Children with HIV living in developed countries can now expect to live with their disease for many years. Advances in early diagnosis and treatment and improved therapeutics against HIV are largely responsible for this longer life expectancy.1 However, improved survival has brought other medical risks associated with this chronic viral illness, and often a life-long exposure to antiretroviral medications has its own toxicities.
Abnormal metabolic conditions associated with HIV infection, and its treatments are some of the most prevalent problems in both adults and children. These conditions include hyperlipidemia, insulin resistance, type 2 diabetes mellitus, and body fat redistribution.2 As patients live longer with HIV, these conditions in adults are now contributing to more serious life-threatening disorders that are associated with atherosclerotic cardiovascular risk, such as myocardial infarction and stroke.3 Although HIV-infected children have risk factors for atherosclerotic cardiovascular disease,4 the prevalence of end-stage events (eg, overt cardiovascular disease) is not yet apparent, in part, because of insufficient follow-up, the overall protective effects of youth and fewer exposures to life-style risk factors (alcohol, smoking, physical inactivity).
The pathogenesis of atherosclerotic cardiovascular disease has been largely linked to a proinflammatory response in the arteries.5 Higher levels of C-reactive protein (CRP), indicating a proinflammatory state, have been associated with cardiovascular risk in adults without HIV infection and has been proposed for clinical use. However, several other biomarkers have also been associated with atherosclerotic cardiovascular risk, including biomarkers associated with inflammation, procoagulation, endothelial dysfunction, and metabolic dysregulation.6 Changes in any of these pathways may increase atherosclerotic cardiovascular risk, and emerging evidence indicates abnormalities in these pathways in HIV-infected adults. Whether these relationships are also found in children who have been exposed to a lifetime of HIV infection and its therapies is unknown.
We determined whether the levels of selected biomarkers associated with vascular inflammation pathways in HIV-infected children differed from those in a similar group of noninfected children. Furthermore, we determined whether there were any disease-specific or treatment-specific factors associated with higher levels of these biomarkers.
For this case-control study, cases were identified from all subjects with HIV infection who were enrolled in a National Heart, Lung, and Blood Institute-supported cohort study on cardiovascular risk in children with HIV infection being conducted at the University of Miami, Miami, FL, from December 2006, through March 2008. HIV infection was confirmed by chart review, documenting repeatedly positive serum enzyme-linked immunosorbent assays confirmed by western blot assays, repeatedly positive HIV RNA or DNA polymerase chain reaction assays, or by HIV culture. At the time of study, HIV-infected children did not have acute infectious illnesses.
A convenience sample of controls were identified from either the siblings of the HIV-infected children and youth or from an urban general pediatric outpatient program of the University of Miami that cares for children and youth of similar socioeconomic backgrounds. None of the controls were known to be HIV infected. Controls were excluded if they had any chronic illness or an acute infectious process.
The Institutional Review Board at the University of Miami approved the research protocol, and informed consent from the parent or guardian and assent from the patient (when appropriate) were obtained.
Clinical data for all children and youth (HIV-infected and controls) at the time of the study visit included age, sex, race, weight, height, and body mass index [BMI: calculated as weight (kg)/height2 (m2)]. Weight, height, and BMI were expressed as Z scores.7 For HIV-infected children, we recorded Center for Disease Control pediatric HIV disease stage,8 percentage of CD4% T-lymphocyte cells, and plasma HIV-1 RNA concentration by quantitative HIV-1 RNA polymerase chain reaction (Amplicor HIV-1 Monitor test, Roche Diagnostic Systems, Branchburg, NJ). Duration of antiretroviral therapy (ART) with nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors (PIs) was documented as the absolute amount of time the subject was exposed to each class of ART. Infected children also underwent dual X-ray absorptiometry testing to quantify body fat and its regionalization (GE/Lunar Prodigy, Madison, WI; enCORE 2006 software version 10.50.086) using standard methods.9 Waist and hip circumferences were measured using a nonstretchable plastic tape measure. Waist circumference was measured at the navel at the end of gentle exhalation. Hip circumference was measured at the maximal extension of the buttocks according to standard methods.10
Biomarkers of Vascular Dysfunction
Fibrinogen and CRP were measured by nephelometry on a Dade-Behring (Deerfield, IL) auto-analyzer using manufacturer's reagents and instructions. Intra-assay and interassay coefficients of variation were 2.6% and 2.7%, respectively, for fibrinogen and 4.4% and 5.7%, respectively, for CRP. Leptin was measured by a double antibody radioimmunoassay (Linco Research, St Charles, MO); intra-assay and interassay coefficients of variation were both less than 5%.
