Low Angiotensin-converting Enzyme 2 Research Articles

Cell type-specific adaptation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (ACE2). However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell-cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

A disintegrin and metalloproteinase domain 9 facilitates SARS-CoV-2 entry into cells with low ACE2 expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus disease-19 (COVID-19) pandemic, utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for virus infection. However, the expression pattern of ACE2 does not coincide with the tissue tropism of SARS-CoV-2, hinting that other host proteins might be involved in facilitating SARS-CoV-2 entry. To explore potential host factors for SARS-CoV-2 entry, we performed an arrayed shRNA screen in H1650 and HEK293T cells. Here, we identified a disintegrin and a metalloproteinase domain 9 (ADAM9) protein as an important host factor for SARS-CoV-2 entry. Our data showed that silencing ADAM9 reduced virus entry, while its overexpression promoted infection. The knockdown of ADAM9 decreased the infectivity of the variants of concern tested-B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron). Furthermore, mechanistic studies indicated that ADAM9 is involved in the binding and endocytosis stages of SARS-CoV-2 entry. Through immunoprecipitation experiments, we demonstrated that ADAM9 binds to the S1 subunit of the SARS-CoV-2 Spike. Additionally, ADAM9 can interact with ACE2, and co-expression of both proteins markedly enhances virus infection. Moreover, the enzymatic activity of ADAM9 facilitates virus entry. Our study reveals an insight into the mechanism of SARS-CoV-2 virus entry and elucidates the role of ADAM9 in virus infection. IMPORTANCE COVID-19, an infectious respiratory disease caused by SARS-CoV-2, has greatly impacted global public health and the economy. Extensive vaccination efforts have been launched worldwide over the last couple of years. However, several variants of concern that reduce the efficacy of vaccines have kept emerging. Thereby, further understanding of the mechanism of SARS-CoV-2 entry is indispensable, which will allow the development of an effective antiviral strategy. Here, we identify a disintegrin and metalloproteinase domain 9 (ADAM9) protein as a co-factor of ACE2 important for SARS-CoV-2 entry, even for the variants of concern, and show that ADAM9 interacts with Spike to aid virus entry. This virus-host interaction could be exploited to develop novel therapeutics against COVID-19.

P-002 SARS-CoV-2 tropism spectrum for human testicular cells

Abstract Study question In human testicular cells, how are SARS-CoV-2 receptors and their priming proteases for the viral spike (S) protein distributed spatially? Summary answer Human testicular tissue expressed both the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) receptors and proteases investigated, key factors for cellular susceptibility to SARS-CoV-2 entry/infection. What is known already Male reproductive function is altered on numerous levels by SARS-CoV-2 and coronavirus disease. Angiotensin-converting enzyme 2 (ACE2) is the main SARS-CoV-2 host membrane receptor but requires transmembrane protease serine 2 (TMPRSS2) for S protein priming (activation by proteolysis). Other proteases, like cathepsin L (CTSL), might nevertheless contribute to SARS-CoV-2 entrance. This mostly happens in cells with low or nonexistent ACE2 expression, which calls for alternative receptors like cluster of differentiation 147 (CD147). Although co-expression of receptors and proteases is essential for successful SARS-CoV-2 infection, it is still unknown how viral receptors and accompanying proteases are distributed geographically in testicular cells. Study design, size, duration To overcome this restriction, we focused on using immunohistochemistry to map the spatial distribution of the SARS-CoV-2 receptors ACE2 and CD147, as well as their priming proteases for the viral spike (S) protein, TMPRSS2 and CTSL, which are necessary for viral fusion with host cells, in human testicular tissue. Participants/materials, setting, methods Laboratory experiments were executed under the Joint Ethics Committee of the Hospital and University (approval number 2020-094 (077-DEFI-078-CE)). Human testicular tissues were obtained from cases presenting Sertoli cell syndrome, maturation arrest, and hypospermatogenesis at the Hospital Pathology department, with five patients per pathology. Formalin-fixed paraffin-embedded tissue sections 4 μm were incubated with anti-antibodies ACE2 (orb638860, 1:100 and 1:150, Byorbit), TMPRSS2 (ab109131, 1:1000, Abcam), CD147 (MA5-29060, 1:2500, Invitrogen) and CTSL (MA5-32602, 1:100, Invitrogen). Main results and the role of chance The present study did not intend to study the virus entry proteins in different testicular pathologies. As spermatogenesis is a complex process involving many intricately linked cells, we selected these syndromes specifically to identify individual cells more clearly, Sertoli cells in Sertoli cell-only syndrome cases, spermatocytes in maturation arrest cases and spermatids in hypospermatogenesis cases. The hypospermatogenesis group consisted of cases whose azoospermia was due to duct obstruction, showing conserved spermatogenesis. Interstitial cells, including endothelium, Leydig and myoid peritubular cells, and the seminiferous epithelium (Sertoli cells, spermatogonia, spermatocytes, and spermatids), were discovered to contain ACE2 and TMPRSS2, demonstrating co-expression of both receptor and protease in all testicular cells. All cell types, with the exception of endothelium and peritubular cells, showed the presence of CD147, whereas CTSL was only found in Leydig, peritubular, and Sertoli cells. As a result, only Leydig and Sertoli cells co-expressed CD147 and CTSL. The findings of proteomic databases indicating testicular cells have SARS-CoV-2 receptors and proteases are supported by our discoveries, demonstrating that testicular cells may be directly infected and injured, halting spermatogenesis and acting as viral vectors for the SARS-CoV-2. Limitations, reasons for caution Our results support viral tropism for human testicular cells and raise the possibility of testicular manifestations, although this does not necessarily mean that the virus infects testicular cells directly and further studies are needed to discern SARS-CoV-2 infection and transmission targeting alternative receptors and to identify the underlying mechanisms. Wider implications of the findings Male reproductive system susceptibility to viral infections has long been demonstrated. To prevent infection by allowing the virus to attach to germ cells, impair sperm production, and increase the risk of meiotic errors and viruses spreading through sperm, our study emphasizes the importance of safe practices in the wider community. Trial registration number not applicable

