Restoring lung renin-angiotensin system balance through blood pressure control

The regulatory systems governing systemic arterial blood pressure (BP), encompassing the kidney, the cardiovascular system, and the central nervous system, operate synergistically to ensure optimal blood flow to all organs throughout the body. At the heart of this regulation is the renin-angiotensin system (RAS), consisting of two main pathways: the classic and the counterregulatory axes.

The classic RAS pathway involves the conversion of angiotensin I into angiotensin II (Ang II) by the angiotensin I-converting enzyme (ACE). Ang II activates the Ang II type 1 receptor (AT1R) in target cells, leading to vasoconstriction, renal sodium reabsorption, and secretion of aldosterone and antidiuretic hormone (ADH), thereby elevating BP [1]. In contrast, the counterregulatory RAS axis operates through the conversion of Ang II into angiotensin-(1–7) by the angiotensin I-converting enzyme 2 (ACE2). Ang-(1–7) then binds to the Mas receptor (MasR), promoting vasodilation and increasing the renal excretion of sodium and water, thereby lowering BP [2,3].

The classic RAS pathway has an established role in the pathogenesis and maintenance of systemic arterial hypertension. It opposes the effects of the counter-regulatory pathway, creating a feedback loop that sustains high BP and leads to inflammation, fibrosis, and oxidative stress in the heart, kidneys, brain, and blood vessels [4-8]. Besides the recognized target organs, abnormal activation of the classic RAS may also result in pulmonary damage in systemic arterial hypertension. Hypertensive individuals have a threefold higher risk of developing pulmonary hypertension, even with normal left-ventricular function [9]. In addition, a single study suggested that SHRs have increased pulmonary artery pressure and morphological features indicative of pulmonary hypertension [10]. However, the association of these phenotypes with pulmonary RAS signaling and their reliance on systemic BP remains unclear.

We have previously demonstrated that male, but not female, SHRs exhibit imbalanced lung classic/counterregulatory RAS due to reduced ACE2 activity, thereby increasing lung Ang II [11]. This phenotype recapitulated what is observed in hypertensive patients, where high expression of ACE shifts the balance toward dysregulated classic RAS signaling [11]. However, whether this imbalance can be modified solely by RAS inhibitors or by controlling BP with other anti-hypertensive agents and its association with lung inflammation remains to be elucidated.

In this study, we investigated whether BP reduction with two different classes of anti-hypertensive medications (either an RAS inhibitor, losartan, or a calcium-channel blocker, amlodipine) in male SHR could rebalance ACE-Ang II/ACE2-Ang-(1–7) in the lungs. Considering the link between systemic ACE2 levels and hypertension described here, we also assessed the effect of BP regulation on circulating ACE2 in individuals with controlled and uncontrolled hypertension.

Animals were kept in a 12:12-h dark–light cycle with food and water ad libitum at the animal facility of the Heart Institute (InCor), Medical School, University of Sao Paulo, Sao Paulo, SP, Brazil. Twelve-week-old male spontaneously hypertensive rats (SHRs) were treated daily with either losartan (100 mg/kg/day, Organon), amlodipine (30 mg/kg/day, Pfizer), or vehicle (water) for 14 days by oral gavage. The doses of losartan and amlodipine administered to SHR were selected based on pilot dose–response studies. The data supporting these dose selections are presented in (Supplemental Table S1). Age-matched control Wistar rats were treated with vehicle. Initially, 12 animals were allocated per group. However, variations in the number of animals used across different experiments occurred due to the limited availability of samples and unforeseen mortality in some SHRs following the gavage procedure. After the treatment, the rats were anesthetized with isoflurane (3–4% for inducement and 2.5–3% for maintenance) for tissue and blood collection. Blood samples were collected from the abdominal aorta and stored in vacuum tubes containing a pro-clotting agent and separator gel (Vacutainer SST Advance, Franklin Lakes, NJ, USA). The right lung was isolated with a surgical suture and frozen in an extraction buffer for molecular analysis. The left lung was inflated with 10% formalin solution for 24 h and used for the histological analysis. Rats were euthanized by pentobarbital overdose (180 mg/kg b.w.). All experimental procedures were approved by the Heart Institute Animal Care and Use Committee.

