The Role of the Endothelium in HPS Pathogenesis and Potential Therapeutic Approaches

American hantaviruses cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect endothelial cells and cause dramatic changes in barrier functions of the endothelium without disrupting the endothelium. Instead hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions of capillaries. The endothelium of arteries, veins, and lymphatic vessels is unique and central to the function of vast pulmonary capillary beds, which regulate pulmonary fluid accumulation. The endothelium maintains vascular barrier functions through a complex series of redundant receptors and signaling pathways that serve to both permit fluid and immune cell efflux into tissues and restrict tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to alter capillary permeability but also defines potential therapeutic targets for regulating acute pulmonary edema and HPS disease. Here we discuss interactions of HPS causing hantaviruses with the endothelium, potential endothelial cell-directed permeability mechanisms, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.

The endothelium of capillaries, veins, and lymphatic vessels are unique and central to discrete functions of vast 2 Advances in Virology renal and pulmonary capillary beds [42,[52][53][54]. Nonlytic viral infection of ECs may disengage one or more fluid barrier regulatory mechanisms, thereby increasing vascular leakage or fluid clearance and as a consequence result in tissue edema [55][56][57][58][59][60]. Although the edematous accumulation of interstitial fluids can result from increased endothelial permeability, a decrease in lymphatic vessel clearance of tissue fluids is also a cause of edema and regulated by unique lymphatic endothelial cells (LECs) [42,53,54,61]. Vascular permeability induced by nonlytic viruses is likely to be multifactorial in nature, resulting from virally altered EC responses, immune cell and platelet functions, hypoxia, or a collaboration of dysregulated interactions that impact normal fluid barrier function [15-18, 20, 27, 62-64]. Failure of the endothelium to regulate fluid accumulation in tissues has pathologic consequences and during HPS results in localized vascular permeability and acute pulmonary edema that contribute to cardiopulmonary insufficiency [4][5][6]9]. Here we focus on the mechanisms by which HPS causing hantavirus infection of ECs induces vascular permeability and acute edema and discuss potential therapeutic mechanisms that may stabilize the endothelium.

Hantavirus Infection and HPS Disease
Hantaviruses are enveloped, tripartite, negative-sense RNA viruses and form their own genus within the Bunyaviridae family [14,65]. Hantaviruses are the only members of the Bunyaviridae that are transmitted to humans by mammalian hosts, and hantaviruses contain highly divergent RNA and protein sequences, which are likely the result of coadaptation with their hosts [13,14,[66][67][68]. Single genes have been exchanged between closely related HPS causing hantaviruses [69]; however, gene reassortment has not permitted the discovery of pathogenic determinants and reverse genetics approaches have thus far proven elusive.
Hantavirus antigen is found predominately in vast pulmonary capillary EC beds but is present in ECs within lymph nodes and throughout the body [8,9,80]. However, cytopathic effects are not evident following hantavirus infection of ECs in vitro or in vivo [9,16,82]. Histologically, the heart, kidneys, brain, and adrenals are grossly normal with pulmonary alveoli filled with acellular proteinaceous fluid, yet the alveolar epithelium remains intact [4-6, 8, 9]. The most striking HPS findings are edematous lungs with up to 8 liters of pleural edema [5,6,8,9]. Pulmonary edema fluid contains few leukocytes, is largely serous in nature, and is consistent with the nearly complete loss of an alveolar capillary fluid barrier [4][5][6]9]. The lack of disrupted endothelium during HPS is similar to edematous pulmonary responses observed in patients with high altitude-induced pulmonary edema [40,60,[110][111][112]. The rapid onset of edematous symptoms during hantavirus infection [6] further suggests the importance of targeting vessel stability in regulating highly lethal hantavirus disease.

