key: cord-0942609-qjpcxruw
authors: Ramos, Simone Gusmão; da Cruz Rattis, Bruna Amanda; Ottaviani, Giulia; Nunes Celes, Mara Rubia; Dias, Eliane Pedra
title: ACE-2 down-regulation may act as a transient molecular disease causing RAAS dysregulation and tissue damage in the microcirculatory environment among COVID-19 patients
date: 2021-05-06
journal: Am J Pathol
DOI: 10.1016/j.ajpath.2021.04.010
sha: 686d8ea783eb19e3e9c073cfb2bfc08d7bd2e7c6
doc_id: 942609
cord_uid: qjpcxruw

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the etiological agent of COVID-19 (coronavirus disease 2019) and the cause of the current pandemic, produces multiform manifestations throughout the body, causing indiscriminate damage to multiple organ systems, particularly the lungs, heart, brain, kidney, and vasculature. The aim of this review is to provide a new look at the data already available for COVID-19, exploring it as a transient molecular disease that causes negative regulation of ACE-2 (angiotensin-converting enzyme 2) and, consequently, deregulates the renin-angiotensin-aldosterone system (RAAS), promoting important changes in the microcirculatory environment. In addition, the authors seek to demonstrate how these microcirculatory changes may be responsible for the wide variety of injury mechanisms observed in different organs in this disease. This new proposed concept of COVID-19 provides a unifying pathophysiological picture of this infection and offers new insights for a rational treatment strategy to combat this new pandemic.

Even a full year after the initial outbreak and spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), its mechanisms of disease are still widely debated. Although the lungs are thought to be the only target organ, other organs and tissues can be affected, leading to a wide variety of clinical conditions in coronavirus disease 2019 (COVID- 19) patients. While the prognosis is favorable in most patients, critical illness, manifested by respiratory distress, thromboembolism, shock, multiorgan failure, and eventually death, has been reported in approximately 5% of cases 1 . Several studies have linked this virus to a defective immune response, with excessive cytokine release as a fundamental aspect of COVID-19 pathogenesis 2 .

However, the way in which this disease causes damage to tissues in various organs is still under investigation.

This article aims to provide an analysis from experienced pathologists regarding the pathophysiological changes resulting from COVID-19. Herein, it is proposed that SARS-CoV-2 provokes a transient molecular disease involving ACE-2 (angiotensin-converting enzyme 2) down-regulation and consequent RAAS (reninangiotensin-aldosterone system) dysregulation, with these phenomena being at least partially responsible for the multifocal tissue damage in the microcirculatory environment and playing a fundamental role in the pathogenesis of this disease.

Entry into host cells is the first step of viral infection. In humans, coronaviruses gain entry into host cells by way of their transmembrane spike (S) GP (glycoprotein), which comprises S1 and S2 subunits. The S1 subunit is responsible for binding to the host cell receptor, and the S2 subunit assists with virus-host cell fusion 3 .

CoV-2 has 10-fold to 20-fold greater binding affinity than SARS-CoV 8 . Therefore, regarding the mechanism of infection, what makes SARS-CoV-2 almost unique (compared with other common respiratory pathogens such as influenza and parainfluenza viruses) is the use of ACE-2, a specific protein, as its receptor.

ACE-2 is a key modulator of the RAAS, an intricate interlinked system that regulates physiological and pathological functions of cardiovascular, renal and pulmonary system [9] [10] [11] . Aside from regulating arterial blood pressure, cardiac function, and fluid balance, the RAAS plays a major role in immunity 12 . Under normal circumstances, ACE-2 terminates the action of angiotensin (Ang) I and Ang II by cleaving these peptides into Ang 1-9 and Ang 1-7, respectively. In the absence of ACE-2 (due to viral blockade and down-regulation), both Ang I and Ang II accumulate.

However, as angiotensin-converting enzyme (ACE) is not engaged by the virus, the conversion of Ang I to Ang II continues unabated, leading to unopposed accumulation of Ang II. Ang II is the biologically active mediator of effects of the RAAS whose functions are controlled by two G protein-coupled receptors (GPCR), AT 1 R (angiotensin-II type 1 receptor) and AT 2 R (angiotensin-II type 2 receptor) [9] [10] [11] . Abnormal activation of the Ang II/AT 1 R component of RAAS has been implicated in several pathologic conditions, including the development of end organ damage through the activation of proinflammatory and profibrotic cascades 13 . Therefore, COVID-19induced ACE/ACE-2 imbalance promoting Ang II upregulation in the microcirculatory environment, may favor local inflammation, capillary leakage, a pro-coagulant state, mitochondrial oxidative damage, ROS (reactive oxygen species) production and IL-6 upregulation, inducing coagulation and an immune response 5, 6, 14 . A detailed review of these mechanisms can be found elsewhere 12, 15-17 . anticoagulants, and the release of neutrophil extracellular traps (NETs) that provide a scaffold procoagulant consisting of DNA, histones, and neutrophil serine proteases.

