ACE2 dysregulation leads to (Micro)vascular dysfunction

Microvascular dysfunction manifesting as vasculitis of the small blood vessels in different organs has been identified in COVID19 patients in a number of studies (e.g. 10.1016/j.ebiom.2020.10318 10.1056/NEJMoa2015432; 10.1016/j.anndiagpath.2020.151645).

Angiotensin Converting Enzyme 2 (ACE2) is recognised as the main receptor for the COVID19 causing SARS-CoV2 virus. In particular the viral spike (S) protein binds to ACE2 mediating viral entry (10.1016/j.cell.2020.02.052; 10.1128/JVI.00127-20) although other receptors or less specific (recognizing glycans on the virion surface of many viruses rather than specific viral protein) to mediators of viral entry have also been suggested (10.1002/rmv.2207; 10.3390/v14112535).

ACE2 is also known to be a cell receptor for SARS-CoV (10.1038/nature02145) although its spike protein shows ~10- to 20-fold lower binding affinity for ACE2 (10.1126/science.abb2507). Evidence from SARS patients is nowhere near that for COVID19, but some evidence of multiorgan injury, including to systemic vacuities has been reported (10.1002/path.1440).

ACE2, is a carboxypeptidase with broad substrate specificity that deactivates or generates active biological peptides such as Angiotensin II (Ang II), Angiotensin 1-7 (Ang1-7), des-Arg9-bradykinin (DABK) but also others (10.1042/BJ20040634; 10.1074/jbc.M200581200; 10.1002/path.2162). These bioactive peptides are involved in regulating vascular tone, inflammation, cell activation/proliferation and tissue remodelling via their receptors Angiotensin II receptor 1 (AT1R), Angiotensin II receptor 2 (AT2R), Mas-Related G Protein-Coupled Receptor (Mas) (10.1152/ajpheart.00723.2018; 10.3389/fnins.2020.586314).

Therefore it is plausible to explore the evidence examining spike protein-mediated dysregulation of ACE2 on aspects of (micro)vascular dysfunction.

The protein profile of blood plasma represents a good indicator of (micro)vascular dysfunction at tissue level.

Increase of soluble ACE2 protein (sACE2) in plasma of SARS-CoV-2 positive patients was correlated with increase of the level of von Willebrand factor (vWF) and coagulation factor VIII comparing COVID19 to healthy controls by ELISA assay (10.1002/jmv.27144). Another study using proteomic/OLING approach and comparing viremic and aviremic COVD19 patients (10.1172/JCI148635) found increase of a larger panel of angiogenesis and endothelial damage related markers, including the vWF, and also coagulation/fibrinolysis related factors: tissue factor III and SERPINE1/Plasminogen activator inhibitor-1 (PAI-1), as well as complement related pathway proteins (PTX3, C1QA). In this study PROC was decreased in the viremic versus aviremic SARS-CoV-2 patients. Importantly, the proteomic study (10.1172/JCI148635) find that the dysregulation of specific endovascular damage markers (ANGPT2, ESM1), coagulation pathway markers (F3/tissue factor, vWF and PROC) and also the complement related pathway proteins (PTX3, C1QA) was specifically associated with SARS-CoV-2 viremia and not with generalised inflammation comparing SARS-CoV2 negative participants with respiratory dysfunction and an inflammatory profile (CRP >10 mg/dL). However, it is not clear whether ACE2 level is also significantly increased specifically in the SARS-CoV2 viremic patients compared to non COVID-19 controls (the supplementary data is not detailed -see sup fig 7). ACE2 mRNA is known to be upregulated by other infections, inflammation, hypoxia and synthetic dsRNA, poly(I:C) (Ziegler, 2020, Smith, 2020; Salka 2021, Zhuang 2020).

Unlike the proteomic study, the (10.1002/jmv.27144) did not find evidence for dysregulation of SERPINE1/PAI-1 in COVID-19 vs healthy controls. In fact, SERPINE1/PAI-1 protein levels were unaffected as were the level of ACE in plasma in COVID-19 patients compared to healthy controls.

