Role of Renin-Angiotensin-Aldosterone System Activation and Other Metabolic Variables in Relation to Arterial Inflammation in HIV

Abstract

Background:

Arterial inflammation remains increased among persons with HIV(PWH) compared to persons without HIV(PWOH) even when controlling for traditional risk factors. We sought to understand whether increased RAAS activation may be related to arterial inflammation in PWH and when compared to PWOH.

Design:

20 PWH and 9 PWOH followed a controlled, standardized low and liberal sodium diet to simulate a RAAS activated and RAAS suppressed state, respectively. We measured serum LpPLA2 concentrations following both conditions to assess the physiologic dynamics of aldosterone in relation to arterial inflammation.

Results:

LpPLA2 levels were significantly higher among PWH vs. PWOH during both the RAAS activated state[5.3(4.2, 6.1) vs. 4.0(3.0, 4.8)nmol/L, median(interquartile range),P=.01] and RAAS suppressed state[4.4(3.9, 5.3) vs. 3.8(3.4, 4.1)nmol/L,P =.01]. Among PWH, but not PWOH, LpPLA2 increased significantly with RAAS activation(P=.03). LpPLA2 levels measured during the RAAS suppressed state among PWH remained relatively higher than LpPLA2 levels under both conditions among PWOH. Log LpPLA2 was related to log aldosterone during the RAAS activated state(r=0.39,P=.04) among all participants. Log LpPLA2 was correlated to visceral fat(r=0.46,P=.04) and log systolic blood pressure(r=0.57,P=.009) during a RAAS activated state when an increase in aldosterone was stimulated in HIV.

Conclusion:

LpPLA2 is increased during a RAAS activated state among PWH, but not among PWOH. Further, LpPLA2 was increased in both RAAS activated and suppressed states in PWH compared to PWOH. These data suggest a biological link between increased aldosterone and arterial inflammation in this population. Future studies should test RAAS blockade on arterial inflammation as a targeted treatment approach in HIV.

Introduction

Atherosclerotic cardiovascular disease (ASCVD) is a major contributor to morbidity and mortality among those persons with HIV (PWH) treated with contemporary antiretroviral therapies (ART). Continuous ART use and other traditional risk modifying therapies do not mitigate CVD risk completely among PWH¹. Large studies have shown that ASCVD is approximately two-fold higher among ART-treated PWH compared to persons without HIV (PWOH) matched for CVD risk factors^2,3. Even those individuals with a long history of HIV infection who are well-treated on ART have evidence of chronic immune activation and persistent systemic inflammation⁴. This state of chronic, systemic inflammation may have localized consequences at the level of vasculature. We have previously utilized ¹⁸F-FDG-PET imaging to measure arterial inflammation and have shown increased aortic inflammation in PWH compared to age- and Framingham Risk Score-matched PWOH⁵. Uptake of ¹⁸F-FDG in the vascular wall reflects an area of highly inflamed endothelium dense with macrophages and prone to plaque development. We have also identified that PWH have more non-calcified plaque, a subtype of plaque characterized as more inflamed and rupture-prone, compared to PWOH^6,7. While there are data available demonstrating inflammatory-mediated CVD among PWH, less is known about the pathogenesis.

Guided by data obtained from our physiology studies probing the renin-angiotensin-aldosterone system (RAAS), we postulated a mechanistic pathway by which excess RAAS activation serves as a mediator of arterial inflammation among PWH. Our prior investigations evaluating aldosterone hormone physiology among PWH have demonstrated increased aldosterone during a RAAS activated state in relation to metabolic disturbances, such as visceral adiposity and insulin resistance, markers of generalized inflammation and monocyte and macrophage activation, and dysregulation of natriuretic peptides^8–10. In this current investigation, we hypothesized a potential link between increased aldosterone, RAAS activation, and arterial inflammation, among PWH. LpPLA2 is a vascular-specific biomarker of inflammation which is produced by macrophages, participates in LDL oxidation, and has a critical role in the pathogenesis of atherosclerotic lesions. In the current study, we sought to probe physiologic changes in LpPLA2 in relation to aldosterone in HIV. To do so, we compared LpPLA2 levels between PWH and PWOH in RAAS activated and suppressed states and further contrasted the changes in LpPLA2 within each group during RAAS activated and RAAS suppressed states.