All participants signed an informed consent form, and the study was approved by the Ethics Committee of the Heart Institute of the University of São Paulo, São Paulo, Brazil. Thirty hypertensive subjects and ten healthy controls were included in this study (Table 1). Hypertension was defined by the presence of at least one of the following criteria: systolic BP > 140 mm Hg, diastolic BP > 90 mm Hg, or chronic treatment with anti-hypertensive medications for at least five years. Patients with a known diagnosis of diabetes mellitus, left ventricular (LV) ejection fraction < 50%, and/or estimated glomerular filtration rate lower than 60 ml/min/1.73 m² were excluded from the study. The ascertainment period was from 2017 to 2021. After enrollment, the serum samples were frozen at −80°C until analysis.

Systolic BP was determined with tail-cuff plethysmography (BP-2000 Blood Pressure Analysis System, Visitech Systems, Apex, NC, USA). Rats were adapted for 2 days and had their BP measured at the beginning and the end of treatment [12]. BP was the average of ten measurements per day acquired on two subsequent days.

Blood samples were centrifuged at 2000×g for 10 min at 4°C and stored at −80°C to obtain serum. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the concentrations of ACE (EKF57865-96T, Biomatik, Cambridge, ON, Canada) and ACE2 (EKF57924 and EKE60212, Biomatik, Cambridge, ON, Canada) according to the manufacturer’s instructions.

Right lungs were processed in ice-cold extraction buffer (150 mM sodium chloride, 2.8 mM monobasic sodium phosphate, 7.2 mM dibasic sodium phosphate, and pH 7.4) supplemented with phosphatase (50 mM sodium fluoride and 15 mM sodium pyrophosphate; Sigma–Aldrich) and protease (1 μM pepstatin A, 1 μM leupeptin, and 230 μM phenylmethylsulfonyl fluoride; Sigma–Aldrich) inhibitors. Homogenates were obtained using a Potter–Elvehjem-style tissue grinder (POLYMIX PX-SR50E, Kinematica, Luzern, Switzerland), followed by centrifugation at 2000×g for 10 min at 4°C (Sorvall ST 16 / 16R Centrifuge, Thermo Scientific). An aliquot of the supernatant was stored at −80°C, while the remaining was centrifuged at 100,000×g for 1 h at 4°C (Optima L-90K Ultracentrifuge, Beckman Coulter). The pellet (membrane fraction) was solubilized in extraction buffer and stored at −80°C. Protein concentration was determined according to Lowry [13].

Lung ACE and ACE2 activity were measured by a fluorometric activity assay (Sigma–Aldrich, CAT #CS0002 and Cat #MAK377) using 2 µg and 20 μg of lung homogenates, respectively. As a negative control, the same assays were performed in the presence or absence of 10 µM of captopril (ACE-selective inhibitor) or 20 μM of ACE2 inhibitor, according to the manufacturer’s instruction. Fluorescence was measured with a spectrophotometer (SpectraMax M5, Molecular Devices, San Jose, CA, U.S.A.) at λ = 320 nm excitation and λ = 420 nm emission for 30 min. All samples were assessed in duplicate. Enzyme activity was expressed as milliunits (mU)/mg.

Equal amounts of lung membrane proteins (5 µg for ACE and 15 µg for ACE2) were solubilized in Laemmli sample buffer and loaded in 10% SDS/PAGE gels. Proteins were transferred to an activated polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Merck Millipore, Darmstadt, Germany) at 350 mA overnight at 4°C. Ponceau staining was performed for 5 min to assess equal protein loading. The membranes were incubated in blocking solution (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and incubated with the primary antibodies against ACE (1:1000; ab254222, Abcam, Cambridge, MA) or ACE 2 (1:1000; ab15348, Abcam) overnight at 4°C. After washing, the membranes were incubated with the respective horseradish-peroxidase (HRP)-conjugated secondary antibodies (1:2000; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature (RT), followed by chemiluminescence visualization of the bands (GE Healthcare, U.K). Band densitometry was performed on ImageJ (National Institutes of Health, Bethesda, MD) and normalized by the respective Ponceau levels. The unedited representative gel images were included within the Supplemental Material (Supplemental Figure S1).