NETosis sequesters platelets in the microcirculation, causing increased fibrin-platelet interactions and the formation of microthrombi 21 . Ordinarily, combat against the pathogen remains restricted to the intravascular compartment, triggering minimal host parenchymal damage. However, on a large scale, immunothrombosis can be a major biological process fostering the pathologies associated with thrombosis in the microvasculature and promoting tissue damage 22,23 .

The mechanisms by which SARS-CoV-2 induces microthrombosis remain incompletely understood. A sequence of events has been proposed as described below. SARS-CoV-2 can directly invade type II pneumocytes 24,25 . Infected cells undergo pyroptosis, leading to the release of danger-associated molecular patterns (DAMPs) and triggering the release of proinflammatory cytokines and chemokines into the environment 26,27 . Then, the activated alveolar endothelium upregulates the expression of VWF (von Willebrand factor) and adhesion molecules, including ICAM (intercellular adhesion molecule)-1, αvβ3 (alpha v beta 3 "vitronectin receptor"), Pselectin and E-selectin, leading to recruitment of platelets and leukocytes and complement activation 25 . Neutrophils release NETs, causing direct activation of the classical complement pathway. Complement activation potentiates these mechanisms by increasing endothelial and monocyte tissue factor (TF), further platelet activation and amplifying endothelial inflammation, which increases the production of proinflammatory cytokines from the endothelium, including IL (interleukin)-1, IL-8, RANTES (regulated on activation, normal T-cell expressed and secreted), IL-6, and MCP (monocyte chemoattractant protein)-1 28,29 . The hypoxic environment can induce HIFs (hypoxia-inducible factors), which upregulate endothelial TF expression. These mechanisms ultimately lead to the unchecked generation of thrombin, resulting in thrombus formation 12,30,31 .

Activated platelets and neutrophils releasing NETs are directly linked to thrombosis and inflammation, causing organ damage and increasing mortality in severe COVID-19 cases 21,32 . Autopsy reports have suggested that COVID-19-induced formation of NETs may contribute to cytokine storms, vascular thrombosis, and acute respiratory distress syndrome (ARDS) 33 . While a limited amount of NET formation at inflammatory sites serves to limit blood loss and prevent the spread of pathogens 18, 19 , NET formation in COVID-19 is clearly dysregulated at multiple intravascular sites, leading to rapid occlusion of microvessels 34 . Therefore, an unfavorable milieu can allow these phenomena to spread uncontrollably in the circulation, promoting multiorgan dysfunction. 56 . These findings may indicate that a procoagulant status may be a hallmark event of COVID-19 and suggest that when they spread widely, these microvascular obstructions may promote tissue damage depending on their quantity, and/or the vulnerability of the affected organ. Altogether, these circumstances strongly point to the vascular system on, both the macro and micro scales, as an important target of COVID-19 57 . However, other organs and tissues are also severely affected by SARS-CoV-2, leading us to pursue a unifying pathophysiological explanation for the multivariate clinical presentation of this unprecedented infection.

