Early HIV infection is characterized by a dramatic depletion of CD4+ T cells in the gut, leading to depletion of the immunity at the gastrointestinal mucosal surface.1 This heightens translocation of microbial products, particularly lipopolysaccharide (LPS), into the systemic circulation.2–4 The resultant immune activation escalates monocyte activation,2,5 often measured by the level of circulating soluble CD14 (sCD14) and soluble CD163 (sCD163). Although these inflammatory markers have been associated with an increased risk of hypertension in the general population,6,7 few data are available on associations between antiretroviral therapy (ART), inflammation, and hypertension in HIV-infected individuals.
Data on the epidemiology of hypertension among HIV-infected persons in Sub-Saharan Africa have demonstrated conflicting findings. Although a number of studies have reported high incidence rates,8–12 others suggest that people with HIV are at lower risk of hypertension than matched HIV-uninfected counterparts.13–16 However, these studies are confounded by the inconsistent inclusion of patients with and without AIDS as well as the variable use and duration of ART in the cohorts.17
It remains unclear whether HIV-mediated systemic inflammation and immune activation increases hypertension risk. If they did, then inflammatory biomarkers may be good candidates for identifying HIV-infected patients at risk of hypertension. Furthermore, a better understanding of these relationships could help identify pathophysiologic mechanisms of cardiovascular disease in this population. We therefore sought to describe blood pressure (BP) changes after ART initiation in a cohort of HIV-infected individuals initiating ART in Uganda, and hypothesized that biomarkers of inflammation [interleukin 6 (IL-6)], indoleamine 2,3-dioxygenase-1 activity as measured by kynurenine/tryptophan ratio, microbial translocation (plasma LPS), monocyte activation (soluble CD14, soluble CD163), and altered coagulation (D-dimer) would be associated with incident hypertension.
We conducted a closed cohort study nested within the Uganda AIDS Rural Treatment Outcomes (UARTO) cohort, which has been described in detail previously.18 The UARTO cohort is a population-based prospective cohort study of HIV-infected adults, aged at least 18 years, living within a 60-kilometer radius around the Mbarara Regional Referral Hospital Immune Suppression Syndrome (ISS) clinic. Participants were enrolled just before initiation of ART and were observed every 3–4 months to collect biologic and socio-behavioral data. By November 2014, 771 individuals were enrolled in UARTO and 94% initiated ART within 4 days of enrollment. Enrollment started in 2005, and follow-up for health events was completed in September 2015. For this analysis, study participants were observed from enrollment into the UARTO cohort until the earliest of the date of hypertension diagnosis or their last recorded clinic visit with a BP measurement. Of note, all UARTO participants received biomarker testing before ART initiation, but only those with HIV viral load suppression (<400 viral copies/mL) at 6 months of ART had repeat biomarker tests performed. Therefore, to describe the mean change in BP, all UARTO cohort data were used. However, only the data of those with pre-ART and on-ART biomarker data were used to determine the association of biomarker levels with incident hypertension in an unmatched case–control design.
The ethical review boards at Mbarara University of Science & Technology, University of California San Francisco, Partners Healthcare, and Uganda National Council of Science and Technology, approved the conduct of this study. All participants signed written informed consent.
At enrollment into UARTO (just before ART initiation) and 6 months later, participants underwent blood draw for CD4+ T-cell count and inflammatory biomarker testing. A trained laboratory technician collected approximately 40 mL of venous blood into sodium heparin-lined vacutainers. All plasma samples were drawn in the morning before 9:30 am. Blood samples were centrifuged immediately after blood draw, and derivative plasma aliquots were then preserved at −80°C until shipment for laboratory testing at the University of California San Francisco, CA.