Effect of COVID-19 on the Role of Renin Enzyme and ACE2 and Hormones in PCOS Females

Polycystic ovary syndrome (PCOS) is the most endocrine problem in women of regenerative age. PCOS women typically belong to an age and sex group which is at higher risk for severe coronavirus disease (COVID-19). COVID-19 targets cells through angiotensin-converting enzyme 2 (ACE2) receptor presents on cells in veins, lungs, heart, digestion tracts, and kidneys. Renin-Angiotensin System (RAS) over activity has likewise been described in metabolic disorders; type 2 diabetes mellitus (T2DM), and conditions shared by women with polycystic ovary condition. The point of this study is to know the job of renin and ACE2 in PCOS and coronavirus and its relationship with hormones and other metabolic parameters related. The study groups consist of 120 sample subjects consisting of 80 PCOS-women divided into two groups: - (40 PCOS-women infected with COVID-19 and 40 PCOS-women non-infected with COVID-19) and 40 healthy-women divided into two groups: - 20 women-COVID-19 infected, and 20 women non-COVID-19 infected, ranging in age from 15–40 years old. Renin and ACE2, FBS, lipid profile, level of insulin, HOMA IR, FSH, LH, and testosterone, were measured for all participants. There was an increase in the levels of renin, ACE2, insulin, HOMA-IR, LH, LH / FSH ratio, and testosterone, except HDL and FSH were low when comparing PCOS patients groups with healthy groups. There was a decrease in renin and ACE2 in PCOS women infected with COVID-19 compared with PCOS women non-infection with COVID-19. Infection of COVID-19 and low ACE2 levels lead to respiratory damage and problem in reproductive in PCOS patients. Also, at the same time ACE2 high level in PCOS makes them more risk for coronavirus and is not involved in the cause of PCOS, however renin enzyme may be affected by BMI associated with PCOS.