Total RNA was extracted from the lung tissue using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) and followed by cDNA synthesis with Super-Script IV Reverse Transcriptase (Thermo Fisher Scientific). Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using SYBR Green PCR Master Mix-PE (Applied Biosystems, Foster City, CA, USA) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems). All samples were assessed in triplicate, and the relative expression was normalized to cyclophilins. Data were analyzed using the 2^(-ΔΔCt) method. The oligonucleotide primers used are described in Table 2.

Left lung samples were fixed in 10% formalin, processed for paraffin embedding, and sectioned into 4-µm-thick cross-sections. For picrosirius staining, sections were heated in a water bath at 60°C for 45 min, deparaffinized, rehydrated, and incubated with a picrosirius stain solution (0.1% red 80 and 0.1% fast green Fcf. dissolved in 1.2% picric acid) for 60 min. For immunohistochemistry, the 4-µm-thick cross-sections were deparaffined, rehydrated, and subjected to antigen retrieval by heating in Tris-EDTA buffer (pH 9.0) in a 140°C water bath for 20 min. After cooling for 30 min at RT, the sections were blocked with 10% goat serum and 0.25% Triton X-100 for 30 min and incubated overnight at 4°C with primary antibodies against CD68 (1:100, ab31630, Abcam) or iNOS (1:100, ab3523, Abcam). The slides were rinsed, treated with 3% H₂O₂ for 10 min at RT to block endogenous peroxidase activity, and incubated with HRP-conjugated secondary antibodies (Invitrogen) for 1 h. Immunodetection was performed using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Zymed) followed by counterstaining with hematoxylin, dehydration, and mounting with Entellan (Merck, Darmstadt, Germany). Darmstadt, Germany). All samples were analyzed using a computerized microscopy image acquisition system (Leica Imaging Systems, Bannockburn, IL, USA). Ten images per sample were captured at 20 × magnification. Macrophage quantification was conducted by two independent examiners blinded to the experimental conditions; results were expressed as the ratio of CD68+ macrophages to the tissue area. The tissue area, defined as the hematoxylin-stained region in each field, was quantified using ImageJ software.

Data are expressed as mean ± standard error mean (SEM) unless otherwise stated. A comparison between the experimental groups was made using a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. P<0.05 was considered for statistical significance. The association between continuous variables was evaluated using Pearson’s correlation. Data points represent biological replicates. All analyses were performed using GraphPad Prism 10.1.1 software (Graph-Pad Software, La Jolla, CA, U.S.A.).

We tested whether BP control would affect the expression of ACE and ACE2, key enzymes in control of the RAS. As expected, vehicle-treated SHRs displayed significantly higher systolic blood pressure (SBP) (184 ± 2 vs. 127 ± 1 mmHg, P<0.0001; Figure 1A) and left-ventricle (LV) weight/tibial length (21.3 ± 0.6 vs. 17.0 ± 0.3 mg/mm, P<0.0001; Figure 1B) than normotensive Wistar rats. Treatment with either losartan (SBP = 124 ± 5 mmHg and = LV weight/tibial length 15.8 ± 0.5 mg/mm) or amlodipine (SBP = 128 ± 3 mmHg and LV weight/tibial length = 17.1 ± 0.5 mg/mm) restored these parameters to control levels. Additionally, vehicle-treated SHRs exhibited higher lung weight/tibial length compared with Wistar control rats (40.5 ± 1.5 vs. 36.1 ± 0.7 mg/mm, P<0.05) and compared with BP-controlled SHRs (40.5 ± 1.5 vs. 32.1 ± 1.1 mg/mm, P<0.0001 in losartan-treated SHR) and (40.5 ± 1.5 vs. 32.8 ± 0.80 mg/mm, P=0.0005 in amlodipine-treated SHR) (Figure 1C). These changes were independent of hypertrophic remodeling of the pulmonary vasculature, as arterial thickness remained unchanged across the groups (Supplemental Figure S1).