Thawed cryopreserved plasma from the pre-ART and 6-month time points was assessed for several biomarkers. Plasma LPS levels were measured from anticoagulant citrate dextrose—anticoagulated plasma with the Limulus amebocyte assay as described previously.19 We used liquid chromatography–tandem mass spectrometry to assess kynurenine and tryptophan levels.20 Cryopreserved plasma was also assessed for soluble CD14 (sCD14, R&D Systems), soluble CD163 (sCD163, Trillium Diagnostics), IL-6 (Human IL-6 Ultra-Sensitive Kit, Meso Scale Diagnostics), and D-dimer (Diagnostico Stago). We chose biomarkers based on previous data which observed important relationships between each of these markers and cardiovascular disease outcomes, mortality, or both.2,21–24
Separate from the UARTO study visits, participants were seen at the Immune Suppression Syndrome (ISS) Clinic at Mbarara Regional Referral Hospital for routine clinical care every 2 weeks to 3 months, as determined by clinical staff. At the ISS clinic, trained nurses measured BP at each visit using an aneroid sphygmomanometer (Welch Allyn Tycos 767 Series; Skaneateles Falls, NY), with normal (22–32 cm) cuff sizes. We defined hypertension as occurrence of a systolic BP ≥140 mm Hg and/or diastolic BP ≥90 mm Hg on at least 3 consecutive clinic visits or prescription of antihypertensive medications after ART initiation.
Weight and height were measured using standardized scales (seca 762, Seca GmbH & Co. KG. Hamburg, Germany) and roll-up measuring stadiometers (seca 206; Seca GmbH & Co. KG. Hamburg, Germany), respectively. Height was measured to the nearest 0.1 cm after removal of shoes. Weight was measured to the nearest kilogram after the participants had removed their shoes, as well as any heavy clothing. We used height and weights to calculate body mass indexes (BMI) as weight (in kilograms) divided by the square of height (in meters squared) for all participants, and categorized BMI as less than 25, 25 to 29.9, or 30 or greater.
We set implausible values as missing, including systolic BP recorded as >300 or <50, diastolic BP recorded as >200 or <30 mm Hg, and weight <25 kg or >200 kg. In the ISS clinic, clinical data were collected on paper forms and subsequently entered into an Open Medical Records System electronic database as part of the International Epidemiologic Databases to Evaluate AIDS Project.25
We conducted 2 principal analyses. In the first, we included all participants in the cohort with biomarker data and at least 1 BP measurement after ART initiation. We fit mixed effects linear regression models with systolic BP as the dependent variable, duration of ART as a fixed effect variable with spline breakpoints at 6 months, 1 year, and 5 years, and a random effect by participant to estimate changes in mean systolic BP over time from after ART initiation.
We then conducted a nested case–control analysis to estimate associations between inflammatory markers at pre-ART and 6-month on-ART time points with incident hypertension. We restricted this analysis to study participants who had 3 or more consecutive visits with high BP during follow-up (cases) or those who had no visits with high BP during follow-up (controls). We plotted mean levels of biomarkers at pre-ART and months 6 to show change in biomarkers over 6 months. We fit logistic regression models to calculate odds ratios for incident hypertension, including traditional risk factors (age, sex, BMI, smoking history, and pretreatment CD4 count); and then added each biomarker at the pre-ART and 6-month visit individually into the model. Models at the pre-ART visit were adjusted for viral load, whereas those at 6 months were not because biomarker testing at 6 months of ART was restricted to participants with a suppressed HIV RNA viral load (<400 copies/mL). Biomarkers were modeled as changes in each interquartile range (IQR) of the log-transformed value.
We performed sensitivity analyses to assess the robustness of our findings. First, we altered our definition of the outcome to include either prevalent or incident hypertension (as opposed to incident hypertension alone). Then, we altered our definition of a case to include any participant who was prescribed any antihypertensive medication at least once during follow-up. All analyses were performed with STATA version 13 (College Station, TX).
As of November 2014, 771 participants had been enrolled into the UARTO cohort study. We excluded 225 participants who had no biomarker testing available and 10 participants with no BP measurements (Fig. 1). Among the remaining 536 participants, women constituted the majority (70%), and the median age at ART initiation was 34 years IQR (29–39) (Table 1).
