Andes Virus Disrupts the Endothelial Cell Barrier by Induction of Vascular Endothelial Growth Factor and Downregulation of VE-Cadherin

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.Hantaviruses, of the family Bunyaviridae, are rodent-borne RNA viruses. Members of the Hantavirus genus have been identified as etiologic agents of two severe human diseases: hemorrhagic fever with renal syndrome (HFRS), which is caused by the Old World hantaviruses, and hantavirus pulmonary syndrome (HPS), which is caused by the New World hantaviruses (38, 39). Sin Nombre virus (SNV) and Andes virus (ANDV) are the main causes of HPS in the Americas. The major hantavirus target in humans is the microvascular endothelium, and the basis of HPS and HFRS is attributed to microvascular leakage (9, 34, 57). Common clinical features of HPS are interstitial pneumonitis with variable amounts of mononuclear cell infiltration, congestion, and both interstitial and alveolar edema (4, 34, 57). Despite the prominent accumulation of viral antigen in the infected vascular endothelium, no evidence of cellular destruction has been observed (57). Absence of a cytopathic effect has also been reported in in vitro studies of hantavirus infection of human primary endothelial cells (35, 46). In general, it is believed that induction of an uncontrolled immune response to the hantavirus infection, rather than the viral infection per se, is the cause of the microvascular leakage and ultimately HPS and HFRS (3, 48, 57). So far, a limited number of in vitro permeability studies have reported either no significant changes in the vascular permeability upon hantavirus infection or a significant increase only when mediators of increased permeability are exogenously added to the hantavirus-infected cells (12, 22, 46).Endothelial cell permeability is a highly regulated process and is maintained by both tight and adherens junctions (47). The disruption of adherens junctions is sufficient to disturb the endothelium barrier function and cause an increase in permeability and formation of edema (25, 47). Adherens junctions are largely composed of vascular endothelial (VE) cadherin (VE-cadherin), an endothelial cell-specific member of the cadherin family of adhesion protein (51, 52). Adherens junctions and in particular VE-cadherin are targets of the signaling pathway of agents that increase vascular permeability (7, 8, 10). Vascular endothelial growth factor (VEGF), one of the most potent vascular permeability agents, exerts its effects after binding to its homologous membrane tyrosine kinase receptor, VEGF-R2, whose expression is restricted to endothelial cells. It is known that VEGF-R2 interacts with VE-cadherin, and together they maintain the endothelial cell barrier (26). When VEGF is present, it binds to VEGF-R2, and that initiates the internalization and degradation of VE-cadherin and disruption of the adherens junctions (10, 54).In general, increase of vascular permeability is an important component of severe disease progression in hemorrhagic fevers (36). A number of studies have investigated the cause of increased vascular permeability in viral hemorrhagic fevers induced by viruses such as Dengue virus or Ebola virus (41, 42, 50, 53, 56). Studies of vascular permeability during hantavirus infection in vitro have mainly been performed in the presence of various inflammatory agents and growth factors (12, 15, 19, 22, 46). A recent study demonstrated that pathogenic hantaviruses sensitize the endothelium and cause hyperpermeability in response to high levels of exogenously added VEGF (12). We show here that VE-cadherin downregulation can be observed in ANDV-infected cells in the absence of exogenous VEGF. The downregulation of VE-cadherin in the absence of exogenous VEGF led us to the discovery that endothelial cells infected with ANDV induce the production of VEGF at early times postinfection. The early increased secretion of VEGF coincides with the initiation of downregulation of the adherent junction protein VE-cadherin and an increase in permeability of endothelial cells. The involvement of VEGF-R2 in VE-cadherin downregulation was demonstrated by antibody blockage of VEGF-R2 that resulted in significant recovery of VE-cadherin levels. These data indicate that the increased vascular permeability seen in HPS could be a direct result of hantavirus infection of the endothelium and may occur through a pathway involving VEGF-induced downregulation of VE-cadherin at early times postinfection.