In This Issue

HomeCirculation ResearchVol. 132, No. 1In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published5 Jan 2023https://doi.org/10.1161/RES.0000000000000587Circulation Research. 2023;132:2Maintenance of Enteral ACE2 Prevents Diabetic Retinopathy in Type 1 Diabetes (p e1)Download figureDownload PowerPointProbiotic delivery of ACE2 improves gut integrity and retinopathy in diabetic mice, show Prasad et al.Type 1 diabetes has a complex etiology and pathology that are not entirely understood. In addition to the destruction of insulin-producing cells, a recently discovered feature of the disease (in both model rodents and humans) is that levels of angiotensin-converting enzyme 2 (ACE2)—a component of the renin-angiotensin system controlling hemodynamics—can be unusually low in certain tissues. Moreover, genetic deficiency of ACE2 in rodents, exacerbates aspects of diabetes such as gut permeability, systemic inflammation and diabetic retinopathy, while boosting ACE2 levels has been shown to ameliorate diabetic retinopathy in mice. Prasad and colleagues now show that ACE2 treatment also improves gut integrity and systemic inflammation as well as retinopathy, and that certain improvements can even be seen once diabetes has long been established. Six months after the onset of diabetes in model mice, oral doses of Lactobacillus paracasei engineered to express humanized ACE2 led to a reversal of the animal’s gut barrier dysfunction and retinopathy, the team showed. Humans with diabetic retinopathy also displayed evidence of increased gut permeability and low ACE2 levels, suggesting they may benefit from a similar probiotic treatment developed for clinical use.Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression (p e22)Download figureDownload PowerPointSuppressing epsin activity could be a new way to treat atherosclerosis, suggest Cui et al.Epsins are a family of plasma membrane proteins that drive endocytosis. They are expressed at varying levels throughout the tissues of the body, and recent research shows they are unusually abundant on macrophages within atherosclerotic lesions. Furthermore, in mice, macrophage-specific epsin loss results in a reduction in foam cell formation and plaque development. Cui and colleagues now show this effect on foam cells is because epsins normally promote the internalization of lipids into macrophages through their endocytic activity. But that’s not all. The proteins also impede cholesterol efflux from macrophages further exacerbating lipid retention, the team found. It turns out, epsins drive the endocytosis and degradation of a cholesterol efflux factor called ABCG1. Importantly, these proatherogenic activities of epsins could be stopped. Using macrophage-targeting nanoparticles carrying epsin-specific silencing RNAs, the team could suppress production of the protein in cultured macrophages, and could reduce the size and number of plaques in atherosclerosis-prone mice (via injection of the nanoparticles). Together the results suggest blocking epsins, via nanotherapy or other means, could be a therapeutic approach to stopping or slowing atherosclerosis progression.Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart (p 10)Download figureDownload PowerPointBlocking 15-PGDH quells fibroblast activation to prevent cardiac fibrosis in mice, report Rubino et al.After a myocardial infarction, the initial scar that forms is essential for protecting the muscle from further damage and rupture. But, if the fibrosis is extensive or prolonged, it can limit muscle function and ultimately lead to heart failure and death. Finding treatments that prevent or reverse fibrosis is thus a desirable clinical goal. To identify inhibitors of profibrotic activity, Rubino and colleagues screened a library of 546 known non-toxic compounds for their ability to block growth factor-induced activation of rat fibroblast cells. Of the nine compounds with such activity, the team focused on one—SW033291—that had previously been shown to prevent lung fibrosis in mice. This compound inhibits the enzyme 15-PGDH, which degrades signaling molecules known as eicosanoids, which are involved in, among other things, tissue repair. Further experiments showed the compound could also switch off the activation state of human cardiac fibroblasts isolated from failing hearts. The team went on to show that infusing SW033291 into mice could ameliorate experimentally induced cardiac fibrosis and diastolic dysfunction. The findings thus identify 15-PDGH as a profibrotic factor that could serve as a therapeutic target for mitigating heart fibrosis and failure. Previous Back to top Next FiguresReferencesRelatedDetails January 6, 2023Vol 132, Issue 1 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000587 Originally publishedJanuary 5, 2023 PDF download Advertisement

ACE2 maybe serve as a prognostic biomarker in breast invasive carcinoma.

BackgroundBreast cancer is a frequently occurring malignant tumor in women. Angiotensin‐converting enzyme 2 (ACE2) is widely expressed in most organs; however, the association of ACE2 with prognosis and immune infiltration in breast invasive carcinoma (BRCA) remains elusive.MethodsWe explored the expression level and prognostic value of ACE2 in patients with BRCA using a series of online bioinformatics analysis databases encompassing Oncomine, UALCAN, Kaplan–Meier plotter, TIMER, LinkedOmics, and GEO. qRT‐PCR was performed to verify our findings.ResultsAngiotensin‐converting enzyme 2 mRNA and protein expression levels were decreased in BRCA tissues, and patients with low ACE2 expression levels had a poor prognosis. DNA promoter methylation of ACE2 significantly downregulated ACE2 expression in BRCA, while the expression of this protein was positively linked to immune infiltration of B cells, CD8+ and CD4+ T cells, neutrophils, and dendritic cells in BRCA tissues. The high expression level of ACE2 in enriched basophils, CD8+ T cells, and type‐2 helper T cells, which showed decreasing levels, indicated a better prognosis for BRCA. Enrichment analyses revealed that NF‐κB, IL‐17, and TNF signaling pathways were highly correlated to ACE2 in BRCA. Verification study revealed that downregulation of ACE2 was associated with a better prognosis in BRCA. Univariate and multivariate analysis confirmed ACE2 expression and clinical stage as independent prognostic factors for breast cancer.ConclusionsAngiotensin‐converting enzyme 2 may be a potential prognostic biomarker and target for BRCA. Nevertheless, future investigations are needed for validating our findings and promoting the clinical application of ACE2 in BRCA.

SARS-CoV-2 susceptibility of domestic and wild mammals: is it all about the similarity of their ACE2 receptor to the human one?