In the total cohort of 536 participants, systolic BP increased significantly at 9.6 mm Hg/yr (95% CI: 7.3 to 11.8) in the first 6 months after ART initiation (Fig. 2). However, BP plateaued thereafter, with total average increase in systolic BP of 2.3 mm Hg/yr (95% CI: 2.1 to 2.5) from years 1 through 5 of observation, and only 0.7 mm Hg/yr thereafter (95% CI: 0.3 to 1.0). We found no difference in mean systolic BP between those with undetectable HIV viremia versus detectable HIV viremia at 6 months (P = 0.18). In contrast, increasing weight from ART initiation to 6-month time point was significantly correlated with increasing systolic BP over first 6 months (R2 = 0.14, P <0.0001).
In the restricted case–control analysis, we excluded 18 participants with prevalent hypertension before ART initiation and 288 participants who had at least 1 but never 3 consecutive visits with hypertension during follow-up. We identified 73 participants who had ≥3 consecutive high BP visits after ART initiation (cases), and 157 participants who never had elevated BP readings on a minimum 3 consecutive visits after ART initiation (controls) (Table 1).
We found significant associations between traditional risk factors and incident hypertension in multivariable logistic regression models for each year increase in age (adjusted odds ratio (AOR) 1.09, 95% CI: 1.04 to 1.13, P < 0.001), male gender (AOR 2.76, 95% CI: 1.34 to 5.68, P = 0.006), overweight (AOR 4.48, 95% CI: 1.83 to 10.97, P = 0.001), obesity (AOR 8.36, 95% CI: 1.27 to 54.79, P = 0.027), and a low pre-ART CD4+ T-cell count (<100 cells) at ART initiation (AOR 3.08, 95% CI: 1.07 to 8.89, P = 0.037) with incident hypertension (Table 2).
At month 6 of ART-mediated viral suppression, all biomarkers had declined from pre-ART levels. The greatest mean change from pre-ART to 6-month levels was observed in D-dimer, kynurenine/tryptophan ratio, IL-6, and sCD163 (Fig. 3). In multivariable logistic regression models adjusted for known cardiovascular risk factors, each unit IQR decrease in D-dimer levels at 6 months (while taking ART) was associated with an increased risk of incident hypertension (AOR 0.61, 95% CI: 0.37 to 0.99, P = 0.049). Although we found no other significant associations between other biomarkers and incident hypertension (Table 2), the estimates indicated that lower levels of IL-6 and sCD14 at 6 months were associated with an increased risk of incident hypertension.
Antihypertensive medications were prescribed at approximately 1% of all observed visits (220/18,698) and were ever prescribed in only 30% (22/74) of patients with 3 or more consecutive visits with high BP; 21 of these were prescribed antihypertensive medication after ART initiation and were already considered cases. In comparison, 7 participants among controls were ever prescribed antihypertensive medication (3 before starting ART and 4 after starting ART), and an additional 18 participants who had been neither classified as cases nor controls in the main models were prescribed antihypertensive medication at least once after starting ART. After reclassifying these 22 participants as cases, we found no significant differences in estimates between models with or without antihypertensive medication use as part of the definition of incident hypertension.
In this study of HIV-infected individuals taking ART in southwestern Uganda, we found that BP increases rapidly in the months immediately after initiation of ART. We hypothesize that this represents an ART-mediated “return to health,”26 which may in turn unmask other mechanisms of hypertension risk such as increased BMI and lipid abnormalities. Although our study did not have an HIV-uninfected group for comparison, the elevations we observed in BP are comparable with those described in HIV cohorts26–28 though greater than what would be expected over the same period in the general population.29 In support of this ART-mediated return to health hypothesis, we observed a positive correlation between weight gain and systolic BP shortly after ART initiation. Alternatively, ART might directly mediate vascular remodeling which has also been demonstrated in other metabolic disorders and is associated with risk of hypertension.17,30
Although we document increasing BP after ART initiation, our finding should be taken in light of population-based surveys from Sub-Saharan Africa, which have suggested that people with HIV seem to be at lower risk of hypertension than matched HIV-uninfected counterparts.13–16 Given that we did not have an HIV-uninfected comparison group in our study, we cannot rule out the possibility that BP remains lower in the HIV-infected population despite the increases we documented. Alternatively, because previous studies tended to assess HIV-infected patients irrespective of ART status, they may not have noted the return to health phenomenon we report here.
