Angiotensin-converting Enzyme Shedding Research Articles

Control of SARS-CoV-2 infection by MT1-MMP-mediated shedding of ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for SARS-CoV-2. The full-length membrane form of ACE2 (memACE2) undergoes ectodomain shedding to generate a shed soluble form (solACE2) that mediates SARS-CoV-2 entry via receptor-mediated endocytosis. Currently, it is not known how the physiological regulation of ACE2 shedding contributes to the etiology of COVID-19 in vivo. The present study identifies Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) as a critical host protease for solACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 infection leads to increased activation of MT1-MMP that is colocalized with ACE2 in human lung epithelium. Mechanistically, MT1-MMP directly cleaves memACE2 at M706-S to release solACE218-706 that binds to the SARS-CoV-2 spike proteins (S), thus facilitating cell entry of SARS-CoV-2. Human solACE218-706 enables SARS-CoV-2 infection in both non-permissive cells and naturally insusceptible C57BL/6 mice. Inhibition of MT1-MMP activities suppresses solACE2-directed entry of SARS-CoV-2 in human organoids and aged mice. Both solACE2 and circulating MT1-MMP are positively correlated in plasma of aged mice and humans. Our findings provide in vivo evidence demonstrating the contribution of ACE2 shedding to the etiology of COVID-19.

A Bitter Experience That Enlightens the Future: COVID-19 Neurological Affection and Perspectives on the Orexigenic System

The history of coronaviruses revealed that these viruses caused multiple outbreaks in the past, including a previous severe acute respiratory syndrome (SARS) outbreak in 2003. In 2019, a novel SARS virus, SARS-CoV-2, started a drastic pandemic that, up till now, keeps peaking in successive waves owing to the mutational ability of the virus versus the short-term immunity against it. Although the angiotensin-converting enzyme 2 (ACE2) is the gate through which the virus gets access to human cells, yet ACE2 is deemed protective in lung injury yielding vasodilator, anti-fibrotic, and anti-inflammatory peptides. The viral-provoked ACE2 downregulation aggravated a subsequent potentially lethal cytokine storm. Both the tumor necrosis factor-alpha (TNF-α) receptor (TNFR), activated by the proinflammatory cytokine, TNF-α, released during coronavirus disease 2019 (COVID-19), and ACE2 are cleaved by tumor necrosis convertase enzyme (TACE) to render respective soluble decoy mediators. Several risk factors were linked to COVID-19 morbidity and neurological affection, including obesity and diabetes mellitus (DM), attributed toACE2 overexpression in obesity, a low-grade inflammatory state with both obesity and DM, and defective lung reparative machinery, added to low tissue-to-lung ACE2 expression in DM. The ACE2 shedding by SARS-CoV-2 upon its entry into the brain, together with the inflammatory cytokines invading the brain, predispose to such neurological affection. However, ACE2 was not sufficient to justify the occurrence of neurological disorders with COVID-19, owing to its lower brain expression, relative to other tissues. Other mediators should have contributed to such neurological disorders, of which, orexins (OXs) are discussed, owing to multiple functional similarities to ACE2. Eventually, this review highlights such similarities selected according to their possible relevance to COVID-19 symptomatology and pathology. Both ACE2 and OXs confer anti-inflammatory benefits, reduce cerebral endothelial dysfunction, promote neuronal survival and neurogenesis, and add to their therapeutic potentiality in sepsis. Both ACE2 and OXs assist in moderating the stress responses and the stress-activated hypothalamic-pituitary-adrenal axis. Both ACE2 and OXs are affected by obesity and DM. The loss of ACE2 and OXs signaling was suggested in neuro-inflammatory and neurodegenerative diseases. Of interest is the abundance of OXs in the dissemination routes to the brain, namely, the peripheral olfactory and the enteric systems. The presumptive role of OXs as analgesics and antipyretics might add to their favorable profile. Advantageously, the availability of OXs agonists and antagonists makes it applicable to corroborate or abrogate the future utility of targeting the orexigenic system in terms of COVID-19 neurological affection. Elaborative work, exploring in vitro and in vivo models, is recommended to identify or deny such perspective involvement.