Knowledge of the underlying biology and physiology of ACE-2 has accumulated over the last 20 years since its discovery and has substantially improved our understanding of the RAAS 10-12 . As ACE-2 contributes critically to the biology of SARS-CoV-2 infection, much attention has been focused on the interplay between COVID-19 and RAAS 58, 59 . The RAAS may also be indirectly involved in the pathophysiology of other respiratory infections, but in SARS-CoV-2 infection, it seems to play an important and direct role in the development and progression of COVID-19 58 . ACE-2 is expressed in almost all human tissues; its expression level is high in the small intestine, testes, adipose tissue, kidneys, heart and thyroid; medium in the lungs, colon, liver, bladder, and adrenal glands; and relatively low in the blood vessels, spleen, bone marrow, brain, and muscle 5, 60 . ACE-2 is expressed as a cell-surface nonraft protein with little intracellular localization, and the protein is not readily internalized. However, binding of a coronavirus spike protein to ACE-2 triggers enzyme internalization, down-regulating its cell-surface activity 61 . Moreover, the viral S subunit contains a cleavage site for furin and other proteases, which accelerates the cellular entry of SARS-CoV-2 12 . In addition to promoting ACE-2 internalization, SARS-CoV promotes the enzymatic shedding of the ACE-2 ectodomain, resulting in both the generation of a soluble form of ACE-2 and an overall reduction in ACE-2 content in the infected cells 30 . As the S proteins of SARS-CoV-2 and SARS-CoV share 76% overall amino acid identity and display similar receptor-binding modules 30 , it is likely that SARS-CoV-2 also induces ACE-2 shedding. ACE-2 serves as an endogenous inhibitor of inflammatory signals associated with four major regulator systems: RAAS, the kallikrein-kinin system (KKS), the J o u r n a l P r e -p r o o f coagulation cascade, and the complement system 12,15,17,61-65 . ACE-2 plays a fundamental role in regulating the RAAS by directly converting Ang II to 10, 12, 13 . Ang II is the main vascular effector of the RAAS and exerts its deleterious effects on the cardiovascular system via angiotensin-II type 1 receptors (AT 1 R) by activating vasoconstrictor, inflammatory and fibrotic pathways. Ang II accumulation also activates ADAM17 activity, thus perpetuating membrane shedding of ACE-2, RAAS overactivation, and inflammation 12,15,65 . In addition, after AT 1 R activation, Ang II can activate the nuclear factor kappa B (NF-κB) pathway 66, 67 via stimulation of the phosphorylation of the p65 subunit of NF-κB 68 . This will lead to increased production of IL-6, TNFα, IL-1β, IL-10 and IL-12 68, 69 . While Ang II induces vasoconstriction and promotes a proinflammatory/prothrombotic phenotype, Ang 1-7 exerts vasodilatory, antiproliferative, anticoagulation, and antifibrotic activity, via its specific Mas receptor (MasR), thus counterbalancing the adverse effects of Ang II mediated by AT 1 R 5,15,61 .

Therefore, Ang 1-7, the main product of ACE-2 in the regulation of RAAS, plays a critical role in maintaining microcirculatory balance through the inhibition of proinflammatory and procoagulant pathways 61 .

The kallikrein-bradykinin (BK) system is intensively interwoven with RAAS through many pathways with complex reciprocal interactions metabolites, especially 70 . DABK is a known pulmonary inflammatory factor 70, 71 . ACE-2 cleaves terminal residue of DABK, resulting in its deactivation 72, 73 . The derangement ACE-2/DABK/Bradykinin B1 Receptor axis activation creates a proinflammatory synergistic effect for SARS-CoV-2 in association with ACE/Ang II/AT 1 R axis activation 15, 62, 63 . The resulting effect would be a more inflammatory state, neutrophil recruitment and enhancement of pathological pulmonary changes in severe COVID-19, including NF-κB activity 62, 63 . The increased production of Ang II and activation of AT 1 R also can occur via activation of the complement cascade, including C5a and C5b-9, pointing to cross-talk between the RAAS and the complement system 74 . Finally, the interaction of ACE-2 with the coagulation system (CS) is indirect and occurs via two mechanisms: 1) catalyzing the production of angiotensin 1-9, which reduces plasminogen activator and increases PAI-1 (Plasminogen Activator Inhibitor-1), thus inhibiting fibrinolysis 75 

In a recent study conducted in patients with COVID-19, the viral load and the severity of lung injury were strongly associated with the circulating levels of Ang II 14 .

Moreover, a cohort of 12 COVID-19 patients showed markedly elevated circulating Ang II levels compared with healthy controls (linearly correlated with viral load), providing a direct link between tissue ACE-2 down-regulation and systemic RAAS imbalance, which favors multiorgan damage from SARS-CoV-2 infection 77 . While respiratory symptoms are predominant, acute cardiac and kidney injuries, arrhythmias, cerebral symptoms, and gut and liver function abnormalities are being reported in infected patients 12 . This is relevant to the pulmonary, cardiac, and renal tissues of infected subjects, especially patients with heart failure, diabetes, pulmonary diseases, and hypertension, whose present clinical settings are associated with RAAS dysregulation 72 . Given the above premises, it seems reasonable to speculate that depletion of ACE-2 and activation of the ACE/Angiotensin II/AT 1 R axis might have a pivotal role in the clinical presentations of COVID-19.

