Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is currently infecting millions of people worldwide and is causing drastic changes in people’s lives. Recent studies have shown that neurological symptoms are a major issue for people infected with SARS-CoV-2. However, the mechanism through which the pathological effects emerge is still unclear. Brain endothelial cells (ECs), one of the components of the blood–brain barrier, are a major hurdle for the entry of pathogenic or infectious agents into the brain. They strongly express angiotensin converting enzyme 2 (ACE2) for its normal physiological function, which is also well-known to be an opportunistic receptor for SARS-CoV-2 spike protein, facilitating their entry into host cells. First, we identified rapid internalization of the receptor-binding domain (RBD) S1 domain (S1) and active trimer (Trimer) of SARS-CoV-2 spike protein through ACE2 in brain ECs. Moreover, internalized S1 increased Rab5, an early endosomal marker while Trimer decreased Rab5 in the brain ECs. Similarly, the permeability of transferrin and dextran was increased in S1 treatment but decreased in Trimer, respectively. Furthermore, S1 and Trimer both induced mitochondrial damage including functional deficits in mitochondrial respiration. Overall, this study shows that SARS-CoV-2 itself has toxic effects on the brain ECs including defective molecular delivery and metabolic function, suggesting a potential pathological mechanism to induce neurological signs in the brain.

Download Full-text

Angiotensin Converting Enzyme 2 (ACE2) Expression in the Visual System

10.21203/rs.3.rs-296818/v1 ◽

2021 ◽

Author(s):

James M. Hill ◽

Christian Clement ◽

L. Arceneaux ◽

Walter Lukiw

Keyword(s):

Signal Processing ◽

Angiotensin Converting Enzyme ◽

Occipital Lobe ◽

Cell Types ◽

Receptor Expression ◽

Host Cells ◽

Visual Signal ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

The Brain

Abstract Background: Multiple lines of evidence currently indicate that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)gains entry into human host cells via a high-affinity interaction with the angiotensin-converting enzyme 2 (ACE2) transmembrane receptor. Research has further shown the widespread expression of the ACE2 receptor on the surface of many different immune, non-immune and neural host cell types, and that SARS-CoV-2 has there markable capability to attack many different types of human-host cells simultaneously. One principal neuroanatomical region for highACE2 expression patterns occurs in the brainstem, an area of the brain containing regulatory centers for respiration, and this may in part explain the predisposition of many COVID-19 patients to respiratory distress. Early studies also indicated extensive ACE2 expression in the whole eye and the brain’s visual circuitry. In this study we analyzed ACE2 receptor expression at the mRNA and protein level in multiple cell types involved in human vision, including cell types of the external eye and several deep brain regions known to be involved in the processing of visual signals.Methods: ACE2 mRNA and protein analysis; multiple eye and brain cells and tissues; gamma32P-adenosine tri-phosphate ([γ-32P]dATP) radiolabeled probes; Northern analysis; ELISA.Results: The four main findings were: (i)that many different optical and neural cell types of the human visual system provide receptors essential for SARS-CoV-2 invasion; (ii)the remarkable ubiquity of ACE2 presence in cells of the eye and anatomical regions of the brain involved in visual signal processing; (iii)that ACE2 receptor expression in different ocular cell types and visual processing centers of the brain provide multiple compartments for SARS-CoV-2 infiltration; and (iv)a gradient of increasing ACE2 expression from the anterior surface of the eye to the visual signal processing areas of the occipital lobe and the primary visual neocortex.Conclusion: A gradient of ACE2 expression from the eye surface to the occipital lobe provide the SARS-CoV-2 virus a novel pathway from the outer eye into deeper anatomical regions of the brain involved in vision. These findings may explain, in part, the many recently reported neuro-ophthalmic manifestations of SARS-CoV-2infection in COVID-19 affected patients.

Download Full-text

The Spatial and Cell-Type Distribution of SARS-CoV-2 Receptor ACE2 in the Human and Mouse Brains

Frontiers in Neurology ◽

10.3389/fneur.2020.573095 ◽

2021 ◽

Vol 11 ◽

Author(s):

Rongrong Chen ◽

Keer Wang ◽

Jie Yu ◽

Derek Howard ◽

Leon French ◽

...

Keyword(s):

Endothelial Cells ◽

Spatial Distribution ◽

Host Cells ◽

Inhibitory Neurons ◽

Middle Temporal Gyrus ◽

Distribution Analysis ◽

Cell Type ◽

Type Distribution ◽

The Brain ◽

Human And Mouse

By engaging angiotensin-converting enzyme 2 (ACE2 or Ace2), the novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades host cells and affects many organs, including the brain. However, the distribution of ACE2 in the brain is still obscure. Here, we investigated the ACE2 expression in the brain by analyzing data from publicly available brain transcriptome databases. According to our spatial distribution analysis, ACE2 was relatively highly expressed in some brain locations, such as the choroid plexus and paraventricular nuclei of the thalamus. According to cell-type distribution analysis, nuclear expression of ACE2 was found in many neurons (both excitatory and inhibitory neurons) and some non-neuron cells (mainly astrocytes, oligodendrocytes, and endothelial cells) in the human middle temporal gyrus and posterior cingulate cortex. A few ACE2-expressing nuclei were found in a hippocampal dataset, and none were detected in the prefrontal cortex. Except for the additional high expression of Ace2 in the olfactory bulb areas for spatial distribution as well as in the pericytes and endothelial cells for cell-type distribution, the distribution of Ace2 in the mouse brain was similar to that in the human brain. Thus, our results reveal an outline of ACE2/Ace2 distribution in the human and mouse brains, which indicates that the brain infection of SARS-CoV-2 may be capable of inducing central nervous system symptoms in coronavirus disease 2019 (COVID-19) patients. Potential species differences should be considered when using mouse models to study the neurological effects of SARS-CoV-2 infection.

Download Full-text

SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.687783 ◽

2021 ◽

Vol 8 ◽

Author(s):

Somasundaram Raghavan ◽

Divya Borsandra Kenchappa ◽

M. Dennis Leo

Keyword(s):

Endothelial Cells ◽

Angiotensin Converting Enzyme ◽

Spike Protein ◽

Endothelial Barrier ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Barrier Integrity ◽

Junctional Proteins ◽

Junction Protein ◽

Junctional Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

Download Full-text

Virtual screening as therapeutic strategy of COVID-19 targeting angiotensin-converting enzyme 2

Frontiers in Molecular Immunology ◽

10.25082/fmi.2021.01.002 ◽

2021 ◽

Vol 2 (1) ◽

pp. 16-27

Author(s):

Zahra Sharifinia ◽

◽

Samira Asadi ◽

Mahyar Irani ◽

Abdollah Allahverdi ◽

...

Keyword(s):

Virtual Screening ◽

Angiotensin Converting Enzyme ◽

Host Cells ◽

Spike Protein ◽

Potential Drug ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Approved Drugs ◽

Fda Approved Drugs

Objective: The receptor-binding domain (RBD) of the S1 domain of the SARS-CoV- 2 Spike protein performs a key role in the interaction with Angiotensin-converting enzyme 2 (ACE2), leading to both subsequent S2 domain-mediated membrane fusion and incorporation of viral RNA in host cells. Methods: In this study, we investigated the inhibitor’s targeted compounds through existing human ACE2 drugs to use as a future viral invasion. 54 FDA approved drugs were selected to assess their binding affinity to the ACE2 receptor. The structurebased methods via computational ones have been used for virtual screening of the best drugs from the drug database. Key Findings: The ligands “Cinacalcet” and “Levomefolic acid” highaffinity scores can be a potential drug preventing Spike protein of SARS-CoV-2 and human ACE2 interaction. Levomefolic acid from vitamin B family was proved to be a potential drug as a spike protein inhibitor in previous clinical and computational studies. Besides that, in this study, the capability of Levomefolic acid to avoid ACE2 and Spike protein of SARS-CoV-2 interaction is indicated. Therefore, it is worth to consider this drug for more in vitro investigations as ACE2 and Spike protein inhibition candidate. Conclusion: The two Cinacalcet and Levomefolic acid are the two ligands that have highest energy binding for human ACE2 blocking among 54 FDA approved drugs.

Download Full-text

Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells

PLoS ONE ◽

10.1371/journal.pone.0249686 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249686

Author(s):

Diána Hudecz ◽

Sara Björk Sigurdardóttir ◽

Sarah Christine Christensen ◽

Casper Hempel ◽

Andrew J. Urquhart ◽

...

Keyword(s):

Endothelial Cells ◽

Transferrin Receptor ◽

Vesicular Transport ◽

Brain Diseases ◽

Brain Endothelial Cells ◽

Binding Studies ◽

Wide Range ◽

Murine Brain ◽

The Brain

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

Download Full-text

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

Science ◽

10.1126/science.abe3255 ◽

2020 ◽

pp. eabe3255 ◽

Cited By ~ 7

Author(s):

Michael Schoof ◽

Bryan Faust ◽

Reuben A. Saunders ◽

Smriti Sangwan ◽

Veronica Rezelj ◽

...

Keyword(s):

Cell Receptor ◽

Affinity Maturation ◽

Host Cells ◽

Spike Protein ◽

Airway Epithelia ◽

Angiotensin Converting Enzyme 2 ◽

Inactive Conformation ◽

Binding Domains ◽

Host Cell Receptor ◽

Yeast Surface

The SARS-CoV-2 virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryogenic electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

Download Full-text

Endogenous THBD (Thrombomodulin) Mediates Angiogenesis in the Ischemic Brain—Brief Report

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.315061 ◽

2020 ◽

Vol 40 (12) ◽

pp. 2837-2844 ◽

Cited By ~ 1

Author(s):

Jan Wenzel ◽

Dimitrios Spyropoulos ◽

Julian Christopher Assmann ◽

Mahtab Ahmad Khan ◽

Ines Stölting ◽

...

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Soluble Form ◽

Stroke Outcome ◽

Brain Endothelial Cells ◽

Ischemic Brain ◽

Infarct Area ◽

The Brain

Objective: THBD (thrombomodulin) is part of the anticoagulant protein C-system that acts at the endothelium and is involved in anti-inflammatory and barrier-stabilizing processes. A recombinant soluble form of THBD was shown to have protective effects in different organs, but how the endogenous THBD is regulated during ischemia, particularly in the brain is not known to date. The aim of this study was to investigate the role of THBD, especially in brain endothelial cells, during ischemic stroke. Approach and Results: To induce ischemic brain damage, we occluded the middle cerebral artery of mice. We found an increased endothelial expression of Thbd in the peri-infarct area, whereas in the core of the ischemic tissue Thbd expression was decreased compared with the contralateral side. We generated a novel Cre/loxP-based mouse line that allows for the inducible deletion of Thbd specifically in brain endothelial cells, which worsened stroke outcome 48 hours after middle cerebral artery occlusion. Unexpectedly, we found no signs of increased coagulation, thrombosis, or inflammation in the brain but decreased vessel diameters and impaired angiogenesis in the peri-infarct area that led to a reduced overall vessel length 1 week after stroke induction. Conclusions: Endogenous THBD acts as a protective factor in the brain during ischemic stroke and enhances vessel diameter and proliferation. These previously unknown properties of THBD could offer new opportunities to affect vessel function after ischemia and thereby improve stroke outcome.

Download Full-text

Cerebral angiogenesis ameliorates pathological disorders in Nemo-deficient mice with small-vessel disease

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x20910522 ◽

2020 ◽

pp. 0271678X2091052

Author(s):

Yun Jiang ◽

Kristin Müller ◽

Mahtab A. Khan ◽

Julian C. Assmann ◽

Josephine Lampe ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Small Vessel Disease ◽

Small Vessel ◽

Incontinentia Pigmenti ◽

Brain Endothelial Cells ◽

Functional Deficits ◽

Intussusceptive Angiogenesis ◽

Inactivating Mutations ◽

Cerebral Small Vessel Diseases

Cerebral small-vessel diseases (SVDs) often follow a progressive course. Little is known about the function of angiogenesis, which potentially induces regression of SVDs. Here, we investigated angiogenesis in a mouse model of incontinentia pigmenti (IP), a genetic disease comprising features of SVD. IP is caused by inactivating mutations of Nemo, the essential component of NF-κB signaling. When deleting Nemo in the majority of brain endothelial cells ( NemobeKO mice), the transcriptional profile of vessels indicated cell proliferation. Brain endothelial cells expressed Ki67 and showed signs of DNA synthesis. In addition to cell proliferation, we observed sprouting and intussusceptive angiogenesis in NemobeKO mice. Angiogenesis occurred in all segments of the vasculature and in proximity to vessel rarefaction and tissue hypoxia. Apparently, NEMO was required for productive angiogenesis because endothelial cells that had escaped Nemo inactivation showed a higher proliferation rate than Nemo-deficient cells. Therefore, newborn endothelial cells were particularly vulnerable to ongoing recombination. When we interfered with productive angiogenesis by inducing ongoing ablation of Nemo, mice did not recover from IP manifestations but rather showed severe functional deficits. In summary, the data demonstrate that angiogenesis is present in this model of SVD and suggest that it may counterbalance the loss of vessels.

Download Full-text

Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum -Infected Erythrocytes to Endothelial Cells

mBio ◽

10.1128/mbio.00615-16 ◽

2016 ◽

Vol 7 (4) ◽

Cited By ~ 43

Author(s):

Marion Avril ◽

Maria Bernabeu ◽

Maxwell Benjamin ◽

Andrew Jay Brazier ◽

Joseph D. Smith

Keyword(s):

Endothelial Cells ◽

Cerebral Malaria ◽

Intercellular Adhesion Molecule ◽

Brain Endothelial Cells ◽

Endothelial Protein C Receptor ◽

Intercellular Adhesion Molecule 1 ◽

Cd36 Expression ◽

Tnf Α ◽

Group A ◽

The Brain

ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum -infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. IMPORTANCE Cerebral malaria is a severe neurological complication of P. falciparum infection associated with infected erythrocyte (IE) binding in cerebral vessels. Yet little is known about the mechanisms by which parasites adhere in the brain or other microvascular sites. Here, we studied parasite lines expressing group A DC13-containing PfEMP1 variants, a subset that has previously been shown to have high brain cell- and other endothelial cell-binding activities. We show that DC13-containing PfEMP1 variants have dual EPCR- and ICAM-1-binding activities and that both receptors are involved in parasite adherence to lung and brain endothelial cells. As both EPCR and ICAM-1 are implicated in cerebral malaria, these findings suggest the possibility that parasites with dual binding activities are involved in parasite sequestration to microvascular beds with low CD36 expression, such as the brain, and we urge more research into the multiadhesive properties of PfEMP1 variants.

Download Full-text

ACE2-expressing endothelial cells in aging mouse brain

10.1101/2020.07.11.198770 ◽

2020 ◽

Author(s):

Su Bin Lim ◽

Valina L. Dawson ◽

Ted M. Dawson ◽

Sung-Ung Kang

Keyword(s):

Endothelial Cells ◽

Mouse Brain ◽

Host Cells ◽

Aging Brain ◽

Systematic Analysis ◽

Angiotensin Converting Enzyme 2 ◽

Functional Changes ◽

Barrier Regulation ◽

Human Brains ◽

Transcriptomic Changes

AbstractAngiotensin-converting enzyme 2 (ACE2) is a key receptor mediating the entry of SARS-CoV-2 into the host cell. Through a systematic analysis of publicly available mouse brain sc/snRNA-seq data, we found that ACE2 is specifically expressed in small sub-populations of endothelial cells and mural cells, namely pericytes and vascular smooth muscle cells. Further, functional changes in viral mRNA transcription and replication, and impaired blood-brain barrier regulation were most prominently implicated in the aged, ACE2-expressing endothelial cells, when compared to the young adult mouse brains. Concordant EC transcriptomic changes were further found in normal aged human brains. Overall, this work reveals an outline of ACE2 distribution in the mouse brain and identify putative brain host cells that may underlie the selective susceptibility of the aging brain to viral infection.

Download Full-text