Modulation of RAAS receptors and miRNAs in COVID-19: implications for disease severity, immune response, and potential therapeutic targets (2025)

Abstract

The SARS-CoV-2 spike protein interacts with ACE2, a key receptor within the renin-angiotensin-aldosterone system (RAAS), which plays a critical role in maintaining vascular homeostasis, regulating blood pressure, and modulating inflammation. An observational study analyzed the gene expression profiles of RAAS receptors and associated miRNAs in 88 hospitalized COVID-19 patients and 20 healthy controls, comparing the acute and post-acute phases to assess their impact on disease severity and recovery. Our findings revealed an association between reduced MAS1 expression in both advanced age (P = 0.03) and the need for oxygen supplementation (P = 0.04). Additionally, reduced ACE expression was associated with worse mortality outcomes (P = 0.01). Notably, ACE2 and TMPRSS2 expression was significantly decreased (P < 0.0001) in individuals requiring oxygen supplementation and in those with diabetes mellitus during both the acute and post-COVID-19 phases, further highlighting the impact of these conditions on RAAS. The miRNA analysis revealed significant downregulation of miR-200c (P = 0.005), miR-let-7 (P = 0.01), and miR-122 (P = 0.03) in acute-phase COVID-19 patients. This dysregulation contributes to the inflammatory response and highlights the interaction between viral entry and immune regulation. These results underscore the significance of the ACE2/Ang-(1–7)/MAS1 axis in inflammation regulation and suggest that targeting this pathway may have therapeutic potential. Our study provides valuable insights into the molecular mechanisms of COVID-19 pathogenesis and identifies the modulation of RAAS receptors and miRNAs as promising biomarkers for disease severity and potential therapeutic interventions.

Clinical trial

Not applicable

Since its emergence in 2019, coronavirus disease (COVID-19) has affected over 770million people globally and has led to more than 7million deaths [1]. Like other respiratory infections, severe COVID-19 can develop in individuals with preexisting cardiovascular diseases and other risk factors, leading to the worsening of underlying chronic pathologies and the onset of new complications [2]. SARS-CoV-2 infection is triggered by the activation of the spike protein by transmembrane serine protease 2 (TMPRSS2). This activation allows the virus to attach to the host receptor angiotensin-converting enzyme 2 (ACE2), which is essential for viral replication [3].

ACE2 plays a crucial role in maintaining the homeostasis of the renin‒angiotensin‒aldosterone (RAA) system, which is responsible for homeostatic regulation of vascular function [4, 5]. Chronic activation of the RAAS can exacerbate pathological processes such as inflammation, fibrosis, apoptosis and aldosterone secretion [6]. ACE2 converts angiotensin II (Ang II) into angiotensin 1–7 (Ang 1–7), which exerts protective effects by activating the MAS1 receptor and counterbalancing the detrimental actions of Ang II [7, 8]. Ang II promotes sodium retention, oxidative stress, fibrosis, inflammation, and aldosterone release, contributing to severe complications such as acute respiratory distress syndrome (ARDS) and multi-organ damage [9, 10, 11].

ACE2 expression can be epigenetically modulated by various transcriptional and post-translational mechanisms, including microRNA (miRNA)-mediated regulation [12]. Recent studies have shown that certain miRNAs, including hsa-miR-125a-5p, hsa-let-7b-5p and members of the miR-200 family, share homology with the 3’ untranslated region of ACE2 mRNA. This enables them to inhibit ACE2 expression [13, 14]. Since ACE2 acts as a counter regulator of the RAAS, repression of ACE2 may lead to pathological consequences, including myocardial fibrosis, inflammation, and cardiovascular dysfunction, in COVID-19 patients [15, 16].

The investigation of differential expression patterns of key receptors within the RAAS during acute infection and subsequent recovery stages is crucial for understanding the molecular and biological mechanisms underlying COVID-19 severity. Comprehensive studies utilizing patient-derived samples are vital to accurately assess the dynamic regulation of these receptors across diverse physiological and pathological contexts. This is particularly significant given the high prevalence of comorbidities such as cardiovascular diseases, hypertension, diabetes, and respiratory disorders, which are known to exacerbate COVID-19 outcomes. Given the critical role of RAAS in COVID-19 pathophysiology and the limited understanding of its dynamic regulation during disease progression, our study aims to elucidate the relationship between RAAS receptor expression in acute and post-COVID-19 phases. By examining its associations with clinical conditions observed throughout hospitalization, we seek to identify potential biomarkers that could aid in predicting disease severity and guiding therapeutic strategies.