Methods

Study Participants

Twenty PWH and ten PWOH participated in a prior study evaluating RAAS activation and suppression physiology⁸. Participants were between ages 18-65 years. Only those PWH on stable antiretroviral therapy (ART) for ≥ 3 months were enrolled. Inclusion and exclusion criteria were similar regardless of serostatus. Participants with known cardiovascular disease, current tobacco use, current antihypertensive or RAAS-blocking medication use, potassium >5.5 mEq/L, creatinine >1.5 mg/dL, or alanine aminotransferase (ALT) >2.5 times the upper limit of normal were not included in the study. Use of estrogen or progesterone-containing hormone replacement therapy within 3 months of screening was also excluded. This study was conducted with Institutional Review Board approval by the Mass General Brigham Research Committee. All participants provided informed consent to participate.

Standardized Diets to Stimulate RAAS Activation

Participants were placed on a protocolized 6-day low sodium diet (10±2 mEq Na⁺, 100±2 mEq K⁺, and 1000±50 mg Ca²⁺) to stimulate RAAS activation. The low sodium diet was prepared by the Metabolic Kitchen at Brigham and Women’s Hospital (BWH) Center for Clinical Investigation. Confirmation of low sodium balance was assessed by a 24 hour urine collection. A 24-hour urine sodium < 50mEq was determined to be consistent with conditions to achieve RAAS activation. Approximately 1 month later, participants were placed on a 6-day liberal sodium diet by augmenting their usual diet with 3 broth packets (47.8 mEq Na⁺ per packet) daily in order to achieve a goal dietary sodium intake of >200mEq Na⁺/day. Confirmation of liberal sodium balance was assessed by a 24 hour urine collection. A 24-hour urine sodium > 200 mEq was determined to be consistent with conditions to achieve RAAS inhibition. To understand the role of RAAS physiology in arterial inflammation, comparisons of aldosterone and LpPLA2 were made during individual conditions of RAAS simulation and suppression.

Characterization of LpPLA2, RAAS, and biomarkers

LpPLA2 was measured by enzyme-linked immunosorbent assay (ELISA) (Diazyme Labs, Inc). Serum aldosterone was assayed by solid-phase radioimmunoassay (RIA) by the Coat-A-Count method (Diagnostics Products Corp). Plasma renin activity was assessed using the GammaCoat [¹²⁵I] RIA kit. Participants were admitted overnight to the BWH Center for Clinical Investigation and directed to lie in a supine position and fast for 12 hours after each of the two standardized diet protocols. Data evaluating RAAS physiology in HIV in relation to visceral fat and other metabolic indices among the same group have been reported^8,9. The current study analyzing the role of RAAS physiology in arterial inflammation has not previously been investigated.

HIV-Related Parameters

Standard clinical assays were used to measure HIV viral load (Ultrasensitive RT PCR, Roche COBAS amplicor, lower level of detection 48 copies/mL) and CD4⁺ T cell counts (flow cytometry).

Adipose Depot Parameters

Magnetic resonance images were obtained. An axial T1-weighted fat suppressed pulse sequence was acquired using a single-slice at the L4 vertebral body. To quantitate abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas, commercial software (Vitrak, Merge e/Film) was utilized to perform an offline analysis of tracings.