Cardiomyocyte-specific knockout of ADAM17 ameliorates left ventricular remodeling and function in diabetic cardiomyopathy of mice

Angiotensin-converting enzyme 2 (ACE2) has proven beneficial in attenuating diabetic cardiomyopathy (DCM) but has been found to be a substrate of a disintegrin and metalloprotease protein-17 (ADAM17). However, whether ADAM17 plays a role in the pathogenesis and intervention of DCM is obscure. In this study, we created cardiomyocyte-specific knockout of ADAM17 (A17α-MHCKO) mice, and left ventricular dimension, function, pathology and molecular biology were assessed in ADAM17fl/fl control, A17α-MHCKO control, ADAM17fl/fl diabetic and A17α-MHCKO diabetic mice. Both differentiated H9c2 cells and neonatal rat cardiomyocytes (NRCMs) were used to explore the molecular mechanisms underlying the effect of ADAM17 on DCM. The results showed that protein expression and activity of ADAM17 were upregulated whereas the protein expression of ACE2 was downregulated in the myocardium of diabetic mice. Cardiomyocyte-specific knockout of ADAM17 mitigated cardiac fibrosis and cardiomyocyte apoptosis and ameliorated cardiac dysfunction in mice with DCM. Bioinformatic analyses detected a number of genes enriched in metabolic pathways, in particular the AMPK signaling pathway, expressed differentially between the hearts of A17α-MHCKO and ADAM17fl/fl diabetic mice. The mechanism may involve activated AMPK pathway, increased autophagosome formation and improved autophagic flux, which reduced the apoptotic response in cardiomyocytes. In addition, hypoxia-inducible factor-1α (HIF-1α) might act as an upstream mediator of upregulated ADAM17 and ADAM17 might affect AMPK signaling via α1 A-adrenergic receptor (ADRA1A). These results indicated that ADAM17 activity and ACE2 shedding were enhanced in DCM, which was reversed by cardiomyocyte-specific ADAM17 knockout. Thus, inhibition of ADAM17 may provide a promising approach to the treatment of DCM.

Early shedding of membrane-bounded ACE2 could be an indicator for disease severity in SARS-CoV-2

SARS-CoV-2 uses membrane bound Angiotensin-Converting Enzyme 2 (ACE2) as a key host receptor for its entry. However, inconsistent results are available in terms of shedding of membrane ACE2 and circulating levels of soluble ACE2 during SARS-CoV-2. To ascertain soluble ACE2 as an effective biomarker for the prediction of COVID-19 outcome, in the present study, we investigated the levels of plasma ACE2 during the early phase of infection in COVID-19 patients. The study involved a total of 42 COVID-19 patients along with 10 healthy controls. Plasma levels of ACE2 was determined using ELISA at the time of admission and on day 7 post admission. The association of sACE2 with D-dimer a marker for hyper-coagulation was performed using a dependence test. Compared to healthy controls, SARS-CoV-2 cases has shown a huge increase in the sACE2 at the time of admission. During the course of infection, we found a significant increase (P ≤ 0.001) in sACE2 in severe cases compared to moderate. There was a strong increase in sACE2 in cases with hypertension and diabetes mellitus. Interestingly, a strong positive correlation (P ≤ 0.001) was obtained between sACE2 and D-dimer. Thus, an excessive shedding of ACE2 during the early phase is a common phenomenon in severe form of the SARS-CoV-2. Along with D-dimer, the sACE2 levels could serve as a clinical biomarker for the prediction of disease outcome. However further studies are needed to ascertain its role in host-virus interplay.

Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity.

ABSTRACTThe spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. The “priming” of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2′ as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2′ cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

ACE2 Shedding and the Role in COVID-19.

Angiotensin converting enzyme 2 (ACE2), a transmembrane glycoprotein, is an important part of the renin-angiotensin system (RAS). In the COVID-19 epidemic, it was found to be the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). ACE2 maintains homeostasis by inhibiting the Ang II-AT1R axis and activating the Ang I (1-7)-MasR axis, protecting against lung, heart and kidney injury. In addition, ACE2 helps transport amino acids across the membrane. ACE2 sheds from the membrane, producing soluble ACE2 (sACE2). Previous studies have pointed out that sACE2 plays a role in the pathology of the disease, but the underlying mechanism is not yet clear. Recent studies have confirmed that sACE2 can also act as the receptor of SARS-COV-2, mediating viral entry into the cell and then spreading to the infective area. Elevated concentrations of sACE2 are more related to disease. Recombinant human ACE2, an exogenous soluble ACE2, can be used to supplement endogenous ACE2. It may represent a potent COVID-19 treatment in the future. However, the specific administration concentration needs to be further investigated.

