ACE2 dysregulation leads to (Micro)vascular dysfunction

Uncertainties:

It is difficult to decipher or generalise which if any particular cell type in the (micro)vasculature represent a key factor in the putative spike-protein mediated (micro)vasculature dysfunction, particularly because the expression of ACE2 varies significantly in the cell systems used, (see Table below). Closer analysis is needed regarding the reagents/antibodies used in these studies and comparative characterisation of the experimental systems, as it appears that ACE2 expression may depend on the dimensionality (2D/3D, co-cultures) but also the dynamics (medium flow or stasis) of the test system.

Notably, ACE2 expression in HBMEC & HUVEC perfusion culture is stimulated by flow (HBMEC < HUVEC) (qRT-PCR); also it is increased by flow intensity and vessel shape in the MCA 3D model of stenosis (immunostaining cells) [10.1161/STROKEAHA.120.032764].

Single­cell RNA­seq analysis of quiescent mouse endothelial cells isolated from different tissues by flow cytometry without the cell culture step, does not identify ace2 enrichment in the ECs from any of the mouse tissues while ace is one of the top-50 marker genes in ECs from arteries in the brain, testes and small intestine. scRNAseq of primary cell cultures from EC from human lung identified similar cell subpopulations and marker genes in mice and human, but interestingly, scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture . (Schupp 2021 - 10.1161/CIRCULATIONAHA.120.052318). 10.1038/s41419-020-03252-9

There are also some apparently conflicting results. For example although [10.1016/j.nbd.2020.105131] shows S-mediated increase of many markers of MV dysfunction, without evidence for any change in ACE2 levels as measured by Western blot of cell culture lysates. This suggests that although the effect is spike protein mediated it may not necessarily be ACE2 mediated and that other receptors for S protein may be involved in MV.

Additional uncertainty in this KER evidence analysis is that most of the studies that use various cultured endothelial cell models, do not specifically measure/identfy ACE2 dysregulation aspects in the same time and binding of S-protein to ACE2 is often assumed. In fact, infection of endothelial cells by SARS-CoV2 is still debated. [10.1021/acscentsci.0c01537] show that ACE2 is expressed at very low level in HUVEC-TERT, immortalised umbilical endothelial cell culture. In contrast two C-type lectin receptors, CD209L (also known as L-SIGN) and CD209 (also known as DC-SIGN), are highly expressed. CD209L and CD209 bind spike RBD domain and mediate SARS-CoV-2 entry into HUVEC-TERT (demonstrated by interference with CD209L). Thus, while endothelial cells may be permissive to SARS-CoV-2 entry/replication, the involvement of ACE2 in these undifferentiated cells in monolayer culture, is uncertain. The same study demonstrates that, when expressed, ACE2 mediates pseudovirus entry more efficiently and in vitro binds S-protein RBD with higher affinity than CD209L and CD209 [10.1021/acscentsci.0c01537].

Sumilarly, [10.3390/ijms231810436 ] that demonstrates effect of spike treatment on a number of markers of endothelial dysfunction in HLMEC alone, or in co-culture with neutrophils also does not characterise the test system for ACE2 status or time concordance with any potential ACE2 dysregulation in either of the cell types in the co-culture. Such characterisation would be helpful in understanding the primary target of the viral or abortive spike-containing particles, as well as the intercellular paracrine interactions driving adversity at tissue level.

Therefore, although in some studies the dependence of aspects of (micro)vacular dysfunction on ACE2 is demonstrated [10.1128/mBio.03185-20], the type of ACE2 dysregulation/effect (upregulation/downregulation) that could be related to the observed aspect of (micro)vascular dysregulation is not clear.

Other plausible mechanisms:

Viral replication and inflammation-related oxidative stress are also explored as significant factors in coagulation and DIC following SARS-CoV-2 infection/stress and may be more direct drivers of the (micro)vascular dysfunction compared to ACE2 dysregulation. Consistent with this, [10.1128/mBio.03185-20] find that primary cultures of scattered endothelial cells derived from microvascular tissue from different organs, express very low levels of ACE2 (if any) and cannot be infected by SARS-CoV2. However, when transduced to overexpress hACE2 they could be infected and the interaction with the replicating virus led to stimulation of mRNA synthesis for coagulation and pro-inflammatory mediators: tissue factor (TF), thrombomodulin (TM), tumour necrosis factor alpha (TNFa), interleukin 6 (IL-6), IL-1b, and E-selectin (qRT-PCR assay following 6h interval time course from 0-24hpi showing significant stimulation for TF and TNFa, as early as 6hpi and for the rest of the mediators  at latter time points). E selectin was also stimulated early with pick at 12hpi. In human heart tissue of COVID-19 infected patients ACE2 immuno-histological staining showed increasing expression towards the small vessels:  capillaries >> arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor [10.1016/j.ebiom.2020.103182]. In the same study, staining of harts from influenza infected patients showed much lower expression of ACE2 even in capillaries, and in small vessels, practically the same in coronary artery.

ACE2 is known to be upregulated by other inflammation, hypoxia and synthetic dsRNA, poly(I:C) (Ziegler, 2020, Smith, 2020; Salka 2021, Zhuang 2020, indicating that inflammation, low oxygen levels and viral replication ma precede or in parallel potentiate ACE2 mediated MV dysfunction by increasing the ACE2 levels in the MV cells which then maintain viral infection cycle but also govern MV dysfunction via spike-protein binding mechanism in the surviving cells.