Macroscopic indicators of dysfunction of the vasculature have also been reported in hACE2 transgenic mice infected with SARS-CoV (PMID 17974127) and SARS-CoV2 (10.1101/2020.08.19.251249 – bioRxiv; 10.1172/jci.insight.142032).  (PMID 17974127) examined different organs and found: in LUNG: oedema, focal haemorrhage, degenerated blood vessels surrounded by monocytic and lymphocytic infiltrates; KIDNY: glomerular capillaries dilated markedly and engorged with lymphocytic infiltrate present in the renal interstitial; BRAIN - vasculitis, and haemorrhage. With SARS-CoV2 multiple intra-alveolar and intra-arteriolar microthrombi adherent to the endovasculature were observed that stained positive for the platelet marker CD61 (glycoprotein IIIa), as well as intra-bronchiolar blood clots and interstitial hemorrhagic patches (10.1101/2020.08.19.251249). Another study with SARS-CoV2 (10.1172/jci.insight.142032) report that vasculitis was the most prominent finding in the lungs of infected transgenic mice.

Wild type mice are resistant to SARS-CoV-2 infection as mouse ACE2 is not binding efficiently to the viral S protein, supporting the role of S-binding to ACE2 (direct or by the virtue of amplification of the stressor) in mediating the observed vascular damage and dysfunction. The findings are also consistent with autopsy findings in COVID-19 patients that showed widespread microthrombi and microangiopathy (/10.1016/S2352-3026(20)30216-7, 10.1007/s00428-020-02886-6, 10.1038/s41379-021-00793-y 10.1161/CIRCRESAHA.120.317447 and references therein).

There is also supporting evidence in lung tissue from autopsies of SARS-CoV2 infected humans compared to controls that died from unrelated causes including car accidents, that indicate increase of ACE2 protein in the lings (by immunostaining) correlated with increase of clotting factor VIII (FVIII) by immunostaining (10.3390/diagnostics10080575; 10.1002/path.5549). Another study identifies gross changes to the vascular component in both the interstitial capillaries and the mid-size vessels, with capillary fibrin micro-thrombi, as well as organized thrombi even in medium-sized arteries, in most cases not related to sources of embolism.

Similarly, increased ACE2 expression associated with multiple fibrin trombi in medium size pulmonary vessels without signs of inflammation is reported in COVID-19 autopsies compared to control [10.1002/path.5549].  Based on CD34 staining authors observe preserved but desquamation of the endothelial cells from the vascular walls, indicating endothelial damage and perturbed intercellular communication 10.1002/path.5549]. These case studies are based on very small number of patients and are not quantitative, but appear to converge on the finding that ACE2 immunohystochemical staining is increased in the lungs of patients with terminal COVID-19, particularly in the regions of small vasculature and alveolar damage. In lung autopsies from COVID-19 patients [10.1093/cvr/cvac097] S-protein collocalised (immunostaining) with the pirocyte marker Neural-glial antigen 2(NG2)/ Chondroitin sulfate proteoglycan 4 (CSP4). Collocalisaton staining was more pronounced in patients with thrombotic complications. Endothelial marker CD144/Cadhirin was also decreased in COVID-19 autopsies compared to age matched controls, and pronounced in patients with thrombotic complications. At the same time WWF factor was increased. Authors note that NG2+ Spike+ cells (actiated pirocytes phenotype) were not lining the endothelium, in particular, cells with high spike staining was associated with ICAM-1 staining suggesting pericyte activation and detachment in terminal cases of COVID-19. Together with the decrease of CD144 these finding strongly suggest dysregulation of the pirocyte-endothelial communication. ACE2 was not evaluated in vovo, but the same study included analisis in human iPCS derived 3D vascular organoids (see section on in vitro data).

10.1016/j.anndiagpath.2020.151645 examined biopsies form different tissues of patients with severe COVID19 (24 out of 26 with fatal outcome). Different degrees of (micro)vascular damage was observed in the different tissues, also without signs of observable inflammation. Small (micro) and bigger thrombi were observed in all tissues in the different size vasculature examined. Endothelial and sub-endothelial microvascular deposition of C3d, C4d and/or C5b-9 was detected by Immunohystochemical staining in the lung, liver, heart and brain autopsies. However, viral RNA was not detected (by in situ hybridisation) in endothelial cells but in cells with the morphology of macrophages. Furthermore, endothelial cells of the microvasculature showed no, or low (~10% of cells), immuno staining for ACE2 in most tissues, except the subcutaneous fat and in the brain where it collocalised with spike protein. Authors suggest spike-containing abortive pseudovirions can be released from infected cells and they can dock on ACE2 positive endothelial cells most prevalent in the skin/subcutaneous fat and brain, leading to activation of the complement pathway/coagulation cascade resulting in thrombogenic vascular dysfunction. In an earlier study focused on skin biopsies of COVID19 patients, [10.1016/j.humpath.2020.10.002] found evidence of endothelial cell injury also largely unaccompanied by significant infiltration of inflammatory cells, where the affected microvascularure in the skin showed significant immunohistochemical staining for C5b-9, C3d, and C4d. C5b-9 colocalized with ACE2 and spike protein.