Based on experimental models of SARS, virus-induced ACE-2 suppression is thought to propagate acute lung injury by leading to increased lung Ang II content 14 . It was also demonstrated in non-SARS experimental studies. ACE-2 knockout (KO) mice showed ARDS/acute lung injury pathology, characterized by increased vascular permeability, increased pulmonary edema, neutrophil accumulation, and deterioration of lung function compared with normal WT (wild type) control mice 14 . ACE deficiency partially rescued the severe lung injury phenotype of mice that had a single mutation in the ACE-2 gene, suggesting that the balance of ACE and ACE-2 levels is the key to lung injury or lung protection during an inflammatory storm 78 . Moreover, the hearts of ACE-2 KO mice showed increased Ang II levels and upregulation of hypoxia-inducible genes, suggesting that cardiac function is modulated by the balance between ACE and ACE-2 and that the increase in local cardiac Ang II is involved in these abnormalities. This is supported by the fact that the cardiac phenotype and increased Ang II levels were completely reversed by concomitant deletion of the ACE gene in ACE-2 KO mice 61 . Recent studies suggest that ACE-2 influences the electrical pathways of the heart.

In ACE-2 transgenic mice, cardiac conduction disturbances were present, and some animals developed lethal ventricular fibrillation 79 . Furthermore, Ang II is increased in damaged tubules, suggesting its possible role as a mediator of renal damage in J o u r n a l P r e -p r o o f experimental and human renal disorders. A disrupted balance between intrarenal ACE and ACE-2 with consequent high levels of Ang II might therefore contribute to progressive renal damage 80 . Collectively, this evidence points to a conceptual framework in which ACE-2 is a central player in normal multiorgan functions, and its negative regulation during infection can be a fundamental event producing disease.

Prof. Pérez-Tamayo, a renowned pathologist, stated that when a structurally normal protein is present but its relative concentration in the different compartments of the organism is perturbed in association with corresponding functional changes, the scenario constitutes a molecular disease; such diseases can be further classified as regulatory (quantitative deviations from the norm) or cq c " x c c " by removal of the responsible agent) 81 Figure 2 .

These microthrombi may be especially prominent wherever there are ACE-2positive cells, especially in the lungs, heart, kidneys, brain and endothelium 58 .

Interestingly, in the pathophysiology of COVID-19, there appears to be enormous spatiotemporal heterogeneity in the involvement of organs 82 . Therefore, histological consequences may be aggravated when occurring simultaneously in patients who already have conditions involving a dysregulation of the RAAS, such as heart diseases, hypertension, diabetes mellitus, chronic diseases, older age and obesity 78, 83 . The damage to the organs will vary depending on the quantity of thrombi, the vulnerability of the tissue, the general conditions of the patient (old age, pre-existing diseases), and genetic conditions (sex, blood type, ethnicity, individual immune response) 16 Unraveling the pathologic basis of COVID-19 is essential for our understanding of the pathophysiology of this intriguing disease.

In the vast majority of conditions defined and/or accompanied by microvascular thrombosis, the diagnosis is typically made clinically, and sometimes, Renin converts Angiotensinogen into Ang I. ACE converts Ang I into Ang II, which binds to its receptors AT 1 R and AT2R. Ang II exerts most of its harmful cardiovascular effects through the ACE/Ang II/AT 1 R axis. Usually, the major part of Ang II is converted by ACE-2 to become Ang 1-7 that activates the MasR signaling pathway with protective effects in the microcirculatory environment. In 

Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China

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Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

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COVID-19: ACE2centric Infective Disease? Hypertension 2020

Intoxication With Endogenous Angiotensin II: A COVID-19 Hypothesis

TMPRSS2 and ADAM17 Cleave ACE2 Differentially and Only Proteolysis by TMPRSS2 Augments Entry Driven by the Severe Acute Respiratory Syndrome Coronavirus Spike Protein

The Reproductive Number of COVID-19 is Higher Compared to SARS Coronavirus

A Human 12

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Sah R: Clinical, Laboratory and Imaging Features of COVID-19: A Systematic Review and Meta-analysis

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SARS-CoV-2 Endothelial Infection Causes COVID-19 Chilblains: Histopathological, Immunohistochemical and Ultrastructural Study of Seven Paediatric Cases

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Endothelial Cell Infection and Endotheliitis in COVID-19

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COVID-19 -A Vascular Disease

Severe Acute Respiratory Syndrome Coronavirus 2, COVID-19, and the Renin-Angiotensin System: Pressing Needs and Best Research Practices

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Expression of the SARS-CoV-2 Cell Receptor Gene ACE2 in a Wide Variety of Human Tissues

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SARS-CoV-2 Receptor is Co-expressed with

Renin-Angiotensin and Coagulation Systems in Alveolar Cells. Sci Rep 2020

Kinins and Chymase: The Forgotten Components of the Renin Angiotensin System and their Implications in COVID-19 Disease