Vascular and Lymphatic Endothelium: Control of Vascular Fluid Barrier Functions
The endothelium lines a series of discrete vessel types that conduct fluids to and from tissues, directs the transfer of nutrients, wastes and oxygen and coordinates tissue responses to changing conditions and pathogens [22,24,27,54,59,[113][114][115][116][117][118][119]. Vascular ECs serve mainly as a conduit in the lining of high pressure arteries but take on a variety of fluid and cellular barrier functions in low pressure veins and capillaries that innervate organs and tissues [54]. Lymphatic vessels have a primary role in draining fluid, proteins, and immune cells from tissues and returning these components to the venous circulation [42,[52][53][54]114]. Depending on their location, lymphatic vessels serve discrete fluid barrier and regulatory functions, keeping pulmonary alveolar spaces dry and clearing fluid influx from the lungs [54,61,120]. These diverse EC settings require discrete EC functions to effect exchange within large capillary beds of the kidney, liver, and lung [27,54]. The EC lining is responsible for controlling vessel damage through a complex mechanism of negative regulation, rapid response and proliferation [22,24,40,53,120,121]. Unless activated, the endothelium normally prevents immune cells and platelets from adhering to its surface [22,122]. Endothelial quiescence is maintained by several mediators, while vascular injury activates clotting factors, platelets, and ECs resulting in the recruitment of platelets to the endothelium [22]. ECs also have angiogenic roles migrating and proliferating to fill in endothelial cell gaps or to rebuild damaged vessels [29,123]. EC migration and vessel remodeling requires changes in cell adherence within the endothelium, and carefully orchestrated receptor signaling responses are required to accomplish this without causing edema.
The endothelial fluid barrier is primarily derived from unique adherens junctions (AJs) composed of an EC-specific vascular-endothelial cadherin (VE-cadherin) [25,30,45,96,117,119,124]. EC barrier functions are increased by the presence of cell surface VE-cadherin and reduced by the dissociation and internalization of VE-cadherin [25,30,117]. Phosphorylated VE-cadherin is internalized by its interaction with intracellular actin complexes and this process is regulated by a variety of cellular receptors and intracellular signaling pathways [25,124,125]. VE-cadherin phosphorylation is downregulated by an EC-specific phosphatase (VE-phosphatase) [124,125] and several pathways that either directly or indirectly induce AJ assembly and EC integrity by returning VE-cadherin to an unphosphorylated resting state [25,117,125]. Chemokines, cytokines, and growth factors indirectly act on EC adherens junctions to increase vascular permeability and thus have the potential to contribute to pathogenic vascular leakage [27,29,96].
VEGF was originally discovered as a potent vascular permeability factor that induces acute edema [29,132]. VEGF reportedly acts within 0.5 mm of its release [133], and circulating soluble VEGFRs prevent VEGF from systemically permeabilizing vessels [39,132]. VEGF is induced by hypoxia, and reduced oxygen levels at high altitudes cause high-altitude-induced pulmonary edema (HAPE) [35,40,113]. This results from activating the hypoxia-induced transcription factor-1α (HIF-1α), which senses oxygen levels and transcriptionally induces VEGF [59,128,134,135]. VEGF further upregulates HIF-1α, forming an autocrine loop, which amplifies hypoxia-mediated VEGF responses and causes HAPE [59,113,136,137]. Although this response fosters increased gas exchange, in continued lowoxygen environments these cellular responses, instead cause pulmonary edema and in HAPE, respiratory distress [40,110,113,128].

Hantavirus Binding to Inactive α v β 3 Integrins Regulates EC Functions and Permeability. Pathogenic hantaviruses bind to inactive, basal conformations of α v β 3 integrin receptors on ECs, while nonpathogenic hantaviruses interact with discrete integrins
One paper suggests that ANDV-infected ECs transiently induce VEGF secretion, VE-cadherin degradation, and increased EC monolayer permeability [21]. However, several studies indicate that monolayers of hantavirus-infected ECs are not permeabilized by infection alone [16,17,82] and instead indicate that pathogenic hantavirus infected ECs are hyperpermeabilized by VEGF [16]. Collectively, these findings demonstrate that cell surface hantaviruses alter normal EC functions that control VEGF-directed vascular permeability [15-18, 62, 153].

Potential Role of LECs in Hantavirus Edema.
Pulmonary lymphatic vessels are responsible for clearing fluid from alveoli and providing a semidry state that permits gas exchange [52,54]. Failure of lymphatic vessels to clear fluids results in lymphedema and suggests an additional mechanism for hantavirus-infected LECs to contribute to acute pulmonary edema during HPS [42,53,54,154]. Analysis of pathology samples from HPS patients indicates that hantavirus antigen is present in LECs of patient lymph nodes [8,9,80]. Although less is known about LECs, as described above, LECs express unique cell surface receptors and their integrity is regulated by both VEGF-A and VEGF-C [42,53,54,61]. Interestingly, LEC VEGFR3 receptors respond to VEGF-C and are associated with reduced tissue edema [42,61], while inhibiting VEGFR3 signaling results in lymphedema [42,131]. Although a recent publication indicates that ANDV infects LECs and alters LEC barrier functions [155], the role of lymphatic vessels and LEC responses remains to be investigated in HPS patients.

