMT progression.
In conclusion, 96-week CIMT progression was not different between ART-naive HIV-infected adults and HIV-uninfected controls. Increased baseline hsCRP was independently predictive of CIMT progression at the CCA and bulb sites in the HIV-infected group supporting the role of inflammation in the progression of atherosclerosis.
The authors would like to thank the patients who participated in this research.
1. Wada N, Jacobson LP, Cohen M, et al.. Cause-specific life expectancies after 35 years of age for human immunodeficiency syndrome-infected and human immunodeficiency syndrome-negative individuals followed simultaneously in long-term cohort studies, 1984–2008. Am J Epidemiol. 2013;177:116–125.
2. Puhan MA, Van Natta ML, Palella FJ, et al.. Excess mortality in patients with AIDS in the era of highly active antiretroviral therapy: temporal changes and risk factors. Clin Infect Dis. 2010;51:947–956.
3. Life expectancy of individuals on combination antiretroviral therapy in high-income countries: a collaborative analysis of 14 cohort studies. Lancet. 2008;372:293–299.
4. Krentz HB, Kliewer G, Gill MJ. Changing mortality rates and causes of death for HIV-infected individuals living in Southern Alberta, Canada from 1984 to 2003. HIV Med. 2005;6:99–106.
5. Triant VA, Lee H, Hadigan C, et al.. Increased acute myocardial infarction rates and cardiovascular risk factors among patients with human immunodeficiency virus disease. J Clin Endocrinol Metab. 2007;92:2506–2512.
6. Petoumenos K, El-Sadr W, Monforte A, et al.. Increased risk of cardiovascular disease with age in men: a comparison of D:A:D with HIV- cardiovascular disease risk equations. Presented at: 20th Conference on Retroviruses and Opportunistic Infections; March 3–6, 2013; Atlanta, Georgia.
7. Stein JH, Brown TT, Ribaudo HJ, et al.. Ultrasonographic measures of cardiovascular disease risk in antiretroviral treatment-naive individuals with HIV infection. AIDS. 2013;27:929–937.
8. Hileman CO, Carman TL, Longenecker CT, et al.. Rate and predictors of carotid artery intima media thickness progression in antiretroviral-naive HIV-infected and uninfected adults: a 48-week matched prospective cohort study. Antivir Ther. 2013;18:921–929.
10. Stein JH, Korcarz CE, Hurst RT, et al.. Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media Thickness Task Force. Endorsed by the Society for Vascular Medicine. J Am Soc Echocardiogr. 2008;21:93–111; quiz 189–190.
11. Hileman C, Longenecker C, Carman T, et al.. Relationship between total bilirubin and endothelial function, inflammation and oxidative stress in HIV-infected adults on stable antiretroviral therapy. HIV Med. 2012;13:609–616.
12. Hileman CO, Carman TL, Storer NJ, et al.. Omega-3 fatty acids do not improve endothelial function in virologically suppressed HIV-infected men: a randomized placebo-controlled trial. AIDS Res Hum Retroviruses. 2012;28:649–655.
13. Hileman CO, Longenecker CT, Carman TL, et al.. Elevated D-dimer is independently associated with endothelial dysfunction: a cross-sectional study in HIV-infected adults on antiretroviral therapy. Antivir Ther. 2012;17:1345–1349.
14. Ross AC, Judd S, Kumari M, et al.. Vitamin D is linked to carotid intima-media thickness and immune reconstitution in HIV-positive individuals. Antivir Ther. 2011;16:555–563.
15. Hsue PY, Lo JC, Franklin A, et al.. Progression of atherosclerosis as assessed by carotid intima-media thickness in patients with HIV infection. Circulation. 2004;109:1603–1608.
16. da Silva EF, Fonseca FA, Franca CN, et al.. Imbalance between endothelial progenitors cells and microparticles in HIV-infected patients naive for antiretroviral therapy. AIDS. 2011;25:1595–1601.
17. Currier JS, Kendall MA, Zackin R, et al.. Carotid artery intima-media thickness and HIV infection: traditional risk factors overshadow impact of protease inhibitor exposure. AIDS. 2005;19:927–933.
