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PLOS ONE logoLink to PLOS ONE
. 2023 Mar 17;18(3):e0282728. doi: 10.1371/journal.pone.0282728

Effect of antiretroviral therapy on decreasing arterial stiffness, metabolic profile, vascular and systemic inflammatory cytokines in treatment-naïve HIV: A one-year prospective study

Pedro Martínez-Ayala 1, Guillermo Adrian Alanis-Sánchez 2, Monserrat Álvarez-Zavala 3, Karina Sánchez-Reyes 3, Vida Verónica Ruiz-Herrera 1, Rodolfo Ismael Cabrera-Silva 3, Luz Alicia González-Hernández 1,3, Carlos Ramos-Becerra 2, Ernesto Cardona-Muñoz 2, Jaime Federico Andrade-Villanueva 1,3,*
Editor: Eliseo A Eugenin4
PMCID: PMC10022802  PMID: 36930649

Abstract

Introduction

Cardiovascular disease is a major cause of death among people living with HIV (PLH). Non-treated PLH show increased levels of inflammation and biomarkers of vascular activation, and arterial stiffness as a prognostic cardiovascular disease risk factor. We investigated the effect of one year of ART on treatment-naïve HIV(+) individuals on arterial stiffness and inflammatory and vascular cytokines.

Methods

We cross-sectionally compared aortic stiffness via tonometry, inflammatory, and vascular serum cytokines on treatment-naïve (n = 20) and HIV (-) (n = 9) matched by age, sex, metabolic profile, and Framingham score. We subsequently followed young, treatment-naïve individuals after 1-year of ART and compared aortic stiffness, metabolic profile, and inflammatory and vascular serum biomarkers to baseline. Inflammatory biomarkers included: hs-CRP, D-Dimer, SAA, sCD163s, MCP-1, IL-8, IL-18, MRP8/14. Vascular cytokines included: myoglobin, NGAL, MPO, Cystatin C, ICAM-1, VCAM-1, and MMP9.

Results

Treatment-naïve individuals were 34.8 years old, mostly males (95%), and with high smoking prevalence (70%). Baseline T CD4+ was 512±324 cells/mcL. cfPWV was similar between HIV(-) and treatment-naïve (6.8 vs 7.3 m/s; p = 0.16) but significantly decreased after ART (-0.52 m/s; 95% CI -0.87 to -0.16; p0.006). Almost all the determined cytokines were significantly higher compared to controls, except for MCP-1, myoglobin, NGAL, cystatin C, and MMP-9. At follow-up, only total cholesterol and triglycerides increased and all inflammatory cytokines significantly decreased. Regarding vascular cytokines, MPO, ICAM-1, and VCAM-1 showed a reduction. D-Dimer tended to decrease (p = 0.06) and hs-CRP did not show a significant reduction (p = 0.17).

Conclusion

One year of ART had a positive effect on reducing inflammatory and vascular cytokines and arterial stiffness.

Introduction

Antiretroviral treatment (ART) has become effective in controlling infections caused by human immunodeficiency virus (HIV). Worldwide, by 2021, the United Nations has estimated that approximately 37.7 million people are currently living with human HIV. In 2020, 73% of patients with HIV had access to ART [1]. In Mexico, until May 2021, 113,788 HIV patients were registered as active for ART [2]. HIV infection itself is associated with the development of cardiovascular disease (CVD) [3,4], atherosclerosis [5], and arterial stiffness [6]. Cardiovascular complications are among the leading causes of morbidity and mortality in patients with HIV. ART and HIV infection have a complex interaction with inflammation, coagulation, and other factors. Higher levels of envelope glycoprotein 120, Nef protein, interleukin-6, high-sensitivity C-reactive protein (hs-CRP), and D-dimer have been associated with endothelial dysfunction, cardiovascular disease, and all-cause mortality [7,8]. In addition, HIV infection has been linked with immune activation and low-grade chronic inflammation [9]. It has been reported that traditional CVD scores (i.e., Framingham Risk Score and Atherosclerotic Cardiovascular Disease Risk Score) systematically underestimate cardiovascular risk in HIV [10].

Aortic arterial stiffness is a predictor of cardiovascular events independent of traditional risk factors [11]. Carotid-femoral pulse wave velocity (cfPWV) is considered the gold standard for measuring aortic stiffness [12]. A recent meta-analysis including 17 studies investigating HIV and arterial stiffness found an overall increased cfPWV in individuals with HIV (+0.44 m/s) [13]. Furthermore, some studies have reported adverse effects of ART on aortic stiffness [14,15], whereas others did not report any association [16,17]. A possible explanation for these discrepancies may be the different combinations of ART regimens, different populations, and methods used to assess arterial stiffness. Most studies on arterial stiffness and HIV are cross-sectional and cannot establish a causal relationship; unfortunately, there is a lack of longitudinal clinical studies that investigate the effect of ART and cfPWV over time with the evaluation of inflammatory and vascular cytokines, especially in developing countries. We have previously reported increased arterial stiffness in treatment-naïve HIV individuals compared to HIV(-) controls [18]. The underlying mechanisms linking HIV infection with arterial stiffness remain unclear. Therefore, the present study aimed to investigate the effect of one year of ART on arterial stiffness, inflammatory and metabolic serum biomarkers in treatment-naïve HIV individuals.

Materials and methods

Study population

Between January 2015 and August 2019, people living with HIV (PLH) were enrolled at the "Antiguo Hospital Civil de Guadalajara" in Guadalajara, Mexico. The study complied with the Declaration of Helsinki and was approved by the ethics committee of the Hospital Civil Fray Antonio Alcalde (Register number: 208/15). After approval from the ethics committee, all individuals who attended the HIV Unit to start ART were invited to participate in the study. Informed consent was obtained from all participants. At study entry, the participants’ medical history and demographic information were obtained using a questionnaire. Inclusion criteria for PLH included: a) Patients 18 years of age or older with confirmed HIV infection and no previous ART, b) Absence of current or previous rheumatological or neoplastic disease or CVD, c) Not taking any vasoactive medication (e.g., antihypertensives, vasopressors, etc.), d) Without opportunistic infections at the time of enrolment. As a pilot prospective study, we analyzed the first 20 PLH from our previous cross-sectional study [18] that achieved and sustained virologic suppression for one year and who had complete data. To avoid selection bias, we confirmed that the demographic and clinical characteristics did not differ from those of the remaining 31 individuals from our cohort, which were not included in the analysis (S1 Table). The control group was paired by age and sex and recruited from our local network of researchers and volunteers within the University of Guadalajara. In addition, general laboratory testing and medical interrogation were performed on control individuals to confirm similar lifestyle as possible (except for the smoking habit which is higher in most HIV cohorts worldwide [19]) and metabolic and cardiovascular risk profiles. Inclusion criteria for the control group included: a) Negative HIV serological test, b) No previous cardiovascular, metabolic, or rheumatological disease, c) Not on any medication, including vasoactive drugs.

Biomarkers

Arterial stiffness

Arterial stiffness was measured by cfPWV as described previously [12] by applanation tonometry (PulsePen, Diatechne, Milan, Italy). cfPWV was calculated as the time delay between the arrival of the pulse wave at the carotid and the femoral artery, divided by the distance between the carotid and femoral arteries, with previous automated subtraction of the segment between the carotid and the sternal notch by the software. All measurements were performed by a single trained technician in a temperature-controlled room. The participants rested in a supine position for 15 minutes before the assessment and were instructed to abstain from smoking, and alcoholic, or caffeinated beverages 12 hours before the evaluation [20]. Systolic (SBP) and diastolic blood pressure (DBP) were measured using an automated sphygmomanometer (Omron HEM-907XL). Mean arterial pressure (MAP) was calculated as MAP = DBP + peripheral pulse pressure (pPP)ˑ0.33.

Viremic control and immunological status

A venous blood sample in an EDTA-tube was obtained from the antecubital vein after 8-hour fasting. CD4+ T-cells count was determined by flow cytometry (FACScalibur System, Becton Dickinson) and HIV-1 viral load was determined using with real-time polymerase chain reaction with retro transcription (Cobas AmpliPrep/Cobas Taqman, Roche Diagnostics) in a federal laboratory.

Metabolic profile

Serum samples were obtained from the antecubital vein after an 8-hour fasting. Serum lipids, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and triglycerides (TG), were determined by colorimetric quantification (AU5800 autoanalyzer, Coulter Beckman, USA). Plasma glucose was determined by photometry (AU5800 autoanalyzer, Beckman Coulter, USA) in a central laboratory.

Cytokine and vascular inflammation proteins quantification by flow cytometry

Three bead-based multiplex assays were employed to quantify cytokines and vascular biomarkers in all participants: LEGENDplexTM Human Inflammation Panel, LEGENDplex™ Human Vascular Inflammation Panel 12P, and finally detection of hs-CRP was performed with the LEGENDplex™ Human Vascular Inflammation Panel 1S/P Plex (BioLegend, Inc., San Diego, CA, USA). All assays were performed in accordance with the manufacturer’s instructions. Data were acquired in an Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA, USA). More than 2,000 events for each analyte were acquired. The files were analyzed using LEGENDplexTM Data Analysis Software. Values are expressed in pg/mL.

D-Dimer and sCD163 quantification

Serum samples were immediately stored at -80°C. Quantification of hs-CRP, D-dimer, and sCD163 was performed using D-Dimer Human SimpleStep and sCD163 (M130) Human, both by ELISA (Abcam®) following the manufacturer’s instructions. Values are expressed as ng/mL.

