Because patients treated with antiretroviral drugs live longer and have to deal with the complications of aging, much attention is turning to the so called “non-AIDS”–related pathologies; a group of conditions generally associated to aging, including cardiovascular disease (CVD), renal impairment, hepatic disease, osteoporosis, and non-AIDS–defining malignancies.1 As the mean age of HIV-infected individuals is progressively increasing, CVD is likely to gain further importance as a cause of mortality in years to come.2 Despite being the focus of intense investigation, the etiology of the increased cardiovascular risk in this population remains unclear, most probably because of a multifactorial pathophysiology.1 Indeed, it is difficult to fully isolate the weight of the different proatherogenic factors in the presence of classical CVD risk factors, which are frequently overrepresented in HIV subjects with respect to the general population. In fact, classical CVD risk factors might be overshadowing the influence of other risk factors driving atherosclerosis within HIV infection.3,4 Although this problem is of extraordinary complexity, many pathways have already been described; among them, there is mounting evidence to support that inflammation and immune activation secondary to the infection are likely to be major drivers of atherosclerosis in HIV-infected patients.5,6 In this context, the study of subclinical atherosclerosis in children and adolescents offers the opportunity to clarify the specific role of antiretroviral treatment (ART), HIV infection, inflammation, T-cell activation, and senescence on the atherogenic process in the absence of classical CVD risk factors. Previous studies have focused on this population using the intima media thickness (IMT) as a surrogate marker of CVD risk and inflammatory biomarkers such as high-sensitivity C-reactive protein (CRP) in rather small cohorts with controversial findings.7–9 Besides, it has been described that HIV-infected children present higher frequencies of activated and senescent CD8+ and CD4+ T-cell subsets,10,11 although its association to disease progression during childhood has been questioned.12 No studies to date have addressed the association between subclinical atherosclerosis and immune activation in this setting. To provide new insight into the relation between subclinical atherosis and HIV-associated variables, chronic inflammation, and immune activation, we performed a cross-sectional analysis in a large cohort of vertically HIV-infected children and adolescents and healthy controls.
Study Design and Eligibility Criteria
We performed a cross-sectional analysis from an ongoing prospective, longitudinal, multicenter observational study evaluating cardiovascular risk in a cohort of HIV-infected children and healthy uninfected controls. Study participants are children and adolescents attending the clinics of 6 different hospitals integrated in the Madrid Cohort of Pediatric HIV-infected children and adolescents. Participants were recruited between June and December 2011. Exclusion criteria included acute or opportunistic infections, chronic inflammatory diseases, diabetes, kidney disease, hypertension, and family history of premature CVD. Healthy volunteers were prospectively enrolled as controls from healthy siblings of the HIV-infected patients, uninfected children born to HIV-infected mothers, healthy volunteers from a high school in the same urban area, and children attending the laboratory for minor surgery purposes or the general pediatric clinics. Additional exclusion criteria for healthy controls included current infectious or inflammatory illnesses, chronic conditions, and current use of medications. Controls were included with the goals of achieving a group with similar age, sex, ethnicity, and body mass index (BMI) (±1 kg/m2).
The study was reviewed and approved by the ethics committee and clinical research of the 6 participating hospitals. All participants, parents, or legal guardians and children older than 12 years gave written informed consent to take part in the study.
Data were collected prospectively, from an interview with the patient's family and the managing pediatrician when necessary, together with a thorough revision of medical records. All children underwent physical examination, including anthropometric and blood pressure measurement, after recommendations of the American Heart Association.13 Weight, height, BMI, and hypertension were adjusted using z score according to the age and gender.14,15 Immunovirological details and previous ART history were collected from the Cohort of Madrid collaborative Pediatric HIV Study database. Time with detectable viral load (VL) summarizes total time (years) with detectable VL during patient's life span. To study the effect of treatment, years of antiretroviral exposure have been analyzed as a continuous variable.