Intricate relationship between SARS-CoV-2-induced shedding and cytokine storm generation: A signaling inflammatory pathway augmenting COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its ability to induce cytokine release syndrome, can set up a generalized inflammatory response together with activating multiple inflammatory pathways, which contributes to a dramatic increase in the number of mortalities and morbidities worldwide. Reportedly, the manipulative nature of coronavirus disease 2019 (COVID-19), which targets the immune system, often focuses on specific inflammation-related pathways, usually confined to interleukins and tumor necrosis factor-α (TNF-α), with a great emphasis on therapeutic approaches targeting the inhibition of these inflammatory mediators. The involvement of a disintegrin and metalloprotease 17 (ADAM-17) and matrix metalloproteinase-9 (MMP-9) in the pathogenesis of COVID-19, through their ability to potentiate the cytokine storm during an episode of SARS-CoV-2 infection, often goes unnoticed. In this review, the intricate relationship between ADAM-17 and MMP-9 together with angiotensin-converting enzyme 2 (ACE-2) as the main target for SARS-CoV-2 is highlighted in detail through a compilation of evidence-based literature; thus, we shed light on a proposed inflammatory pathway that COVID-19 may exploit to provoke an inflammatory response of a complex nature. Conclusively, our proposed mechanism acts as a means to developing a therapeutic approach aimed at modulating the intricate communication between ADAM-17 and MMP-9, where a great emphasis on the role of ACE-2 shedding and subsequent elevation in angiotensin II (Ang-II) levels is crucial to understanding the awry inflammatory response in patients with COVID-19. From this concept, designing a therapeutic strategy targeting multiple inflammatory mediators and enzymes simultaneously is another approach to unravel this global pandemic.

Calcium Signaling Pathway Is Involved in the Shedding of ACE2 Catalytic Ectodomain: New Insights for Clinical and Therapeutic Applications of ACE2 for COVID-19.

The angiotensin-converting enzyme 2 (ACE2) is a type I integral membrane that exists in two forms: the first is a transmembrane protein; the second is a soluble catalytic ectodomain of ACE2. The catalytic ectodomain of ACE2 undergoes shedding by a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), in which calmodulin mediates the calcium signaling pathway that is involved in ACE2 release, resulting in a soluble catalytic ectodomain of ACE2 that can be measured as soluble ACE2 plasma activity. The shedding of the ACE2 catalytic ectodomain plays a role in cardiac remodeling and endothelial dysfunction and is a predictor of all-cause mortality, including cardiovascular mortality. Moreover, considerable evidence supports that the ACE2 catalytic ectodomain is an essential entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Additionally, endotoxins and the pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-alpha (TNFα) all enhanced soluble catalytic ectodomain ACE2 shedding from the airway epithelia, suggesting that the shedding of ACE2 may represent a mechanism by which viral entry and infection may be controlled such as some types of betacoronavirus. In this regard, ACE2 plays an important role in inflammation and thrombotic response, and its down-regulation may aggravate COVID-19 via the renin-angiotensin system, including by promoting pathological changes in lung injury. Soluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. Furthermore, given that vitamin D enhanced the shedding of ACE2, some studies reported that vitamin D treatment is associated with prognosis improvement in COVID-19. This is an updated review on the evidence, clinical, and therapeutic applications of ACE2 for COVID-19.

ADAM17/MMP inhibition prevents neutrophilia and lung injury in a mouse model of COVID-19.

Severe coronavirus disease 2019 (COVID‐19) is characterized by lung injury, cytokine storm, and increased neutrophil‐to‐lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID‐19 in infected individuals. Angiotensin‐converting enzyme‐2 (ACE2) is the receptor for SARS‐CoV‐2, the virus causing COVID‐19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID‐19‐related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor‐binding domain of the SARS‐CoV‐2 spike protein (RBD‐S) to mimic lung damage associated with COVID‐19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID‐19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI‐1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM‐1 and VCAM‐1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS‐CoV‐2‐infected individuals to prevent the progression toward severe COVID‐19.

