Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation

Chiam, Rachael; Šmid, Lojze; Kydd, Julia H.; Smith, Ken; Platt, A.J.; Davis-Poynter, Nicholas

doi:10.1016/j.vetmic.2005.11.005

Cited by 11 publications

(9 citation statements)

References 28 publications

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“…Recent comparative sequencing of complete genomes of a representative EHV-1 abortigenic and neurotropic strain revealed no candidate gene for pathogenicity, but identified that a single mutation in the viral DNA-polymerase gene might correlate with neuropathogenic potential. To date, however, it has been difficult to model variations in neuropathogenicity in animal models or in vitro systems (Smith et al 1992(Smith et al , 2000van Woensel et al 1995;Chiam et al 2006;Allen 2006), although a definite correlation has been described between this genetic marker and outbreaks of EAND vs. abortion and the capacity of high levels of viraemia combined with high replication rates leading to high viral loads (Nugent et al 2001;Allen 2006; A l l e n and Breathnach 2006).…”

Section: Virologymentioning

confidence: 99%

“…Another critical step is the cellto-cell transmission of virus into the endothelial cells of the CNS, which involves the expression of adhesion molecules on the surface of endothelial cells and PBL. The interaction between viraemic leucocytes and endothelial cells might be tissueselective, with local factors such as chemokines, cytokines, hormones and immune complexes influencing endothelial cell activation and therefore surface membrane receptors available for attraction of virus-infected leucocytes (Smith et al , 2002Chiam et al 2006). Chemokines have been shown to play an important role in inflammation.…”

Section: Viraemiamentioning

confidence: 99%

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Pathogenesis of equine herpesvirus-associated neurological disease: a revised explanation

Borchers¹,

Thein

Sterner-Kock

2010

Equine Veterinary Journal

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Section: Virologymentioning

confidence: 99%

Section: Viraemiamentioning

confidence: 99%

Pathogenesis of equine herpesvirus-associated neurological disease: a revised explanation

Borchers¹,

Thein

Sterner-Kock

2010

Equine Veterinary Journal

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“…So far, several in vitro systems have demonstrated the potential utility of cultured EC in the study of the pathogenesis of EHV-1 (16,17). Studies have shown that the infection of EC located in the vasculature of the late-gravid uterus or CNS was mediated by cellto-cell contacts between infected PBMC and EC and occurred even in the presence of virus-neutralizing antibodies (18,19).…”

mentioning

confidence: 99%

Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a ⁺ Monocytic Cells upon Adhesion to Endothelial Cells

Laval

Favoreel

Poelaert

et al. 2015

J Virol

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Equine herpesvirus type 1 (EHV-1) is a main cause of respiratory disease, abortion, and encephalomyelopathy in horses. Monocytic cells (CD172a ؉ ) are the main carrier cells of EHV-1 during primary infection and are proposed to serve as a "Trojan horse" to facilitate the dissemination of EHV-1 to target organs. However, the mechanism by which EHV-1 is transferred from CD172a ؉ cells to endothelial cells (EC) remains unclear. The aim of this study was to investigate EHV-1 transmission between these two cell types. We hypothesized that EHV-1 employs specific strategies to promote the adhesion of infected CD172a ؉ cells to EC to facilitate EHV-1 spread. Here, we demonstrated that EHV-1 infection of CD172a؉ cells resulted in a 3-to 5-fold increase in adhesion to EC. Antibody blocking experiments indicated that ␣ 4 ␤ 1 , ␣ L ␤ 2 , and ␣ V ␤ 3 integrins mediated adhesion of infected CD172a ؉ cells to EC. We showed that integrin-mediated phosphatidylinositol 3-kinase (PI3K) and ERK/MAPK signaling pathways were involved in EHV-1-induced CD172a؉ cell adhesion at early times of infection. EHV-1 replication was enhanced in adherent CD172a؉ cells, which correlates with the production of tumor necrosis factor alpha (TNF-␣). In the presence of neutralizing antibodies, approximately 20% of infected CD172a؉ cells transferred cytoplasmic material to uninfected EC and 0.01% of infected CD172a؉ cells transmitted infectious virus to neighboring cells. Our results demonstrated that EHV-1 infection induces adhesion of CD172a؉ cells to EC, which enhances viral replication, but that transfer of viral material from CD172a ؉ cells to EC is a very specific and rare event. These findings give new insights into the complex pathogenesis of EHV-1. IMPORTANCEEquine herpesvirus type 1 (EHV-1) is a highly prevalent pathogen worldwide, causing frequent outbreaks of abortion and myeloencephalopathy, even in vaccinated horses. After primary replication in the respiratory tract, EHV-1 disseminates via cell-associated viremia in peripheral blood mononuclear cells (PBMC) and subsequently infects the endothelial cells (EC) of the pregnant uterus or central nervous system, leading in some cases to abortion and/or neurological disorders. Recently, we demonstrated that CD172a؉ monocytic carrier cells serve as a "Trojan horse" to facilitate EHV-1 spread from blood to target organs. Here, we investigated the mechanism underlying the transmission of EHV-1 from CD172a ؉ cells to EC. We demonstrated that EHV-1 infection induces cellular changes in CD172a؉ cells, promoting their adhesion to EC. We found that both cell-to-cell contacts and the secretion of soluble factors by EC activate EHV-1 replication in CD172a ؉ cells. This facilitates transfer of cytoplasmic viral material to EC, resulting mainly in a nonproductive infection. Our findings give new insights into how EHV-1 may spread to EC of target organs in vaccinated horses. E quine herpesvirus type 1 (EHV-1), a member of the subfamily Alphaherpesvirinae, is a ubiquitous pathogen in horses, causing...

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“…This study presents the first in vitro angiogenesis assay of equine endothelial cells. In the past, equine endothelial cells were isolated from the aorta [51,82], pulmonary artery [51,56,82], umbilical vein [22], fatty omentum [16], carotid artery [13,37], or brain [37]. These cultures were used in studies focusing on clinical aspects related to viral infections or endotoxemia without addressing the angiogenesis cascade.…”

Section: Discussionmentioning

confidence: 99%

Isolation of equine endothelial cells and life cell angiogenesis assay

Dietze

Slosarek

Fuhrmann-Selter

et al. 2014

Clinical Hemorheology and Microcirculation

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Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis.

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Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation

Cited by 11 publications

References 28 publications

Pathogenesis of equine herpesvirus-associated neurological disease: a revised explanation

Pathogenesis of equine herpesvirus-associated neurological disease: a revised explanation

Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a ⁺ Monocytic Cells upon Adhesion to Endothelial Cells

Isolation of equine endothelial cells and life cell angiogenesis assay

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Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation

Cited by 11 publications

References 28 publications

Pathogenesis of equine herpesvirus-associated neurological disease: a revised explanation

Pathogenesis of equine herpesvirus-associated neurological disease: a revised explanation

Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a + Monocytic Cells upon Adhesion to Endothelial Cells

Isolation of equine endothelial cells and life cell angiogenesis assay

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Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a ⁺ Monocytic Cells upon Adhesion to Endothelial Cells