Fasting blood samples were drawn from the 150 patients and 97 controls for real-time measurements of insulin and glucose levels and lipid profile [total cholesterol, high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol] that were determined in the different participating hospitals using standard enzymatic methods. Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR = fasting insulin (microU/mL) × fasting glucose (g/dL)/405).16
In the HIV-infected group, plasma HIV-1 VL was quantified using the Cobas TaqMan HIV-1 assay (Roche Diagnostics Systems, Inc, Branchburg, NJ) with a detection limit of 50 copies per cubic millimeter.
Absolute and percentage of CD4 and CD8 T-cell counts were concomitantly measured with standard flow cytometric methods. A sample of every participant was sent to the Pediatric HIV BioBank integrated in the Spanish AIDS Research Network (RIS) processed and stored at −80° using standard procedures for subsequent determinations.17
T-cell activation and senescence were measured by immunophenotyping performed at the Immune-Biology Laboratory of the Gregorio Marañón Hospital, from fresh samples or cryopreserved peripheral blood mononuclear cells, thawed using methods that have been optimized and validated. Peripheral blood mononuclear cells were stained using different monoclonal antibodies: CD45-RO-phycoerythrin-Cy7 (PE-Cy7) (Becton Dikinson, San Jose, CA), CD3-allophycocyanin-Cy7 (APC-Cy7), CD4-peridinin chlorophyll protein complex, CD8-PE-Cy7, CD38-PE, HLA-DR-APC, CD57-fluorescein isothiocyanate, and PD-1-PE (Beckman Coulter, Miami, FL). Cellular activation was characterized by HLA-DR+ and CD38+ expression and senescence by CD28− CD57+ expression. Stained cells were run on a Gallios flow cytometer (Beckman Coulter, Inc, Münster, Germany) and data analyzed using Kaluza software (Beckman Coulter, Inc).
High-sensitivity CRP was analyzed from frozen samples with a commercial instant ELISA kit (eBioscience, Inc, San Diego, CA), with a detection threshold of 3 pg/μL, and an intraassay and interassay coefficient of variation of 6.9% and 13.1%, respectively.
Carotid Artery Ultrasound
IMT was examined using ultrasonography (CX50 portable equipment, Philips Medical Systems, Inc, Eindhoven, the Netherlands). Specific IMT detection software was previously calibrated using QLab (Philips Medical Systems, Inc, Eindhoven, the Netherlands).18 Measurements were made bilaterally at the common carotid artery (1–2 cm proximal to the bulb) following the Mannheim criteria.19 More than 400 measurements of a 10-mm segment of the far wall of the artery were performed and digitalized for each patient, and the median value was used for the statistical analysis as previously described.20 Images were read by an experienced cardiologist blind to the HIV status. Median value of the right and left measurement was then calculated and used for the analysis. All carotid ultrasounds were performed by 2 trained technicians who had previously participated in a pilot study (repeated and blinded measurements performed in a random sample of 36 uninfected healthy children and adolescents) that showed no evidence of systematic observer bias. The intraclass correlation coefficient was >0.90.
Qualitative variables were reported as a frequency distribution, whereas normally distributed quantitative variables were described as mean and SD. The continuous nonnormally distributed variables were reported as median and interquartile ranges (IQRs). Means for variables with a normal distribution were compared using the Student t test. Nonparametric variables were examined using the Mann–Whitney and Kruskal–Wallis tests. Given the small dispersion of the independent variable—IMT—and the absence of pediatric reference values; we classified subjects as having increased IMT when they showed a value above the healthy subjects' median (0.42 mm). Simultaneous independent associations between IMT and a subset of independent variables, including cardiovascular risk factors (sex, age, tobacco use, and BMI) and HIV seropositivity were evaluated by logistic regression analysis. Then, a second multivariate analysis was performed only in HIV-infected patients to explore the independent associations with IMT. This logistic regression model included the following variables: lipodystrophy, CD4 nadir, time with detectable VL, and cumulative exposure to ART and to protease inhibitors (PI), CD4 and CD8 T-cell count. To corroborate results, the multivariate analysis was reapplied to nonsmoker perinatally infected children. Finally, linear and logistic regression models were built to explore associations between IMT, high-sensitivity C-reactive protein (hsCRP), and the percentage of HLA-DR+ CD38+ and CD28− CD57+ T cells.