You have accessMoreSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Cite this article Di Guardo Giovanni 2022SARS-CoV-2 susceptibility of domestic and wild mammals: is it all about the similarity of their ACE2 receptor to the human one?Proc. R. Soc. B.2892021256020212560http://doi.org/10.1098/rspb.2021.2560SectionYou have accessCommentSARS-CoV-2 susceptibility of domestic and wild mammals: is it all about the similarity of their ACE2 receptor to the human one? Giovanni Di Guardo† Giovanni Di Guardo http://orcid.org/0000-0003-4592-1084 Retired Professor of General Pathology and Veterinary Pathophysiology, Veterinary Medical Faculty, University of Teramo, Località Piano d'Accio, 64100 Teramo, Italy [email protected] Contribution: Writing – original draft Google Scholar Find this author on PubMed Search for more papers by this author Giovanni Di Guardo† Giovanni Di Guardo http://orcid.org/0000-0003-4592-1084 Retired Professor of General Pathology and Veterinary Pathophysiology, Veterinary Medical Faculty, University of Teramo, Località Piano d'Accio, 64100 Teramo, Italy [email protected] Contribution: Writing – original draft Google Scholar Find this author on PubMed Search for more papers by this author Published:02 February 2022https://doi.org/10.1098/rspb.2021.2560This article comments on the following:Research ArticlePredicting the zoonotic capacity of mammals to transmit SARS-CoV-2https://doi.org/10.1098/rspb.2021.1651 Ilya R. Fischhoff, Adrian A. Castellanos, João P. G. L. M. Rodrigues, Arvind Varsani and Barbara A. Han volume 288issue 1963Proceedings of the Royal Society B: Biological Sciences17 November 2021I read with great interest the article by Dr Ilya Fischhoff and co-workers, in which an elegant machine learning-based approach was successfully employed to predict the susceptibility of mammals to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), along with the zoonotic hazard posed by them. The three-dimensional model described by the aforementioned colleagues enabled prediction of the zoonotic potential of SARS-CoV-2 for more than 5000 domestic and wild mammals, with the obtained predictions being additionally corroborated by in vivo studies [1]. One of the main reasons underlying this work was represented by the comparatively much fewer mammalian species for which the primary amino acid sequences of their angiotensin-converting enzyme 2 (ACE2) viral host cell receptor are currently available.In this respect, however, adequate emphasis should be also placed upon the fact that a number of discrepancies have been reported between ACE2-expressing cells, on one side, and tissue tropism of SARS-CoV-2, on the other, with very low ACE2 expression levels having been found in respiratory and olfactory epithelial cells, despite their extremely frequent, if not constant, targeting by this viral pathogen [2].Noteworthy, two independent studies published in November 2020 in Science have shown that neuropilin-1 (NRP1), which is consistently expressed by respiratory, olfactory epithelial and endothelial cells, binds (similarly to ACE2) the furin-cleaved S1 fragment of SARS-CoV-2 spike glycoprotein, thereby potentiating infectivity and allowing viral entry into host cells [3,4]. This could also explain why a number of viruses such as human T-cell lymphotropic virus type 1 [5] and Epstein–Barr virus [6] use NRP1 for gaining entrance into susceptible host cells, given its high expression on epithelia facing the external environment and its role in enabling cell, vascular and tissue colonization.Within this framework, it would be interesting to comparatively investigate if and to what extent the different body cells and tissues from humans as well as from a number of SARS-CoV-2-susceptible mammals—either naturally/experimentally or suspectedly, based upon the herein commented machine learning approach [1]—express ACE2 and NRP1 simultaneously. Furthermore, the comparative study of the degree of primary amino acid sequence homology between human and animal ACE2 and NRP1 viral host cell receptors/coreceptors would probably clarify the SARS-CoV-2 susceptibility of the different species under investigation. This, in turn, could also allow a given animal species to act as a potentially valuable model in the comparative pathogenetic study of human SARS-CoV-2 infection, thus elucidating past and future ‘trajectories’ of this threatening viral agent, with special emphasis on its origin and on its ‘spillovers and spillbacks' from animals to mankind (and vice versa).Data accessibilityThis article has no additional data.Authors' contributionsG.D.G.: writing—original draft.Competing interestsI declare I have no competing interests.FundingI received no funding for this study.Footnotes†Present address: Viale Pasteur 77, 00144 EUR Rome, Italy.© 2022 The Author(s)Published by the Royal Society. All rights reserved.

Rapid de novo discovery of peptidomimetic affinity reagents for human angiotensin converting enzyme 2

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Angiotensin converting enzyme 2 (ACE2) has recently been identified as a cardiovascular disease biomarker and the primary receptor utilized by the severe acute respiratory syndrome coronavirus 2. In this study, we report the discovery of high affinity peptidomimetic binders to ACE2 via affinity selection-mass spectrometry (AS-MS). Multiple high affinity ACE2-binding peptides (ABP) were identified by selection from canonical and noncanonical peptidomimetic libraries containing 200 million members (dissociation constant, KD = 19–123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (KD = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (KD = 26 nM). Picomolar to low nanomolar ACE2 concentrations in human serum were detected selectively using ABP N1 in an enzyme-linked immunosorbent assay. The discovery of serum-stable noncanonical peptidomimetics like ABP N1 from a single-pass selection demonstrates the utility of advanced AS-MS for accelerated development of affinity reagents to protein targets.