A number of in vitro studies are available that use particularly recombinant spike protein (or its S1 or S2 subdomains) to explore the mechanistic drivers of the relationship between ACE2 dysregulation elicited by the binding of the spike protein and any aspect of the observed (micro)vascular disfunction at tissue/organ level. Mainly, different endothelial cell cultures (primary and immortalised) are used, but also 2D and 3D co-cultures.

Different types of dysregulation are explored and reported depending on the focus on the researchers, methods used for detection of specific markers of dysregulation and cell systems available:

•Loss of the barrier integrity based on: Electric Cell-Substrate Impedance sensing (ECIS), trans-endothelial electrical resistance (TEER), FITC-dextran or or FITC-BSA permeability assays. [10.1016/j.nbd.2020.105131; 10.1152/ajplung.00223.2021; 10.3389/fcvm.2021.687783;  10.4081/monaldi.2022.2213].

•Cell death and apoptosis preferentially of pericytes and to a lesser extent of ECs [10.1093/cvr/cvac097] – 72hpt

•Surface expression of adhesion proteins (↑ICAM-1, ↑VCAM-1) based on FACS-SCAN assay [10.1016/j.nbd.2020.105131 – significant outcome detected as early as 4hpt; (↓JAM-A, ↓Connexin-43, ↓PECAM based on Western blotting, time not specified) - 10.3389/fcvm.2021.687783]. No effect on mRNA for VCAM and E-selection  have also been  reported monitored 24hpt [10.4081/monaldi.2022.2213].

• Up-regulation of pro-inflammatory cytokines (IL6, IL1, CCL5, CXCL10) (qRT-PCR) [10.1016/j.nbd.2020.105131 significant outcome detected as early as 4hpt]. IL8, CXCL10, TGFbeta, MCP-1/CCL2 (qRTPCR) [10.4081/monaldi.2022.2213 measured only at 24hpt, CCL5 and IL6 not significantly changed in this study].

• Up-regulation of matrix metalloproteinases (e.g.MMP2, MMP3, MMP9, MMP1) and their regulators (e.g. MMP inhibitor TIMP1 (qRT-PCR) [10.1016/j.nbd.2020.105131 significant outcome detected as early as 4hpt];

• Up-regulation of anti-inflammatory cytokines (e.g. IL4, IL11, IL10RB) and down-regulation of inflammasome (NLRP3) and apoptotic pathways (CCL17) (Nanostring nCounter Assay and qRT-PCR for selected mRNAs to confirm) [10.1161/STROKEAHA.120.032764 – study monitored outcomes at 24hpt]

• Production of coagulation and/or fibrinolytic factors such as the plasminogen activator inhibitor 1(PAI-1), the inhibitor of the pro-fibrinolytic factors plasminogen activator (tPA) and urokinase (uPA). [10.1165/rcmb.2020-0544OC – study monitored outcome 24hpt at protein level by western blotting of cell lysates with anti PAI-1 antibody). Time course study in a co-culture test system showed increase of synthesis and secretion of coagulation factors: tissue factor (TF), factor (F)-V, thrombin, and fibrinogen with decrease in tissue factor pathway inhibitor (TFPI). mRNA, but also protein synthesis and secretion was measured) [10.3390/ijms231810436 – effect seen as early as 6hpt with two variants of S-protein].

The above findings have been obtained with different cell systems:

10.1016/j.nbd.2020.105131 - 2D&3D primary culture models with human brain microvascular endothelial cells (hBMVECs/D2) and immortalised hCMEC (telomerase-immortalized human brain endothelial cell line ) 3D perfused cultures. Assays done 4hpt.