All statistical analyses were performed using the SPSS 18.0 statistical package (SPSS, Inc, Chicago, IL). The level of significance for all analyses was set at 0.05.
Study Population and Between Group Comparison of Clinical Characteristics
The study population included 150 HIV-infected children and adolescents and 150 healthy volunteers. Main characteristics of both cohorts are summarized in Table 1. Globally, mean age was 14.8 ± 4.6 and range was 2.5–23.8. Most subjects were female (64%) and of white origin (78%). Most of HIV-infected patients (96.7%) had acquired HIV from mother-to-child transmission and all except 4 were receiving ART at the time of study evaluation. However, only 76.2% had achieved viral suppression. Median VL for the unsuppressed group was 4607 copies/mL (230–27,600) and median logVL 3.66 copies/mL (2.36–4.44). HIV infection characteristics are described in Table 1.
HIV-infected and -uninfected subjects showed similar age, gender, blood pressure, and frequency of hypertension according to z score adjusted by age and height. Although both groups had similar BMI, z score adjusted BMI was higher in the control group. On the contrary, waist-to-hip ratio was higher in the HIV-infected group. In addition, HIV-infected patients displayed significantly higher glycemia and homeostasis model assessment scores and a worse lipid profile, with decreased levels of HDLc and higher low-density lipoprotein cholesterol, total cholesterol/HDLc ratio, and triglycerides. None of the subjects had been previously diagnosed with diabetes, hypertension, or family history of premature CVD, and none was taking hypoglycemic agents, antihypertensives, statins, or fibrates. No patient was taking trimethoprim–sulfamethoxazole.
Between Group Comparison of IMT Measurements
First of all, we compared IMT between HIV-infected and -uninfected subjects. IMT was higher in the HIV-infected children compared with the HIV-uninfected group (P < 0.001; Fig. 1A) in both unadjusted and adjusted analysis. This difference remained statistically significant when the analysis included only those patients on stable ART who had achieved undetectable VL (P < 0.001; Fig. 1B). To specifically address the effect of viral suppression, we compared IMT between virally suppressed and unsuppressed patients, and no statistically significant differences were found (P = 0.349; Fig. 1B). A multivariate analysis was built to simultaneously analyze the effect of diverse exposure variables on IMT. After adjustment by age, gender, tobacco use, BMI, non-HDL cholesterol, and triglycerides, HIV status was the only variable independently associated with higher IMT (P = 0.007; Table 2), demonstrating its independent predictive value, with a borderline significance for BMI (P = 0.057).
Subsequently, a second multivariate regression logistic analysis was performed, including only HIV-infected patients and adjusting by specific HIV-related variables. A lower CD4 nadir was the only variable associated to a higher IMT (Table 3; odds ratio (OR): 0.82 per 100 cell/mL increase; P = 0.033), with a borderline statistical significance for PI exposure (Table 3; OR: 0.89; P = 0.072). To corroborate these results, a subanalysis restricted to nonsmokers and perinatally infected children was done. Again, a lower CD4 nadir was associated to higher IMT (Table 3; OR: 0.77; P = 0.019) and PI exposure reached statistical significance (Table 3; OR: 0.84; P = 0.023). Results of both analyses are shown in Table 3.
Between Group Comparison of High-Sensitivity CRP Measurements
CRP was determined in a representative subgroup of 64 HIV-infected patients (51 aviremic and 15 viremic) and 30 controls. No differences were found when analyzing HIV-infected subject versus HIV-uninfected controls (Table 1) or patients with detectable versus undetectable VL. No subject in the study showed markedly increased levels of CRP, suggesting acute infection. Finally, no statistical association was found between IMT measurement and CRP values (data not shown).
Between Group Comparison of T-Cell Activation and Senescence Markers
To analyze influence of immune activation on the atherogenic process, T-cell activation and senescence markers were studied in a random subgroup of 11 controls and 38 patients (29 aviremic and 9 viremic) that underwent immunophenotyping by flow cytometry. This sample was representative of the subjects of the study (median age: 16.7 years, 66.7% women, 70.1% white, all of them vertically HIV-infected and on treatment).