Interleukin-6 (IL-6), monocyte chemoattractant protein −1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM), soluble intracellular cell adhesion molecule-1 (sICAM), soluble E-selectin, and soluble P-Selectin were measured by ELISA using reagents manufactured by R&D Systems (Minneapolis, MN). Intra-assay and interassay coefficients of variation were, respectively; IL-6, 6.8% and 9.4%; MCP-1 4.0% and less than 7.5%; sVCAM-1, 5.9% and 10.2%; sICAM-1, 4.8% and 10.1%; E-Selectin, 5.0% and 8.8%; and P-Selectin, 4.2% and 9.8%.
Demographic and anthropometric characteristics of the HIV-infected children and controls were summarized using means and standard deviations or percentages, as appropriate, and compared using t tests and Fisher exact tests, respectively. Biomarkers for the 2 study groups were log-transformed because of right-skewed distributions, and the log-transformed values were used for all analyses.
Log-transformed biomarkers were compared between HIV-infected and control children using repeated measures linear regression. The regression model allowed for exchangeable correlation between siblings and was adjusted for sex, race/ethnicity (black, Hispanic, white/other), quartiles of age, and quartiles of BMI Z scores. For interpretability on the natural measured scale, the effect estimate from the regression model was exponentiated to produce a geometric mean. The geometric mean is similar to an ordinary mean but is less influenced by outliers. After adjustment for the regression covariates, the geometric mean represents the typical value of the inflammatory marker for an average child.
For the biomarkers that we found to be higher in the HIV-infected children than in controls, we wanted to further examine whether the extent of elevation was related to the extent of disease or to the extent of anthropometric changes linked to HIV. These analyses were carried out in the HIV children only and, because there were no siblings within the HIV cohort, simple correlation coefficients were initially used to measure the relationships between the log-transformed biomarker and each of the disease and anthropometric measures. From these initial results, we found 3 disease measures (CD4%; log-transformed viral load; and duration of highly active antiretroviral therapy (HAART) and 4 anthropometric measures (waist-to-hip ratio; dual X-ray absorptiometry testing body fat %; trunk fat %; and height Z score) that were considered for inclusion in multivariable models. For each log-transformed biomarker marker, a linear regression model was run that included sex, race/ethnicity, age, and the most significant disease and anthropometric measures. The effect estimates from the regression model were exponentiated to express the relative change in the biomarker that could be expected due to differences in the predictors.
Demographic and Clinical Characteristics
We enrolled 106 HIV-infected children and 55 normal control children (25 siblings of the cases and 30 unrelated) (Table 1). HIV infection was acquired perinatally in 101 subjects and horizontally in 5 subjects. Infected children were significantly older than controls (14.8 vs 12.3 years). Sex, weight, and BMI were similar between the groups, although the HIV group had slightly more Black non-Hispanics and were shorter than controls. Among children with HIV, 73% were Center for Disease Control stage B/C and had a median viral load of 882 copies/mL (interquartile range = 50-11,500); 86% were receiving HAART for a mean of 6 years.
Differences in Biomarkers of Vascular Dysfunction
Table 2 shows the comparison of vascular biomarkers between the HIV-infected group and controls. After adjustment for sex, race, age, and BMI, 2 biomarkers of endothelial dysfunction, sICAM and sVCAM, were greater in the HIV group than in the controls. There were also differences in measures of inflammation where IL-6, MCP-1, and CRP were all higher in the HIV group compared with controls, although the difference for CRP was not statistically significant (P = 0.08). Fibrinogen (a measure of coagulant dysfunction but also an acute phase reactant) was also significantly greater in the children with HIV infection.
