Andes virus disrupts the endothelial cell barrier by induction of vascular endothelial growth factor and downregulation of VE-cadherin - PubMed (original) (raw)
Andes virus disrupts the endothelial cell barrier by induction of vascular endothelial growth factor and downregulation of VE-cadherin
Punya Shrivastava-Ranjan et al. J Virol. 2010 Nov.
Abstract
Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantavirus infection. High levels of virus replication occur in microvascular endothelial cells but without a virus-induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the vascular endothelial growth factor (VEGF) receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin are seen at early times postinfection of human primary lung endothelial cells with an HPS-associated hantavirus, Andes virus. Furthermore, active virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and in VE-cadherin activation, internalization, and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes virus-induced VE-cadherin reduction. These data implicate virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS and suggest potential immunotherapeutic targets for the treatment of the disease.
Figures
FIG. 1.
Downregulation of VE-cadherin by ANDV and SNV infection. (Left and middle blots) Confluent monolayers of HMVEC-L cells were infected with ANDV at an MOI of 1, were treated with inactivated ANDV, or were left untreated (mock). Cell lysates were harvested at 12, 16, 24, or 72 h postinfection and analyzed. (Right blot) Cells infected with SNV at an MOI of 0.2, treated with inactivated SNV, of untreated were analyzed 72 h postinfection for expression of VE-cadherin, virus nucleocapsid, and cellular actin by Western blotting.
FIG. 2.
Immunofluorescent staining of HMVEC-L cells infected with ANDV. HMVEC-L cells grown on coverslips were infected with ANDV at an MOI of 0.5. After 12, 16, or 24 h (A) or 72 h (B) postinfection, cells were fixed and triple stained with VE-cadherin-specific mouse monoclonal antibody (green), anti-ANDV rabbit polyclonal antibody (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). The arrow indicates VE-cadherin endocytosed at higher rates in the ANDV-infected cells. For controls, cells were either treated with inactivated ANDV or left untreated.
FIG. 3.
A VEGF-R2-neutralizing antibody inhibits ANDV-induced VE-cadherin downregulation. Confluent monolayers of HMVEC-L cells were infected with ANDV at an MOI of 1, were treated with inactivated ANDV, or were left untreated (mock). After 1 h of virus absorption, cells were either treated with a neutralizing monoclonal antibody to VEGF-R2 (500 ng/ml) or with control mouse IgG (500 ng/ml). Cell lysates were harvested at 16 or 24 h postinfection and analyzed for expression of VE-cadherin, virus nucleocapsid, and cellular actin by Western blotting.
FIG. 4.
ANDV infection of HMVEC-L cells results in increased secretion of free VEGF. Confluent monolayers of HMVEC-L cells were infected with ANDV at an MOI of 1, were treated with inactivated ANDV, or were left untreated (mock). Supernatants were harvested at 12, 16, or 24 h postinfection, and the amount of free VEGF was determined by ELISA. The data represent the means and standard deviations of duplicate samples. Results shown are representative of five independent experiments.
FIG. 5.
ANDV infection increases endothelial cell permeability. (A) Confluent monolayers of HMVEC-L cells grown on transwell inserts were infected with ANDV at an MOI of 1, were treated with inactivated ANDV, or were left untreated. After 12, 16, 24, or 72 h postinfection, FITC-dextran was added to the upper chambers of transwell inserts. The passage of FITC-dextran to the lower chambers was assayed after 30 min of incubation at room temperature as described in Materials and Methods. The data represent the means and standard deviations of quadruplicate samples. (B) Cells were infected with SNV at an MOI of 0.2, treated with inactivated SNV, or left untreated and were assayed after 72 h postinfection as described for panel A.
FIG. 6.
Increased levels of free VEGF in sera from HPS patients. Acute-phase sera from hospitalized patients exhibiting ARDS-like symptoms were analyzed for free VEGF levels. Nine patients were laboratory confirmed as HPS cases, while the severe respiratory disease in the other six cases was undiagnosed but determined to be not hantavirus related.
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