Binding to ACE2 leads to ACE2 dysregulation

Evidence Collection Strategy

The literature search is not comprehensive or systematic and needs continuous improvement.

In all of the studies reviewed here, ACE2 dysregulation following exposure to recombinant S-protein and replicating virus is considered as equivalent for the prpose of evaluationg ACE2 dysregulation. However, it should be noted that the literature screening step showed that inflammation and exposure to other stressors (nanomaterials and other viruses, like influenza) were also associated with ACE2 dysregulation, indicating that aspects of the stressor multiplication and/or persistency, additional to the S-protein binding, may be responsible for the observed ACE2 dyregulation. These references were tagged but not further analysed and not included in this KER that is focused on S-protein binding. They can be informative if this KER is eventually modified and potentially split into two: (i) direct/adjacent KER (studies examining ACE2 dysregulation after direct and short term exposure to S-protein binding or non-replicating pseudoviruses) and (ii) indirect/nonadjacent KER (studies with replication virus and persistent infection).

Focus on GI tract relevant evidence (published in https://doi.org/10.3390/jcm11185400)

Both ACE2 down and up regulation are observed in the gut as in the other test systems (see gut unreated evidence table). The apparent inconsistences regarding the direction and magnitude of ACE2 dysregulation in the different studies may reflect the dynamic, temporal components of the dysregulation driven not only by the interaction of ACE2 with the surface viral components i.e. S-protein binding, but also by the interaction of the replicating viral components with the innate immunity response elements, particularly in the test systems with replicating viruses used with the GI-tract-derived organoids.

ACE2 mRNA down-regulation in SARS-COV2 treated GI-derived organoids is reported in one study using scRANseq analysis (Triana et al., (2021). The down-regulation was specific to enterocytes actively replication the virus. Another study Nataf & Pays (2021) also reports profound but transient ACE mRNA down regulation.

Evidence for SARS-COV2 mediated up-regulation of ACE2 mRNA in GI-tract derived organoids is also available (Lamers et al., 2020; Lee at all (2020); Heuberger et al., 2021) and consistent with the similar studies in many other tissue/organ systems (see evidence table for other organs above). This is also consistent with the finding that ace2 is an Interferon Stimulated Gene (IFG) in airway epithelial cells (Ziegler, 2020) and also in colon enterocytes (Heuberger et al., 2021). In fact, all there studies also demonstrate a time concordance of ACE2 mRNA up-regulation with stimulation of ISG response in the infected organoids (Lamers et al., 2020; Lee at all (2020); Heuberger et al., 2021). Interestingly, even the scRNAseq study by Triana et al., 2021 which zoomed on specific cells within the organoid found that SARS-COV2 treatment induced distinct proinflammatory and ISG expression profiles in infected and bystander cells in the organoid. Namely, expression of interferon-stimulated genes was pronounced in bystander cells, while the infected cells showed strong NFkB/TNF-mediated pro-inflammatory response but a limited production of ISGs, suggesting that while SARS-CoV-2 may activate ISG by paracrine signaling, it may suppress the autocrine action of interferon i.e inducton of IGS including ACE2 in infected cells. This would be consistent with down-regulation of ACE2 in the infected cells observed in this study. In addition, this may explain why in some studies down-regulation of ACE2 mRNA can be observed under certain conditions (e.g. bulk mRNA measurements) and at some (earlier) time points of replication. Furthermore, the relationship of an observed increase of ACE2 mRNA to dysregulation at protein and enzymatic level remains to be elucidated. Indeed, most recently Harnik et al. 2021 (10.1038/s42255-021-00504-6) examined the spatial discordances between mRNAs and proteins in the intestinal epithelium and their significance for interpretation of transcriptomic data. Such apparent discordances have also been reported in the heart and lung tissue in mice and human (KE1854).

Identification of alternative forms of ACE2 mRNA and protein, an N-terminus truncated dACE2, which appears to have distinct transcriptional regulation profile compared to flACE2 (Onabajo et al, 2020, Janakowski et al (2021) - 0.1016/ j.isci.2021.102928) may also account for some of the observed inconsistences. However, a detailed analysis of experimental conditions in past, and careful design of probes and primers in future studies is necessary. Interestingly, concomitant down and up regulation of 97kD and 80kD anti-ACE2 polyclonal Ab-reacting proteins has been detected in differentiating human colon adenocarcinoma cell line HT29 (Bártová et al. (2020) 10.18632/aging.202221). Considering only one form of ACE2 relevant (97KD the only form detected in HEK293 and A549), the authors conclude that ACE2 is down-regulated in mature differntiatiatiated enterocytes compared to undifferentiated ones. This is in contrast to all mRNA and even cytoimmunostaining studies described above which demonstrate that a highest level of ACE2, both mRNA and protein is detected in the mature enterocytes and at the brush borders of the intestine and 3D organoids (eg. Lamers et all., 2020, Lee et al., 2020; Hauberge et al., 2021; Triana et al., 2021). Notably, the initial analysis by Onabajo et al, 2020 found that dACE2 mRNA is enriched in in squamous tumors of the respiratory, gastrointestinal and urogenital tracts.

The uncertainties and uncosnstances disused above illustrate clearly the need for careful characterization of the test systems to facilitate robust interpretation of the results.

In addition, we note that to date (to our knowledge), the majority of studies related to SARS-COV2-mediated ACE2 dysregulation, focus on ACE2 mRNA expression while structural/protein and functional studies are lacking, particularly in the gastro intestinal system. The novel gut-derived organoid systems can help address this gap by monitoring level and cell distribution of ACE2 protein as well as its function as B⁰AT1chaperon, via monitoring the membrane expression and/or the transporter function of B⁰AT1 itself. In addition, treatment with S-protein and/or non-replicating SARS-COV2 pseudo-viruses [Minghai & Zhang (2021) 10.7150/ijbs.59184], may help address better any potential direct effect of S-binding on ACE2 dysregulation. Finally, a development of more complex organoid systems that would also include microbiota or elements of the immune and/or vascular system are needed to better examine ACE2 dysregulation by SARS-COV2 but also the effects of such dysreulation at higher organizational level and in conjunction with the other elements of the RAS system.

Finally, evidence on up- or down-regulation of ACE2 in the GI tract of SARS-COV infected patients is not available to our knowledge. Examining the GI specific transcriptomic, proteomic and biomarker databases of COVID19 patents may help address some of these uncertainties.