Statistical analyses

Depending on their distribution, data are presented as mean± standard deviation and median [interquartile range]. Parametric and non-parametric tests for independent and paired variables were used accordingly. Chi-square was used for non-paired and McNemar´s test for paired categorical data. Statistical significance was set at a two-tailed p-value of <0.05. Cliff’s delta is a non-parametric effect size estimate that was calculated to assess the effect of ART on inflammatory and vascular cytokines [21]. Effect size thresholds for Cliff’s delta were <0.147 for negligible, 0.148–0.33 for small, 0.334–0.474 for medium, and 0.475 for large effects. Based on previous data from our lab, we calculated a Pearson correlation coefficient between cfPWV paired data of 0.81 and the sample size necessary to detect a paired difference of 0.6 m/s and pooled SD 1.1, which resulted in a sample size of 12 individuals with 80% power. In addition, the effect size of ART on IL-10 after a 12-month ART was 5.1 requiring 6 patients in total [22], and 0.96 for VCAM, requiring 19 patients in total [23]. Statistical analysis and graphical representations were performed with GraphPad Prism 6. Cliff’s delta was calculated using RStudio v.1.3.1073 (Vienna, Austria) with the package “effsize” (v0.8.0, Torchiano, 2020) [24]. Sample size calculation was performed using G*Power V.3.1.9.6 (Universität Kiel, Germany). Due to the explorative nature of this study and the small sample size, a correlation analysis was not performed. Future studies with sufficient statistical power to establish an association between cytokine levels and arterial stiffness changes are required.

Results

Demographic data

Patient´s characteristics at enrolment are shown in Table 1. Age and sex distributions were similar between the HIV(-) and treatment-naïve HIV(+) groups, with a higher prevalence of males in both groups. ART was initiated within one month of enrolment, fourteen patients (73.6%) with EFV/TDF/FTC, two patients (10.5%) with ATV/r/TDF/FTC, and three patients (15.7%) with DVR/r/TDF/FTC. Among modifiable CVD risk factors, there was a significantly higher proportion of active smokers in the treatment-naïve HIV(+) group, with no changes in smoking at follow-up. BMI was similar between HIV(-) and treatment-naïve HIV(+) groups and did not change after one year of ART.

Table 1. Demographic, metabolic, HIV and vascular data from HIV negative and treatment-naïve HIV individuals at baseline and 1-year post ART follow-up (Post-ART).

HIV (+)
HIV (-)
(n = 9)
Naïve
(n = 20)
Post-ART
(n = 20)
Demographics
    Age, years 34.4 ± 8.2 34.8 ± 10.1 -
    Male sex, % 8 (88.8) 19 (95) -
    Cigarette smoking, % 1 (11.1) 14 (70)* 14 (70)
    BMI, kg/m2 24.3 ± 1.8 25.3 ± 4.1 25.9 ± 4.2
Metabolic profile
    Glucose, mg/dL 87.5 ± 5.5 84.4 ± 8.7 85.4 ± 7.2
    TC, mg/dL 185.6 ± 41.0 167.0 ± 41.5 185.6 ± 44.6†
    c-LDL, mg/dL 116.7 ± 37.1 100.2 ± 32.5 112.8 ± 38.3
    c-HDL, mg/dL 50.2 ± 10.3 33.7 ± 9.4* 35.3 ± 8.5
    TG, mg/dL 93.0 ± 40.3 167.0 ± 67.5* 176.3 ± 72.7*
    Framingham score, % 2.8 [1–3.3] 3.3 (1.9–7.9) 3.3 (2.3–7.0)
HIV status
    T CD4+, cells/mcL - 512 ± 324 727 ± 306†
    T CD8+, cells/mcL - 977 (733–11374) 859 (572–1180)
    CD4+/CD8+ ratio - 0.39 (0.27–0.44) 0.73 (0.39–1.48) †
    Viral load ≥50 cop/mL, n (%) - 20 (100) 0 (0) ††
Hemodynamics
    SBP, mmHg 111 ± 11 108 ± 13 116 ± 12†
    DBP, mmHg 66 ± 8 67 ± 8 67 ± 10
    MAP, mmHg 81 ± 7 80 ± 8 83 ± 10
    pPP, mmHg 45 ± 14 39 ± 11 49 ± 11†
    cfPWV, median, m/s 6.8 (5.8–7.7) 7.3 (6.7–8.1) 6.8 (5.8–7.8) †

Values are presented as median (IQR) and mean ± SD. BMI, body mass index; TC, total cholesterol; c-LDL, low-density lipoprotein cholesterol; c-HDL, high-density lipoprotein cholesterol; TG, triglycerides; T CD4+, lymphocytes T CD4; T CD8+, lymphocytes T CD8+; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; pPP, peripheral pulse pressure; cfPWV, carotid-femoral pulse wave velocity. *p<0.05 vs HIV(-), † vs HIV(+) naïve. *p<0.01 vs HIV(-), †p<0.01 vs HIV naïve.

Metabolic data

Regarding the lipid profile, the treatment-naïve HIV(+) group exhibited higher TG (p = 0.003) and lower c-HDL (p<0.01) compared to HIV(-), with no significant changes at one-year follow-up. Although TC and c-LDL levels were similar between groups at baseline, in the treatment-naïve HIV(+) group, TC significantly increased (p = 0.011) and c-LDL showed a trend toward a significant increase post-ART (p = 0.055). Plasma glucose levels were not significantly different between HIV(-) and treatment-naïve HIV(+) and did not change with ART. Both HIV(-) and treatment-naïve HIV(+) exhibited similar Framingham scores (p = 0.145), with no changes post-ART (p = 0.400).

HIV variables

All treatment-naïve HIV(+) individuals achieved virologic suppression (viral load <50 copies/mL). CD4+ T-cell count increased by 36% from baseline (p = 0.003), CD8+ T-cells did not change (p = 0.14), but the CD4+/CD8+ ratio increased at follow-up (p<0.001).

Blood pressure and arterial stiffness

The treatment-naïve HIV(+) group presented similar SBP, DBP, MAP, and pPP at baseline (all p>0.05), compared to HIV(-), with a significant increase only in SBP (p = 0.04) and pPP (p = 0.005) at 1-year follow-up. Baseline cfPWV was similar between treatment-naïve HIV(+) and HIV(-) (p = 0.16); however, the post-ART group showed a significant reduction (-0.52 m/s; 95% CI -0.87 to -0.16; p = 0.006) (Fig 1).

Fig 1. Carotid-femoral pulse wave velocity (cfPWV) in HIV(-), treatment-naïve HIV(+), and after one-year follow-up of antiretroviral therapy (post-ART).

Fig 1

Inflammation Biomarkers

Inflammation biomarkers are shown in Table 2. At baseline, SAA, sCD163, IL-8, IL-18, calprotectin (i.e., MRP8/14), hs-CRP, and D-dimer levels were higher in treatment-naïve HIV(+) compared to HIV(-) (all p<0.05), while MCP-1 levels were similar (p = 0.247). After ART, most biomarkers of inflammation significantly decreased (p<0.01), except for D-dimer which showed a trend toward a significant decrease (p = 0.063). Similarly, hs-CRP (p = 0.187) was reduced to almost half its baseline value, but the difference was not statistically significant. Despite ART, hs-CRP levels remained significantly higher in the post-ART group than in the control group. The effect size of ART on inflammation biomarkers is shown in Fig 2A. Cliff’s delta showed a medium effect on SAA (-0.38; 95% CI -0.10 to -0.65) and a large on sCD163 (-0.63, 95% CI -0.27 to -0.83), IL-8 (-0.62; 95% CI 0.27 to 0.82), IL-18 (-0.54; 95% CI -0.16 to -0.77), calprotectin (-0.52; 95% CI -0.16 to -0.76), and MCP-1 (-0.53; 95% CI -0.16 to -0.77).

Table 2. Inflammatory, vascular, and metabolic cytokines in controls, HIV+ naïve and HIV+ treated.

HIV (+)
Cytokines HIV (-)
(n = 9)
Naïve
(n = 20)
Post-ART
(n = 20)
Inflammatory
    SAA, ng/mL 143.8 (84.9–445.5) 916.7 (440.5–1714)* 307.6 (212.6–996.9)
    sCD163s, ng/mL 40683 (34261–45137) 81701 (54210–116553)* 45768 (40090–75773)
    MCP-1, pg/mL 812.1 (608.8–1178) 972.2 (781.8–1471) 653.8 (362.4–793.7)
    IL-8, pg/mL 4.9 (3.2–36.8) 189.1 (68.8–519.1)* 12.2 (3.1–127.9)
    IL-18, pg/mL 171.4 (74.1–296.4) 357.4 (166.1–707.5)* 99.6 (82.0–237.8)
    MRP8/14, ng/mL 4569 (2182–7312) 6988 (5214–11153)* 4398 (2939–5593)
    hs-CRP, mg/dL 0.4 (0.2–0.6) 2.8 (0.6–6.6)* 1.1 (0.7–2.2)*
    D-Dimer, ng/mL 225 (27–370) 694 (211–1886)* 366 (205–570)
Vascular
            Myoglobin, ng/mL 20.6 (14.7–48.2) 30.5 (22.2–44.0) 24.5 (13.5–49.2)
    NGAL, ng/mL 261.5 (107.0–380.2) 381.1 (240.7–569) 5.8 (2.5–197.8)*
    MPO, ng/mL 45.1 (12.5–107.7) 132.6 (91.5–233.8)* 45.1 (8.1–82.2)
            Cystatin C, mg/mL 0.22 ± 0.09 0.25 ± 0.10 0.20 ± 0.10
    ICAM-1, ng/mL 3.14 (1.9–4.0) 6.56 (5.5–8.1)* 4.3 (1.9–6.6)
    VCAM-1, ng/mL 1107 (1040–1172) 2541 (2067–2881)* 791.6 (17.8–1530)
    MMP-9, ng/mL 563.6 ± 295.7 693.6 ± 408.7 681 ± 379.6

Values are presented as median (IQR) and mean ± SD. SAA, serum amyloid A; sCD163, soluble CD163; MCP-1, monocyte chemoattractant protein-1; IL-8, interleukin 8; IL-18; interleukin 18; MRP8/14, myeloid-related protein 8/14 calprotectin; hs-CRP, high-sensitivity C-reactive protein; NGAL, neutrophil gelatinase-associated lipocalin; MPO, myeloperoxidase; ICAM, intercellular adhesion molecule 1; VCAM, vascular cell adhesion molecule 1; MMP-9, matrix metalloproteinase-9. *p<0.05 vs HIV(-), † vs HIV(+) treatment-naïve.