Compared with uninfected subjects, HIV-infected patients had higher levels of HLA-DR+ CD38+ CD4 T cells (Fig. 2A). This difference remained significant when the comparison included only those HIV-infected patients on ART who had achieved undetectable VL (Fig. 2B). Differences in the frequency of activated CD8 T cells did not reach statistical significance (Fig. 2C). However, viremic patients showed significantly higher frequency of HLA-DR+ CD38+ CD8 T cells (Fig. 2D). The frequency of CD28− CD57+ T cells was similar between HIV-uninfected and HIV-infected subjects, both for CD4 and CD8 subsets (Figs. 2E, G). Again, viremic patients showed an increase in the percentage of CD28− CD57+ CD8 T cells (Fig. 2H).
We did not detect any significant association between markers of T-cell activation/senescence and IMT in the univariate analysis. Also, linear and logistic regression models were built to explore associations between the frequencies of activated and senescent T cells and IMT, and no association was found. Similarly, relations between both variables and hsCRP were explored, and no significant associations were detected (data not shown).
Results of our study corroborate the hypothesis that structural changes of the arterial wall appear in the first decades of life during HIV infection, highlighting the need of a prompt diagnosis and treatment of cardiovascular risk factors during childhood. Our findings support a role for the HIV infection itself rather than for HIV-related factors or ART as suggested by other pediatric studies.7,8 Immune activation and senescence are present since childhood during HIV infection, often related to persistence of detectable VLs, although no relation to subclinical atherosclerosis was detected. To the best of our knowledge, this is the largest study analyzing factors associated with subclinical atherosclerosis in HIV-infected children and adolescents.
Although clinical manifestations of atherosclerosis do not present during childhood, the atherogenic process already begins since earliest stages of life.21 In our study, we used high-resolution ultrasound measurement of the carotid IMT to assess structural changes of the arterial wall. It is probably the most validated surrogate marker of subclinical atherosclerosis and is considered an independent predictor of adverse cardiovascular events in adults.22 Several studies have demonstrated increase of IMT in children with known cardiovascular risk factors, such as diabetes, obesity, or metabolic syndrome,23–26 and many others support the concept that anatomic changes of the arterial wall may improve over time with appropriate intervention.27–29 Different groups have evaluated atherosclerosis in HIV-infected children by means of IMT measurement.6,8,9,30–32 Most, but not all these studies showed increased IMT in HIV-infected children compared with uninfected controls, with differences in mean IMT values ranging from 0.02 to 0.15 mm. Although these rather small differences in IMT are of uncertain clinical relevance, we understand that the presence of detectable structural changes of the arterial wall in HIV-infected children is worrisome because these subjects face a long life span to develop CVD. Huge heterogeneity regarding technical aspects of IMT measurement within the studies, inclusion of rather small sample sizes, and the lack of consensus concerning characteristics of the ideal control group may explain some of these divergences among results. The only longitudinal study performed in children published to date has not been able to fully enlighten this complex question to the scientific community32. Our findings corroborate the hypothesis that structural changes of the arterial wall appear since childhood in vertically HIV-infected patients, underlining the fact that preventive measures are to be implemented in this high-risk population.
As previously mentioned, the impact of cardiovascular and HIV-related factors on CVD remains controversial as well. Although the effect of antiretroviral therapy on the vasculature has been widely studied in adult population, the existence of a correlation between cardiovascular events and ART exposure (especially PI) remains uncertain.33–35 Results of some of pediatric studies have suggested an increase of IMT associated to ART, especially PI or Stavudine exposure.6,8,31 Nevertheless, results from our study in which cumulative exposure to different antiretroviral regimens was quantified and included in the multivariate analysis, show no relation between PI or ART exposure and subclinical atherosclerosis. Moreover, PI exposure seems to be more likely a protective factor than a proatherogenic one in this large cohort of vertically HIV-infected patients. Among other HIV-related variables, CD4 T-cell count and CD4 nadir have been proposed to be the most robust risk factor for increased IMT in the adult population.36,37 Similarly, CD4 nadir has shown to be associated with thicker IMT in our study, supporting the hypothesis that immunity might play a strong role on the atherogenic process during HIV infection.