Angiotensin II Induced Proteolytic Cleavage of Myocardial ACE2 is Mediated by TACE/ADAM-17: A Positive Feedback Mechanism in the RAS

Proinflammatory Actions of Angiotensins

Angiotensin II Induces Nuclear Factor (NF)-κB1 Isoforms to Bind the Angiotensinogen Gene Acute-Phase Response Element: A Stimulus-Specific Pathway for NF-κB Activation

Angiotensin II Stimulates The Release of Interleukin-6 and Interleukin-8 from Cultured Human Adipocytes by Activation of NF-κB

Interleukin-6 Family of Cytokines Mediate Angiotensin II-induced Cardiac Hypertrophy in Rodent Cardiomyocytes

Angiotensin II Cell Signaling: Physiological and Pathological Effects in the Cardiovascular System

MAP Kinases in the Immune Response

Mitogen-activated Protein Kinases in Innate Immunity

Letter to the Editor: Angiotensinconverting enzyme 2: An Ally or a Trojan Horse? Implications to SARS-CoV-2-Related Cardiovascular Complications

The Kinin B1 Receptor Mediates Alloknesis in a Murine Model of Inflammation

Severe Hypertension with Renal Thrombotic Microangiopathy: What Happened to the Usual Suspect?

Angiotensin-(1-9) Enhances Stasis-induced Venous Thrombosis in the Rat Because of the Impairment of Fibrinolysis

Vederas JC: Plasma Kallikrein Cleaves and Inactivates Apelin

Apelin-17 Analogs as Metabolically Stable Blood Pressure-lowering Agents

Clinical and Biochemical Indexes from 2019-nCoV Infected Patients Linked. Sci China Life Sci

Angiotensin-converting enzyme 2 Protects from Severe Acute Lung Failure

Heart Block, Ventricular Tachycardia, and Sudden Death in ACE2 Transgenic Mice with Downregulated Connexins

Combination Therapy with ACE Inhibitors and Angiotensin II Receptor Blockers to Halt Progression of Chronic Renal Disease: Pathophysiology and Indications

Mechanisms of disease: An Introduction to Pathology. 2 nd Edition. Edited by Year Book Medical Publishers, INC

SARS-CoV-2 and the Pathophysiology of Coronavirus Disease 2019 (COVID-19)

Clinical Characteristics of Coronavirus Disease 2019 in China

Angiotensin-Converting Enzyme Gene Polymorphism and Severe Lung Injury in Patients with Coronavirus Disease

The Case Fatality Rate in COVID-19 Patients With Cardiovascular Disease: Global Health Challenge and Paradigm in the Current Pandemic

Endothelial Activation and Dysfunction in COVID-19: from Basic Mechanisms to Potential Therapeutic Approaches

Spatial and Temporal Dynamics of the Endothelium

Calls for Immediate Attention: The Emerging Roles of the Endothelium in Inflammation Caused by SARS-CoV-2

The Interactions Between Inflammation and Coagulation

Dynamics of Leukocyte-platelet Adhesion in Whole Blood

Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19

Van Der Poll T: Sepsis and Thrombosis

The Role of Leukocytes in Thrombosis

Blood Coagulation in Immunothrombosis-At the Frontline of Intravascular Immunity

Manipulation of ACE2 Expression in COVID-19

AT2R, angiotensin-II type 2 receptor; BKB-1 R, bradykinin B1 receptor

BKB-2 R, bradykinin B1 receptor

DABK, des-Arg9 bradykinin

Ang 1-7 receptor Mas; NF-κB, nuclear factor kappa B; PAI-1

PRR, pattern recognition receptor

SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Created with BioRender.com, Toronto, Canada. Figure 2. Schematic representation showing the pathological consequences of negative regulation of ACE-2 in COVID-19

The downregulation of ACE-2 leads to RAAS dysregulation, which associated with the exacerbated innate immunity response, favors the appearance of immunothromboses in the microcirculation. These immunothrombi result from the activation of inflammatory and coagulation pathways through a cytokine storm, resulting in endothelial dysregulation, leukocyte activation, NETs generation, complement deposition and platelet consumption. Pre-existing dysregulation of the RAAS in elderly patients, heart disease, hypertension, diabetes mellitus, chronic diseases

Tissue damage can occur through a wide range of mechanisms, including tissue hypoxia, damage by ROS, ischemia/necrosis, ischemia-reperfusion injury, hemorrhage, and/or thromboembolism. These changes, if left untreated, can lead to multiorgan dysfunction and death

NETs, neutrophil extracellular traps; RAAS, reninangiotensin-aldosterone system

ROS, reactive oxygen species, and SARS