There are two main characteristics of hantavirus infection in humans: endothelial cell tropism and increased vascular permeability (4
). The mechanism(s) by which hantaviruses cause vascular permeability is unknown. Several studies have indicated that hantavirus infection may exert changes in the barrier function of the vascular endothelium indirectly by inducing endothelial cell and leukocyte activation and triggering synthesis and release of proinflammatory lymphokines (1
). Most of the in vitro
studies examining hantavirus-induced vascular permeability have been performed with the addition of recombinant expressed factors, such as tumor necrosis factor alpha and VEGF (12
). Here, we showed for the first time that ANDV infection alone can directly increase the vascular permeability of human lung endothelial cells at early times postinfection.
VE-cadherin plays a prominent role in the establishment of the endothelial barrier (7
). Disruption of VE-cadherin function has been shown to result in interstitial edema and accumulation of inflammatory cells in the heart and lung microcirculation (7
). Our observation that the enhanced permeability in endothelial cells resulting from ANDV infection correlated with downregulation of VE-cadherin suggests that disruption of cell-cell adhesion mediated by VE-cadherin is sufficient to induce interendothelial gap formation and hence play a controlling role in the perturbation of the endothelial barrier. VE-cadherin degradation is known to involve extensive internalization, ubiquitination, and proteosomal degradation (54
Hantaviruses are not the only viruses that cause downregulation of VE-cadherin and increases in vascular permeability. Dengue virus (the cause of dengue hemorrhagic fever) and Karposi's sarcoma-associated herpesvirus (KSHV) have also been shown to downregulate VE-cadherin (21
). In the case of KSHV it has been demonstrated that a viral protein which functions as a ubiquitin ligase targets VE-cadherin and causes its downregulation (29
). The mechanism by which VE-cadherin is degraded by ANDV infection remains to be determined.
A previous study showed that infection with a pathogenic hantavirus sensitized cells to the addition of recombinant VEGF and suggested a role for VEGF in vascular leakage and disease (12
). However, it was unclear what the source of VEGF might be during hantavirus infections. Here we showed detection of free secreted VEGF as a direct result of hantavirus infection of lung endothelial cells.
VEGF is a potent vascular permeability factor that has been shown to act directly on endothelial cells by binding to VEGF-R2 (26
). VEGF can induce an increase in vascular permeability by a variety of means (49
). In endothelial cells, VEGF-R2 associates with VE-cadherin and provides regulation of cell-cell junctional integrity (40
). When VEGF binds to VEGF-R2, VEGF-R2 is activated and endocytosed and a signaling cascade is initiated, leading to the induction of VE-cadherin degradation and an increase in junctional permeability (10
). The involvement of VEGF-R2 in VE-cadherin downregulation in ANDV-infected human lung endothelial cells is supported by the demonstration that a neutralizing antibody to VEGF-R2 (55
) inhibits the virus-induced reduction of VE-cadherin. In further support, a very recent study demonstrated an increase in VEGF-R2 phosphorylation and internalization of VE-cadherin in hantavirus-infected cells treated with high levels of exogenous VEGF (14
VEGF could also cause changes in the vascular permeability by the activation of integrins, which have been characterized as receptors for hantaviruses (5
). However, this mechanism does not appear to play a major role, based on our observation that treatment of human lung endothelial cells with inactivated ANDV caused only a small transient increase in secreted VEGF and did not alter the total amounts of VE-cadherin or increase vascular permeability. Further studies are needed to precisely determine the mechanism of induction of the secreted VEGF in ANDV-infected cells.
Not only does hantavirus infection of endothelial cells result in secretion of VEGF (as shown here), but previous studies also demonstrated increases in permeability of these cells relative to uninfected endothelial cells in response to exogenously added VEGF (12
). The synergy between VEGF production by hantavirus-infected endothelial cells and the sensitization of these cells to the effects of VEGF could result in an amplifying cascade that leads to enhanced endothelial cell leakage.
What happens in vivo
is still poorly understood. The progression to HPS is possibly a multifactorial process with contributions from other elements of the immune response to the viral infection (19
). Is the induction of VEGF from the vascular endothelium enough to cause pulmonary edema? Pulmonary edema is a rapidly evolving process (24
). Studies of animal models of both high-permeability and cardiogenic pulmonary edema have shown increases in VEGF expression and its linkage to the vascular leakage (24
). In one of the initial studies characterizing the role of VEGF in pulmonary edema, Kaner and colleagues demonstrated that overexpression of VEGF in mouse lungs induced high-permeability pulmonary edema (20
). Here, we have shown that human primary lung endothelial cells infected with ANDV secrete VEGF at early times postinfection. We have also found increased secretion of VEGF from ANDV-infected primary human macrophages (unpublished data). Additional VEGF could be secreted by ANDV-activated T cells and platelets in vivo
). It is possible that VEGF from such sources would achieve high concentrations in the microvasculature of the lung. The increase in vascular permeability observed solely in response to virus infection is unlikely to be sufficient to be the whole explanation for HPS. However, virus infection per se
will initiate vascular leakage and the cascade of cell signaling events which lead to hyperpermeability and disease.
Angiopoetin 1 is known to counteract induced increases in vascular permeability resulting from physiologic levels of VEGF (11
). Interestingly, reduced expression of angiopoetin 1 has been recently reported in patients with HFRS (19
). This may represent an additional means by which endothelial cell hyperpermeability could be induced during hantavirus infection in humans.
While the number of HPS cases analyzed in our study is limited, the finding that the HPS patient sera had much higher free VEGF levels than those seen in the non-hantavirus-infected control patients correlated well with our in vitro
experimental results and suggested that the increased levels of VEGF seen in the sera of HPS patients may be linked to the vascular leakage typical of HPS cases. In dengue hemorrhagic fever, levels of free secreted VEGF and VEGF-R2 have been shown to correlate with the severity of the disease (41
). Analysis of a greater number of HPS cases and more precise clinical records detailing the severity of disease will allow determination of whether a similar correlation exists for HPS. A complementary approach will be to analyze the significance of VEGF in disease development in the Syrian hamster animal model for HPS induced by ANDV infection (17
The novel aspect and importance of the study presented here is the demonstration of a direct virus effect on vascular endothelium which had been overlooked in previous studies. This observation modifies the established paradigm that HPS is solely an immune-modulated disease. We demonstrate for the first time that ANDV and to a lesser extent SNV infection of human primary lung endothelial cells directly results in the downregulation of VE-cadherin, and this downregulation correlates with increased secretion of VEGF and increased endothelial cell permeability. Our data showing that antibody neutralization of VEGF-R2 blocks virus-induced VE-cadherin downregulation implicates VEGF-R2 in this process. Identification of the precise steps leading to hantavirus-induced endothelial cell leakage will facilitate the development of novel immunotherapeutics for the treatment of patients with HPS.