Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

Ackermann, Maximilian; Verleden, Stijn E.; Kuehnel, Mark; Haverich, Axel; Welte, Tobias; Laenger, Florian; Vanstapel, Arno; Werlein, Christopher; Stark, Helge; Tzankov, Alexandar; Li, William W.; Li, Vincent W.; Mentzer, Steven J.; Jonigk, Danny

doi:10.1056/nejmoa2015432

Cited by 4,994 publications

(6,606 citation statements)

References 22 publications

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“…Only at that time point, the arterial and venous endothelium of animals in both groups was swollen and vacuolated, with necrotic endothelial cells separated from the underlying basement membrane by the presence of subendothelial lymphocytes and neutrophils (Fig. S2C, left), consistent with what has been described as endothelialitis in human SARS-CoV-2 infection 29 . At 7 dpi, recovery as indicated by marked hyperplasia of bronchial epithelial cells and type II alveolar epithelial cells were seen in both groups (Fig.…”

Section: Main Textsupporting

confidence: 84%

Age-dependent progression of SARS-CoV-2 infection in Syrian hamsters

Osterrieder

Bertzbach

Dietert

et al. 2020

Preprint

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22In late 2019, an outbreak of a severe respiratory disease caused by an emerging 23 coronavirus, SARS-CoV-2, resulted in high morbidity and mortality in infected humans 1 . 24Complete understanding of COVID-19, the multi-faceted disease caused by SARS-CoV-25 2, requires suitable small animal models, as does the development and evaluation of 26 vaccines and antivirals 2 . Because age-dependent differences of COVID-19 were identified 27 in humans 3 , we compared the course of SARS-CoV-2 infection in young and aged Syrian 28 hamsters. We show that virus replication in the upper and lower respiratory tract was 29 independent of the age of the animals. However, older hamsters exhibited more 30 pronounced and consistent weight loss. In situ hybridization in the lungs identified viral 31 RNA in bronchial epithelium, alveolar epithelial cells type I and II, and macrophages. 32Histopathology revealed clear age-dependent differences, with young hamsters launching 33 earlier and stronger immune cell influx than aged hamsters. The latter developed 34 conspicuous alveolar and perivascular edema, indicating vascular leakage. In contrast, 35we observed rapid lung recovery at day 14 after infection only in young hamsters. We 36propose that comparative assessment in young versus aged hamsters of SARS-CoV-2 37 vaccines and treatments may yield valuable information as this small-animal model 38 appears to mirror age-dependent differences in human patients.

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Section: Main Textsupporting

confidence: 84%

Age-dependent progression of SARS-CoV-2 infection in Syrian hamsters

Osterrieder

Bertzbach

Dietert

et al. 2020

Preprint

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“…S1 binding to NRP1 may therefore contribute to viral entry and survival within the host cell. Interstingly, gene expression analysis of lung tissue from COVID-19 patients recently revealed an up-regulation of NRP1 and NRP2 (16) .…”

Section: (Human Lung Cancer) Cells Incubation Of These Cells With Rementioning

confidence: 99%

Neuropilin-1 is a host factor for SARS-CoV-2 infection

Daly

Simonetti

Antón-Plágaro

et al. 2020

Preprint

255

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¶ These authors contributed equally. ∬ These authors contributed equally. § These authors jointly supervised this work and are joint corresponding authors. SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease thathas infected more than 6.6 million people and caused over 390,000 deaths worldwide 1,2 . The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2 (3,4) . For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors 5 . Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID- 19.A striking difference in the S protein of SARS-CoV-2 and SARS-CoV is the presence, in the former, of a polybasic sequence motif, RRAR, at the S1/S2 boundary. It provides a cleavage site for a proprotein convertase, furin, a membrane-bound host cell protease [3][4][5] (Figure 1A). The resulting two proteins, S1 and S2, remain noncovalently associated, with the serine protease TMPRSS2 further priming the S2 protein by proteolytic cleavage 6 . Several observations indicate that the furinmediated processing of the S protein increases the infection and affects the tropism of SARS-CoV-2 (3)(4)(5) . Proprotein convertase inhibitors that target furin robustly diminish SARS-CoV-2 entry into Calu-3 and HeLa cells exogenously expressing ACE2 (7) . Moreover, furin knockdown impairs S processing, and abrogation of the polybasic site in the S reduces syncytia formation in infected cells 4,7 .We noticed that the C-terminus of the S1 protein generated by furin has a polybasic amino acid sequence ( 682 RRAR 685 ), that conforms to a [R/K]XX[R/K] motif, termed the 'C-end rule' (CendR) (Figure 1B) 8 . CendR motifs bind to neuropilin-1 (NRP1) and NRP2, dimeric transmembrane receptors that regulate pleiotropic biological processes, including axon guidance, angiogenesis and vascular permeability 8-10 .To explore the possible association of the SARS-CoV-2 S1 protein with neuropilins we engineered a HEK293T cell line to stably express SARS-CoV-2 S protein. In this line we transiently expressed full length NRP1 tagged at the C-terminus with GFP were treated with the particle-mesh Ewald's method and a long-range dispersion co...

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“…In fact, recent studies have found increased serum levels of angiotensin II in COVID-19 patients 28 . ACE2 is expressed on the vasculature 2 and recent studies of COVID-19 patients have shown virus like particles in pulmonary 29 and kidney 30 endothelium. ACE2 is also expressed on the human cerebral vasculature 19,31 , which we also confirm herein.…”

Section: Introductionmentioning

confidence: 99%

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood–brain barrier

Buzhdygan

DeOre

Baldwin‐Leclair

et al. 2020

Preprint

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As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

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Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

Cited by 4,994 publications

References 22 publications

Age-dependent progression of SARS-CoV-2 infection in Syrian hamsters

Age-dependent progression of SARS-CoV-2 infection in Syrian hamsters

Neuropilin-1 is a host factor for SARS-CoV-2 infection

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood–brain barrier

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