Correlates of Elevated Biomarkers of Vascular Dysfunction
For the HIV-infected children only, we analyzed clinical correlates of 5 of the 9 biomarkers that differed significantly between groups (Table 3). Not only were there no significant differences between HIV and control children with respect to P-selectin, E-selectin, and leptin but within the cohort of HIV-infected children, there was no significant relationship to either % CD4 or viral load. We also analyzed CRP because the difference was almost significant and because it is easily measured in clinical settings. Results of the unadjusted correlations are shown in Table 3. Higher levels of sVCAM, a measure of endothelial dysfunction, correlated with greater disease severity (as defined by CD4 percent and viral load). Measures of inflammation (MCP-1, CRP) had greater correlations with anthropometric and body fat measures. For example, higher levels of MCP-1 correlated with greater waist to hip ratio and CRP correlated with higher body fat. Furthermore, higher measures of inflammation (MCP-1, CRP, and IL-6) also correlated with greater disease severity (lower CD4 percent), as examples. Higher levels of the procoagulant fibrinogen were associated with adiposity (waist to hip ratios and body fat percentage) and disease severity (CD4 percent). No significant relationships were found for age, weight, BMI, waist size, hip size, or any specific antiretroviral therapy (PI, nucleoside reverse transcriptase inhibitor, or nonnucleoside reverse transcriptase inhibitor).
Each of the 6 biomarkers of vascular dysfunction was then considered as an outcome in a multivariate analysis (Table 4). In general, there were few associations between any of these biomarkers and demographic variables, including age, sex, and race, with the exception the girls had 31% lower levels of sICAM than boys. Considering all anthropometric and body composition measures, waist to hip ratio was most consistently associated with higher levels of biomarkers for inflammation and endothelial dysfunction. For example, for each increase of 1 standard deviation in waist to hip ratio, sICAM increased by 17% and CRP by 59%. CD4% level, as a measure of disease severity, was most consistently associated with inflammation and procoagulation. Each increase of 1 standard deviation in CD4 percent was correlated with anywhere from an 18% (MCP-1) to 34% (CRP) lower level of inflammation. A higher viral load was more predictive of higher sVCAM (22% increase per 1 SD increase in viral load; P < 0.001).
Our study shows that HIV-infected children have higher levels of the biomarkers of vascular dysfunction than do an urban cohort of otherwise healthy children. These biomarkers have been associated with obesity, insulin resistance, and atherosclerotic cardiovascular risk in adults.6 In particular, biomarkers associated with inflammation, endothelial dysfunction, and procoagulation were higher in infected children than in controls. Clinical factors associated with these biomarkers of vascular dysfunction were more related to HIV disease severity (low CD4 counts and higher viral load) than to antiretroviral drug exposure. Furthermore, abdominal adiposity, as reflected by higher waist to hip ratio, was the best anthropometric correlate of these biomarkers.
Since the introduction of HAART, HIV disease in developed nations has transitioned from an almost uniformly fatal illness to a disease in which therapies are now targeted toward indefinite viral suppression.11 Although certain cardiovascular risk factors, such as hyperlipidemia, diabetes, and endothelial dysfunction, were present before the advent of HAART,12,13 they have increased both in prevalence and severity.14 These cardiovascular risks and metabolic complications have been better described in adults,2,3,14 more limited data are available on children.15-19 Potential contributors to increased cardiovascular risk in adults include HIV alone, specific types of HAART therapy, sex, life style habits (exercise, smoking), and successful viral suppression.3,20,21 In children, PI therapy is associated with several cardiovascular risk factors.4
For HIV-infected children, the true impact of these cardiovascular disease risk factors can only be appreciated after years of follow-up: most have not aged sufficiently to reach the associated endpoints. However, the multinational Data Collection on Adverse Events of Anti-HIV Drugs (DAD) Study Group in adults with HIV infection found an incidence of coronary heart disease of 3.5 per 1000 person-years and 11% of all recorded patient deaths caused by myocardial infarction, stroke, or other cardiovascular events.3 Relative risks up to 25% higher than those in the general population have been cited3,22 and substantiated.23,24
Several studies of subjects without HIV infection found that biomarkers of vascular dysfunction predict adverse cardiovascular events.25,26 Increasing evidence indicates the importance of vascular inflammation or dysfunction in the pathogenesis of cardiovascular disease in HIV-infected individuals.27 Vascular dysfunction may be a result of the direct cytopathic effects of the virus on the endothelial cell or of the inflammation and oxidative stress28,29 associated with chronic immune activation induced by the virus.30 Further, dysfunction may be induced by the metabolic consequences of the disease and its therapies or by a combination of both.