A cohort of 88 individuals with a confirmed diagnosis of COVID-19 was enrolled, all of whom were admitted to the Centro Hospitalar da COVID-19, Instituto Nacional de Infectologia Evandro Chagas (INI-FIOCRUZ), Rio de Janeiro, Brazil, between June 2020 and December 2021. Participants were enrolled in the study based on their hospital admission sequence, with additional eligibility criteria including the absence of RAAS inhibitor use, age above 18 years, non-pregnancy, and provision of informed consent. The study involved two time points: an acute phase (D0) at the time of admission (n = 88), and a post-acute phase approximately 300 days after symptom onset, with a subset of 55 patients available for follow-up. Demographic and clinical information was collected during each patient’s initial visit and no participant had received a COVID-19 vaccine. Blood samples from healthy individuals collected before the COVID-19 pandemic (n = 20) were used as controls. All samples were de-identified before analysis to protect participant confidentiality. The study was approved by the local Ethics Committee (CAAE: 32449420.4.1001.5262) and followed the Declaration of Helsinki, as well as all relevant guidelines and regulations.

Samples

Blood samples were centrifuged at room temperature, and the separated plasma was frozen at -80°C. Peripheral blood mononuclear cells (PBMCs) were isolated via Histopaque 1077 density gradient centrifugation (Sigma‒Aldrich, USA). The isolated cells were subsequently cryopreserved in fetal bovine serum with 10% DMSO and stored in liquid nitrogen until further use.

RNA and miRNA isolation

The cryopreserved PBMCs were thawed, and their viability was assessed via automated cell counting with trypan blue staining. Only vials with a cell viability above 90% were used for the subsequent steps. PureLink RNA Mini Kit columns (Invitrogen) were used for samples D0, D300, and the controls. The mirVana™ isolation kit (Thermo Fisher Scientific, USA) was used for miRNA extraction from the D0 and control samples following the manufacturer’s instructions. The purity (260/280 ratio) and concentration of the RNA and miRNA samples were evaluated via a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Integrity was assessed via the RNA Integrity Number (Agilent).

Real-time quantitative RT‒PCR

To quantify the expression of ACE, ACE2, AT1R, AT2R, MAS1, and TMPRSS2, cDNA was obtained from the extracted RNA via a high-capacity cDNA reverse transcription kit with an RNase inhibitor (Applied Biosystems). TaqMan Universal Master Mix II (Applied Biosystems™) was used, along with TaqMan^® Gene Expression Assays (IDs: Hs01104600–Hs02786624), which included the targets of interest and endogenous controls β-actin or ACTB and glyceraldehyde-3-phosphate dehydrogenase or GAPDH.

For cDNA synthesis of miRNAs, the TaqMan^® Advanced miRNA cDNA Synthesis Kit (Applied Biosystems™) was used as per the manufacturer’s recommendations. The expression of miRNAs involved in ACE2 regulation, including hsa-miR-200c-3p (ID: 002300), hsa-let-7-5p (ID: 002619), and hsa-miR-122-5p (ID: 002245), was quantified via TaqMan^® Advanced miRNA Assays (Applied Biosystems™) in conjunction with TaqMan^® Advanced Master Mix (Applied Biosystems™). hsa-miR-26-a (ID: Hs04231546_s1), located on chromosome 12:57824609, was used as an endogenous gene to normalize gene expression, as reported by Ragni et al. and Timoneda et al. [17, 18].

Amplifications were carried out via the 7500 Real-Time PCR System (Life Technologies). RQ Manager Software 1.2 was used to calculate the threshold cycle (CT) values. The expression levels of the genes of interest were calculated from the difference between the CT values of the gene and the endogenous housekeeping genes; ΔCT = (CT _Target – CT _Endogenous). Higher ΔCT values indicate a greater number of cycles (CTs) required to quantify the target in the samples, which corresponds to lower levels of gene expression [19]. To calculate relative changes in gene expression between COVID-19 samples and the reference control group [20], we used the comparative CT method, also known as the fold change method, relative quantification (RQ), or its formula 2^−ΔΔCt, via Expression Suite software version 1.3.