To the best of our knowledge, ours is the first report from a large cohort demonstrating no statistical association between biomarkers of immune activation and risk of incident hypertension after adjusting for traditional risk factors. Indeed, we found that D-dimer levels at month 6 of ART (IL-6 and sCD14 to a lesser extent) were inversely associated with risk of incident hypertension. These findings contrast those from a Norwegian HIV cohort that showed increased inflammation (LPS and sCD14) predicted hypertension.31 However, participants in that study had less advanced HIV disease at ART initiation compared with those in our study. Our findings, therefore, suggest that—if anything—lower inflammation is associated with higher BP, implying distinct pathophysiologic mechanisms of hypertension in this population. Whereas HIV-associated inflammation and immune activation are known to contribute to arterial stiffness32 and atheroma formation,22,33,34 similar pathways are not known to be primarily responsible for hypertension. In fact, certain immune activation pathways that are increased by microbial products in HIV infection like the kynurenine pathway of tryptophan catabolism may be causally associated with vasodilation.35
Our findings of increasing age, male gender, overweight and obesity, and lower pre-ART CD4+ T-cell counts are consistent with previous studies.13,36–38 These data reaffirm that, unlike atherosclerosis, risk factors for hypertension among those with HIV are largely similar to the known risk factors in those without HIV.8,13,39 We did find a paradoxical association between low pre-ART CD4+ T-cell count and hypertension. This suggests that the mechanism by which low pre-ART CD4 is associated with hypertension may not be mediated by chronic immune activation.40,41 Whereas further data will be required to help elucidate these mechanisms, it could be explained by the possibility that immunodeficiency and immune activation affect hypertension risk through fundamentally distinct pathways.14,42,43
Our data should be interpreted in the context of the study design. Our results could be biased by both exposure and outcome misclassification. We attempted to minimize this through rigorous laboratory quality assurance protocols and by using strict criteria for our definitions of both cases and controls, respectively. Differential misclassification of inflammatory markers is also unlikely because laboratory testing of biomarkers was performed without knowledge of participant BP results, which further mitigates risk of systematic bias in our results.
Our study was observational without a randomized intervention, and thus our results could be susceptible to both measured and unmeasured confounders. Confounding by other conditions such as diabetes mellitus,37 dyslipidemia, and chronic kidney disease, which were not captured in the database, offers a particularly important challenge in the context of hypertension. These conditions are known to directly or indirectly cause hypertension and may also be associated with immune activation. We attempted to mitigate such confounding by adjusting for surrogates common to the above potentially confounding conditions (eg, age, sex, and BMI). However, we lacked data on renal function or glucose intolerance, and so we were unable to assess other putative pathways between inflammation and hypertension risk. The findings described are largely generalizable to those HIV-infected individuals achieving viral suppression within 6 months of ART.
We found significant elevations in BP among HIV-infected persons in the first 6 months after initiation of ART. This effect might be mediated by an ART-induced return to health, as evidenced by our observation that the risk of incident hypertension was positively associated with traditional risk factors (age, male gender, and BMI), but not elevated levels of biomarkers of immune activation. In fact, we found evidence that HIV-associated immune activation, inflammation, and altered coagulation may be inversely related to risk of incident hypertension, such that lower levels of D-dimer at 6 months after ART initiation were associated with an increased risk of incident hypertension. Taken together, the lack of an association between incident hypertension and inflammatory markers may suggest that traditional cardiovascular risk factors are the main predictors of hypertension in this population. If corroborated, our results support the integration of strategies for hypertension prevention and management into routine HIV care. Focusing attention on traditional cardiovascular risk factors might be the most appropriate method to affect hypertension-related morbidity and mortality in HIV-infected individuals in this setting.
The authors would like to thank the ISS clinic patients at Mbarara Regional Referral Hospital and Bernard Lown Scholars in Cardiovascular Health Program at Harvard T. H Chan School of Public Health.
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