Several studies in adults report associations between biomarkers of endothelial dysfunction such as ICAM, VCAM, E-selectin, and von Willebrand Factor (VWF), and the platelet factor, P-selectin, among others, with HIV disease severity.31,32 These findings suggest that HIV itself may cause immune activation and be an important mechanism for cardiovascular risk,31 and effective antiretroviral therapy decreases these vascular inflammatory biomarkers.32 Finally, the HIV tat and nef proteins induce VCAM-1, ICAM-1, MCP-1, and other inflammatory chemokines that disrupt the vascular endothelium in coronary vessels.13,33,34 In addition, MCP-1 has been proposed to activate viral infection.35
Alternatively, HIV-infected patients have metabolic conditions often related to antiretroviral therapy, including dyslipidemia, insulin resistance, and abnormal fat distribution, that can contribute to the activation or injury of the endothelium.27 Certain antiretroviral agents also seem to damage endothelial mitochondrial DNA and are toxic to the endothelial cell itself, including the ability to induce apoptosis.36-38 PIs can increase mitochondrial production of reactive oxygen species,38 increase endothelial cell permeability,39 and leukocyte adhesion40 in cell culture. Johnson et al41 reported higher levels of pro-inflammatory cytokines in the subcutaneous adipose tissue of HIV-infected individuals with lipodystrophy, when compared with those without. Others have found associations between these biomarkers, HAART, and metabolic dysfunction.42 Thus, antiretroviral therapy could directly or indirectly (through changes in the metabolic profile) increase levels of these biomarkers.
Studies on vascular inflammatory pathways and vascular dysfunction (ie, vessel compliance, distensibility, and structure) in HIV-infected children have been limited.43-46 McComsey et al46 showed greater carotid intima-media thickness and higher levels of certain biomarkers among 31 HIV-infected children. A study of 49 children found that vascular dysfunction (stiffness) was greater in HIV-infected children than in controls, independent of known cardiovascular risk factors and antiretroviral therapy.43 However, other studies show carotid intima thicknesses are similar to controls.46 Charakida et al45 found among 83 HIV-infected children, carotid intima thickness was greater than it was in controls, as were the abnormalities in flow-mediated vasodilation. These differences were more pronounced in children receiving PIs, and lipid abnormalities did not account for these differences. Interestingly, some pre-HAART studies in children showed increased coronary artery calcifications,47 suggesting the contribution of baseline immune activation to cardiovascular risk.
Limitations of the Study
As with many studies of cardiovascular risk in children, finding risk may not always equate to adverse outcomes. Only continued longitudinal follow-up of children will definitively confirm that our findings translate to cardiovascular disease events. Although we tried to limit the number of outcomes to those that differed between cases and controls and the number of predictors that were linked to progressive HIV disease, we cannot rule out false positive findings secondary to multiple statistical testing. Confirmatory studies are needed. We have not evaluated the association of other metabolic parameters (lipids, insulin resistance) or other known risk factors such as family history, smoking exposure, and alcohol intake on our outcomes because of incomplete data on the entire sample. These metabolic factors and health behaviors need to be critically evaluated as potential causative factors in the future. However, early data in another study48 suggest that there are minimal relationships. Finally, our control group was slightly unbalanced in age and race. However, all of our analyses were adjusted for these demographic factors.