Fig 2.

Fig 2

Non-parametric effect size (Cliff’s delta) and 95% confidence interval (error bars) of the effect of ART on a) inflammatory cytokines and b) markers of vascular inflammation. sCD163, soluble CD163; MCP-1, monocyte chemoattractant protein-1; SAA, serum amyloid A; IL-8, interleukin 8; IL-18; interleukin 18; MRP8/14, myeloid-related protein 8/14 (calprotectin); NGAL, neutrophil gelatinase-associated lipocalin; MPO, myeloperoxidase; Cys C, cystatin C; ICAM, intercellular adhesion molecule VCAM, vascular cell adhesion molecule 1; MMP-9, matrix metalloproteinase-9. *p<0.05 significant reduction compared to baseline.

Vascular inflammation biomarkers

The vascular inflammation biomarkers are shown in Table 2. Treatment-naïve HIV(+) had higher D-dimer, ICAM-1, VCAM-1, and MPO levels, compared to HIV(-) (all p<0.05), while neutrophil gelatinase-associated lipocalin (NGAL) tended to be higher (p = 0.08). Myoglobin, Cystatin C, and MMP-9 levels were similar between the treatment-naïve HIV(+) and HIV(-) groups. At follow-up, treatment-naïve HIV(+) showed a significant reduction in ICAM-1, VCAM-1, neutrophil gelatinase-associated lipocalin (NGAL), and MPO. On the other hand, MPO, ICAM-1, and VCAM1 were not significantly different between the post-ART and HIV(-), except for NGAL which was lower than HIV(-) (p = 0.029). The effect size of ART on vascular inflammation biomarkers is shown in Fig 2B. Cliff´s delta effect size showed a large effect on NGAL (-0.66; 95% CI -0.30 to -0.85), MPO (-0.65; 95% CI -0.29 to -0.85), and VCAM (-0.78; 95% CI -0.45 to -0.92).

Discussion

In this study, we investigated the effects of one-year ART on arterial stiffness and inflammatory and vascular cytokines levels in non-elderly, treatment-naïve PLH. First, we observed that the treatment-naïve HIV(+) group presented higher levels of inflammatory, vascular cytokines, and arterial stiffness compared to HIV(-) controls. Second, after a one-year follow-up, cfPWV and some cytokines significantly decreased from baseline levels (before ART) and reached levels similar to those in the control group. However, hs-CRP remained higher post-ART, which may suggest a persistent low-grade inflammatory state despite ART.

As previously reported by Smith et al., [25] we observed a higher smoking prevalence among the HIV population (70%). We also found that the treatment-naïve HIV(+) group presented the typical dyslipidemia pattern in PLH, characterized by high TG and low HDL levels [26]. In our population, the most common treatment was EFV/TDF/FTC (70%), which showed an unfavorable lipid profile outcome, as previously reported by Daar et al. [27].

The arterial system acts as a conduit for blood and as a buffering system to deliver a steady flow to key organs such as the brain and kidneys. Their buffering capacity depends on how elastic/stiff arteries are; in this sense, arterial elasticity is influenced by the vasomotor tone and structural characteristics of the arterial wall (elastin and collagen content). The vasomotor tone is regulated by the endothelium, sympathetic tone, and vasoactive hormones such as the renin-angiotensin-aldosterone system (RAAS). Nitric oxide (NO) is a key molecule responsible for regulating vasomotor tone. During inflammation, released cytokines can induce endothelial dysfunction by limiting the availability of tetrahydrobiopterin, the precursor of NO [28], and decrease the capacity of endothelial NO synthase (eNOS) to produce NO [29], subsequently limiting the capacity to reduce vasomotor tone. In HIV, several factors can affect the vasculature, including endothelial dysfunction [30], RAAS hyperactivation, infection of vascular smooth muscle cells, increased coagulation, chronic immune activation (by the virus itself and by microbial translocation caused by enteropathy), abnormal cholesterol metabolism, lipoprotein transportation, and platelet activation [31,32]. In our study, we found a significant decrease in several inflammatory and vascular cytokines, a halving of hs-CRP levels, and a decrease in PWV in the post-ART group. Our findings differ from those of Rose et al., [33] who did not observe a decrease in PWV after 1-year of ART. Conversely, Maia-Leite et al., [34] reported that in their ART-experienced group, 87.9% of individuals virologically suppressed that aortic stiffness was similar to HIV(-) controls, implying the importance of viral suppression in arterial health. Our findings suggest that there is an improvement in arterial function, based on lower inflammation and a better environment for recovering normal arterial function. Nevertheless, we consider that future studies with complementary arterial evaluation techniques (e.g., flow mediation dilation) could provide better insights into the mechanisms responsible for recovering normal arterial function in inflammatory states.

The data obtained by measuring the vascular inflammation biomarkers in our study were consistent with previously reported data. Teasdale et al., [35] reported a significant decrease in D-dimer levels after 6 months of ART; [36,37]. In our study, we observed an overall trend toward a significant decrease in D-dimer post-ART. However, patients treated with ART based on transcriptase inhibitors (both non-nucleoside analogue and nucleoside analogue) presented a reduction (79%) in D-dimer levels, while patients with ritonavir-boosted PI-based ART D-dimer increased by 1.35 times. These data are consistent with the association of ritonavir-boosted PI exposure time with CVD risk and suggest that it can appear as soon as a year of ritonavir-boosted PI-based ART [36].

In the present study, MPO concentrations decreased after ART and could be associated with a PWV improvement. El-Bejanni et al. [38] reported a negative correlation between MPO and CD4+ T-cell counts. Thus, immune reconstitution in these individuals is accompanied by a decrease in MPO and PWV. MPO and aortic stiffness have been related to plaque instability in atherosclerotic disease [37,39,40]. Therefore, it could be considered that the global effect of one year on ART is reflected by an improvement in arterial health and probably less CVD risk.

Another interesting vascular marker is NGAL, which is associated with inflammation, leukocyte migration, carotid stenosis, endothelial dysfunction, plaque formation, acute myocardial infarction, and chronic heart failure [41,42]. It has been demonstrated that NGAL can be expressed in macrophages, smooth muscle cells, and endothelial cells; moreover, it can activate the NF-κB pathway, promoting the expression of several proinflammatory cytokines such as IL-8, MCP-1, TNF-α, and IL-1β [43]. We found a significant decrease in NGAL levels, which could be proposed as a novel cardiovascular biomarker in PLWH.

ICAM-1 and VCAM-1 have been associated with atherosclerosis [44] and CVD risk even in HIV(-) population [30,35,37,38]. In our study, we observed a substantial decrease in the levels of VCAM-1 after ART, as reported by other groups [45], reaching similar levels to the HIV(-) population, as well as ICAM-1 which showed a reduction although non statistically significant. MPO and aortic stiffness have been related to plaque instability in atherosclerotic disease [37,39,40]. In the present study, MPO concentrations also decreased after ART and could be associated with a PWV improvement. El-Bejanni et al. [38] reported a negative correlation between MPO and CD4+ T-cell counts. Thus, the immune reconstitution of these individuals is accompanied by a decrease in both variables; therefore, it could be considered that the global effect of one year on ART is reflected in an improvement in arterial health and probably less CVD risk.

It has been reported that PLH exacerbates the production of proinflammatory cytokines, such as IL-1β, IL-8, and TNF-α, and molecules related to non-canonical activation pathways of coagulation, such as IL-6, hs-CRP, SSA, and D-dimer [38,4649]. In line with those studies, we observed that the concentrations of SAA, sCD163, MCP-1, IL-8, and IL-18 were increased in PLH, but after a year of treatment, most HIV (+) patients reached concentrations similar to controls, except for SAA and sCD163. These results suggest that ART reduced the degree of systemic inflammation and improved arterial stiffness after one year of treatment.

It is well described that ART does not completely suppress viral load, particularly in the viral reservoirs. Hence, low-grade chronic inflammation persists and contributes to an increased risk of non-AIDS-defining events such as CVD [50,51]. As we previously mentioned, there was a reduction of SAA, sCD163, and calprotectin after a year of successful ART; however, the levels remained higher compared to controls. SAA, like CRP, is an acute-phase protein produced by the liver. It is known that SAA can establish an inflammatory atherosclerotic and thrombotic microenvironment that impacts immune dysfunction and promotes CVD [52,53]. In our study, we observed that SAA was higher in treatment-naïve compared to controls and reduced post-ART to levels similar to those in HIV(-). High concentrations of calprotectin were detected before ART; however, serum levels normalized after the virologic control, this pattern is consistent with previous data reported since calprotectin is one of the first proteins to increase in plasma, even earlier than other markers of myocardial necrosis. Finally, calprotectin promotes atherosclerosis, and elevated plasma concentrations of calprotectin are considered predictors of future CV events [42,43,46].

As PLH live longer, new widely available biomarkers are necessary to properly evaluate the overall health of this population and to prepare physicians for the more plausible complications their patients may have. This study has some prospectively analyzed biomarkers that could be of use; however, they require further investigation to evaluate their long-term effectiveness in the daily clinical field.

These results are consistent with the idea that PLH develops a low-grade inflammation state and metabolic imbalance from the beginning of HIV infection, which remains despite ART. Furthermore, it seems that besides the current therapy goals (i.e., CD4+ T-cell count and HIV RNA levels) it would be advisable to monitor arterial health with different available techniques to try to lower the CV risk in this population, which increases with age and ART exposure.