Statistical analysis

Absolute frequencies were calculated for qualitative variables, whereas arithmetic means and relative frequencies were calculated for quantitative variables. Spearman’s rank correlation coefficient was used to correlate age with the expression level of each receptor. A generalized linear mixed model with a gamma distribution assumption was employed, adjusting for confounding variables (age and sex). Mann-Whitney U test was used to evaluate the ΔCT values and clinical, sociodemographic, and symptomatic data. To assess the likelihood that the observed differences in gene expression are not due to chance alone, the software conducts an unpaired, two-tailed Student’s t-test. The Wilcoxon test was performed for the paired analyses of RAAS receptors in the acute COVID-19 and post-COVID-19 samples. Unpaired t-tests were used to compare the control and acute groups for miRNA analyses. The analyses were conducted via GraphPad Prism version 8.0 and R software (https://www.r-project.org/about.html), with a predetermined significance level of 0.05 (alpha).

Demographic and clinical characteristics

The mean age of acute COVID-19 patients was 59.7 years (± 12.4), while that of the control group was 37.8 years (± 10.1); however, no statistically significant difference in age was observed between groups. Among the COVID-19 group, 57.5% (50) were male and 42.5% (38) were female, compared to 45% (9) male in the control group, with no statistical difference in gender distribution. Clinical characteristics on the first day of hospital admission showed that 41% of COVID-19 patients had systemic arterial hypertension (without RAAS inhibitors), 33% had diabetes mellitus, and 82% required oxygen supplementation or ventilatory support. Study outcomes showed a 24% mortality rate and a 76% discharge rate, as detailed in Supplementary Table 1.

Correlation between receptor expression levels and clinical/sociodemographic factors in COVID-19 Patients

After conducting a comprehensive analysis of the potential correlations between participants’ age, gender, and receptor expression levels, we found a significant association between the average age of participants and MAS1 receptor expression (P = 0.04) (Fig.1A). We observed a significant correlation between downregulation of the MAS1 receptor (indicated by higher ΔCT values) and individuals over 60 years of age (P = 0.03). Additionally, participants who required oxygen supplementation during hospitalization also showed lower expression for MAS1 (P = 0.04). Our analysis also revealed that individuals with oxygen saturation below 95% during hospitalization had significantly lower expression TMPRSS2 compared to those with normal oxygen saturation (P < 0.01). Furthermore, participants who died during hospitalization exhibited downregulation of ACE expression compared to those who were discharged (P < 0.01).

Differential gene expression

The analysis of receptor gene expression demonstrated significant upregulation of ACE, ACE2, and MAS1 in participants without comorbidities during both the acute COVID-19 and the post-COVID-19 phase compared to the healthy control group (Fig.2A). In contrast, TMPRSS2 expression was downregulated, while no significant changes were observed in AT1R and AT2R expression levels.

To gain deeper insights into the influence of health conditions such as diabetes mellitus (DM), hypertension, oxygen requirements during hospitalization, and RAAS modulation on disease progression, we analyzed relative expression data based on participants’ health status. In COVID-19 patients with diabetes mellitus (DM), ACE2 expression was found to be downregulated, with a significant difference observed between the acute and post-COVID-19 phases (Fig.2B). The group of participants with systemic arterial hypertension exhibited upregulation of TMPRSS2 compared to the healthy control group. Additionally, MAS1 upregulation showed a statistically significant difference between the acute and post-COVID-19 phases (Fig.2C). Compared to healthy controls, participants requiring oxygen supplementation during hospitalization exhibited a gene expression profile akin to that of individuals with diabetes mellitus (DM), characterized by downregulation of ACE2. A significant difference in TMPRSS2 expression was observed between the acute and post-COVID-19 phases (Fig.2D). These findings underscore the impact of underlying conditions on RAAS modulation during COVID-19, suggesting that ACE2 downregulation may serve as a potential biomarker for disease severity, long-term sequelae, in patients with DM or those requiring oxygen supplementation.

MicroRNA analysis

Compared to the control group, all the miRNAs studied were significantly downregulated in the acute infection group: miR-200c (P = 0.005), miR-let-7b (P = 0.01), and miR-122 (P = 0.03) (Fig.3A). The downregulation of these miRNAs may reflect underlying inflammatory and immune dysregulation mechanisms, with potential implications for disease severity, recovery, and the development of miRNA-based biomarkers for monitoring disease progression and therapeutic response.