Celecoxib, Glipizide, Lapatinib, and Sitagliptin as potential suspects of aggravating SARS-CoV-2 (COVID-19) infection: a computational approach

COVID-19 caused by SARS-CoV-2 has emerged as a potential threat to human life, especially to people suffering from chronic diseases. In this study, we investigated the ability of selected FDA-approved drugs to inhibit TACE (tumor necrosis factor α converting enzyme), which is responsible for the shedding of membrane-bound ACE2 (angiotensin-converting enzyme2) receptors into soluble ACE2. The inhibition of TACE would lead to an increased population of membrane-bound ACE2, which would facilitate ACE2-Spike protein interaction and viral entry. A total of 50 drugs prescribed in treating various chronic diseases in Saudi Arabia were screened by performing molecular docking using AutoDock4.2. Based on docking energy (≤ −9.00 kcal mol−1), four drugs (Celecoxib, Glipizide, Lapatinib, and Sitagliptin) were identified as potential inhibitors of TACE, with binding affinities up to 106–107 M −1. Analysis of the molecular docking suggests that these drugs were bound to TACE's catalytic domain and interact with the key residues such as His405, Glu406, and His415, which are involved in active site Zn2+ ion chelation. Molecular dynamics simulation was performed to confirm the stability of TACE-drugs complexes. RMSD (root mean square deviation), RMSF (root mean square fluctuation), Rg (radius of gyration), and SASA (solvent accessible surface area) were within the acceptable limits. Free energy calculations using Prime-MM/GBSA suggest that Celecoxib formed the most stable complex with TACE, followed by Glipizide, Sitagliptin, and Lapatinib. The finding of this study suggests a mechanism for drugs to aggravate SARS-CoV-2 infection and hence high mortality in patients suffering from chronic diseases. Communicated by Ramaswamy H. Sarma

Diminazen Aceturate Protects Pulmonary Ischemia-Reperfusion Injury via Inhibition of ADAM17-Mediated Angiotensin-Converting Enzyme 2 Shedding.

Lung ischemia-reperfusion (IR) injury is induced by pulmonary artery occlusion and reperfusion. Lung IR injury commonly happens after weaning from extracorporeal circulation, lung transplantation, and pulmonary thromboendarterectomy; it is a lethal perioperative complication. A definite therapeutic intervention remains to be determined. It is known that the enzyme activity of angiotensin-converting enzyme 2 (ACE2) is critical in maintaining pulmonary vascular tone and epithelial integrity. In a noxious environment to the lungs, inactivation of ACE2 is mainly due to a disintegrin and metalloprotease 17 (ADAM17) protein-mediated ACE2 shedding. Thus, we assumed that protection of local ACE2 in the lung against ADAM17-mediated shedding would be a therapeutic target for lung IR injury. In this study, we established both in vivo and in vitro models to demonstrate that the damage degree of lung IR injury depends on the loss of ACE2 and ACE2 enzyme dysfunction in lung tissue. Treatment with ACE2 protectant diminazen aceturate (DIZE) maintained higher ACE2 enzyme activity and reduced angiotensin II, angiotensin type 1 receptor, and ADAM17 levels in the lung tissue. Concurrently, DIZE-inhibited oxidative stress and nitrosative stress via p38MAPK and NF-κB pathways consequently reduced release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. The underlying molecular mechanism of DIZE contributed to its protective effect against lung IR injury and resulted in the improvement of oxygenation index and ameliorating pulmonary pathological damage. We concluded that DIZE protects the lungs from IR injury via inhibition of ADAM17-mediated ACE2 shedding.