Hantavirus-Endothelial Edemagenic Mechanisms.
Prominent pulmonary and renal dysfunction are components of both HPS and HFRS diseases and likely stem from hantavirus infection of ECs, which line vast alveolar and renal capillary beds [4-6, 8, 9, 156, 157]. HPS patients are often young adults that arrive at hospitals in acute respiratory distress [4]. Acute pulmonary edema is a hallmark of HPS, with bilateral fluid infiltrates accumulating at up to a liter per hour resulting in pulmonary insufficiency and patient hypoxia during a critical phase of the disease [4,6,8,9]. The cause of acute edema following hantavirus infection is likely to be multifactorial [6, 15-18, 64, 153, 155, 158] but revolves around the ability of the hantaviruses to infect ECs within alveolar capillary beds that normally regulate edema and gas exchange within the lung.
Clues to the mechanism of hantavirus-induced edema come from disparate findings on the role of hypoxia in acute pulmonary edema and the role of α v β 3 and VEGFR2 EC responses, which are uniquely altered by pathogenic hantaviruses [6,15,16,20,155]. Hypoxia is a prominent component of HPS patients and directs VEGF secretion from endothelial, epithelial, and immune cells [5,6,8,9]. Consistent with the enhanced permeability of hantavirus-infected ECs in response to VEGF [16], HPS may be the result of hypoxia-induced VEGF that leads to acute pulmonary edema and may be exacerbated by reduced lymphatic vessel fluid clearance [155]. In fact, HPS patient VEGF levels were markedly elevated in pulmonary edema fluid and PBMCs in acute early phases of HPS [159]. Although a demonstrated role for hypoxia in hantavirus-induced permeability has yet to be conclusively defined, the ability of extracorporeal membrane oxygenation (ECMO) to reduce HPS patient mortality [4,6] strongly suggests a role for hypoxia and VEGF in the acute pulmonary edema of HPS patients.

Animal HPS Model
Only ANDV infection of Syrian hamsters (Mesocricetus auratus) serves as a model of hantavirus pathogenesis that mimics Advances in Virology 5 human HPS in onset symptoms and lethal acute respiratory disease [19,160,161]. Inoculation of Syrian hamsters with ANDV, but not SNV or other HPS causing hantaviruses, induces pathology approximating human disease. ANDV causes a fatal infection of Syrian hamsters with an LD 50 of 8 plaque-forming units. The disease is characterized by large pleural effusions, congested lungs, and interstitial pneumonitis in the absence of disrupted endothelium [19,160,161]. The onset of pulmonary edema coincides with a rapid increase in viremia on day 6, and large inclusion bodies and vacuoles in ultrastructural studies of infected pulmonary ECs [160,161]. Viral antigen was localized to capillary ECs, alveolar macrophages, and splenic follicular marginal zones populated by dendritic cells. Interestingly, depletion of CD4 and CD8 T-cells had no effect on the onset, course, symptoms, or outcome of ANDV infection and indicates the absence of T-cell responses [19]. Consistent with the potential involvement of β 3 integrins and VEGF in this process, ANDV binds to conserved residues within PSI domains of both human and hamster β 3 integrins [20,79]. Thus the mechanism of pathogenesis caused by ANDV is consistent with hypoxia-VEGF-directed acute pulmonary edema that occurs in the absence of T-cellmediated pathology [19]. These findings differ from a report associating T-cell responses with HPS disease, although the same data support a lack of T-cell involvement, since half of HPS patients had no elevated T-cell responses regardless of disease severity [64]. Observed T-cell responses may instead correlate with viral clearance [63,162]. The mechanism of pathogenesis may be further elucidated by studies in Syrian hamsters and thus provides a model of ANDV pathogenesis that permits the evaluation of therapeutics that target barrier functions of the endothelium.