Gene expression analysis demonstrated a threefold higher expression of Ace (303 ± 34 vs. 100 ± 15%, P<0.0001; Figure 2A) and an approximate 50% lower expression of Ace2 (54 ± 2 vs. 100 ± 10%, P<0.001; Figure 2B) in the lungs of SHR compared with Wistar rats. BP control with either losartan or amlodipine down-regulated Ace (73 ± 15 vs. 303 ± 34% in losartan-treated vs. vehicle-treated SHR, P<0.0001 and 72 ± 13 vs. 303 ± 34% in amlodipine-treated vs. vehicle-treated SHR, P<0.0001) and up-regulated Ace2 expression in SHR lungs (54 ± 2 vs. 93 ± 4% in losartan-treated SHR, P<0.0001 and 54 ± 2 vs. 95 ± 8% in amlodipine-treated SHR, P<0.0001) (Figure 2C).

At the protein level, the amount of ACE in lung membrane extracts remained comparable in all experimental groups (Figure 2D). In contrast, ACE2 was lower in SHR vehicle membrane extracts compared to Wistar (64 ± 4 vs. 100 ± 2%, P<0.0001) and restored similarly by either losartan (122 ± 3 vs. 100 ± 2%, ns) or amlodipine (113 ± 7 vs. 100 ± 2%, not significant (n.s.)) (Figure 2E). Similar findings were obtained in sections stained for ACE (Figure 2F) and ACE2 (Figure 2G).

The enzymatic activity of ACE and ACE2 followed a similar pattern to their protein content. ACE activity was constant across the experimental groups (Figure 3A). In contrast, ACE2 activity was about 45% lower in vehicle-treated SHR compared with Wistar rats (40 ± 2 vs. 71 ± 4 mU/mg, P<0.0001) and restored to Wistar levels upon treatment with losartan (62 ± 5 vs. 71 ± 4 mU/mg, n.s.) or amlodipine (63 ± 5 vs. 71 ± 4 mU/mg, n.s.) (Figure 3B). Thus, the ACE/ACE2 ratio showed a prevalence of ACE in SHR-vehicle, and both anti-hypertensive drugs normalized ACE/ACE2 activity to similar normotensive values (Figure 3C). Similarly, lung Ang II and Ang-(1–7) quantifications showed that Ang II concentration was higher (158 ± 14 vs. 35 ± 2 pg/g, P<0.0001) (Figure 3D) and Ang-(1–7) was lower (44 ± 3 vs. 72 ± 4 pg/g, P<0.01) in the vehicle-treated SHR than in Wistar rats (Figure 3E). Losartan and amlodipine reversed this phenotype and attenuated the prevalent Ang II/Ang-(1–7) ratio observed in vehicle-treated SHR (Figure 3F). Collectively, these findings demonstrate that BP control restores the equilibrium between ACE and ACE2 in hypertensive lungs at the transcription, enzyme activation, and peptide content levels (Figures 2C and 3C, and F).