MicroRNA‑28‑3p inhibits angiotensin‑converting enzyme2 ectodomain shedding in 293T cells treated with the spike protein of severe acute respiratory syndrome coronavirus2 by targeting A disintegrin and metalloproteinase 17.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019. Angiotensin-converting enzyme 2 (ACE2) is the SARS-CoV binding site and is ubiquitously expressed in endothelial cells of several organs, with the highest levels in the cardiovascular system, kidney and lungs. A disintegrin and metalloproteinase 17 (ADAM17) is involved in ectodomain shedding of ACE2. In the present study, reverse-transcription-quantitative PCR, transfection, TUNNEL assay, dual-luciferase activity assay and western blotting were conducted to investigate the effects of microRNA (miR)-28-3p on ADAM17-dependent shedding of the ACE2 ectodomain following treatment with the spike protein (S-protein) of SARS-CoV-2. It was found that miR-28-3p was significantly downregulated in 293T cells treated with 100 ng/ml of S-protein for 24 h at 37°C, which led to upregulation of ADAM17. In addition, the expression of ADAM17 and miR-28-3p were negatively correlated based on Pearson's correlation test in 293T cells treated with S-protein for 24 h. Overexpression of miR-28-3p and inhibition of ADAM17 regulated 293T cell viability, apoptosis and ACE2 ectodomain shedding. It was also demonstrated that ADAM17 was the target gene of miR-28-3p and that miR-28-3p negatively regulated ADAM17 expression. Notably, the inhibition of ADAM17 expression blocked the effects of miR-28-3p inhibitor on proliferation, apoptosis and ACE2 ectodomain shedding in 293T cells treated with S-protein. The findings of the present study suggested that miR-28-3p inhibits ADAM17-dependent ACE2 ectodomain shedding in 293T cells treated with the S-protein of SARS-CoV-2, which suggested the potential therapeutic role of miR-28-3p mimic in the prevention and treatment of patients with SARS-CoV-2.

A model for COVID-19-induced dysregulation of ACE2 shedding by ADAM17

The angiotensin Converting Enzyme 2 (ACE2) receptor is a key component of the renin-angiotensin-aldesterone system (RAAS) that mediates numerous effects in the cardiovascular system. It is also the cellular point of contact for the coronavirus spike protein. Cleavage of the receptor is both important to its physiological function as well as being necessary for cell entry by the virus. Shedding of ACE2 by the metalloprotease ADAM17 releases a catalytically active soluble form of ACE2, but cleavage by the serine protease TMPRSS2 is necessary for virion internalization. Complicating the issue is the observation that circulating ACE2 can also bind to the virus effectively blocking attachment to the membrane-bound receptor. This work investigates the possibility that the inflammatory response to coronavirus infection can abrogate shedding by ADAM17, thereby favoring cleavage by TMPRSS2 and thus cell entry by the virion.

Angiotensin‐II type 2 receptor activation modulates renal neprilysin and coronavirus receptor angiotensin converting enzyme‐2 expression and activity in obese rats fed high salt diet

Neprilysin and coronavirus receptor angiotensin‐converting enzyme‐2 (ACE2) are peptidases critically involved in the breakdown of deleterious angiotensin‐II and the formation of protective angiotensin‐(1‐7). A disintegrin and metalloproteinase‐17 (ADAM17) is involved in the shedding of neprilysin and ACE2. We have reported the increase in renal angiotensin‐(1‐7) formation upon activation of angiotensin‐II type 2 receptor (AT2R). However, the modulation of expression and activity of these peptidases upon stimulation of AT2R in the kidney under the normal, obese, and high‐salt diet (HSD)‐fed condition is not known. Hence, we have treated, male, lean and obese Zucker rats (OZR) for 14 days without or with AT2R agonist C21 (1 mg/kg/day, via osmotic pumps) while fed HSD or normal diet. We have observed that the activity of neprilysin and ACE2 is decreased in the kidney of OZR fed either diet, however, only neprilysin activity has been found increased in the plasma (p<0.05). The C21‐treatment increased renal neprilysin expression and activity in the HSD‐fed OZR reflecting an increase in the urinary but decreased plasma activity (p<0.05). Likewise, The renal ACE2 expression is also increased upon C21‐treatment in HSD‐fed OZR and lean rats (p<0.05), but not the activity. These changes may be ascribed to ADAM17, as the renal expression of ADAM17 is paradoxically decreased upon HSD feeding in OZR (p<0.05) which is further reduced by C21‐treatment. Results indicate that AT2R activation differentially affects renal neprilysin and ACE2 which is critical in reference to current coronavirus pandemic.