Extensive research has highlighted the pivotal role of the RAAS in blood pressure regulation and its involvement in the pathogenesis of COVID-19 [4, 7, 21, 22, 23, 24]. Our findings suggest that advanced age, comorbidities, and receptor expression are closely tied to COVID-19 severity, underscoring the intricate relationship between the virus-induced immunoinflammatory response and RAAS pathway disruption. Decreased expression of receptors such as MAS1 and ACE may be an early indicator of risk, while the relationship between TMPRSS2 and oxygenation highlights the direct impact of respiratory function on disease progression. Additionally, genetic variations within the RAAS have been explored as potential contributors to COVID-19 severity [25]. We compared the expression of RAAS receptors between COVID-19 severe patients and healthy controls, observing a significant reduction in TMPRSS2 mRNA expression during the acute phase of infection in participants without comorbidities, which persisted up to 300 days post-COVID-19. These findings reveal a dynamic modulation of TMPRSS2 expression, which is crucial for viral activation [26]. This modulation may exacerbate disease progression by enhancing viral load, aligning with previous studies linking TMPRSS2 expression to COVID-19 severity [27]. Taking together, these findings suggest that TMPRSS2 holds potential as a valuable prognostic biomarker.

Although an increase in MAS1 expression was observed in individuals with COVID-19 compared to healthy controls, we conclude that the presence of this receptor should not be considered an optimal biomarker, as its expression is closely linked to age. However, a significant elevation in MAS1 expression was observed in the COVID-19 group compared to the healthy control group, indicating persistent RAAS modulation even 300 days after acute infection. This finding aligns with existing evidence that RAAS function is regulated by a delicate balance between the classical vasoconstrictive pathway and the opposing vasorelaxant pathway [28, 29, 30]. Consequently, the observed dysregulation in the expression of MAS1, ACE2, and ACE indicates a prolonged disruption of RAAS activity extending beyond the acute phase of the disease. These findings suggest that the disruption of homeostasis may adversely affect the recovery of COVID-19 patients.

Notably, we identified a significant association between reduced ACE expression and mortality during the acute phase of infection, suggesting that diminished ACE gene expression may profoundly impair physiological RAAS balance. While therapeutic inhibition of ACE via medication has the potential to regulate RAAS, this raises the question of whether this pathway can be effectively targeted with ACE inhibitors in the context of severe COVID-19, without a comprehensive evaluation of RAAS receptor expression profiles in patients. The decreased expression of ACE has already been associated with severe COVID-19, as previously demonstrated by Garvin et al. in their research involving bronchoalveolar fluid cells from COVID-19 patients [31]. These findings suggest that this expression profile is responsible for the worsening of respiratory symptoms due to increased vascular permeability mediated by bradykinin, thereby promoting an inflammatory condition.

According to the literature, SARS-CoV-2 infection may cause downregulation of receptors due to internalization after binding to the spike protein [32], this could explain the decrease in ACE2 and TMPRSS2. Complementary findings from other studies indicate that elevated blood glucose levels and glycation products in individuals with diabetes exacerbate RAAS activity [33], a fact that could synergistically aggravate the RAAS imbalance and contributes to impaired insulin secretion, accelerated pancreatic cell damage, and a heightened risk of diabetic ketoacidosis, ultimately worsening clinical outcomes in patients with diabetes following SARS-CoV-2 infection.

The inclusion of a control group consisting of individuals with diabetes and/or hypertension, but without COVID-19, is crucial for accurately evaluating the true impact of SARS-CoV-2 on individuals with these comorbidities. This is particularly important given the limitation of our analysis, which only compared RAAS expression during infection to participants who were healthy prior to COVID-19 pandemic. Additionally, miRNA analysis was not performed post-COVID-19, leaving uncertainty regarding whether miRNA modulation reverted to baseline patterns, as seen in the control group. Nevertheless, the miRNA data from the acute COVID-19 phase provided valuable insights into ACE2 gene expression, contributing meaningfully to our understanding of the disease’s molecular impact.

Studies have demonstrated that miRNAs exhibit diverse functions, even among members of the same family [13, 14, 22, 34, 35, 36]. In addition to regulating TMPRSS2 and ACE2 expressions, miRNA let-7-5p plays a role in amplifying the inflammatory response [37]. In line with our findings, Wang et al. reported that hsa-let-7-5p expression levels were downregulated in both mild and severe COVID-19 patients [38]. Notably, hsa-let-7-5p has also been shown to target IL6R [39], suggesting that the let-7 family may inhibit the translation of both IL6 and IL6R. According to the study by Khanal et al. hepatocytes transfected with let-7-5p exhibited a significant downregulation of TMPRSS2 mRNA and protein levels [40], which aligns with our findings and further supports the potential interplay between miRNA regulation, viral entry, and the severity of COVID-19.