We evaluated early mechanistic factors for cardiovascular disease in children with HIV infection. Elevations of the biomarkers of vascular dysfunction in HIV-infected children provide strong evidence of ongoing cardiovascular risk in this population. Disease severity and truncal adiposity were both independently associated with higher levels of these biomarkers of vascular dysfunction, suggesting that these biomarkers and other cardiovascular risk factors should be closely monitored in children with those characteristics. There were no associations with antiretroviral therapy. Long-term evaluation of cardiovascular risk, incorporating complete metabolic screens, and interventions to modify this risk should be considered.
1. Laufer M, Scott GB. Medical management of HIV disease in children. Pediatr Clin North Am
2. Carr A, Samaras K, Chisholm DJ, et al. Abnormal fat distribution and use of protease inhibitors. Lancet
3. Friis-Moller N, Sabin CA, Weber R, et al. Combination antiretroviral therapy and the risk of myocardial infarction. N Engl J Med
4. Miller TL, Orav EJ, Lipshultz SE, et al. Risk factors for cardiovascular disease in children infected with human immunodeficiency virus-1. J Pediatr
5. Libby P. Inflammation in atherosclerosis. Nature
6. Ikonomidis I, Stamatelopoulos K, Lekakis J, et al. Inflammatory and non-invasive vascular markers: the multimarker approach for risk stratification in coronary artery disease. Atherosclerosis
7. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, et al. CDC growth charts: United States. Adv Data
8. Caldwell MB, Oxtoby MJ, Simonds RJ, et al. 1994 Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR CDC Surveill Summ
9. Margulies L, Horlick M, Thornton JC, et al. Reproducibility of pediatric whole body bone and body composition measures by dual-energy X-ray absorptiometry using the GE Lunar Prodigy. J Clin Densitom
11. Powderly WG, Landay A, Lederman MM. Recovery of the immune system with antiretroviral therapy: the end of opportunism? JAMA
12. Teitel JM, Shore A, Read SE, et al. Immune function of vascular endothelial cells is impaired by HIV. J Infect Dis
13. Dhawan S, Puri RK, Kumar A, et al. Human immunodeficiency virus-1-tat protein induces the cell surface expression of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in human endothelial cells. Blood
14. Fisher SD, Miller TL, Lipshultz SE. Impact of HIV and highly active antiretroviral therapy on leukocyte adhesion molecules, arterial inflammation, dyslipidemia, and atherosclerosis. Atherosclerosis
15. Bitnun A, Sochett E, Dick PT, et al. Insulin sensitivity and beta-cell function in protease inhibitor-treated and -naive human immunodeficiency virus-infected children. J Clin Endocrinol Metab
16. Miller TL. Nutritional aspects of HIV-infected children receiving highly active antiretroviral therapy. AIDS
. 2003;17(Suppl 1):S130-S140.
17. Arpadi SM, Bethel J, Horlick M, et al. Longitudinal changes in regional fat content in HIV-infected children and adolescents. AIDS
18. Miller TL, Grant YT, Almeida DN, et al. Cardiometabolic disease in human immunodeficiency virus-infected children. J Cardiometab Syndr
19. Miller TL, Mawn BE, Orav EJ, et al. The effect of protease inhibitor therapy on growth and body composition in human immunodeficiency virus type 1-infected children. Pediatrics
20. Hadigan C, Meigs JB, Corcoran C, et al. Metabolic abnormalities and cardiovascular disease risk factors in adults with human immunodeficiency virus infection and lipodystrophy. Clin Infect Dis
21. Grinspoon S, Carr A. Cardiovascular risk and body-fat abnormalities in HIV-infected adults. N Engl J Med
22. Friis-Moller N, Reiss P, Sabin CA, et al. Class of antiretroviral drugs and the risk of myocardial infarction. N Engl J Med
23. Martinez E, Larrousse M, Gatell JM. Cardiovascular disease and HIV infection: host, virus, or drugs? Curr Opin Infect Dis
24. 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
25. Ridker PM, Hennekens CH, Buring JE, et al. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med