ADAM17‐enriched Exosomes Contribute to Neuronal Activation in Hypertension

ADAM17 is the prime mediator of Angiotensin Converting Enzyme 2 (ACE2) shedding, a process by which the ACE2 ectodomain is cleaved from the cell membrane and released in the surrounding milieu. ACE2 is a compensatory enzyme converting Angiotensin (Ang)-II to Ang-(1-7). Recent studies have reported the presence of activated ADAM17 on exosomes, suggesting the possibility of shedding propagation from one cell to another. Here, we hypothesized that in hypertensive conditions, exosomes are enriched in ADAM17 and contribute to neuronal activation, possibly leading to enhanced sympathetic activity. Human plasma and cerebrospinal fluid were collected from both African Americans (AA) and Caucasian Americans (CA), normotensive and hypertensive patients (n=3/group). Exosomes were extracted using total exosome isolation kit (Invitrogen) and quantified by estimation of proteins (BCA kit). The ADAM17 expression in exosomes was measured by capillary western (protein Simple) and the exosomes’ morphology was studied using electron microscopy (Cryo-TEM). To investigate the role of these exosomes, Neuro2A cells were treated with phorbol myristate, an ADAM17 activator, or Ang-II (100 nM) for 4 hours, followed by extraction of exosomes (Invitrogen) and estimation of their ADAM17 levels (capillary western). After 24 h, Neuro2A cells were transfected with GCaMP6s, a fluorescent calcium indicator, and exposed for 4 h with PBS (control), Ang-II (100 nM) or exosomes isolated from hypertensive patients before Ca++-mediated fluorescence was quantified (Cytation 5). Exosomes extracted from patients’ plasma showed a significant 3-fold increase in ADAM17 expression in hypertensive individuals when compared to normotensive counterparts (Normotensive AA: 3.1 ±0.6, CA: 4.5 ±0.3; Hypertensive AA: 7.1 ±0.2, CA: 7.0 ±0.5, au: arbitrary units; n=3: one-way ANOVA, P< 0.01 and P< 0.001 vs. normotensive). In normotensive patients ADAM17 content was higher in CA but was similar in AA and CA under hypertensive conditions. Exosomes extracted from Neuro2A cells showed a 3-fold increase in ADAM17 expression in both Ang-II- and PMA-treated groups when compared to control, thereby confirming the expression of ADAM17 in exosomes from stimulated neuronal cells. The Ca++-mediated fluorescence intensity in Neuro2A cells treated with exosomes from hypertensive patients or Ang-II was significantly increased when compared to control cells (control: 4.0 ±0.6, Ang-II: 20 ±0.2, exosomes: 15.1 ±0.2, au, n=3, one-way ANOVA, P<0.001 vs. control, n=3). Our data show that upon activation the mature form of ADAM17 is released in the form of exosomes in the brain or plasma from hypertensive patients, and in cells following Ang-II stimulation, possibly contributing to the enhanced ACE2 shedding in those individuals. In addition, neurons exposed to Ang-II and ADAM17-enriched exosomes (i.e. isolated from hypertensive patients) show increased Ca++-mediated fluorescence confirming an activated neuron phenotype that could contribute to elevated excitatory activity on neuronal cells and ultimately to an enhanced sympathetic activity.

Effects of SARS-CoV-2 on Cardiovascular System: The Dual Role of Angiotensin-Converting Enzyme 2 (ACE2) as the Virus Receptor and Homeostasis Regulator-Review.

Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of Coronavirus Disease-2019 (COVID-19) in humans. ACE-2 is a type I transmembrane metallocarboxypeptidase expressed in vascular endothelial cells, alveolar type 2 lung epithelial cells, renal tubular epithelium, Leydig cells in testes and gastrointestinal tract. ACE2 mediates the interaction between host cells and SARS-CoV-2 spike (S) protein. However, ACE2 is not only a SARS-CoV-2 receptor, but it has also an important homeostatic function regulating renin-angiotensin system (RAS), which is pivotal for both the cardiovascular and immune systems. Therefore, ACE2 is the key link between SARS-CoV-2 infection, cardiovascular diseases (CVDs) and immune response. Susceptibility to SARS-CoV-2 seems to be tightly associated with ACE2 availability, which in turn is determined by genetics, age, gender and comorbidities. Severe COVID-19 is due to an uncontrolled and excessive immune response, which leads to acute respiratory distress syndrome (ARDS) and multi-organ failure. In spite of a lower ACE2 expression on cells surface, patients with CVDs have a higher COVID-19 mortality rate, which is likely driven by the imbalance between ADAM metallopeptidase domain 17 (ADAM17) protein (which is required for cleavage of ACE-2 ectodomain resulting in increased ACE2 shedding), and TMPRSS2 (which is required for spike glycoprotein priming). To date, ACE inhibitors and Angiotensin II Receptor Blockers (ARBs) treatment interruption in patients with chronic comorbidities appears unjustified. The rollout of COVID-19 vaccines provides opportunities to study the effects of different COVID-19 vaccines on ACE2 in patients on treatment with ACEi/ARB.