ACE and ACE2 can be enzymatically shed from the plasma membrane, leading to their presence in the serum and other biological fluids [14]. In contrast to its membrane levels, serum ACE was higher in vehicle-treated SHR than in Wistar (320 ± 31 vs. 84 ± 8 ng/ml, P<0.0001) and lowered to levels similar to Wistar controls with losartan (126 ± 11 vs. 84 ± 8, ns) and amlodipine (121 ± 10 vs. 84 ± 8 ng/ml, n.s.) (Figure 4A). Similar observations were made for ACE2: it was increased in SHR serum compared with Wistar rats (4.4 ± 0.9 vs. 1.0 ± 0.1 ng/ml, P<0.01) and was reduced to levels similar to controls with losartan (2.0 ± 0.3 vs. 1.0 ± 0.1 ng/ml, n.s.) and amlodipine (1.9 ± 0.2 vs. 1.0 ± 0.1 ng/ml, ns) (Figure 4B). Unlike in the lungs, the serum ACE/ACE2 ratio remained unchanged across all experimental groups (Figure 4C).

Dysregulated activation of the classic RAS pathway (as observed in uncontrolled hypertension) is commonly associated with inflammation, a signaling feedback that ultimately leads to tissue damage and fibrosis. While this is true for the heart, the brain, and the kidneys, to this date, no evidence of lung inflammation in the setting of uncontrolled hypertension has been reported. To test this hypothesis, lung sections were stained for CD68 (a macrophage marker) as a proxy for lung inflammation. We observed that vehicle-treated SHRs had a higher number of macrophages in the lungs when compared with Wistar controls (Figure 5). As BP control restored the ACE/ACE2 balance in the lungs, we tested the hypothesis that it would also lead to the resolution of lung inflammation. In agreement, macrophage quantity was normalized to Wistar levels with either losartan or amlodipine (Figure 5A and B). Similarly, the inflammatory genes Ccr2, Nos2, and Tnf were up-regulated in vehicle-treated SHRs and down-regulated to Wistar levels with losartan or amlodipine (Figure 5C–E). Differential expression of the M1 macrophage marker iNOS (encoded by the Nos2 gene) was qualitatively validated by immunohistochemistry. Staining was observed in SHRs compared with Wistar rats, with reduced or undetectable staining following BP control with either amlodipine or losartan ( Supplemental Figure S2). Collectively, these findings suggest that the lungs are also a target organ in hypertension, as it is associated with an inflammatory response that is mitigated by rebalancing ACE/ACE2.

As ACE2 retention in the lungs improves upon BP control and ACE2 has been demonstrated to protect against pulmonary diseases [15-18], we explored whether serum ACE2 levels could predict the expression of inflammatory genes and lung inflammation. Using Pearson’s correlation test, we observed a positive correlation between ACE2 and the genes Ccr2 (r = 0.8121, P<0.0001), Nos2 (r = 0.8778, P<0.0001), and Tnf (r = 0.8669, P<0.0001) (Figure 6A–C), demonstrating that the higher the expression of these genes, the higher the circulating levels of ACE2, in agreement with the dependence of inflammation for ACE2 shedding and its presence in the serum [14].

We next sought to evaluate whether our experimental observations could be recapitulated in normotensive and hypertensive subjects. Participants' characteristics are found in Table 1. Analysis of circulating ACE and ACE2 demonstrated higher levels in individuals with uncontrolled hypertension when compared with normotensive subjects, while people with the diagnosis of hypertension reporting the use of either losartan or amlodipine had reduced ACE2 levels than those with uncontrolled hypertension (Figure 7AB). Similar to what was observed in SHRs, the ACE/ACE2 ratio remained unchanged (Figure 7C).

In the present study, we show that not only losartan but also amlodipine restores the balance between classic and counterregulatory RAS and reduces the presence of macrophage in SHR lungs. We found a positive correlation between circulating ACE2 levels and the expression of inflammatory genes in the lungs, suggesting that serum ACE2 could be a biomarker of lung-related RAS-associated inflammation. Furthermore, the analysis of serum obtained from individuals with controlled and uncontrolled hypertension further strengthens our experimental findings, providing insight into the relationship between RAS signaling and pulmonary diseases.