Statistical Analysis

The distribution of variables was evaluated by the Anderson-Darling test. Data are reported as mean ± standard deviation (SD) for variables of normal distribution or median (interquartile range [IQR]) for variables of non-normal distribution. Categorical variables are shown as percentages. The Tukey Method was performed for LpPLA2 values which appeared to be potential outliers upon visual inspection of the data. A value demonstrated to be greater or less than 1.5*IQR was considered an outlier and was not included in the analyses. One participant was determined to be an outlier and was not included in the analyses. The study was based on a convenience sample from a prior study assessing effects of RAAS activation on inflammatory parameters in PWH⁸. Comparisons within serostatus group (PWH or PWOH) across low and ad libitum sodium conditions were made using the Wilcoxon signed rank. Comparisons between serostatus group (PWH compared to PWOH) at the low and ab libitum sodium conditions were made using the Kruskal-Wallis test. Univariate analysis of LpPLA2 was assessed using Pearson’s correlation coefficient within each group after log transformation was applied to non-normally distributed variables. The following variables were log transformed prior to applying Pearson’s correlation coefficient due to non-normal distribution: LpPLA2, aldosterone, SAT, systolic blood pressure and diastolic blood pressure. In order to represent variables in a clinically relevant manner, data are reported prior to log transformation. Statistical significance was defined as P≤.05. Analyses were performed using SAS JMP (version 15.0).

Results

Clinical Characteristics

As shown in Table 1, age and sex were similar across both groups. PWH had good immunologic control, as indicated by CD4⁺ T-cell count (571 ± 325 cells/mL, mean ± SD) and log HIV viral load (1.8 ± 0.9 copies/mL). In addition, there was a long-term history of HIV infection (18 ± 7 years) and ART use (11 ± 5 years) in the HIV group (Table 1). Both groups had similar mean BMI, in the overweight category. Body composition did not differ by SAT [209 (107, 342) vs. 222 (130, 287) cm², median (IQR), HIV vs. non-HIV] or VAT (140 ± 88 vs. 140 ± 79 cm², PWH vs. PWOH). Body composition following stratification by sex in shown in Supplemental Table 1. Additional metabolic parameters including systolic blood pressure (SBP) and diastolic blood pressure (DBP) were similar among PWH and PWOH. Thirty percent of PWH had current statin use compared to 11% of PWOH (P=.25). (Table 1)

Table 1.

Baseline Demographic and Clinical Characteristics of Persons with and without HIV

	Persons without HIV (n=9)	Persons with HIV (n=20)	P Value
Demographics
Age (years)	51 ± 7	49 ± 7	0.39
Race (%)			0.22
Caucasian	78	45
African American	22	35
Other	0	20
Male Sex (%)	67	65	0.93
HIV Parameters
CD4+ T cell count (cells/μL)	N/A	571 ± 325	--
Log HIV RNA Viral Load (copies/mL)	N/A	1.8 ± 0.9	--
Duration HIV (years)	N/A	18 ± 7	--
Duration ART use (years)	N/A	11 ± 5	--
Current PI use (%)	N/A	60	--
Duration PI (years)	N/A	9 ± 5	--
Current NRTI use (%)	N/A	90	--
Duration NRTI (years)	N/A	11 ± 5	--
Current NNRTI use (%)	N/A	25	--
Duration NNRTI (years)	N/A	9 ± 5	--
History of HCV infection (%)	0	20	0.07
Body Composition and Metabolic Parameters
Iliac Waist Circumference (cm)	91 ± 13	94 ± 16	0.53
BMI (kg/m2)	26 ± 3	26 ± 6	0.88
VAT (cm2)	140 ± 79	140 ± 88	1.00
SAT (cm2)	222 (130, 287)	209 (107, 342)	1.00
Systolic Blood Pressure (mmHg)	119 (115, 125)	124 (116,138)	0.28
Diastolic Blood Pressure (mmHg)	80 (69, 82)	79 (68, 84)	0.71
Total Cholesterol (mmol/L)	4.5 ± 1.0	4.7 ± 0.9	0.65
Current Statin Use (%)	11	30	0.25
HOMA-IR	1.6 ± 1.2	1.5 ± 0.6	0.88
Creatinine (μmol/dL)	70.4 ± 11.7	74.9 ± 13.4	0.37
Potassium (mmol/L)	4.1 (3.9, 4.4)	4.0 (3.9, 4.1)	0.44

Data reported as mean ± standard deviation, percentage, or median (interquartile range).