PLASMA ACE2 ACTIVITY IS INCREASED IN PATIENTS RECOVERED FROM SARS-COV-2 INFECTION: IMPLICATIONS FOR THE PROLONGED CONSEQUENCES OF COVID-19

Objective: COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), utilises the catalytic site of membrane-bound angiotensin converting enzyme 2 (ACE2) for cell entry. It is thought that endocytosis of ACE2 results in a decrease in membrane bound ACE2 expression, and disruption of the local tissue renin angiotensin system protection. In this study, we hypothesised that SARS-CoV-2 infection would be associated with shedding of ACE2 leading to increased plasma ACE2 activity. Design and method: Australians aged &gt; 18 years (n = 66) who had recovered from SARS-CoV-2 infection (positive result by PCR testing) and uninfected controls (n = 70) were recruited. Serial samples were available in 23 recovered SARS-CoV-2 patients. Plasma ACE2 activity was measured using a fluorescent substrate-based assay and levels were compared using the Mann-Whitney or Kruskal–Wallis test. Serial ACE2 activity were analysed using the Friedman test for repeated measures. Post-hoc analysis was performed with a Bonferroni correction. Two-tailed P-values &lt; 0.05 were considered significant. Results: Controls and SARS-CoV-2 recovered patients were matched for age (mean ± SD, 54 ± 11 vs. 53 ± 14 years, p = 0.47) and gender (53% vs. 59% male, p = 0.49). There were no significant differences (p &gt; 0.05) in the proportion of hypertension, obesity, diabetes, cardiovascular disease, or use of anti hypertensive, lipid lowering, and anti-platelet medications between the controls and SARS-CoV-2 patients. Plasma ACE2 activity at median 35 days post-infection [interquartile range 30–38 days] was 97-fold higher in SARS-CoV-2 patients compared to controls (5.8 [2–11.3] vs. 0.06 [0.02–2.2] pmol/min/ml, p &lt; 0.0001). Plasma ACE2 activity was significantly different across disease severity (p = 0.033), with severe COVID-19 associated with higher ACE2 activity compared to mild disease (11.2 [8.3–23.2] vs. 5.4 [1.8–9.0] pmol/min/ml, p = 0.027). Men recovered from SARS-CoV-2 had higher ACE2 levels compared to women (9.2 [5.8–15.3] vs. 2.1 [0.2–5.1] pmol/min/ml, p &lt; 0.0001). In 23 patients who had serial blood samples at 63 [56–65] and 114 [111–125] days post infection, median ACE2 activity remained persistently elevated with no differences between time-points (p &gt; 0.05). Conclusions: Plasma ACE2 activity is elevated after SARS-CoV-2 infection and remains elevated post-infection. Our findings indicate the need for ongoing investigation to determine if ACE2 levels identify people at risk of prolonged illness following COVID-19.