Interactions of renin-angiotensin system and COVID-19: the importance of daily rhythms in ACE2, ADAM17 and TMPRSS2 expression.

Angiotensin-converting enzyme 2 (ACE2) was identified as a molecule that mediates the cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several membrane molecules of the host cell must cooperate in this process. While ACE2 serves in a membrane receptor-mediating interaction with the surface spike (S) glycoprotein of SARS-CoV-2 located on the virus envelope, enzyme A disintegrin and metalloproteinase 17 (ADAM17) regulates ACE2 availability on the membrane and transmembrane protease serine 2 (TMPRSS2) facilitates virus-cell membrane fusion. Interestingly, ACE2, ADAM17 and TMPRSS2 show a daily rhythm of expression in at least some mammalian tissue. The circadian system can also modulate COVID-19 progression via circadian control of the immune system (direct, as well as melatonin-mediated) and blood coagulation. Virus/ACE2 interaction causes ACE2 internalization into the cell, which is associated with suppressed activity of ACE2. As a major role of ACE2 is to form vasodilatory angiotensin 1-7 from angiotensin II (Ang II), suppressed ACE2 levels in the lung can contribute to secondary COVID-19 complications caused by up-regulated, pro-inflammatory vasoconstrictor Ang II. This is supported by the positive association of hypertension and negative COVID-19 prognosis although this relationship is dependent on numerous comorbidities. Hypertension treatment with inhibitors of renin-angiotensin system does not negatively influence prognosis of COVID-19 patients. It seems that tissue susceptibility to SARS-CoV-2 shows negative correlation to ACE2 expression. However, in lungs of infected patient, a high ACE2 expression is associated with better outcome, compared to low ACE2 expression. Manipulation of soluble ACE2 levels is a promising COVID-19 therapeutic strategy.

Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction

BackgroundCOVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood.MethodsWe report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue.ResultsWe demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2.ConclusionsOur results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

O-142 COVID19-free endometrium: Undetectable viral RNA in endometrial biopsies from positive symptomatic SARS-CoV-2 women

Abstract Study question Does SARS-CoV-2 infect the endometrial tissue in women with coronavirus disease 2019 (COVID-19)? Summary answer Symptomatic women with COVID-19 report no presence, in the short term, of viral RNA from SARS-CoV-2 in the endometrium. What is known already The recent emergence of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has not allowed yet to establish putative relations between this disorder and other existing pathologies. It is the case with fertility problems and the reproductive organs, including a possible endometrial disorder caused by the virus. Thus, an important step is to elucidate the viral presence in different human tissues to improve diagnostics, prevention and/or treatment. The hypothesis of the possible infection of the endometrial tissue comes from the described expression of ACE2 protein in the human endometrium, mainly in stromal cells during the secretory phase. Study design, size, duration 15 endometrial biopsies from symptomatic and hospitalized women with COVID-19 were collected. Endometrial samples were obtained from August to November 2020 at the Hospital Universitari i Politècnic La Fe (Valencia, Spain); the project received the approval of the hospital’s medical ethics committee (registration number: 2020-268-1). The main objective was to study by real-time PCR (RT-PCR) the presence of viral RNA from SARS-CoV-2 as well as the expression of ACE2 receptor on the endometrial tissue. Participants/materials, setting, methods 15 women in the reproductive age (24-46 years) accepted to participate in the study and signed the informed consent. All these patients tested positive for SARS-CoV-2 by RT-PCR of nasopharyngeal swabs (1-17 days before the biopsy collection) and were hospitalized due to health complications (pneumonia) derived from COVID-19. Endometrial biopsies were taken by aspiration and preserved in RNA-later until -80ºC cryopreservation in a biobank; RNA was extracted for RT-PCR for N1, N2, and ACE2 genes. Main results and the role of chance The 15 recruited patients represented the different phases of the menstrual cycle: proliferative (n = 3) and secretory (n = 10); 2 patients had amenorrhea. The viral RNA for SARS-CoV-2, measured by the detection of N1 and N2 gene targets (fragments of N gene, from the viral nucleocapsid) by RT-PCR methodology, was undetectable in all the endometrial biopsies analyzed (n = 15). In all the cases the housekeeping gene RPP30 was used as positive control and to check RNA integrity. To correlate the presence or absence of SARS-CoV-2 with the organ-specific expression of ACE2 (angiotensin-converting enzyme 2), the main postulated entry receptor of SARS-CoV-2, the endometrial RNA was also analyzed by RT-PCR for the ACE2 receptor gene. This gene was only detectable in 10 of the 15 biopsies, and the levels ranged from 28.65 to 36.19 Ct values, revealing a very low expression of ACE2 in the tissue. Moreover, ACE2 results did not report any correlation with the phase of the menstrual cycle. Limitations, reasons for caution These results imply endometrium is safe from SARS-CoV-2 infection, at least in the short term. All the endometrial samples were taken at maximum of 17 days after a positive test by RT-PCR of nasopharyngeal swabs (to note that all were hospitalized during the early stages of the disease). Wider implications of the findings In conclusion, the SARS-CoV-2 RNA is not present in the human endometrial tissue of positive patients. This hypothesis was reinforced by the low ACE2 receptor levels. However, an in-depth genetic analysis comparing to a negative control group could elucidate a systemic affectation of the endometrium, despite the negative RT-PCR results. Trial registration number not applicable