Abbreviations: ART, antiretroviral therapy; N/A, not applicable; PI, protease inhibitor; NRTI, nucleoside/nucleotide reverse transcriptase inhibitors; NNRTI, non-nucleoside reverse transcriptase inhibitors; HCV, hepatitis C virus; BMI, body mass index; VAT, visceral adipose tissue; SAT, subcutaneous adipose tissue; HOMA-IR, homeostatic model assessment of insulin resistance

Evaluation of LpPLA2 during the RAAS Activated and RAAS Suppressed State

Both groups demonstrated a significant increase in aldosterone levels in the low sodium RAAS-activated state compared to the RAAS-suppressed state, but aldosterone levels were significantly higher among PWH than PWOH in a RAAS-activated state [384 (268, 857) vs. 254 (205, 354) pmol/L, P = .02] (Table 2). In this context, we further assessed LpPLA2. LpPLA2 was significantly higher among PWH compared to PWOH during both the RAAS activated state [5.3 (4.2, 6.1) vs. 4.0 (3.0, 4.8) nmol/L, P = .01] and RAAS suppressed state [4.4 (3.9, 5.3) vs. 3.8 (3.4, 4.1) nmol/L, P = .01] (Table 2). The difference in LpPLA2 during the RAAS activated state compared to the RAAS suppressed state was only significant in the HIV group (P =.03) (Table 2). In addition, the LpPLA2 level measured during the RAAS suppressed state among PWH remained relatively higher than the LpPLA2 levels under both conditions among PWOH. The analyses were repeated including the outlier and the results evaluating LpPLA2 during the RAAS activated and RAAS suppressed state among PWH and PWOH remained generally similar (see Supplemental Table 2). Among all participants, log LpPLA2 was significantly related to log aldosterone during the RAAS activated state (r=0.39, P=.04).

Table 2.

Comparison of RAAS Activation between Ad Libitum and Low Sodium Conditions Within and Between Groups of Persons with and without HIV

	Low Sodium Conditions	Ad Libitum Sodium Conditions	Change Between Ad Libitum and Low Conditions	Within Group Change P Value
LpPLA2 (nmol/L)
Persons without HIV (n=9)	4.0 (3.0, 4.8)	3.8 (3.4, 4.1)	0.3 (−0.2, 0.7)	0.30
Persons with HIV (n=20)	5.3 (4.2, 6.1)	4.4 (3.9, 5.3)	0.6 (−0.1, 1.1)	0.03
Between Group P Value	0.01	0.01
Aldosterone (pmol/L)
Persons without HIV (n=9)	254 (205, 354)	69 (69, 110)	150 (132, 259)	0.004
Persons with HIV (n=20)	384 (268, 857)	72 (69, 97)	282 (198, 731)	<0.0001
Between Group P Value	0.02	0.92

Data reported as median (interquartile range).

RAAS-related Parameters during the RAAS Activated and RAAS Suppressed State

The median 24-hour urine sodium during the low sodium diet was below 50 mmol for both groups [14 (11, 34) mmol for PWH and 18 (13, 26) mmol for PWOH). The median 24-hour urine sodium during the liberal sodium diet was above 250 mmol for both groups [270 (189, 358) mmol for PWH and 264 (218, 437) mmol for PWOH). Overall, there were no differences between groups in 24-hour urine sodium levels, mean arterial pressure, or serum potassium levels during either low or liberal sodium diets. (Table 3)

Table 3.

RAAS Parameters During Low Sodium and Ad Libitum Sodium Conditions among Persons with and without HIV

	Persons without HIV (n=9)	Persons with HIV (n=20)	P Value
RAAS Parameters During Low Sodium Conditions
24 hour Urine Sodium (mmol/24 hour)	18 (13, 26)	14 (11, 34)	0.49
Mean Arterial Pressure (mmHg)	86 (82, 88)	86 (80, 89)	0.87
Potassium (mmol/L)	4.1 (4.0, 4.3)	4.1 (4.0, 4.4)	0.60
RAAS Parameters During Ad Libitum Sodium Conditions
24 hour Urine Sodium (mmol/24 hour)	264 (218, 437)	270 (189, 358)	0.65
Mean Arterial Pressure (mmHg)	87 (86, 100)	87 (83, 93)	0.38
Potassium (mmol/L)	4.1 (4.0, 4.3)	4.1 (3.9, 4.3)	0.86

Data reported as median (interquartile range).