Strengths and limitations

This study has some limitations. Despite our aim to isolate the effect of HIV on arterial stiffness by choosing PLH patients without any other comorbidities and comparing them with healthy controls, there may be a series of genetic, lifestyle, social, and behavioral factors that differ between the populations studied, which could not be controlled for. Our study sample included mostly males, and sex differences could play a role in the response of arterial stiffness and levels of inflammatory and vascular biomarkers to ART. Another limitation of the present study was the absence of cytokine reference values for the Mexican population to determine whether HIV-infected individuals achieved normal cytokine levels after ART. The sample size of the control group was smaller than that of the HIV(+) group, which may have been underpowered for some biomarkers. Lastly, we only measured PWV twice (baseline and at 12 months); thus, we could not determine how far into the treatment the PWV and cytokines started to decrease. Nevertheless, the strength of this study is the prospective evaluation of arterial health and serum biomarkers of inflammation which allowed us to study the effects of ART.

Conclusion

One year of ART had a positive effect on reducing both inflammatory and vascular cytokines, and arterial stiffness.

Supporting information

S1 Table. Demographic and clinical characteristics of patients included and not included in the final analysis.

(DOCX)

Acknowledgments

This article is dedicated to the memory of Dr. Silvia G. Esquivel Razo, whose kindness and passion for science and service were an inspiration for all of us. Her legacy will remain in our hearts and minds.

Data Availability

Data are available from Harvard Database at https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/MGQGKG.

Funding Statement

The author(s) received no specific funding for this work.

References

  • 1.Global HIV & AIDS statistics—Fact sheet | UNAIDS. [cited 31 Jul 2022]. Available: https://www.unaids.org/en/resources/fact-sheet.
  • 2.Noticia—Estadísticas a propósito del Día mundial de la lucha contra el VIH/SIDA (1 de diciembre). [cited 31 Jul 2022]. Available: https://www.inegi.org.mx/app/saladeprensa/noticia.html?id=6976.
  • 3.Hsue PY, Waters DD. HIV infection and coronary heart disease: mechanisms and management. Nat Rev Cardiol. 2019;16: 745–759. doi: 10.1038/s41569-019-0219-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Vachiat A, McCutcheon K, Tsabedze N, Zachariah D, Manga P. HIV and Ischemic Heart Disease. J Am Coll Cardiol. 2017;69: 73–82. doi: 10.1016/j.jacc.2016.09.979 [DOI] [PubMed] [Google Scholar]
  • 5.Di Yacovo S, Saumoy M, Sánchez-Quesada JL, Navarro A, Sviridov D, Javaloyas M, et al. Lipids, biomarkers, and subclinical atherosclerosis in treatment-naive HIV patients starting or not starting antiretroviral therapy: Comparison with a healthy control group in a 2-year prospective study. PLoS One. 2020;15. doi: 10.1371/journal.pone.0237739 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Seaberg EC, Benning L, Sharrett AR, Lazar JM, Hodis HN, MacK WJ, et al. Association between human immunodeficiency virus infection and stiffness of the common carotid artery. Stroke. 2010;41. doi: 10.1161/STROKEAHA.110.583856 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Jiang J, Fu W, Wang X, Lin PH, Yao Q, Chen C. HIV gp120 induces endothelial dysfunction in tumour necrosis factor-alpha-activated porcine and human endothelial cells. Cardiovasc Res. 2010;87: 366–74. doi: 10.1093/cvr/cvq013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Wang T, Green LA, Gupta SK, Kim C, Wang L, Almodovar S, et al. Transfer of intracellular HIV Nef to endothelium causes endothelial dysfunction. PLoS One. 2014;9: e91063. doi: 10.1371/journal.pone.0091063 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lewis DE, Couturier JP. Chronic Inflammation in HIV Pathogenesis: Effects on Immune Cells, Organ Systems, and Systemic Consequences. Translational Inflammation. Elsevier; 2019. pp. 111–131. doi: [DOI] [Google Scholar]
  • 10.Triant VA, Perez J, Regan S, Massaro JM, Meigs JB, Grinspoon SK, et al. Cardiovascular Risk Prediction Functions Underestimate Risk in HIV Infection. Circulation. 2018;137: 2203–2214. doi: 10.1161/CIRCULATIONAHA.117.028975 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Willum-Hansen T, Staessen JA, Torp-Pedersen C, Rasmussen S, Thijs L, Ibsen H, et al. Prognostic value of aortic pulse wave velocity as index of arterial stiffness in the general population. Circulation. 2006;113: 664–70. doi: 10.1161/CIRCULATIONAHA.105.579342 [DOI] [PubMed] [Google Scholar]
  • 12.Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D, et al. Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J. 2006;27: 2588–605. doi: 10.1093/eurheartj/ehl254 [DOI] [PubMed] [Google Scholar]
  • 13.Kuate Defo A, Chalati MD, Labos C, Fellows LK, Mayo NE, Daskalopoulou SS. Association of HIV Infection and Antiretroviral Therapy With Arterial Stiffness: A Systematic Review and Meta-Analysis. Hypertension. 2021/06/29. 2021;78: 320–332. doi: 10.1161/HYPERTENSIONAHA.121.17093 [DOI] [PubMed] [Google Scholar]
  • 14.Lopez-Sublet M, Honoré P, Bentata M, Bratis C, Rouges F, Krivitzky A, et al. Protease inhibitor treatment effect on aortic stiffness in normotensive patients with human immunodeficiency virus infection. J Mal Vasc. 2012;37: 179–85. doi: 10.1016/j.jmv.2012.05.001 [DOI] [PubMed] [Google Scholar]
  • 15.Eira M, Bensenor IM, Dorea EL, Cunha RS, Mill JG, Lotufo PA. Potent antiretroviral therapy for human immunodeficiency virus infection increases aortic stiffness. Arq Bras Cardiol. 2012;99: 1100–1107. doi: 10.1590/s0066-782x2012005000110 [DOI] [PubMed] [Google Scholar]
  • 16.van Vonderen MGA, Hassink EAM, van Agtmael MA, Stehouwer CDA, Danner SA, Reiss P, et al. Increase in carotid artery intima-media thickness and arterial stiffness but improvement in several markers of endothelial function after initiation of antiretroviral therapy. J Infect Dis. 2009;199: 1186–94. doi: 10.1086/597475 [DOI] [PubMed] [Google Scholar]
  • 17.Maloberti A, Dozio D, Betelli M, Bandera A, Squillace N, Gori A, et al. Brachial and central blood pressure in HIV-infected subjects. Hypertens Res. 2015;38: 405–412. doi: 10.1038/hr.2015.25 [DOI] [PubMed] [Google Scholar]
  • 18.Martínez-Ayala P, Alanis-Sánchez GA, González-Hernández LA, Álvarez-Zavala M, Cabrera-Silva RI, Andrade-Villanueva JF, et al. Aortic stiffness and central hemodynamics in treatment-naïve HIV infection: a cross-sectional study. BMC Cardiovasc Disord. 2020;20: 440. doi: 10.1186/s12872-020-01722-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Rahmanian S, Wewers ME, Koletar S, Reynolds N, Ferketich A, Diaz P. Cigarette smoking in the HIV-infected population. Proc Am Thorac Soc. 2011;8: 313–9. doi: 10.1513/pats.201009-058WR [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Townsend RR, Wilkinson IB, Schiffrin EL, Avolio AP, Chirinos JA, Cockcroft JR, et al. Recommendations for Improving and Standardizing Vascular Research on Arterial Stiffness: A Scientific Statement from the American Heart Association. Hypertension. 2015;66. doi: 10.1161/HYP.0000000000000033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Cliff N. Dominance statistics: Ordinal analyses to answer ordinal questions. Psychol Bull. 1993;114. doi: 10.1037/0033-2909.114.3.494 [DOI] [Google Scholar]
  • 22.Osuji FN, Onyenekwe CC, Ahaneku JE, Ukibe NR. The effects of highly active antiretroviral therapy on the serum levels of pro-inflammatory and anti-inflammatory cytokines in HIV infected subjects. J Biomed Sci. 2018;25: 88. doi: 10.1186/s12929-018-0490-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.O’Halloran JA, Dunne E, Gurwith M, Lambert JS, Sheehan GJ, Feeney ER, et al. The effect of initiation of antiretroviral therapy on monocyte, endothelial and platelet function in HIV-1 infection. HIV Med. 2015;16: 608–19. doi: 10.1111/hiv.12270 [DOI] [PubMed] [Google Scholar]
  • 24.Torchiano M. effsize: Efficient effect size computation. R package version 08. 2020;1. [Google Scholar]
  • 25.Smith J. C, Sabin A. C, Lundgren D. J, Thiebaut R, Weber R, Law M, et al. Factors associated with specific causes of death amongst HIV-positive individuals in the D:A:D study. AIDS. 2010;24. doi: 10.1097/qad.0b013e32833a0918 [DOI] [PubMed] [Google Scholar]
  • 26.Grunfeld C, Pang M, Doerrler W, Shigenaga JK, Jensen P, Feingold KR. Lipids, lipoproteins, triglyceride clearance, and cytokines in human immunodeficiency virus infection and the acquired immunodeficiency syndrome. J Clin Endocrinol Metab. 1992;74: 1045–52. doi: 10.1210/jcem.74.5.1373735 [DOI] [PubMed] [Google Scholar]
  • 27.Daar ES, Tierney C, Fischl MA, Sax PE, Mollan K, Budhathoki C, et al. Atazanavir plus ritonavir or efavirenz as part of a 3-drug regimen for initial treatment of HIV-1. Ann Intern Med. 2011;154: 445–56. doi: 10.7326/0003-4819-154-7-201104050-00316 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Gunnett CA, Lund DD, McDowell AK, Faraci FM, Heistad DD. Mechanisms of inducible nitric oxide synthase-mediated vascular dysfunction. Arterioscler Thromb Vasc Biol. 2005;25: 1617–22. doi: 10.1161/01.ATV.0000172626.00296.ba [DOI] [PubMed] [Google Scholar]
  • 29.Venugopal SK, Devaraj S, Yuhanna I, Shaul P, Jialal I. Demonstration that C-reactive protein decreases eNOS expression and bioactivity in human aortic endothelial cells. Circulation. 2002;106: 1439–41. doi: 10.1161/01.cir.0000033116.22237.f9 [DOI] [PubMed] [Google Scholar]
  • 30.Solages A, Vita JA, Thornton DJ, Murray J, Heeren T, Craven DE, et al. Endothelial function in HIV-infected persons. Clin Infect Dis. 2006;42: 1325–32. doi: 10.1086/503261 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Mu H, Chai H, Lin PH, Yao Q, Chen C. Current update on HIV-associated vascular disease and endothelial dysfunction. World J Surg. 2007;31: 632–43. doi: 10.1007/s00268-006-0730-0 [DOI] [PubMed] [Google Scholar]
  • 32.Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347: 1557–65. doi: 10.1056/NEJMoa021993 [DOI] [PubMed] [Google Scholar]
  • 33.Rose H, Low H, Dewar E, Bukrinsky M, Hoy J, Dart A, et al. The effect of HIV infection on atherosclerosis and lipoprotein metabolism: a one year prospective study. Atherosclerosis. 2013;229: 206–11. doi: 10.1016/j.atherosclerosis.2013.04.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Maia-Leite LH, Catez E, Boyd A, Haddour N, Curjol A, Lang S, et al. Aortic stiffness aging is influenced by past profound immunodeficiency in HIV-infected individuals: results from the EVAS-HIV (EValuation of Aortic Stiffness in HIV-infected individuals). J Hypertens. 2016;34: 1338–46. doi: 10.1097/HJH.0000000000000957 [DOI] [PubMed] [Google Scholar]
  • 35.Teasdale CA, Hernandez C, Zerbe A, Chege D, Hawken M, El-Sadr WM. Changes in D-dimer after initiation of antiretroviral therapy in adults living with HIV in Kenya. BMC Infect Dis. 2020;20. doi: 10.1186/S12879-020-05213-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Martínez E, D’Albuquerque PM, Llibre JM, Gutierrez F, Podzamczer D, Antela A, et al. Changes in cardiovascular biomarkers in HIV-infected patients switching from ritonavir-boosted protease inhibitors to raltegravir. AIDS. 2012;26. doi: 10.1097/QAD.0b013e328359f29c [DOI] [PubMed] [Google Scholar]
  • 37.Neuhaus J, Jacobs DR, Baker JV, Calmy A, Duprez D, La Rosa A, et al. Markers of inflammation, coagulation, and renal function are elevated in adults with HIV infection. J Infect Dis. 2010;201: 1788–95. doi: 10.1086/652749 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.El-Bejjani D, Hazen SL, Mackay W, Glass NE, Hulgan T, Tungsiripat M, et al. Higher plasma myeloperoxidase levels are not associated with an increased risk for cardiovascular events in HIV-infected adults. HIV Clin Trials. 2008;9: 207–211. doi: 10.1310/hct0903-207 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.O’Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996/02/15. 1996;93: 672–682. doi: 10.1161/01.cir.93.4.672 [DOI] [PubMed] [Google Scholar]
  • 40.Hwang SJ, Ballantyne CM, Sharrett AR, Smith LC, Davis CE, Gotto AM Jr., 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. 1998/01/07. 1997;96: 4219–4225. doi: 10.1161/01.cir.96.12.4219 [DOI] [PubMed] [Google Scholar]
  • 41.Papasavvas E, Azzoni L, Pistilli M, Hancock A, Reynolds G, Gallo C, et al. Increased soluble vascular cell adhesion molecule-1 plasma levels and soluble intercellular adhesion molecule-1 during antiretroviral therapy interruption and retention of elevated soluble vascular cellular adhesion molecule-1 levels following resumption of antiretroviral therapy. AIDS. 2008/06/06. 2008;22: 1153–1161. doi: 10.1097/QAD.0b013e328303be2a [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Healy AM, Pickard MD, Pradhan AD, Wang Y, Chen Z, Croce K, et al. Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events. Circulation. 2006;113: 2278–84. doi: 10.1161/CIRCULATIONAHA.105.607333 [DOI] [PubMed] [Google Scholar]
  • 43.Croce K, Gao H, Wang Y, Mooroka T, Sakuma M, Shi C, et al. Myeloid-related protein-8/14 is critical for the biological response to vascular injury. Circulation. 2009;120: 427–36. doi: 10.1161/CIRCULATIONAHA.108.814582 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Murdaca G, Colombo BM, Cagnati P, Gulli R, Spanò F, Puppo F. Endothelial dysfunction in rheumatic autoimmune diseases. Atherosclerosis. 2012;224: 309–17. doi: 10.1016/j.atherosclerosis.2012.05.013 [DOI] [PubMed] [Google Scholar]
  • 45.Martínez E, D’Albuquerque PM, Llibre JM, Gutierrez F, Podzamczer D, Antela A, et al. Changes in cardiovascular biomarkers in HIV-infected patients switching from ritonavir-boosted protease inhibitors to raltegravir. AIDS. 2012/09/29. 2012;26: 2315–2326. doi: 10.1097/QAD.0b013e328359f29c [DOI] [PubMed] [Google Scholar]
  • 46.Pedersen L, Nybo M, Poulsen MK, Henriksen JE, Dahl J, Rasmussen LM. Plasma calprotectin and its association with cardiovascular disease manifestations, obesity and the metabolic syndrome in type 2 diabetes mellitus patients. BMC Cardiovasc Disord. 2014;14: 196. doi: 10.1186/1471-2261-14-196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Soylu K, Nar G, Aksan G, Gedikli Ö, İnci S, Yuksel S, et al. Serum neutrophil gelatinase-associated lipocalin levels and aortic stiffness in noncritical coronary artery disease. Cardiorenal Med. 2014;4: 147–54. doi: 10.1159/000365200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Shrestha K, Borowski AG, Troughton RW, Klein AL, Tang WHW. Association between systemic neutrophil gelatinase-associated lipocalin and anemia, relative hypochromia, and inflammation in chronic systolic heart failure. Congest Heart Fail. 2012;18: 239–244. doi: 10.1111/j.1751-7133.2012.00287.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Eilenberg W, Stojkovic S, Piechota-Polanczyk A, Kaun C, Rauscher S, Gröger M, et al. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro. Eur J Vasc Endovasc Surg. 2016;51: 623–31. doi: 10.1016/j.ejvs.2016.01.009 [DOI] [PubMed] [Google Scholar]
  • 50.Rajasuriar R, Wright E, Lewin SR. Impact of antiretroviral therapy (ART) timing on chronic immune activation/inflammation and end-organ damage. Curr Opin HIV AIDS. 2015;10: 35–42. doi: 10.1097/COH.0000000000000118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Višković K, Židovec Lepej S, Gorenec A, Grgić I, Lukas D, Zekan Š, et al. Cardiovascular markers of inflammation and serum lipid levels in HIV-infected patients with undetectable viremia. Sci Rep. 2018;8: 6113. doi: 10.1038/s41598-018-24446-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Malle E, Steinmetz A, Raynes JG. Serum amyloid A (SAA): an acute phase protein and apolipoprotein. Atherosclerosis. 1993;102: 131–146. doi: 10.1016/0021-9150(93)90155-n [DOI] [PubMed] [Google Scholar]
  • 53.Samikkannu T, Rao KVK, Arias AY, Kalaichezian A, Sagar V, Yoo C, et al. HIV infection and drugs of abuse: role of acute phase proteins. J Neuroinflammation. 2013;10: 113. doi: 10.1186/1742-2094-10-113 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Eliseo A Eugenin