18. Currier JS, Kendall MA, Henry WK, et al.. Progression of carotid artery intima-media thickening in HIV-infected and uninfected adults. AIDS. 2007;21:1137–1145.
19. Hsue PY, Scherzer R, Hunt PW, et al.. Carotid intima-media thickness progression in HIV-infected adults occurs preferentially at the carotid bifurcation and is predicted by inflammation. J Am Heart Assoc. 2012;1:e000422.
20. Boulware DR, Hullsiek KH, Puronen CE, et al.. Higher levels of CRP, D-dimer, IL-6, and hyaluronic acid before initiation of antiretroviral therapy (ART) are associated with increased risk of AIDS or death. J Infect Dis. 2011;203:1637–1646.
21. Feldman JG, Goldwasser P, Holman S, et al.. C-reactive protein is an independent predictor of mortality in women with HIV-1 infection. J Acquir Immune Defic Syndr. 2003;32:210–214.
22. Lau B, Sharrett AR, Kingsley LA, et al.. C-reactive protein is a marker for human immunodeficiency virus disease progression. Arch Intern Med. 2006;166:64–70.
23. Tien PC, Choi AI, Zolopa AR, et al.. Inflammation and mortality in HIV-infected adults: analysis of the FRAM study cohort. J Acquir Immune Defic Syndr. 2010;55:316–322.
24. Ledwaba L, Tavel JA, Khabo P, et al.. Pre-ART levels of inflammation and coagulation markers are strong predictors of death in a South African cohort with advanced HIV disease. PLoS One. 2012;7:e24243.
25. Mangili A, Polak JF, Quach LA, et al.. Markers of atherosclerosis and inflammation and mortality in patients with HIV infection. Atherosclerosis. 2011;214:468–473.
26. Duprez DA, Neuhaus J, Kuller LH, et al.. Inflammation, coagulation and cardiovascular disease in HIV-infected individuals. PLoS One. 2012;7:e44454.
27. Triant VA, Meigs JB, Grinspoon SK. Association of C-reactive protein and HIV infection with acute myocardial infarction. J Acquir Immune Defic Syndr. 2009;51:268–273.
28. Danesh J, Wheeler JG, Hirschfield GM, et al.. C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med. 2004;350:1387–1397.
29. Kaptoge S, Di Angelantonio E, Pennells L, et al.. C-reactive protein, fibrinogen, and cardiovascular disease prediction. N Engl J Med. 2012;367:1310–1320.
30. Greenland P, Alpert JS, Beller GA, et al.. 2010 ACCF/AHA guideline for assessment of cardiovascular risk in asymptomatic adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2010;56:e50–e103.
31. Pearson TA, Mensah GA, Alexander RW, et al.. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation. 2003;107:499–511.
32. Bisoendial RJ, Boekholdt SM, Vergeer M, et al.. C-reactive protein is a mediator of cardiovascular disease. Eur Heart J. 2010;31:2087–2091.
33. Bisoendial RJ, Kastelein JJ, Stroes ES. C-reactive protein and atherogenesis: from fatty streak to clinical event. Atherosclerosis. 2007;195:e10–e18.
34. Hirschfield GM, Pepys MB. C-reactive protein and cardiovascular disease: new insights from an old molecule. QJM. 2003;96:793–807.
35. Torzewski J, Torzewski M, Bowyer DE, et al.. C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries. Arterioscler Thromb Vasc Biol. 1998;18:1386–1392.
36. Yasojima K, Schwab C, McGeer EG, et al.. Generation of C-reactive protein and complement components in atherosclerotic plaques. Am J Pathol. 2001;158:1039–1051.
37. Devaraj S, Kumaresan PR, Jialal I. Effect of C-reactive protein on chemokine expression in human aortic endothelial cells. J Mol Cell Cardiol. 2004;36:405–410.
38. Bisoendial RJ, Kastelein JJ, Peters SL, et al.. Effects of CRP infusion on endothelial function and coagulation in normocholesterolemic and hypercholesterolemic subjects. J Lipid Res. 2007;48:952–960.