Relationships of LpPLA2 during the RAAS Activated State

Among PWH, log LpPLA2 was significantly correlated to VAT (r = 0.46, P = .04) and log SBP (r = 0.57, P = .009) and tended to be related to log DBP (r = 0.39, P = .09) with RAAS activation and increased aldosterone. There were no significant correlations demonstrated among PWOH during the RAAS activated state (Table 4). The analyses were repeated including the outlier and relationships were generally similar (Supplemental Table 3).

Table 4.

Relationship of Body Composition and Metabolic Parameters to log LpPLA2 Among Persons with and without HIV During the RAAS Activated State

	All Participants		Persons without HIV (n=9)		Persons with HIV (n=20)
	r	P	r	P	r	P
Age (years)	−0.21	0.27	0.03	0.94	−0.25	0.29
Iliac Waist Circumference (cm)	0.30	0.11	0.23	0.54	0.32	0.17
BMI (kg/m2)	0.23	0.22	0.09	0.81	0.33	0.15
VAT (cm2)	0.33	0.08	0.24	0.54	0.46	0.04
Log SAT (cm2)	0.11	0.56	0.33	0.39	0.04	0.86
Log Systolic Blood Pressure (mmHg)	0.45	0.01	0.25	0.51	0.57	0.009
Log Diastolic Blood Pressure (mmHg)	0.23	0.23	0.06	0.87	0.39	0.09

Relationships were assessed by Pearson’s Correlation Coefficient.

Abbreviations: BMI, body mass index; VAT, visceral adipose tissue; SAT, subcutaneous adipose tissue

Discussion

In the current study, we demonstrate a novel relationship between the RAAS and LpPLA2 among PWH. These data show that LpPLA2 is increased with RAAS activation in PWH and when compared to PWOH. In support of this, LpPLA2 correlated with aldosterone during the RAAS activated state. These data suggest a unique biologic association between aldosterone and arterial inflammation in PWH.

A mechanism focused on RAAS activation as a contributor to increased arterial inflammation among PWH is plausible given aldosterone has been shown to promote vascular inflammation and endothelial dysfunction^11,12. In line with this, prior literature suggests components of the RAAS can be found on immune type cells and the vasculature^13,14, and activation of mineralocorticoid receptor predisposes to vascular injury and promotes an inflamed environment¹². In murine models, exposure to an aldosterone infusion led to formation of inflammatory coronary lesions with evidence of perivascular macrophage infiltration¹⁵. Following vascular injury, macrophages recruited into the arterial intima transform into foam cells and promote the formation of atherosclerotic plaque. One group also demonstrated that exposing monocytes to aldosterone stimulates immunologic imprinting, such that these monocytes develop a persistent proinflammatory phenotype when transformed into macrophages^16,17. Similarly, patients with primary hyperaldosteronism, a condition of autonomous aldosterone production, are reported to have increased uptake at the arterial wall on ¹⁸F-FDG-PET imaging¹⁸. Few data are available assessing a direct relationship of aldosterone and LpPLA2, even in human studies of PWOH. A study of patients with aldosterone-producing adenomas had relatively higher LpPLA2 levels when compared to those with essential hypertension, though a difference in LpPLA2 between both groups was not detected¹⁹. It was recently shown that Ang-II stimulation upregulates PLA2g7, the identified gene of LpPLA2, in macrophages localized in the heart, and subsequently application of an emerging LpPLA2 inhibitor reduces Ang-II mediated inflammation in a murine model²⁰. In addition, some evidence suggests Ang-II may increase LDL oxidation in macrophages²¹ and stimulate atherosclerotic plaque formation. Taken together, a RAAS-driven pathway may have a key contribution to HIV-related CVD, as PWH has been shown to have relatively increased RAAS activation in relation to generalized markers of inflammation⁸, markers of macrophage/monocyte activation⁹, and now arterial inflammation. To add to this, Eckard et al. have shown that LpPLA2 correlates with other systemic and vascular markers of inflammation in HIV, such including sTNFR-II, sICAM-1, sVCAM-1 and sCD14,²² suggesting a broader link to inflammation.