Lower Gene Expression of Angiotensin Converting Enzyme 2 Receptor in Lung Tissues of Smokers with COVID-19 Pneumonia.

Angiotensin-converting enzyme 2 (ACE-2) is the main cell entry receptor for severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2), thus playing a critical role in causing Coronavirus disease 2019 (COVID-19). The role of smoking habit in the susceptibility to infection is still controversial. In this study we correlated lung ACE-2 gene expression with several clinical/pathological data to explore susceptibility to infection. This is a retrospective observational study on 29 consecutive COVID-19 autopsies. SARS-CoV-2 genome and ACE-2 mRNA expression were evaluated by real-time polymerase chain reaction in lung tissue samples and correlated with several data with focus on smoking habit. Smoking was less frequent in high than low ACE-2 expressors (p = 0.014). A Bayesian regression also including age, gender, hypertension, and virus quantity confirmed that smoking was the most probable risk factor associated with low ACE-2 expression in the model. A direct relation was found between viral quantity and ACE-2 expression (p = 0.028). Finally, high ACE-2 expressors more frequently showed a prevalent pattern of vascular injury than low expressors (p = 0.049). In conclusion, ACE-2 levels were decreased in the lung tissue of smokers with severe COVID-19 pneumonia. These results point out complex biological interactions between SARS-CoV-2 and ACE-2 particularly concerning the aspect of smoking habit and need larger prospective case series and translational studies.

Can SARS-CoV-2 infect the eye? An overview of the receptor status in ocular tissue.

Is the new coronavirus SARS-CoV‑2 able to infect ocular tissue and thus poses a risk of infection through the tissue in addition to the risk of contact? This is the question that has occupied ophthalmologists since the beginning of the outbreak. In order to infect a certain type of tissue specific receptors for each virus and sometimes also coreceptors or other proteins must be present. The aim of this review was to summarize and reflect the current state of research with the help of the currently available literature as of 28 May 2020. At the time of the research, angiotensin-converting enzyme 2 (ACE2) was clearly identified as the receptor and transmembrane serine protease 2 (TMPRSS2) as the necessary protease to enable the infection of human cells with SARS-CoV‑2. In the eye both ACE2 and TMPRSS2 are expressed, although sometimes very weakly and with varying degrees in different tissues. It is noteworthy that very different results were obtained with different methods. Several reasons can account for this effect: Firstly, the method of detection or preservation of the tissue, secondly, the possibly different expression of the tested tissue samples and thirdly, a possibly rapid loss of receptor expression post-mortem. Therefore, an infection of the eye seems possible, which has already been reported in various publications. The amount of virus or receptor expression necessary to cause an infection is not known. According to current state of knowledge the eye is not considered to be a high-risk tissue due to the low ACE2 and TMPRSS2 expression. Nevertheless, appropriate protective measures are necessary for both medical personnel and patients. In cases of corneal transplantation an infection of the donor tissue with SARS-CoV‑2 must be excluded.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Can SARS-CoV-2 infect the eye?-An overview of the receptor status in ocular tissue