31 Oct 2022

PONE-D-22-26706Effect of antiretrovirals on the regression of arterial stiffness, metabolic, vascular, and systemic inflammatory cytokines at one year of virologic controlPLOS ONE

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Reviewer #1: Effect of antiretrovirals on the regression of arterial stiffness, metabolic, vascular, and systemic inflammatory cytokines at one year of virologic control

Pedro Martínez-Ayala PlosOne

HIV creates an increase inflammation with vascular consequences and been only partially reversed by ART. Such inflammation is driven by HIV itself and also by leaky gut allowing microbial translocation of bacterial and fungus products.

Investigators assessed the effect of one-year treatment-naïve HIV individuals on arterial stiffness and inflammatory and vascular cytokines. tonometry, inflammatory, and vascular serum cytokines on treatment-naïve (n=20) and HIV uninfected (n=9) age matched control with metabolic profile, and Framingham score evaluation.

Study findings indicate that ART had a significant effect on reducing inflammatory and most vascular cytokines and arterial stiffness.

Comments:

As nearly half of participants receive and NNRT vs PI comparison of these 2 groups will be of interest as PI increased cholesterol and have a cumulative risk for CV events.

The difficulty of such studies with many markers only a few fit with the study hypothesis. Here the 2 inflammatory markers linked with non-AIDS events d-dimers and IL-6 were not different. Discussion on these 2 validated markers for CVV risks should be discussed in detail.

LPS, sCD14 and beta-d-glucan play an important role in HIV-related inflammation and are linked with CV risk by imaging. Measurement of these markers will add value to the manuscript

Isnard S, et al. Circulating β-d-Glucan as a Marker of Subclinical Coronary Plaque in Antiretroviral Therapy-Treated People With Human Immunodeficiency Virus. Open Forum Infect Dis. 2021 Mar 7;8(6):ofab109.

Isnard S, et al. Gut Leakage of Fungal-Related Products: Turning Up the Heat for HIV Infection. Front Immunol. 2021 Apr 12;12:656414.

In addition, the stress cytokine GDF-15 emerges as one of the best markers in CV disorders: ischemic conditions, atrial fibrillation and cardiac insufficiency and in 2022 in HIV. Assessment of one the best CV markers will be welcome for this study.