Angiotensin receptor blockers (ARBs) and calcium channel blockers (CCBs) are first-line drugs for the treatment of hypertension, according to international guidelines [19]. Here, we took advantage of the distinct mechanisms of action of these molecules – losartan, an AT1R blocker (ARB), and amlodipine, a blocker of voltage-dependent L-type calcium channels (CCB) – to establish a direct relationship between lowering BP and the balance between classic and counterregulatory RAS in the lungs. While it is anticipated that blocking AT1R would alter this balance [1,20-22], the precise mechanism linking amlodipine to RAS signaling remains unclear. Classic RAS signaling triggers calcium influx in the heart and the vasculature [23], and Ang II promotes the expression of the LTCC α1C subunit in an atrial myocyte cell lineage [24]. These findings suggest that the BP-lowering effects of a CCB might modulate RAS tissue balance, potentially linking chronic exposure to high BP itself as a driver of expression tissue ACE and ACE2. Indeed, other studies have shown that amlodipine shifts RAS signaling toward the counterregulatory axis in hypertensive rats' kidneys, the aorta, and the heart [25-27], suggesting a shared mechanism across different organs.

For years, the lungs have been understudied as a target of systemic arterial hypertension. Drawing from our prior research revealing imbalanced classic RAS in the lungs of male SHRs [11], we posited that, akin to the heart, brain, and vasculature, this imbalance correlated with tissue inflammation. Indeed, an increased presence of macrophages was observed in the lungs of untreated SHRs, as confirmed by iNOS staining, which was mitigated by BP control. These findings align with observations of heightened leukocyte presence in the lungs of SHRs exhibiting pulmonary hypertension features [10] and with the established interplay between ACE and ACE2 in lung inflammation. ACE is up-regulated in the bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome (ARDS) [28], interstitial lung disease, pneumonia, and sarcoidosis [29]. Moreover, its levels correlate with mortality in ARDS [30] and increase the risk of ARDS and pneumonia in patients with polymorphisms that enhance ACE expression [31]. Conversely, patients taking ACE inhibitors chronically have reduced risk [32]. On the other hand, experimental loss of ACE2 exacerbates lung inflammation in response to infection and acid aspiration [15-18], suggesting that ACE2 may act as a gatekeeper for tissue damage and inflammation by shifting the balance toward the counterregulatory RAS [15,16]. We observed that while a relationship exists between ACE and ACE2 levels (represented as the ACE/ACE2 ratio) at the tissue level, this relationship does not extend to serum ACE and ACE2 levels. These findings support our hypothesis that tissue ACE2 counterbalances ACE activity to prevent tissue damage.

In cardiometabolic diseases, inflammation plays a role in promoting ACE2 shedding, an additional regulatory mechanism of local ACE2 activity. This process involves matrix metalloproteinases removing ACE2 from the plasma membrane, resulting in increased serum ACE2 observed in our untreated SHRs and compromising the local protective function of membrane-bound ACE2. Notably, increased ACE2 shedding has been reported in response to endotoxin and TNF-α in airway epithelial cells [14]. Intriguingly, TNF-α and the matrix metalloproteinases ADAM10 and ADAM17, which are known to shed ACE2 from the plasma membrane [14], are induced by classic RAS signaling [1]. The absence of tissue ACE2-dependent protection may facilitate inflammatory cell infiltration, contributing to the observed macrophage accumulation and the expression of inflammatory genes in the lungs of untreated SHRs. Consistent with these findings, we observed a direct correlation between circulating ACE2 and the expression of inflammatory genes in the lungs. The normalization of these parameters by amlodipine aligns with literature evidence demonstrating amlodipine’s ability to reduce the tissue expression of MCP-1 and its receptor CCR2 in monocytes, thereby attenuating inflammation and oxidative stress in atherosclerotic rats [27].