Our studies were performed under rigorous conditions with a standardized sodium and posture protocol, which are key to assessing the RAAS in a controlled manner²³. Ordinarily, we would expect increased sodium intake to be associated with hypertension and contribute to vascular injury²⁴. On the contrary, our data show that LpPLA2 increased in relationship to aldosterone and blood pressure during a sodium restrictive state, simulating RAAS activation. These data suggest that aldosterone, as opposed to the sodium, is the mediator of arterial inflammation. Excess aldosterone could potentiate abnormal hemodynamics and endothelial dysfunction, arterial stiffness, or vascular remodeling and contribute to the pathogenesis of atherosclerotic disease in this way²⁵. In this regard, it is possible that increased aldosterone may have multiple effects on the vasculature, both direct and indirect consequences. Increasing LpPLA2 with RAAS activation may be one mechanism of vascular injury in PWH.

We have shown that PWH with increased visceral fat have increased aldosterone during a RAAS activated state when compared to those PWH without increased visceral fat and those PWOH with and without increased visceral fat⁸. In a separate investigation among PWH, we also demonstrated an association between visceral fat and LpPLA2²⁶, and now in the current study, we highlight a potential relationship drawing together aldosterone, arterial inflammation, and visceral adiposity. The visceral adipose depot is a highly inflamed and dysfunctional adipose depot^27,28, which has been linked to arterial inflammation and is a predictor of CVD events in the general population²⁹. Some data suggest the adipose depot has components of the RAAS³⁰. Interestingly, LpPLA2 was not completely dampened among PWH during a RAAS suppressed state to those levels among PWOH, so we could postulate autonomous production of aldosterone which could be linked to the adipose depot, a factor which would not be acutely modifiable by sodium intake. However, VAT did not differ significantly between groups as would be expected if it were to account for the differences in LpPLA2 among PWH vs. PWOH. In this way, the RAAS may be directly implicated in arterial inflammation regardless of VAT or there may be other metabolic drivers of LpPLA2 that may relate to VAT.

There are a few limitations of the study. Our current study leverages PWOH as a comparator group and demonstrates a unique physiology relating the RAAS to LpPLA2 among PWH. However, there were a smaller number of participants without HIV, and we may not have been able to detect significant relationships of RAAS activation to LpPLA2 among the PWOH group. It would be important to perform larger studies to further address this question. As the main aim of the study was to perform a detailed investigation of the RAAS physiology, the scope of this exploratory investigation did not include radiologic assessments to quantify arterial inflammation. In that regard, these preliminary findings will be more rigorously tested through an ongoing 12 month double-blinded, placebo-controlled randomized control trial (MIRABELLA HIV substudy, NCT02740179) among PWH which aims to test mineralocorticoid blockade on the specific endpoint of aortic inflammation quantified via target-to-background ratio on ¹⁸F-FDG-PET/CT. This interventional study will test this hypothesized mechanism further by assessing RAAS activation in relation to aortic inflammation and whether subsequent RAAS blockade will reduce aortic inflammation.

In summary, we demonstrate LpPLA2 is increased during a RAAS activated state among PWH, but not among PWOH. Further LpPLA2 was increased in both RAAS activated and suppressed states in PWH compared to PWOH. These data suggest a potential unique biologic association between aldosterone and arterial inflammation in this population at increased risk for CVD. These data support the need for future studies investigating the efficacy of RAAS blockade on arterial inflammation as a targeted treatment approach for inflammatory-mediated CVD in HIV.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.