Kann das neue Coronavirus SARS-CoV‑2 okulares Geweben infizieren und damit neben dem Kontaktrisiko ein Infektionsrisiko durch das Gewebe darstellen, ist die Frage, die die Augenheilkunde seit Beginn der Infektion beschäftigt. Um einen bestimmten Gewebetyp zu infizieren, muss jedes Virus seinen bestimmten Rezeptor und manchmal auch Korezeptoren oder andere Proteine vorfinden. Ziel dieser Übersichtsarbeit war es, mithilfe der aktuellen, zugänglichen Literatur (Stand 28.05.2020) den aktuellen Forschungsstatus zusammenzufassen und wiederzugeben. Zum Zeitpunkt der Recherche waren eindeutig ACE2 als Rezeptor und TMPRSS2 als notwendige Protease identifiziert, um die Infektion einer humanen Zelle mit SARS-CoV‑2 zu ermöglichen. Im Auge werden sowohl ACE2 als auch TMPRSS2 exprimiert, wenn auch teilweise sehr schwach und unterschiedlich schwach in verschiedenen Geweben. Beachtenswert ist, dass mit verschiedenen Methoden sehr unterschiedliche Ergebnisse erzielt wurden. Hierfür kommen mehrere Gründe infrage: zum einen die Methode der Detektion bzw. der Konservierung des Gewebes, zum anderen die möglicherweise unterschiedliche Expression der getesteten Gewebeproben, des Weiteren ein möglicherweise rascher Rezeptorexpressionsverlusts post mortem. Eine Infektion des Auges erscheint daher möglich, worüber auch schon in diversen Publikationen berichtet wurde. Welche Virusmenge bzw. welche Rezeptorexpression notwendig ist, um eine Infektion zu ermöglichen, ist ungeklärt. Aufgrund der geringeren ACE2- und TMPRSS2-Expression ist das Auge – nach aktuellem Wissenstand – aber nicht als Hochrisikogewebe anzusehen. Trotzdem sind entsprechende Schutzmaßnahmen sowohl für das medizinische Personal als auch für die Patienten notwendig. Bei Hornhauttransplantation ist eine Infektion des Spendergewebes mit SARS-Cov‑2 auszuschließen.

A special on epidemic prevention and control: analysis on expression of 2019-nCoV related ACE2 and TMPRSS2 in eye tissues

This article was published ahead of print on the official website of Chinese Journal of Ophthalmolog on Apirl 22,2020. Objective: Angiotensin converting enzyme 2 (ACE2) and Transmembrane serine protease 2 (TMPRSS2) are the key proteins for 2019-nCoV entry into host cells. To evaluate the potential infection risk of 2019-nCoV on ocular surface, we compared ACE2 and TMPRSS2 expression among different eye tissues. Methods: Experimental study. Thirty mice were assigned to male, female, aged, diabetic and non-diabetic groups, with 6 mice in each group. Real-time PCR was performed to quantify ACE2 and TMPRSS2 gene expression in conjunctiva, cornea, lacrimal gland, iris, lens, retina, lung, heart, kidney, and liver from male mice. Immunohistochemistry staining was applied to visualize the distribution of the two proteins in different mice tissues, and in human corneal and conjunctival sections. Published transcriptome datasets were extracted to generate the expression comparasion of ACE2 and TMPRSS2 between human conjunctival and corneal tissues, and results were analyzed using Mann-Whitney U test. Female mice, aged mice, STZ-induced diabetic mice, diabetic group control mice were also subjected to ACE2 expression analysis. Results were analyzed using Student's t-test. Results: The expression of ACE2 and TMPRSS2 genes were the highest in conjunctiva among all the six mice eye tissues explored. The expression of these two genes in conjunctiva were lower than that in kidney and lung. ACE2 and TMPRSS2 shared similar expression pattern with the staining concentrated in corneal epithelium, conjunctival epithelium and lacrimal gland serous cells. The expression levels of ACE2 showed gender difference. Female mice had lower ACE2 in conjunctiva and cornea than male mice, with the expression levels being only 43% (t=3.269, P=0.031) and 63% (t=4.080, P=0.015) of that in the male conjunctiva and cornea, respectively. Diabetic mice expressed more ACE2 in conjunctiva (1.21-fold, P>0.05) and lacrimal gland (1.10-fold, P>0.05) compared with the control group. No significant difference on ACE2 expression was found between the aged and young adult mice. The expression level of human conjunctiva ACE2 and TMPRSS2 were significantly higher than that in the cornea (P=0.007), with 5.74-fold and 12.84-fold higher in the conjunctiva than in the corneal epithelium cells, which resembled the situation in mice. Conclusion: The observation of high-level ACE2 and TMPRSS2 expression in conjunctiva among the 6 eye tissues examined suggests that conjunctiva serves as an infection target tissue of 2019-nCoV. (Chin J Ophthalmol, 2020, 56:438-446).

Serum angiotensin-converting enzyme 2 is an independent risk factor for in-hospital mortality following open surgical repair of ruptured abdominal aortic aneurysm.