ACE2 independent (though spike-mediated) mechanisms leading to microvasuclar disfunction (TEER disruption), have also been suggested [10.1038/s41467-022-34910-5]. In this study human pulmonary microvascular endothelial cells (HPMEC) were used as a representative endothelial cell relevant to COVID-19 pathology that do not endogenously express ACE2 and are not permissive to SARS-CoV-2 infection (as demonstrated by the authors in their study). That allowed them to separate viral replication from S-mediated dysfunction and compare them to transfected ACE2 overexpressing line of HPMEC and also HPMEC in which ace2 gene was disrupted by CRISPR-Cas9. The alternate mechanism appears to involve perturbation of surface levels of key endothelial glycocalyx layer (EGL) components, including sialic acid (SIA), heparan sulfate (HS), hyaluronic acid (HA), and chondroitin sulfate (CS) (immunofluorescence staining) and EGL-degrading enzymes such as hyaluronidase and neuraminidase. Integrins and TGFbeta sugnaling also appar essencial for tis mechanism. Intradermal and intranasal administration of recombinant spike protein also appeared to disrupt endothelial barrier function in vivo in leak models in WT C57BL/6 mice which do not express human ACE2 and are not permissive to infection by most SARS-CoV-2 variants. Binding of spike protein to alternative (co)receptors including integrins and heparin sulfate has been implicated as well [10.3390/v13040645; 10.1016/j.cell.2020.09.033]. It should be noted that in some test systems ACE2 mRNA was up-regulated as a result of spike treatment in vitro (eg. 80% confluent HUVEC cell [10.1016/j.jbc.2022.101695s]) even though the initial spike interaction was likely to an alternative receptors leading to activation of the quiescent endothelium. Resulting ACE2 up-regulation and/or activation of quiescent endothelium could initiate additional and parallel ACE2 dependent perturbations leading to vascular dysfunction [10.1021/acscentsci.0c01537].Contribution of the effects of the activation of the adaptive immune response to spike and other viral components or endogenous host prpteins relevant to (micro)vascular dysfunction  is possible but not explored sufficiently by this evidence collection strategy, although it has been indicated [10.3390/pathophysiology29020021; 10.3389/fimmu.2022.906063]

Gaps:

Overall, the studies with more differentiated 2D/3D cultures and recombinant S protein as a stressor, strongly suggest that spike-mediated ACE2 dysregulation could also, in part lead to aspects of (micro)vascular dysfunction. The initial target cells still remain to be elucidated. This may vary in different organs. Notably, a very recent cell atlas of adult human myocardium made by single nuclear transcriptome analysis found that the highest level of ACE2 mRNA in the heart was found in the pericytes a type of perivascular mural cells [10.1093/cvr/cvaa078]. Therefore, more complex and better characterised in vitro, optimally microfluidic dynamic systems, are needed to better understand molecular and cellular aspects of SARS-CoV-2, spike protein-mediated or more general toxicity (including general inflammation) -mediated (micro)vascular dysfunction. One potentially very useful system has already been described. [10.1016/j.cell.2020.04.004] developed human capillary organoids from induced pluripotent stem cells (iPSCs). They resembled human capillaries with a lumen, CD31+ endothelial lining, PDGFR+ pericyte coverage, as well as formation of a basal membrane. Consistent with [10.1021/acscentsci.0c01537} these organoids were permissive to SARS-CoV-2 infection and replication that could be significantly but only partially blocked by soluble human recombinant ACE2 in a dose dependent manner. However, the target cell and the ACE2 status of the different cells in the organoids are not reported and authors discuss the possibility that there might be other co-receptors/auxiliary proteins or even other mechanisms by which viruses can enter cell.

However, in another iPCS derived organoid system with characterised ACE2 expression, authors show that perycites express significantly higher levels of ACE2 also show preferential uptake of viral particles. In addition productive infection of perycites has been found in primary cultures of cardiac perycites in vitro and in hart tissue from COVID-19 patents [10.1016/j.jacbts.2022.09.001].

In addition, the role of microparticles (MPs) and microvesicles (MV) in mediating intercellular communication or its perturbation need to be examined in more detail.  [10.1152/ajpheart.00409.2022] show that plasma MV from platelet and white blood cell origin contain high amounts of ACE2 that binds the spike protein of SARS-CoV-2. MVs also contain other adhesion moolecules (eg. Tissue Factor- CD152 ) mediating EC adhesion and alternative entry pathway by endocytosis for viral or abortive spike-containing particles with affinity for ACE2, ultimately leading to activation and dysfunction of the EC and micro(vsculature). It is interesting that [10.1152/ajpheart.00409.2022] found difference in the ACE2 containing MVs between normal and diabetic patients which have increased incidence of severe and fulminant COVID-19.

Extracellular vesicles (EVs) including microvesicles (MVs) and exosomes can also mediate cell–cell signalling in a paracrine and/or even endocrine mechanism via their protein and RNA cargo (10.1093/nar/gky985; 10.1016/j.cell.2016.01.043). Non coding RNAs such as microRNAs (miRNA or miR) have been implicated in various biological processes including angiogenesis and vascular dysfunction (10.1530/VB-19-0009). A number of miRs (eg miR122, miR143, miR155, miR181a and miR1246, miR421) have also been implicated in regulation of ACE2 in different tissues and plasma (10.1016/j.ncrna.2020.09.001; 10.1016/j.yjmcc.2020.08.017; 10.1016/j.omtn.2022.06.006; 10.1042/CS20130420). Therefore interference with the expression of relevant miRs by exogenous stressors, including RNA viruses, LPS or dysregulated endogenous signalling molecules (eg. vasoactive peptides) can potentially lead to ACE2 dysregulation and (micro)vascular dysfunction and need to be examined in greater detail.

Finally, the distribution and role in (micro)vascular dysfunction, of the receptors for the substrates and products of ACE2 need to be investigated in these systems to better elucidate a potential link with up- or down-regulation of ACE2 function locally and/or systemically. Test systems that could monitor activation of the complement system proteins would also be valuable.