26. Hwang SJ, Ballantyne CM, Sharrett AR, et al. Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study. Circulation
27. Grinspoon SK, Grunfeld C, Kotler DP, et al. State of the science conference: Initiative to decrease cardiovascular risk and increase quality of care for patients living with HIV/AIDS: executive summary. Circulation
28. Pace GW, Leaf CD. The role of oxidative stress in HIV disease. Free Radic Biol Med
29. Lo J, Grinspoon S. Cardiovascular disease in HIV-infected patients: does HIV infection in and of itself increase cardiovascular risk? Curr Opin HIV AIDS
30. Triant VA, Meigs JB, Grinspoon SK. Association of C-reactive protein and HIV infection with acute myocardial infarction. J Acquir Immune Defic Syndr
31. de Larranaga GF, Bocassi AR, Puga LM, et al. Endothelial markers and HIV infection in the era of highly active antiretroviral treatment. Thromb Res
32. Wolf K, Tsakiris DA, Weber R, et al. Antiretroviral therapy reduces markers of endothelial and coagulation activation in patients infected with human immunodeficiency virus type 1. J Infect Dis
33. Swingler S, Mann A, Jacque J, et al. HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages. Nat Med
34. Cota-Gomez A, Flores NC, Cruz C, et al. The human immunodeficiency virus-1 Tat protein activates human umbilical vein endothelial cell E-selectin expression via an NF-kappa B-dependent mechanism. J Biol Chem
35. Vicenzi E, Alfano M, Ghezzi S, et al. Divergent regulation of HIV-1 replication in PBMC of infected individuals by CC chemokines: suppression by RANTES, MIP-1alpha, and MCP-3, and enhancement by MCP-1. J Leukoc Biol
36. Zhong DS, Lu XH, Conklin BS, et al. HIV protease inhibitor ritonavir induces cytotoxicity of human endothelial cells. Arterioscler Thromb Vasc Biol
37. Jiang B, Hebert VY, Li Y, et al. HIV antiretroviral drug combination induces endothelial mitochondrial dysfunction and reactive oxygen species production, but not apoptosis. Toxicol Appl Pharmacol
38. Hebert VY, Crenshaw BL, Romanoff RL, et al. Effects of HIV drug combinations on endothelin-1 and vascular cell proliferation. Cardiovasc Toxicol
39. Chen C, Lu XH, Yan S, et al. HIV protease inhibitor ritonavir increases endothelial monolayer permeability. Biochem Biophys Res Commun
40. Mondal D, Pradhan L, Ali M, et al. HAART drugs induce oxidative stress in human endothelial cells and increase endothelial recruitment of mononuclear cells: exacerbation by inflammatory cytokines and amelioration by antioxidants. Cardiovasc Toxicol
41. Johnson JA, Albu JB, Engelson ES, et al. Increased systemic and adipose tissue cytokines in patients with HIV-associated lipodystrophy. Am J Physiol Endocrinol Metab
42. de Gaetano Donati K, Rabagliati R, Tumbarello M, et al. Increased soluble markers of endothelial dysfunction in HIV-positive patients under highly active antiretroviral therapy. AIDS
43. Bonnet D, Aggoun Y, Szezepanski I, et al. Arterial stiffness and endothelial dysfunction in HIV-infected children. AIDS
44. Giuliano Ide C, de Freitas SF, de Souza M, et al. Subclinic atherosclerosis and cardiovascular risk factors in HIV-infected children: PERI study. Coron Artery Dis
45. Charakida M, Donald AE, Green H, et al. Early structural and functional changes of the vasculature in HIV-infected children: impact of disease and antiretroviral therapy. Circulation
46. McComsey GA, O'Riordan M, Hazen SL, et al. Increased carotid intima media thickness and cardiac biomarkers in HIV infected children. AIDS
47. Perez-Atayde AR, Kearney DI, Bricker JT, et al. Cardiac, aortic, and pulmonary arteriopathy in HIV-infected children: the Prospective P2C2 HIV Multicenter Study. Pediatr Dev Pathol
48. Miller T, Jacobson D, Mendez A, et al. Biomarkers of vascular dysfunction in HIV-infected children with and without hyperlipidemia. Presented at: 16th Conference on Retroviruses and Opportunistic Infections (CROI 2009); February 8-11, 2009; Montreal, Canada. 2009. Abstract #917.