These data support the wide body of evidence suggesting that HIV itself plays a role in the development of atherosclerosis.38–41 Among underlying mechanisms, chronic inflammation and immune activation have been proposed to be at least partially responsible of the atherogenic process.6 Recent studies in adults have shown that HIV-associated T-cell changes are associated with subclinical carotid artery abnormalities; higher frequencies of activated CD4 and CD8 T cells and senescent CD8 T cells are associated with an increased prevalence of carotid artery lesions, although no relation to IMT could be demonstrated.5 To date, very few studies have focused on the effect that increased activation/senescence may have on premature clinical aging in children. In our study, higher frequencies of activated CD4 T cells were found in HIV-infected children when compared with uninfected controls. Frequencies of activated and senescent CD8 T cells were markedly increased only in those patients with detectable VL, along with findings from previous studies in children,10,11 pointing out the important effect of achieving viral suppression on the CD8 T-cell subsets. However, no relation could be established with IMT thickness. Although the minimal dispersion of IMT values in our study population may have underpowered the analyses to detect statistically significant associations with markers of T-cell activation, these findings are consistent with the recently published results from other pediatric studies, which suggest that T-cell activation is not associated to disease progression in vertically HIV-infected children.12,42 Levels of T-cell activation were rather low in our ART-treated group. A possible explanation for that finding is the fact that CMV coinfection may not be as prevalent in HIV-infected children as it is in adults. CMV seems to act synergistically to increase immune activation during HIV infection because both viruses coexist in most patients and in fact, CMV infection has been linked in HIV-infected adults to T-cell activation and atherosclerosis.43,44 Unfortunately, in our study, CMV serostatus of the participating children was not determined, and thus, effect of CMV infection could not be isolated. Whether there is a real association between HIV-induced T-cell activation and senescence and IMT remains to be answered.
To minimize the weight of classical cardiovascular risk factors, controls were enrolled aiming to achieve a group with similar age, sex, ethnicity, and BMI. However, the nature of the control group might be a limitation of the study. Uninfected ART exposed children and siblings have been proposed as the optimal control group, as socioeconomical background determines many cardiovascular risk factors. Nevertheless, it is not well known the effect that intrautero exposure to ART might have on cardiovascular parameters, and thus, recruitment of the ideal control group remains to be a challenge. In our study, no differences were found regarding absolute values of BMI between groups; however, HIV-infected children showed a statistically significant lower z score adjusted BMI. We understand that this fact could have led to underestimate the difference in IMT values between both cohorts because there is a positive correlation between IMT and BMI and provides with an additional value to the difference found.
In conclusion, we herein corroborate in a large cohort of patients that structural changes of the vasculature present early in HIV-infected subjects. CD4 nadir but not ART seems to be related to the described increased in cardiovascular risk. Most studies showing increased risk of cardiovascular events during HIV infection include adults of age around 50 years old who have been living along with the virus for a median of 3–8 years.34,45 By the time they grow that old, our vertically HIV-infected children will have been living with the infection for a period of 5–10 times longer. Thus, these patients should be carefully monitored for the prompt detection and early treatment of noninfectious disorders, and strategies are urgent to be defined for the prevention of CVD in this unique population. Long-term longitudinal follow-up of the patients included in this study is ongoing for a better understanding of the vascular changes and to ascertain the contribution of different HIV-related factors to cardiovascular risk.
Associations between clinical factors, markers of inflammation, immune activation/senescence, and carotid IMT are analyzed in 150 HIV-infected children and 150 HIV-uninfected controls.
The authors want to particularly acknowledge all patients and healthy volunteers and their families for their participation in this study. The authors thank all the professionals involved in the study and especially those integrating the Spanish Pediatric HIV infection Network (CORISPE) and the Pediatric HIV BioBank integrated in the Spanish AIDS Research Network (RIS). The authors would like to particularly thank S. Jimenez de Ory and J. M. Bellón for his kind help with the statistical analyses and database management.
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Keywords:© 2014 by Lippincott Williams & Wilkins
HIV; adolescents; cardiovascular risk factors; IMT; immune activation