Royston L, . Growth differentiation factor-15 as a biomarker of atherosclerotic coronary plaque: Value in people living with and without HIV. Front Cardiovasc Med. 2022 Aug 26;9:964650.

Discussion is much too long and should not be structured, no subchapters are necessary. Investigators should focus only on novelty as many data are confirming previous findings.

Increased in Cholesterol and TG are considered to augment as return to health at one year as generally patients gain 2 kg initially taken for HIV. Therefore, early changes are not linked to inflammation or PI effects on lipids after one year of ART.

Limitations: absence of cellular markers of adaptative and innate immunity.

As cells were not assessed a small paragraph on the role of adaptive immunity cells, such as CD4 cells are acknowledged to participate to CVD pathogenesis (Emeson et al., 1996; Zhou et al., 2005). CD4 cells were found infiltrated in atherosclerotic plaques (Saigusa et al., 2020). Recent evidence indicates that T helper 1 (Th1) cells have pro-atherogenic roles, whereas regulatory T cells (Tregs) can play a dual role being both anti-atherogenic or pro-atherogenic (George et al., 2012; Maganto-Garcia et al., 2011).

Similarly in HIV, innate immunity: Elevated frequency of circulating non-classical monocytes (CD14dimCD16++) (Gu et al., 1998) and the intermediate CD14+CD16+ monocyte counts were associated to subclinical atherosclerosis (Hanna et al., 2017) and the expression of CX3CR1 on CD16+ monocytes predicted carotid artery thickness (Westhorpe et al., 2014)

Reviewer #2: This is important research. The study provides novel, prospective data in the field of HIV-associated CVD and warrants publication, even considering the relatively small number of participants. I do however think the manuscript could be improved before publication.

Major Comments:

My biggest concern is how the group of 20 HIV infected persons were recruited. The authors mention prior work with 51 individuals. Were these persons included from the prior cohort or are the participants all new? How were they selected if previously enrolled? Was everyone that was recruited followed up fully and included? Attrition? This needs to be discussed that possible bias may be evaluated. If they were chosen based on viral suppression alone, comparison with a virally unsuppressed group will be valuable and should be strongly considered.

A significant limitation is the fact that the study essentially only included males. This should be discussed as a significant limitation as sex-differences were not evaluated and females are already significantly under-represented in CV research.

There were isolated delta (change over time) correlations mentioned , however, I miss a dedicated correlation analysis. Relevant correlations (or lack thereof) needs to be reported. Viral load, CD4 count, smoking, inflammatory markers, vascular markers and aortic stiffness?

Varying ways of referring to HIV infected persons and the HIV cohorts are used in manuscript. Decide on terminology and use this consistently.

NB: Mean CD4 count in table 1 is different to what is written in the abstract and the manuscript text.

The discussion section is too long and reads as quite meandering. Every section should have a clear message and build on the message of the article. The final message of the article should be clear, and I think the authors should work on the conclusion (both in the abstract and the manuscript) to clearly summarise what they think their findings mean.

Comments:

Introduction:

line 58-59: Unreferenced, inaccurate statement. CVD is not the leading cause of death in HIV. Rather say one of the leading causes of CVD.

line 65 onward: Needs rewrite to clarify. There are sweeping statements without references and should be avoided. Smoking (as the authors mention later) has been associated with HIV in certain studies. This sentence reads as contradictory.

line 68: reference please

line 69 onward: Tonometry-based cfPWV is frequently employed in clinical research, but is not considered to be the definitive method of PWV measurement as it is implied in the text. The statement in the manuscript should be corrected to only read: 'cfPWV is considered the gold standard of aortic stiffness measurement.' Consensus has not been reached as to the definitive method to measure cfPWV and various techniques are well described.

read

Rajzer, Marek W; Wojciechowska, Wiktoria; Klocek, Marek; Palka, Ilona; Brzozowska-Kiszka, Małgorzata; Kawecka-Jaszcz, Kalina (2008). Comparison of aortic pulse wave velocity measured by three techniques: Complior, SphygmoCor and Arteriograph. Journal of Hypertension, 26(10), 2001–2007. doi:10.1097/hjh.0b013e32830a4a25

line 77: Sparse work is available from low- and middle income countries, however, recent data have shown similar findings in sub-Saharan Africa. This may serve to strengthen your rationale for the research:

read

Robbertse PS, Doubell AF, Innes S, et al. Pulse wave velocity demonstrates increased aortic stiffness in newly diagnosed, antiretroviral naïve HIV infected adults: A case-control study. Medicine. 2022; 101:e29721.

line 113, 148 and other locations: Define all abbreviations with first use.

line 150: p-value of <0.05

line 153-155: This does not seem correct. Paired samples t-test with 80% power to detect a 0.6m/s difference and the quoted SD with alpha=0.05, calculates to 29 pairs on SPSS. This is more than double the number quoted in the text.

Results:

Were any of the patients of vasoactive mediation? I think this is a worthwhile to mention, even if no-one was on these medicines.

I find the BMI findings the inverse of what I would expect when compared to the controls. Any ideas why this may be the case in your study?

Not all abbreviations used in the tables are found in the legends. Please double check. Tables should be able to be able to be freestanding from the article and still be easy to interpret.

line 186: p=0.055 is not significant according to your study’s predefined level of significance. Rather state a trend toward significance for accuracy.

line 205: Again, consistent use of wording to refer to the study groups. Post-ART should rather be ART-group or ART-experienced group.

line 208: ‘In the beginning’. Do the authors mean at baseline?

Dedicated correlation analysis is an omission in my opinion as discussed earlier.

Discussion:

I would restructure the first paragraph to emphasise your own novel, longitudinal findings first. The mention of lack of longitudinal studies belong in the introduction. Rather state own strengths, than others weaknesses in this critical paragraph.

line 254: ‘slightly or did not decrease’. Reword this to communicate your findings clearly. Median hs-CRP essentially halved after ART. I would say something like ‘The hs-CRP showed a strong trend towards decrease, however, this did not reach statistical significance. hs-CRP, despite ART, remained significantly higher in ART-experienced persons when compared to the control group.’

line 265-270: This is all true. However, I think the reader would benefit from better packaging of these facts using our current theoretical framework of the factors that underpin cfPWV (especially in light of your young cohort, with likely negligible amounts of atherosclerotic disease). Pressure amplification by peripheral arterial tone. Vasomotor arterial tone is modulated by endothelial function, sympathetic tone, and the RAAS system. The manuscript would benefit from the incorporation of key concepts from the following articles (In general and in the setting of HIV). Use your excellent data to examine some of these concepts and what you think actually drive the increased (and decreased on ART) PWV mechanistically. Your study is explorative and should make the best use of your data.

read:

1. Cavalcante JL, Lima JAC, Redheuil A, et al. Aortic Stiffness. J Am Coll Cardiol. 2011;57:1511–22.

2. Robbertse PS, Doubell AF, Innes S, et al. Pulse wave velocity demonstrates increased aortic stiffness in newly diagnosed, antiretroviral naïve HIV infected adults: A case-control study. Medicine. 2022; 101:e29721.

line 319: reference

line 320: contrasting what? Use clear language.

line 367-368: I would be careful with these strong statements. Your findings do not quite support this and I suggest rewording this statement. I would focus on what you could show: that was that ART had an overall positive effect (decreased vascular pathology markers, decreased markers of inflammation, and decreased cfPWV). The observation that aortic stiffness decreased with ART is important, as this means (as you stated) that a reversible component of aortic stiffness remains and there is likely a window before this becomes irreversible (collagen deposition, degradation of elastin etc). When this window is, remains unknown.

Furthermore, if cfPWV is used a surrogate of CV risk, you demonstrated increased risk compared to HIV uninfected persons at baseline. As cfPWV decreased at one year on ART (in a small group of virally supressed individuals), I would see this as a relative decrease in CV risk. Yet another reason to give ART. The residual risk despite ART is still of concern, and as you mentioned stratification may be an issue, as people on ART have higher CVD compared to those without HIV.

Limitations:

The sample is small and explorative and should be stated in no uncertain terms. Selection of the cohort needs to be more detailed, as there is a perceived risk of inclusion bias.

Typo’s, spelling, and language: Various errors present. I would suggest involving a proof-reader before re-submission.

line 30. PWHIV

line 36. young, treatment-naïve. Not treatment-naïve young.

line 60: envelop

line 211: inflammation. rather biomarkers of inflammation

Minor comments for your discretion:

Title: I miss the word HIV, even though antiretrovirals imply this. I do not agree with the word "regression". Consider rewriting the title to refer to a decrease in aortic stiffness.

line 61: We generally refer to hs-CRP as high-sensitivity CRP, not highly sensitive CRP

line 62: remove increased

line 64: non-related should be unrelated

---

I look forward to the amended version of the manuscript and the inevitable publication of this important research.

**********

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Reviewer #2: No

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PLoS One. 2023 Mar 17;18(3):e0282728. doi: 10.1371/journal.pone.0282728.r002

Author response to Decision Letter 0


20 Jan 2023

RESPONSE TO REVIEWERS

We wish to thank the reviewers for their insightful and kind comments. After doing the necessary modifications, this manuscript improved substantially.

Reviewer #1: Effect of antiretrovirals on the regression of arterial stiffness, metabolic, vascular, and systemic inflammatory cytokines at one year of virologic control

Pedro Martínez-Ayala PlosOne

HIV creates an increase inflammation with vascular consequences and been only partially reversed by ART. Such inflammation is driven by HIV itself and also by leaky gut allowing microbial translocation of bacterial and fungus products.

Investigators assessed the effect of one-year treatment-naïve HIV individuals on arterial stiffness and inflammatory and vascular cytokines. tonometry, inflammatory, and vascular serum cytokines on treatment-naïve (n=20) and HIV uninfected (n=9) age matched control with metabolic profile, and Framingham score evaluation.

Study findings indicate that ART had a significant effect on reducing inflammatory and most vascular cytokines and arterial stiffness.