Open surgical repair (OSR) is a conventional surgical method used in the repair a ruptured abdominal aortic aneurysm (AAA); however, OSR results in high perioperative mortality rates. The level of serum angiotensin-converting enzyme 2 (ACE2) has been reported to be an independent risk factor for postoperative in-hospital mortality following major cardiopulmonary surgery. In the present study, the association of serum ACE2 levels with postoperative in-hospital mortality was investigated in patients undergoing OSR for ruptured AAA. The study enrolled 84 consecutive patients underwent OSR for ruptured AAA and were subsequently treated in the intensive care unit. Patients who succumbed postoperatively during hospitalization were defined as non-survivors. Serum ACE2 levels were measured in all patients prior to and following the surgery using ELISA kits. The results indicated that non-survivors showed significantly lower mean preoperative and postoperative serum ACE2 levels when compared with those in survivors. Multivariate logistic regression analysis also showed that, subsequent to adjusting for potential confounders, the serum ACE2 level on preoperative day 1 showed a significant negative association with the postoperative in-hospital mortality. This was confirmed by multivariate hazard ratio analysis, which showed that, subsequent to adjusting for the various potential confounders, the risk of postoperative in-hospital mortality remained significantly higher in the two lowest serum ACE2 level quartiles compared with that in the highest quartile on preoperative day 1. In conclusion, the present study provided the first evidence supporting that the serum ACE2 level is an independent risk factor for the in-hospital mortality following OSR for ruptured AAA. Furthermore, low serum ACE2 levels on preoperative day 1 were found to be associated with increased postoperative in-hospital mortality. Therefore, the serum ACE2 level on preoperative day 1 may be a potential biomarker or prognostic factor for in-hospital mortality following OSR for ruptured AAA.

Effect of Astragali Radix in improving early renal damage in metabolic syndrome rats through ACE2/Mas pathway

To study the expression of angiotensin converting enzyme 2 (ACE2) and angiotensin (Ang) 1-7 specific receptor Mas protain in renal blood vessels of metabolic syndrome ( MS) rats and its anti-oxidative effect. A total of 80 male SD rats were divided into four groups: the normal control group (NC, the same volume of normal saline), the MS group (high fat diet), the MS + Astragali Radix group (MS + HQ, 6 g x kg(-1) x d(-1) in gavage) and the MS + Valsartan group (MS + XST, 30 mg x kg(-1) x d(-1) in gavage). After four weeks of intervention, their general indexes, biochemical indexes and blood pressure were measured; plasma and renal tissue Ang II, malondialdehyde (MDA) and superoxide demutase (SOD) levels were measured with radioimmunoassay. The protein expressions of Mas receptor, AT1R, ACE and ACE2 were detected by western blot analysis. According to the result, compared with the NC group, the MS group and the MS + HQ group showed significant increases in systolic and diastolic pressures, body weight, fasting glucose, fasting insulin, triglycerides, free fatty acid and Ang II level of MS rats (P < 0.05). The MS + XST group showed notable decreases in systolic and diastolic pressures than that of the MS group. The MS group showed significant increases in the SOD activity and NO level and decrease in the MDA level after being intervened with Astragali Radix. ACE and AT1R protein expressions in renal tissues of the MS group were higher than that in the NC group, but with lower ACE2 and -Mas receptor expressions (all P < 0.05). Compared with the MS group, the MS + HQ group showed significant increase in Mas receptor expression in renal tissues, whereas the MS + XST group showed notable decrease in AT1R (all P < 0.05). In conclusion, Astragali Radix can increase the Mas receptor expressions in renal tissues, decrease ACE expression and change local Ang II, MDA, NO and SOD in kidneys, so as to protect early damages in renal tissues.

Association between serum angiotensin-converting enzyme 2 levels and postoperative myocardial infarction following coronary artery bypass grafting.

Myocardial infarction (MI) is a predominant and severe complication in patients that undergo aortocoronary bypass surgery. Angiotensin-converting enzyme 2 (ACE2) activation is reportedly a protective mechanism in MI; therefore, in the present study, the association between serum ACE2 levels and postoperative MI following coronary artery bypass grafting (CABG) was investigated. Preoperative and postoperative serum ACE2 levels in 136 subjects undergoing CABG were observed and the serum ACE2 levels, 1 h post surgery, were divided into quartile categories. Following adjustment for age, gender, body mass index, hypertension, previous MI, current smoking status, hyperlipidemia, diabetes mellitus, Gensini score, aortic clamp time, number of grafts and pre-CABG medications; the risk of developing postoperative MI following CABG was observed to be significantly higher in the lowest serum ACE2 level quartile than when compared with the highest quartile (hazard ratio, 2.94; 95% confidence interval, 1.85–4.16; P=0.009). The subjects that exhibited a serum ACE2 level ≤1.06 ng/ml showed significantly higher rates of postoperative MI, arrhythmia and reduced cardiac output in addition to increased instances of in-hospital mortality post CABG, compared with those exhibiting a serum ACE2 level >1.06 ng/ml. A significant negative correlation was observed between serum ACE2 and serum cardiac troponin I levels, however, no significant association was identified between the serum ACE2 level quartiles and the ACE2 gene polymorphisms. The present study indicated that a low serum ACE2 level, 1 h post CABG was independently associated with an increased risk of postoperative MI. Thus, the serum ACE2 level may be a potential novel prognostic factor for postoperative MI following CABG.