10.1093/cvr/cvac097 – vascular organoids derived from human-induced pluripotent stem cells consisting of endothelial cells and pirocytes

10.1152/ajplung.00223.2021; 10.3390/ijms231810436 - human lung microvascular endothelial cells (HLMEC), alone or in co culture with neutrophils

10.1161/STROKEAHA.120.032764 -human brain microvascular endothelial cells (HBMECs) & human umbilical vascular endothelial cells HUVECs. This study also identified some differences between the activation profiles of HBMECs and HUVECs. Characteristically different in HBMEC was the observed ↑ in C3, CCL8, IL15, IL9 that was not observed in HUVECs.

10.4081/monaldi.2022.2213 - human umbilical vascular endothelial cells HUVEC

10.1165/rcmb.2020-0544OC - Human pulmonary microvascular endothelial cell (HPMECs)

In vitro 3D vascular organoid system [10.1093/cvr/cvac097] also supported studies with replicating virus. Organoids infected with SARS-CoV2 (0.5 MOI for 48h), spike protein immunofluoresecence (imaging) co-localised 2x more with pyrocyte markers (CD140b(PFGFR)+/NG2+ cells)  than with UAE1-positive ECs. Pirocyte apploptosis was also significantly higher (TUNEL assay). Presumably, UAE1 rather than CD144 was chosen as a “housekeeping” EC marker as ACE2 (spike receptor) co-localised 2x less with EC (CD144+) cells than with perycites (CD140b(PFGFR)+/NG2+ cells) in uninfected organoids. Taken together, the results strongly suggest that that SARS-COV2 is preferentially taken up by pirocytes via the ACE2.  Viral entry was associated with increased cell apoptosis (TUNEL assay) which was significantly higher in pericytes (markers) compared to ECs (markers).

Given the preferencial infection and death of pericytes, the relatively low EC death (TUNEL) and the observed CD144 decrease (immunostaining) suggest that ACE2-dependent virus entry into pyrocites could lead to cell death and further to vascular dysfunction due to loss of functional pirocyte-EC communication.

In the same organoid test system [10.1093/cvr/cvac097], treatment with recombinant viral S or N proteins (72h) decreased the percentage of live pirocytes and ECs (FACS scan for live/total CD140b(PFGFR)/NG2+ and CD144+/CD31+ cells, respectively). In the case of ECs the % decrease of live cells appears comparable after treatment with S, N and combination of S+N protein. In the case of perycites, effect of S alone and in combination with N appears much more pronounced.  For both cell types, treatment with proinflmatory IL1-beta togather with S, N or both antigens, did not have additional cytotoxic effect. IL1beta alone, had greater cytotoxic effect on ECs compared to pirocytes.   However, neither EC nor pirocytes appeared to be activated by the S or N protein treatment as judged by the lack of significant change of CD31 or CD140b(PFGFR)/NG2+/ICAM-1+  cells, respectively.  Similarly expression of WVF, IL6 or IL8 (ELISA of supernatants) was not affected by S or N treatment of ECs. In contrast, IL1-beta alone activated both cell types (as judged by increase of ICAM-1+ cells of both types) and also induced of WVF, IL6 or IL8 production by ECs.There was no difference between the teratemnt with IL1-beta alone or with the recombinant viral proteins, suggesting that viral replication rather than interaction with the viral spike (or N) protein may be driver of cell activation observed in the patients lung authopsies . At mRNA level, joined S + N treatment induced ACE2 expression (qRT-PCR) as early as 4hpt, peak at 24hpt, maintained up to 72hpt. Individual antigen treatment is not tested (or shown?).

Taken together thehe lines of evidence from testing systems of different complexity [10.1093/cvr/cvac097]  suggests that in vivo the most likely primary target for viral infection or uptake of S-containing abortive pseudoviral particles in he (micro)vasculature are the pericytes. Infection/antigen uptake induce pericyte damage which dysregulates the intercellular crosstalk with ECs which in turn dysregulates overall vascular permeability (one potential aspect by interference with CD144 expression/function). In a parallel process EC may be also be activated by the inflammatory response in vivo, leading to significant increase in vascular permeability and microvasuclar dysfunction. Neutrophils are not present in this test system where apparently different results were obtained [10.3390/ijms231810436], where on the other hand pericytes are not present.

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.