Comments:

As nearly half of participants receive and NNRT vs PI comparison of these 2 groups will be of interest as PI increased cholesterol and have a cumulative risk for CV events.

The difficulty of such studies with many markers only a few fit with the study hypothesis. Here the 2 inflammatory markers linked with non-AIDS events d-dimers and IL-6 were not different. Discussion on these 2 validated markers for CVV risks should be discussed in detail.

- In our population IL-6 has great variability and for this reason, has not been useful as a stable biomarker. Thus, we preferred to use hsCRP, which besides being a surrogate marker of IL-6 it is widely available in all laboratory settings in our country. Furthermore, a cut-off value for hsCRP is known for adverse cardiovascular outcomes in HIV. (http://www.biomedcentral.com/1471-2334/13/414)

LPS, sCD14 and beta-d-glucan play an important role in HIV-related inflammation and are linked with CV risk by imaging. Measurement of these markers will add value to the manuscript.

Isnard S, et al. Circulating β-d-Glucan as a Marker of Subclinical Coronary Plaque in Antiretroviral Therapy-Treated People With Human Immunodeficiency Virus. Open Forum Infect Dis. 2021 Mar 7;8(6):ofab109.

Isnard S, et al. Gut Leakage of Fungal-Related Products: Turning Up the Heat for HIV Infection. Front Immunol. 2021 Apr 12;12:656414.

In addition, the stress cytokine GDF-15 emerges as one of the best markers in CV disorders: ischemic conditions, atrial fibrillation and cardiac insufficiency and in 2022 in HIV. Assessment of one the best CV markers will be welcome for this study.

Royston L, . Growth differentiation factor-15 as a biomarker of atherosclerotic coronary plaque: Value in people living with and without HIV. Front Cardiovasc Med. 2022 Aug 26;9:964650.

-Great suggestion. However, sCD163 could act as a surrogate biomarker of CD14. sCD163 provides information on monocyte-macrophage activation. Both LDS and B-D-glucan have been indeed widely investigated but these markers were not included in the original study design. https://doi.org/10.3389/fimmu.2020.560381, https://pubmed.ncbi.nlm.nih.gov/25362192/

Discussion is much too long and should not be structured, no subchapters are necessary. Investigators should focus only on novelty as many data are confirming previous findings.

-We have eliminated the subchapters' headings and rewrote the discussion making it more concise and focusing more on the new findings.

Increased in Cholesterol and TG are considered to augment as return to health at one year as generally patients gain 2 kg initially taken for HIV. Therefore, early changes are not linked to inflammation or PI effects on lipids after one year of ART.

Limitations: absence of cellular markers of adaptative and innate immunity.

-We agree and even though both cholesterol and TG had a statistically significant increase, the final post-ART mean levels were not clinically significant and did not require pharmacological treatment.

As cells were not assessed a small paragraph on the role of adaptive immunity cells, such as CD4 cells are acknowledged to participate to CVD pathogenesis (Emeson et al., 1996; Zhou et al., 2005).

CD4 cells were found infiltrated in atherosclerotic plaques (Saigusa et al., 2020). Recent evidence indicates that T helper 1 (Th1) cells have pro-atherogenic roles, whereas regulatory T cells (Tregs) can play a dual role being both anti-atherogenic or pro-atherogenic (George et al., 2012; Maganto-Garcia et al., 2011).Similarly in HIV, innate immunity: Elevated frequency of circulating non-classical monocytes (CD14dimCD16++) (Gu et al., 1998) and the intermediate CD14+CD16+ monocyte counts were associated to subclinical atherosclerosis (Hanna et al., 2017) and the expression of CX3CR1 on CD16+ monocytes predicted carotid artery thickness (Westhorpe et al., 2014)

-We acknowledge that CD4 do play a role in atherogenesis, and it will be taken into account in the next study we conduct. Nonetheless, given that arteriosclerosis is different from atherosclerosis and given the length of our discussion, we believe it would be more appropriate if we had measured atherosclerosis (e.g., intima media thickness).

Reviewer #2: This is important research. The study provides novel, prospective data in the field of HIV-associated CVD and warrants publication, even considering the relatively small number of participants. I do however think the manuscript could be improved before publication.

Major Comments:

My biggest concern is how the group of 20 HIV infected persons were recruited. The authors mention prior work with 51 individuals. Were these persons included from the prior cohort or are the participants all new? How were they selected if previously enrolled? Was everyone that was recruited followed up fully and included? Attrition? This needs to be discussed that possible bias may be evaluated. If they were chosen based on viral suppression alone, comparison with a virally unsuppressed group will be valuable and should be strongly considered.

All patients that attended the HIV Unit of the hospital were invited to participate in the study. In the beginning, we initially enrolled all 51 participants from our previous cross-sectional study. Unfortunately, due to the high drop-out rate (20%) and missing follow-up, we were only able to include 20 participants for the analysis from whom we had complete longitudinal data.

A significant limitation is the fact that the study essentially only included males. This should be discussed as a significant limitation as sex-differences were not evaluated and females are already significantly under-represented in CV research.

-Included in the limitations.

There were isolated delta (change over time) correlations mentioned , however, I miss a dedicated correlation analysis. Relevant correlations (or lack thereof) needs to be reported. Viral load, CD4 count, smoking, inflammatory markers, vascular markers and aortic stiffness?

-We believe that it was better to remove the isolated delta we reported. We think this could have been a type 1 error given the number of comparisons performed and the absence p-value adjustment. Moreover, given the small sample size, the risk of false negatives would be high to report an absence of association. We are looking to increase our sample size to determine the presence or absence of associations, especially with arterial stiffness.

However, after expressing our thoughts if both reviewers think a correlation analysis would be of benefit, we will happily add a correlation analysis section that could go as a Supplementary file.

Varying ways of referring to HIV infected persons and the HIV cohorts are used in the manuscript. Decide on terminology and use this consistently.

-We decided to use the term PLH to refer to the HIV population in general.

NB: Mean CD4 count in table 1 is different to what is written in the abstract and the manuscript text.

-Corrected.

The discussion section is too long and reads as quite meandering. Every section should have a clear message and build on the message of the article. The final message of the article should be clear, and I think the authors should work on the conclusion (both in the abstract and the manuscript) to clearly summarise what they think their findings mean.

- We have substantially modified the discussion and did some changes to the conclusion.

Comments:

Introduction:

line 58-59: Unreferenced, inaccurate statement. CVD is not the leading cause of death in HIV. Rather say one of the leading causes of CVD.

-Corrected. It now reads “Cardiovascular complications are one of the leading causes of morbidity…”.

line 65 onward: Needs rewrite to clarify. There are sweeping statements without references and should be avoided. Smoking (as the authors mention later) has been associated with HIV in certain studies. This sentence reads as contradictory.

- Good observation. We removed that last sentence. We believe it is more important to emphasize that traditional risk calculators used in the general population may not be accurate in the HIV population.

line 68: reference please

- Reference added.

line 69 onward: Tonometry-based cfPWV is frequently employed in clinical research, but is not considered to be the definitive method of PWV measurement as it is implied in the text. The statement in the manuscript should be corrected to only read: 'cfPWV is considered the gold standard of aortic stiffness measurement.' Consensus has not been reached as to the definitive method to measure cfPWV and various techniques are well described.

read

Rajzer, Marek W; Wojciechowska, Wiktoria; Klocek, Marek; Palka, Ilona; Brzozowska-Kiszka, Małgorzata; Kawecka-Jaszcz, Kalina (2008). Comparison of aortic pulse wave velocity measured by three techniques: Complior, SphygmoCor and Arteriograph. Journal of Hypertension, 26(10), 2001–2007. doi:10.1097/hjh.0b013e32830a4a25

- Your comment is right. We modified it to state that carotid-femoral pulse wave velocity is the gold standard for aortic stiffness measurement without specifying the technique (i.e, oscillometric, mechanotransducer or tonometry).

line 77: Sparse work is available from low- and middle income countries, however, recent data have shown similar findings in sub-Saharan Africa. This may serve to strengthen your rationale for the research:

read

Robbertse PS, Doubell AF, Innes S, et al. Pulse wave velocity demonstrates increased aortic stiffness in newly diagnosed, antiretroviral naïve HIV infected adults: A case-control study. Medicine. 2022; 101:e29721.

- We added at the end of the sentence that there is limited data in developing countries.

line 113, 148 and other locations: Define all abbreviations with first use.

Those abbreviations and the rest have been defined.

line 150: p-value of <0.05

-Corrected

line 153-155: This does not seem correct. Paired samples t-test with 80% power to detect a 0.6m/s difference and the quoted SD with alpha=0.05, calculates to 29 pairs on SPSS. This is more than double the number quoted in the text.

Yes, you are correct. We forgot to mention that for this calculation it is necessary to include a correlation coefficient for paired data. Given that we were not able to find available such data in the literature, we used paired cfPWV from previous paired data in our lab in people living with HIV. We obtained a Pearson correlation coefficient of 0.81 and aimed to detect a 0.6 m/s difference and pooled 1.1 SD, as reported in our previous cross-sectional study between PLH and HIV(-). We used GPowet to do the sample size calculation.

Results:

Were any of the patients of vasoactive mediation? I think this is a worthwhile to mention, even if no-one was on these medicines.

- None of the study participants were on vasoactive medications. We added as part of the inclusion criteria for both PLWIH and controls.

I find the BMI findings the inverse of what I would expect when compared to the controls. Any ideas why this may be the case in your study?

- We believe that it could be an incidental finding due to the small sample size and/or even though they were treatment-naïve, 90% of HIV individuals had CD4 levels >200; thus, wasting syndrome was less likely in our sample.

Not all abbreviations used in the tables are found in the legends. Please double check. Tables should be able to be able to be freestanding from the article and still be easy to interpret.