In addition to its role in ACE2 regulation, miR-122 influences the progression of cardiovascular diseases such as myocardial infarction, heart failure, and atherosclerosis [41]. The suppression of this miRNA has been shown to mitigate aortic remodeling and fibrosis in rats via apelin, a molecule known to counteract adverse myocardial remodeling and dysfunction mediated by Ang II [42, 43]. This observation may provide a mechanistic basis for the downregulation of miR-122 during the acute phase of COVID-19, suggesting that this negative regulation could play a role in exacerbating the severity of the disease.

Our differential gene expression data were normalized with data from healthy individuals, revealing agreement with previous studies, which also found a significant increase in ACE2 expression [44, 45]. Our observations indicate that miRNAs involved in the regulation of ACE2 expression exhibit consistent patterns in healthy individuals, which are markedly altered in individuals with COVID-19. In the latter, a negative regulatory shift was observed, with downregulation of miRNAs leading to the overexpression of ACE2. Specifically, we identified the downregulation of miR-200c, along with other miRNAs (hsa-let-7b-5p and hsa-miR-122-5p) during the acute phase, suggesting their modulation by SARS-CoV-2. These findings align with prior research, which reports a decrease in these miRNAs at disease onset [46].

Therefore, the impact of epigenetics on the regulation of RAAS receptors could improve screening for COVID-19 severity. Collectively, these findings offer valuable insights into the interactions between clinical presentations in the acute phase of the disease and the RAAS, thus enhancing our understanding of COVID-19 pathogenesis and supporting the development of more targeted and effective therapeutic strategies.

ACE:

Angiotensin-converting enzyme

ACE2:

Angiotensin-converting enzyme 2

ACTB:

Beta-actin (endogenous control)

Ang II:

Angiotensin II

ARDS:

Acute respiratory distress syndrome

AT1R:

Angiotensin II type 1 receptor

AT2R:

Angiotensin II type 2 receptor

COVID-19:

Coronavirus disease 2019

CT:

Cycle threshold

DM:

Diabetes mellitus

DMSO:

Dimethyl sulfoxide

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase (endogenous control)

IL6R:

Interleukin 6 receptor

MAS1:

Mitochondrial assembly

miRNA:

MicroRNA

Ox.supple:

Oxygen supplementation

PBMC:

Peripheral blood mononuclear cell

RAAS:

Renin-angiotensin-aldosterone system

RQ:

Relative quantification

SARS-CoV-2:

Severe acute respiratory syndrome coronavirus 2

SAH:

Systemic arterial hypertension

TMPRSS2:

Transmembrane serine protease 2

Authors and Affiliations

1. Laboratório de AIDS e Imunologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil

   Thais Freitas Barreto Fernandes,Jose Henrique Pilotto,Priscila Alves Cezar,Fernanda Heloise Côrtes,Mariza G. Morgado,Carmem Beatriz W. Giacoia-Gripp,Nathalia Beatriz Ramos De Sá,Andressa Da Silva Cazote,Agatha Freixinho Neves,Marcel De Souza Borges Quintana&Dalziza Victalina De Almeida

2. Instituto Nacional de Infectologia Evandro Chagas, FIOCRUZ, Rio de Janeiro, Brasil

   Maria Pia Diniz Ribeiro,Sandra Wagner Cardoso,Valdiléa G. Veloso&Beatriz Grinsztejn

Contributions

Conceptualization: DVA, BGData curation: MSBQFormal Analysis: MSBQ, TFBF, DVAFunding acquisition: DVA, MGM, FHC, BG, VGVInvestigation: FHC, CBWG, NBRDS, MSBQ, SWC, BG, VGV, JHP, DVAMethodology: TFBF, PAC, FHC, CBWG, NBRDS, AC, AFN, DVAProject administration: MPDR, SWC, BG, VGV, DVAResources: MGM, BG, VGVSupervision: MGM, BG, VGV, JHP, DVAValidation: TFBF, DVAVisualization: TFBF, DVAWriting – original draft: TFBF, DVAWriting – review & editing: DVA, JHP, FHC, MGM.

Corresponding author

Correspondence to Dalziza Victalina De Almeida.

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Instituto Nacional de Infectologia Evandro Chagas [INI]/FIOCRUZ, Rio de Janeiro, Brazil, under the approval number CAAE 32449420.4.1001.5262. All participants or their legal representatives signed an informed consent form prior to enrollment in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions