The human brain vasculature shows a distinct expression pattern of SARS-CoV-2 entry factors

ABSTRACTA large number of hospitalized COVID-19 patients show neurological symptoms such as ischemic- and hemorrhagic stroke as well as encephalitis, and SARS-CoV-2 can directly infect endothelial cells leading to endotheliitis across multiple vascular beds. These findings suggest an involvement of the brain- and peripheral vasculature in COVID-19, but the underlying molecular mechanisms remain obscure. To understand the potential mechanisms underlying SARS-CoV-2 tropism for brain vasculature, we constructed a molecular atlas of the expression patterns of SARS-CoV-2 viral entry-associated genes (receptors and proteases) and SARS-CoV-2 interaction partners in human (and mouse) adult and fetal brain as well as in multiple non-CNS tissues in single-cell RNA-sequencing data across various datasets. We observed a distinct expression pattern of the cathepsins B (CTSB) and -L (CTSL) - which are able to substitute for the ACE2 co-receptor TMPRSS2 - in the human vasculature with CTSB being mainly expressed in the brain vasculature and CTSL predominantly in the peripheral vasculature, and these observations were confirmed at the protein level in the Human Protein Atlas and using immunofluorescence stainings. This expression pattern of SARS-CoV-2 viral-entry associated proteases and SARS-CoV-2 interaction partners was also present in endothelial cells and microglia in the fetal brain, suggesting a developmentally established SARS-CoV-2 entry machinery in the human vasculature. At both the adult and fetal stages, we detected a distinct pattern of SARS-CoV-2 entry associated genes’ transcripts in brain vascular endothelial cells and microglia, providing a potential explanation for an inflammatory response in the brain endothelium upon SARS-CoV-2 infection. Moreover, CTSB was co-expressed in adult and fetal brain endothelial cells with genes and pathways involved in innate immunity and inflammation, angiogenesis, blood-brain-barrier permeability, vascular metabolism, and coagulation, providing a potential explanation for the role of brain endothelial cells in clinically observed (neuro)vascular symptoms in COVID-19 patients. Our study serves as a publicly available single-cell atlas of SARS-CoV-2 related entry factors and interaction partners in human and mouse brain endothelial- and perivascular cells, which can be employed for future studies in clinical samples of COVID-19 patients.

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Established, New and Emerging Concepts in Brain Vascular Development

Frontiers in Physiology ◽

10.3389/fphys.2021.636736 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ankan Gupta ◽

Kevin R. Rarick ◽

Ramani Ramchandran

Keyword(s):

Endothelial Cells ◽

Model Systems ◽

Embryonic Brain ◽

Brain Endothelial Cells ◽

Vascular Patterning ◽

Brain Vasculature ◽

Single Cell Rna Sequencing ◽

Recent Developments ◽

Distinguishing Features ◽

The Brain

In this review, we discuss the state of our knowledge as it relates to embryonic brain vascular patterning in model systems zebrafish and mouse. We focus on the origins of endothelial cell and the distinguishing features of brain endothelial cells compared to non-brain endothelial cells, which is revealed by single cell RNA-sequencing methodologies. We also discuss the cross talk between brain endothelial cells and neural stem cells, and their effect on each other. In terms of mechanisms, we focus exclusively on Wnt signaling and the recent developments associated with this signaling network in brain vascular patterning, and the benefits and challenges associated with strategies for targeting the brain vasculature. We end the review with a discussion on the emerging areas of meningeal lymphatics, endothelial cilia biology and novel cerebrovascular structures identified in vertebrates.

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Nonlytic exocytosis of Cryptococcus neoformans from neutrophils in the brain vasculature

Cell Communication and Signaling ◽

10.1186/s12964-019-0429-0 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 3

Author(s):

Xiaofan Yang ◽

Huijuan Wang ◽

Fan Hu ◽

Xichen Chen ◽

Mingshun Zhang

Keyword(s):

Endothelial Cells ◽

Cryptococcus Neoformans ◽

Actin Polymerization ◽

Brain Endothelial Cells ◽

Brain Vasculature ◽

Mouse Brain Endothelial Cells ◽

Immunodeficiency Syndrome ◽

Brain Microvasculature ◽

The Brain

Abstract Background Cryptococcus neoformans (C. neoformans) is an encapsulated budding yeast that causes life-threatening meningoencephalitis in immunocompromised individuals, especially those with acquired immunodeficiency syndrome (AIDS). To cause meningoencephalitis, C. neoformans circulating in the bloodstream must first be arrested in the brain microvasculature. Neutrophils, the most abundant phagocytes in the bloodstream and the first leukocytes to be recruited to an infection site, can ingest C. neoformans. Little is known about how neutrophils interact with arrested fungal cells in the brain microvasculature. Methods A blood-brain barrier (BBB) in vitro model was established. The interactions between neutrophils adhering to brain endothelial cells and fungi were observed under a live cell imaging microscope. A flow cytometry assay was developed to explore the mechanisms. Immunofluorescence staining of brain tissues was utilized to validate the in vitro phenomena. Results Using real-time imaging, we observed that neutrophils adhered to a monolayer of mouse brain endothelial cells could expel ingested C. neoformans without lysis of the neutrophils or fungi in vitro, demonstrating nonlytic exocytosis of fungal cells from neutrophils. Furthermore, nonlytic exocytosis of C. neoformans from neutrophils was influenced by either the fungus (capsule and viability) or the neutrophil (phagosomal pH and actin polymerization). Moreover, nonlytic exocytosis of C. neoformans from neutrophils was recorded in brain tissue. Conclusion These results highlight a novel function by which neutrophils extrude C. neoformans in the brain vasculature. Graphical abstract

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Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation

Journal of Neuroinflammation ◽

10.1186/s12974-021-02324-7 ◽

2022 ◽

Vol 19 (1) ◽

Author(s):

Marta Machado-Pereira ◽

Cláudia Saraiva ◽

Liliana Bernardino ◽

Ana C. Cristóvão ◽

Raquel Ferreira

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Primary Cultures ◽

Neurovascular Unit ◽

Brain Endothelial Cells ◽

Neuronal Function ◽

Nitric Oxide Release ◽

Argonaute 2 ◽

Brain Vasculature ◽

The Brain

Abstract Background The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial–glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types. Methods Mouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function. Results Herein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood–brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function. Conclusions Ago2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

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Abstract MP17: Upregulating E2F1 Mitigates Senescence-Associated Phenotypes in Cultured Primary Endothelial Cells

Stroke ◽

10.1161/str.52.suppl_1.mp17 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Michael E Maniskas ◽

Yun-ju Lai ◽

Sean P Marrelli ◽

Louise D McCullough ◽

Jose F Moruno-manchon

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Neurovascular Unit ◽

Glucose Deprivation ◽

Cerebral Hypoperfusion ◽

Aged Mouse ◽

Aged Mice ◽

Brain Vasculature ◽

Bilateral Carotid ◽

The Brain

Vascular contributions to cognitive impairment and dementia (VCID) includes multiple disorders that are identified by cognitive deficits secondary to cerebrovascular pathology. The risk of VCID is higher in people after the age of 70, and, currently, there is no effective treatment. Vascular endothelial cells (VEC) are critical components of the brain vasculature and neurovascular unit and their health is vital to the capacity of the brain vasculature to respond to stressors. However, aged VEC may enter an irreversible replicative-arrest state (senescence), which has been associated with dementia. E2F transcription factor 1 (E2F1) regulates cell cycle progression and DNA damage repair. Importantly, E2F1 deficiency is associated with cell senescence. We hypothesized that E2F1 downregulation contributes to senescence in the cerebral endothelium during aging. We used cultured primary VEC from young (4-months old, mo) and aged (18-mo) male and female mice for RNA sequencing, plasmid-based gene delivery, high-resolution microscopy, and (4-, 12-, and 18-mo) mice of the bilateral carotid artery stenosis (BCAS) model, which produces chronic cerebral hypoperfusion and recapitulates some of the features seen in patients with VCID. We found that overexpression of E2F1 reduced the levels of senescence-associated phenotypes in cultured VEC from young mice that were exposed to oxygen and glucose deprivation (p<0.001), which induces endothelial senescence. Our RNA seq data showed that the expression of E2f1 was reduced (~40%) in cultured primary VEC from aged mouse brains compared with young cells (p<0.001). E2F1 levels were reduced in the brains of aged mice. Interestingly, we found sex differences in E2F1 levels, with less protein levels (~30%) in males vs females (p<0.05), independently of age. Also, aged BCAS mice (1 month after surgery) had more severe senescence phenotypes, reduced cerebral blood flow, and worse memory deficits compared with control mice (p<0.05). The effect of BCAS was more prominent in aged mice compared with younger (4- and 12-mo) mice. In conclusion , our study identifies E2F1 as a potential regulator of endothelial senescence in mice and highlights the contribution of aging as an important factor in losing endothelial resilience.

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Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

10.1101/2021.02.18.431829 ◽

2021 ◽

Author(s):

Robert W. Robey ◽

Andrea N. Robinson ◽

Fatima Ali-Rahmani ◽

Lyn M. Huff ◽

Sabrina Lusvarghi ◽

...

Keyword(s):

Endothelial Cells ◽

Multidrug Transporter ◽

Glycoprotein P ◽

Tissue Localization ◽

Brain Vasculature ◽

P Glycoprotein ◽

Capillary Endothelial Cells ◽

The Brain ◽

Viable Model

ABSTRACTGiven its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

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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.

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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.

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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.

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Differential Expression of CD31 and Von Willebrand Factor on Endothelial Cells in Different Regions of the Human Brain: Potential Implications for Cerebral Malaria Pathogenesis

Brain Sciences ◽

10.3390/brainsci10010031 ◽

2020 ◽

Vol 10 (1) ◽

pp. 31 ◽

Cited By ~ 4

Author(s):

Smart Ikechukwu Mbagwu ◽

Luis Filgueira

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Human Brain ◽

Expression Pattern ◽

Von Willebrand Factor ◽

Brain Regions ◽

And Function ◽

Von Willebrand ◽

Willebrand Factor ◽

The Brain

Cerebral microvascular endothelial cells (CMVECs) line the vascular system of the brain and are the chief cells in the formation and function of the blood brain barrier (BBB). These cells are heterogeneous along the cerebral vasculature and any dysfunctional state in these cells can result in a local loss of function of the BBB in any region of the brain. There is currently no report on the distribution and variation of the CMVECs in different brain regions in humans. This study investigated microcirculation in the adult human brain by the characterization of the expression pattern of brain endothelial cell markers in different brain regions. Five different brain regions consisting of the visual cortex, the hippocampus, the precentral gyrus, the postcentral gyrus, and the rhinal cortex obtained from three normal adult human brain specimens were studied and analyzed for the expression of the endothelial cell markers: cluster of differentiation 31 (CD31) and von-Willebrand-Factor (vWF) through immunohistochemistry. We observed differences in the expression pattern of CD31 and vWF between the gray matter and the white matter in the brain regions. Furthermore, there were also regional variations in the pattern of expression of the endothelial cell biomarkers. Thus, this suggests differences in the nature of vascularization in various regions of the human brain. These observations also suggest the existence of variation in structure and function of different brain regions, which could reflect in the pathophysiological outcomes in a diseased state.

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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.

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