During the COVID-19 pandemic, ACE2, the receptor for the SARS-CoV-2 virus [33], garnered significant attention, as its circulating levels were associated with disease severity and mortality [34,35]. SARS-CoV-2 binding decreases membrane-bound ACE2 and, via ADAM17 induction [36,37], increases circulating ACE2, disrupting the ACE/ACE2 balance in the lungs. Hypertension was identified as a risk factor for poor COVID-19 outcomes [38,39], and our prior research indicated that BP influences lung ACE2 levels [11]. In this study, we investigated whether controlled and uncontrolled hypertension affects circulating ACE2 levels, similar to observations in SHRs. Our findings demonstrated that individuals with uncontrolled hypertension exhibited higher serum ACE2 levels compared with normotensive individuals. In contrast, patients treated with either losartan or amlodipine had lower circulating ACE2 levels than those with uncontrolled hypertension, consistent with our experimental data. These results suggest that BP reduction using calcium channel blockers (CCBs) or angiotensin receptor blockers (ARBs) may shift the balance toward the counter-regulatory RAS axis [1,20-22], potentially reducing inflammatory mechanisms driving ACE2 shedding. Further research is needed to determine whether this shift protects against COVID-19-related lung inflammation.

In summary, we provided evidence that BP control restores the balance between classic and counterregulatory RAS axes in the lungs of male SHRs, thereby impacting inflammatory cell infiltration and the expression of inflammatory genes. These findings demonstrate, for the first time, that similar to the kidney, the brain, the heart, and blood vessels, the lungs also represent a target organ for systemic arterial hypertension. At the molecular level, BP control promoted the retention of ACE2 in the plasma membrane, thus reducing the levels of circulating ACE2, which were correlated with inflammatory genes. As a proof of concept, analysis in the sera of people with controlled and uncontrolled hypertension demonstrated a role for BP control in circulating ACE2 levels, as hypertensive people taking amlodipine or losartan have less circulating ACE2 than those with uncontrolled hypertension. These findings emphasize the clinical relevance of our results and underscore the need for further research into how restoring the ACE/ACE2 balance could prevent the development and progression of conditions where hypertension and inflammation synergistically affect ACE2 levels, tissue function, organ damage, and disease outcomes.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Caio A. M. Tavares reported receiving presenter fees from Novo Nordisk outside the submitted work. All the other authors have no conflicts of interest to declare.

This work was supported by grants 2020/05338–3 and 2021/14534–3 from the São Paulo Research Foundation (FAPESP) and the National Council for Scientific and Technological Development (CNPq 307156/2018–4). Joao Carlos Ribeiro-Silva is supported by a Postdoctoral Diversity Supplement under the National Institutes of Health grant number R21-EY034277 awarded to AS Viczian.

Gabriela Catao: Data Curation, Formal analysis, Investigation; Joao Carlos Ribeiro-Silva: Formal analysis; Writing – original draft, Writing – review & editing; Andreia Boaro: Data Curation, Formal analysis, Investigation; Flavia L. Martins: Formal analysis, Investigation, Visualization; Thais Mauad: Supervision; Caio A.M. Tavares: Formal analysis, Investigation, Writing – review & editing; Lisete Ribeiro Teixeira: Investigation; Bruno Caramelli: Resources, Validation; Adriana C.C. Girardi: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing– review & editing.

ACE2

Angiotensin I-Converting Enzyme 2

ACE

Angiotensin I-Converting Enzyme

ADH

Antidiuretic Hormone

ANOVA

One-Way Analysis of Variance

ARBs

Angiotensin Receptor Blockers

ARDS

Acute Respiratory Distress Syndrome

AT1R

Ang II Type 1 Receptor

Ang II

Angiotensin II

BP

Blood Pressure

CCBs

Calcium Channel Blockers

DAB

3,3'-Diaminobenzidine Tetrahydrochloride

ELISA

Enzyme-Linked Immunosorbent Assay

HRP

Horseradish-Peroxidase

HTN

Hypertension

LV

Left Ventricle

MasR

Mas Receptor

PVDF

Polyvinylidene Difluoride

RAS

Renin-Angiotensin System

RT

Room Temperature

RT-PCR

Reverse Transcription-Polymerase Chain Reaction

SBP

Systolic Blood Pressure

SHRs

Spontaneously Hypertensive Rats