ACE2 Shedding and Furin Abundance in Target Organs may Influence the Efficiency of SARS-CoV-2 Entry

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lineage B coronavirus, causing the worldwide outbreak of Corona Virus Disease 2019 (COVID-19). Despite genetically closed to SARS-CoV, SARS-CoV-2 seems to possess enhanced infectivity and subtle different clinical features, which may hamper the early screening of suspected patients as well as the control of virus transmission. Unfortunately, there are few tools to predict the potential target organ damage and possible clinical manifestations caused by such novel coronavirus. Methods: To solve this problem, we use the online single-cell sequence datasets to analyze the expression of the major receptor in host cells that mediates the virus entry, including angiotensin converting enzyme 2 (ACE2), and its co-expressed membrane endopeptidases. Results: The results indicated the differential expression of ADAM10 and ADAM17 might contribute to the ACE2 shedding and affect the membrane ACE2 abundance. We further confirm a putative furin-cleavage site reported recently in the spike protein of SARS-CoV-2, which may facilitate the virus-cell fusion. Based on these findings, we develop an approach that comprehensively analyzed the virus receptor expression, ACE2 shedding, membrane fusion activity, virus uptake and virus replication to evaluate the infectivity of SARS-CoV-2 to different human organs. Conclusion: Our results indicate that, in addition to airway epithelia, cardiac tissue and enteric canals are susceptible to SARS-CoV-2 as well.

Effects of Angiotensin II Type 1A Receptor on ACE2, Neprilysin and KIM-1 in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension.

Activation of the renin angiotensin system plays a pivotal role in the regulation of blood pressure, which is mainly attributed to the formation of angiotensin-II (Ang II). The actions of Ang II are mediated through binding to the Ang-II type 1 receptor (AT1R) which leads to increased blood pressure, fluid retention, and aldosterone secretion. In addition, Ang II is also involved in cell injury, vascular remodeling, and inflammation. The actions of Ang II could be antagonized by its conversion to the vasodilator peptide Ang (1–7), partly generated by the action of angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Previous studies demonstrated increased urinary ACE2 shedding in the db/db mouse model of diabetic kidney disease. The aim of the study was to investigate whether renal and urinary ACE2 and NEP are altered in the 2K1C Goldblatt hypertensive mice. Since AT1R is highly expressed in the kidney, we also researched the effect of global deletion of AT1R on renal and urinary ACE2, NEP, and kidney injury marker (KIM-1). Hypertension and albuminuria were induced in AT1R knock out (AT1RKO) and WT mice by unilateral constriction of the renal artery of one kidney. The 24 h mean arterial blood pressure (MAP) was measured using radio-telemetry. Two weeks after 2K1C surgery, MAP and albuminuria were significantly increased in WT mice compared to AT1RKO mice. Results demonstrated a correlation between MAP and albuminuria. Unlike db/db diabetic mice, ACE2 and NEP expression and activities were significantly decreased in the clipped kidney of WT and AT1RKO compared with the contralateral kidney and sham control (p < 0.05). There was no detectable urinary ACE2 and NEP expression and activity in 2K1C mice. KIM-1 was significantly increased in the clipped kidney of WT and AT1KO (p < 0.05). Deletion of AT1R has no effect on the increased urinary KIM-1 excretion detected in 2K1C mice. In conclusion, renal injury in 2K1C Goldblatt mouse model is associated with loss of renal ACE2 and NEP expression and activity. Urinary KIM-1 could serve as an early indicator of acute kidney injury. Deletion of AT1R attenuates albuminuria and hypertension without affecting renal ACE2, NEP, and KIM-1 expression.

Angiotensin-converting enzyme 2 and COVID-19 in cardiorenal diseases.

The rapid spread of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought into focus the key role of angiotensin-converting enzyme 2 (ACE2), which serves as a cell surface receptor required for the virus to enter cells. SARS-CoV-2 can decrease cell surface ACE2 directly by internalization of ACE2 bound to the virus and indirectly by increased ADAM17 (a disintegrin and metalloproteinase 17)-mediated shedding of ACE2. ACE2 is widely expressed in the heart, lungs, vasculature, kidney and the gastrointestinal (GI) tract, where it counteracts the deleterious effects of angiotensin II (AngII) by catalyzing the conversion of AngII into the vasodilator peptide angiotensin-(1-7) (Ang-(1-7)). The down-regulation of ACE2 by SARS-CoV-2 can be detrimental to the cardiovascular system and kidneys. Further, decreased ACE2 can cause gut dysbiosis, inflammation and potentially worsen the systemic inflammatory response and coagulopathy associated with SARS-CoV-2. This review aims to elucidate the crucial role of ACE2 both as a regulator of the renin–angiotensin system and a receptor for SARS-CoV-2 as well as the implications for Coronavirus disease 19 and its associated cardiovascular and renal complications.