Abbreviations added to the table legends.

line 186: p=0.055 is not significant according to your study’s predefined level of significance. Rather state a trend toward significance for accuracy.

- It now reads: “c-LDL showed a trend towards a significant increase post-ART.”

line 205: Again, consistent use of wording to refer to the study groups. Post-ART should rather be ART-group or ART-experienced group.

-We changed it to ART-group.

line 208: ‘In the beginning’. Do the authors mean at baseline?

- Changed to “At baseline”.

Dedicated correlation analysis is an omission in my opinion as discussed earlier.

Discussion:

I would restructure the first paragraph to emphasise your own novel, longitudinal findings first. The mention of lack of longitudinal studies belong in the introduction. Rather state own strengths, than others weaknesses in this critical paragraph.

- The first paragraph was modified.

line 254: ‘slightly or did not decrease’. Reword this to communicate your findings clearly. Median hs-CRP essentially halved after ART. I would say something like ‘The hs-CRP showed a strong trend towards decrease, however, this did not reach statistical significance. hs-CRP, despite ART, remained significantly higher in ART-experienced persons when compared to the control group.’

- We rephrased that sentence.

line 265-270: This is all true. However, I think the reader would benefit from better packaging of these facts using our current theoretical framework of the factors that underpin cfPWV (especially in light of your young cohort, with likely negligible amounts of atherosclerotic disease). Pressure amplification by peripheral arterial tone. Vasomotor arterial tone is modulated by endothelial function, sympathetic tone, and the RAAS system. The manuscript would benefit from the incorporation of key concepts from the following articles (In general and in the setting of HIV). Use your excellent data to examine some of these concepts and what you think actually drive the increased (and decreased on ART) PWV mechanistically. Your study is explorative and should make the best use of your data.

read:

1. Cavalcante JL, Lima JAC, Redheuil A, et al. Aortic Stiffness. J Am Coll Cardiol. 2011;57:1511–22.

2. Robbertse PS, Doubell AF, Innes S, et al. Pulse wave velocity demonstrates increased aortic stiffness in newly diagnosed, antiretroviral naïve HIV infected adults: A case-control study. Medicine. 2022; 101:e29721.

- We rewrote the arterial stiffness paragraph to give a short introduction of arterial stiffness, how it is regulated and a possible mechanism that could explain our results from an inflammatory point of view.

line 319: reference

- We removed that part of the discussion.

line 320: contrasting what? Use clear language.

- We modified this paragraph.

line 367-368: I would be careful with these strong statements. Your findings do not quite support this and I suggest rewording this statement. I would focus on what you could show: that was that ART had an overall positive effect (decreased vascular pathology markers, decreased markers of inflammation, and decreased cfPWV). The observation that aortic stiffness decreased with ART is important, as this means (as you stated) that a reversible component of aortic stiffness remains and there is likely a window before this becomes irreversible (collagen deposition, degradation of elastin etc). When this window is, remains unknown.

Furthermore, if cfPWV is used a surrogate of CV risk, you demonstrated increased risk compared to HIV uninfected persons at baseline. As cfPWV decreased at one year on ART (in a small group of virally supressed individuals), I would see this as a relative decrease in CV risk. Yet another reason to give ART. The residual risk despite ART is still of concern, and as you mentioned stratification may be an issue, as people on ART have higher CVD compared to those without HIV.

- We modified this last paragraph of the discussion.

Limitations:

The sample is small and explorative and should be stated in no uncertain terms. Selection of the cohort needs to be more detailed, as there is a perceived risk of inclusion bias.

Typo’s, spelling, and language: Various errors present. I would suggest involving a proof-reader before re-submission.

line 30. PWHIV

-Corrected. We decided to use the abbreviation PLH across the manuscript.

line 36. young, treatment-naïve. Not treatment-naïve young.

-Corrected.

line 60: envelop

-Corrected.

line 211: inflammation. rather biomarkers of inflammation

-Corrected.

Minor comments for your discretion:

Title: I miss the word HIV, even though antiretrovirals imply this. I do not agree with the word "regression". Consider rewriting the title to refer to a decrease in aortic stiffness.

- We changed the title to: “Effect of antiretroviral therapy on decreasing of arterial stiffness, metabolic profile, vascular and systemic inflammatory cytokines in treatment-naïve HIV: A one-year prospective study.”

line 61: We generally refer to hs-CRP as high-sensitivity CRP, not highly sensitive CRP

- Corrected

line 62: remove increased

- Removed.

line 64: non-related should be unrelated

-Modified that last sentence.

Decision Letter 1

Eliseo A Eugenin

13 Feb 2023

PONE-D-22-26706R1Effect of antiretroviral therapy on decreasing of arterial stiffness,  metabolic profile, vascular and systemic inflammatory cytokines in treatment-naïve HIV: A one-year prospective study.PLOS ONE

Dear Dr. Andrade,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: issues have been addressed including statistics and discussion conclusion.

Prospective data with relatively advanced patients add to current knowledge

Reviewer #2: Thank you for the author's responses to my comments. I am mostly satisfied with the responses and the amendments to the manuscript. The new title is excellent.

I discuss a few comments that still need to be adequately addressed below:

Original comment 1: Recruitment

Thank you for clarifying. Selection bias is be a factor that needs consideration by the reader and merits careful explanation. If the original cohort had 51 participants, the completion rate is 39%. (higher attrition than most HIV research, granted that PLWH are known to have high attrition). The quoted 20% dropout rate is therefore confusing, as the numbers do not add up. Please state how many were lost, withdrew, died, or had incomplete records. I would state this high attrition rate as a limitation of the study - it's higher than to be expected and likely influenced your results.

Original comment 3: Correlation analysis

I see the merit in the argument and agree that a future, larger sample size would be more appropriate for this. Please state explicitly in the methodology that a correlation analysis was not undertaken due to the small sample size in this explorative work.

New comments:

1. Limitations section: "Despite that we aimed to isolate the effect of HIV on arterial stiffness by choosing PLH without any other comorbidities and comparing them with healthy controls, there may be a series of genetic, lifestyle..."

Please improve the language here: My suggestion: "Despite our aim to isolate the effect HIV on arterial stiffness by choosing PLH without any other comorbidities and comparing them with healthy controls, there may be ..."

2. There are new, untracked additions to the manuscript. All changes should be stated or better yet, clearly tracked/highlighted for the editor.

Of note: "The literature vastly describes that ART does not completely suppress viral load, particularly in the viral reservoirs".

This is not in the academic style of writing. Rephrase. I suggest: "It is well described that ART does not completely suppress viral load..."

3. Define all abbreviations with first use: hs-CRP is never defined. hsCRP vs hs-CRP are both used in the manuscript.

4. I still feel the article could receive additional language manicuring. An academic proof reader should be considered.

I look forward to your work in print.

Regards,

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Jean-Pierre Routy

Reviewer #2: No

**********

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PLoS One. 2023 Mar 17;18(3):e0282728. doi: 10.1371/journal.pone.0282728.r004

Author response to Decision Letter 1


20 Feb 2023

Original comment 1: Recruitment

Thank you for clarifying. Selection bias is be a factor that needs consideration by the reader and merits careful explanation. If the original cohort had 51 participants, the completion rate is 39%. (higher attrition than most HIV research, granted that PLWH are known to have high attrition). The quoted 20% dropout rate is therefore confusing, as the numbers do not add up. Please state how many were lost, withdrew, died, or had incomplete records. I would state this high attrition rate as a limitation of the study - it's higher than to be expected and likely influenced your results.

- Thank you for your comment. Indeed, it is very important that we explain and make sure there was no selection bias. As stated in the revised manuscript, we selected the first 20 patients, with no selection of any kind, who had completed one year of viral suppression and had complete baseline and final data. We compared 31 patients from our cohort that were not included in the analysis to ensure that there were no differences in clinical or demographic characteristics.

Original comment 3: Correlation analysis

I see the merit in the argument and agree that a future, larger sample size would be more appropriate for this. Please state explicitly in the methodology that a correlation analysis was not undertaken due to the small sample size in this explorative work.

-Included in the methods section.

New comments:

1. Limitations section: "Despite that we aimed to isolate the effect of HIV on arterial stiffness by choosing PLH without any other comorbidities and comparing them with healthy controls, there may be a series of genetic, lifestyle..."

Please improve the language here: My suggestion: "Despite our aim to isolate the effect HIV on arterial stiffness by choosing PLH without any other comorbidities and comparing them with healthy controls, there may be ..."

- Changed as suggested.

2. There are new, untracked additions to the manuscript. All changes should be stated or better yet, clearly tracked/highlighted for the editor.

Of note: "The literature vastly describes that ART does not completely suppress viral load, particularly in the viral reservoirs".

This is not in the academic style of writing. Rephrase. I suggest: "It is well described that ART does not completely suppress viral load..."

- Changed as suggested by the reviewer.

3. Define all abbreviations with first use: hs-CRP is never defined. hsCRP vs hs-CRP are both used in the manuscript.

- All hs-CRP abbreviations are now consistent.

4. I still feel the article could receive additional language manicuring. An academic proof reader should be considered.

- We have several minor corrections to the grammar.

Decision Letter 2

Eliseo A Eugenin

22 Feb 2023

Effect of antiretroviral therapy on decreasing arterial stiffness,  metabolic profile, vascular and systemic inflammatory cytokines in treatment-naïve HIV: A one-year prospective study.

PONE-D-22-26706R2

Dear Dr. Andrade,

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Additional Editor Comments (optional):

Dear Dr. Andrade

Thank you for submitting your manuscript to PLOSone and include the changes requested

Eliseo Eugenin

Reviewers' comments:

Acceptance letter

Eliseo A Eugenin

10 Mar 2023

PONE-D-22-26706R2

Effect of antiretroviral therapy on decreasing arterial stiffness,  metabolic profile, vascular and systemic inflammatory cytokines in treatment-naïve HIV: A one-year prospective study.

Dear Dr. Andrade-Villanueva:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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