Targeted Therapeutic Approaches for Stabilizing the Endothelium
Currently, there are no effective therapeutics for hantavirus infections or disease. Antiviral effects of interferon or the nucleoside analog ribavirin are only effective prophylactically or at very early times postinfection [14,163]. They appear to target early viral replication but neither is effective 1-2 weeks postinfection after the onset of HPS symptoms [4][5][6]163]. An alternative approach against viruses with a long disease onset may be to therapeutically target the acute pathologic response instead of viral replication. Since hantaviruses infect and alter fluid barrier functions of the endothelium, targeting EC responses that transiently stabilize the vasculature has the potential to reduce the severity and mortality of HPS [50,129,164]. This approach also has the advantage of being implemented at the onset of symptoms where antiviral approaches appear to be ineffective [163]. Intracellular signaling pathways coordinately regulate the adherence of ECs to the extracellular matrix, anchor receptors to cytoskeletal elements, and induce growth factor directed migration, proliferation and permeability responses [18,35,41,43,50,96,116,165,166]. The complexity of VEGF induced permeability is further demonstrated by the reported ability of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) signaling responses, to block VEGF-induced microvascular permeability [167][168][169][170][171]. This multifactorial coordination indicates why so many factors are capable of permeabilizing or stabilizing the endothelium and rationalizes their potential roles in pathogen-induced capillary leakage.
Antibody to VEGFR2 reportedly suppresses VEGFinduced pulmonary edema and suggests the potential of therapeutically antagonizing VEGFR2-Src-VE-cadherin signaling pathways as a means of reducing acute pulmonary edema during HPS [18,25,39,50,[172][173][174][175][176]. Several wellstudied VEGFR2 and Src inhibitors are in human clinical trials or are used therapeutically to treat human cancers and have the potential to reduce the severity of viral permeability-based diseases [18,42,50,173,174,[177][178][179]. In vitro, angiopoietin-1 (Ang-1), sphingosine-1-phosphate (S1P), pazopanib, and dasatinib inhibited EC permeability directed by pathogenic hantaviruses [16,18]. Ang-1 is an ECspecific growth factor that transdominantly blocks VEGFR2directed permeability in vitro and in vivo by binding to Tie-2 receptors [180][181][182][183]. S1P is a platelet derived lipid mediator, which enhances vascular barrier functions by binding to Edg-1 receptors on the endothelium [47,172,173,179,184], while pazopanib and dasatinib are drugs that inhibit VEGFR2-Src signaling [174,185]. Pazopanib, dasatinib, and the S1P analog FTY720 are already in clinical trials or used clinically for other purposes [34,186]. Targeting EC responses provides a potential means of stabilizing HPS patient vessels and reducing edema. The use of S1P receptor agonists has also been shown to regulate the pathogenesis of influenza virus infection by acting on ECs and reducing immune cell recruitment and entry into the lung [172]. These findings suggest the targeting of EC functions as a means of increasing capillary barrier functions and regulating immune responses that contribute to viral pathogenesis.
The regulation of additional EC receptors that stabilize interendothelial cell AJs and fluid barrier functions of the endothelium may be considered as therapeutic targets. The Robo4 receptor has been shown to inhibit VEGFR2 responses, stabilize vessels and block vascular permeability [48,148,152]. This new potential target is highly expressed by lung microvascular ECs and is currently being evaluated as a therapeutic for a variety of vascular disorders [149,152]. However, Robo4 directed stability of interendothelial cell junctions may also be applicable to reducing HPS severity.
Several additional EC receptors that bind to VEGFR2 ectodomains positively or negatively regulate α v β 3 -VEGFR2 functions and may provide additional therapeutic targets for regulating vascular permeability. Potential responses which need to be investigated as therapeutic targets include: NRP1, Syndecan1 (sdc1), and the insulin-like growth factor1 receptor (IGF1R), which are recruited to α v β 3 ectodomain complexes [49,141,142,144,175,187,188]: Surfen, a heparan sulfate containing protein that reportedly blocks EC permeability [189], and Fibulin-5, a matrix protein that reportedly promotes EC adherence by binding α v β 3 and is associated with emphysema [190][191][192]. However, inhibiting β 3 receptors that are present on both platelets and ECs may exacerbate permeability and thus the choice of therapeutic targets is likely to be critical to increasing fluid barrier functions of the endothelium. Targeting the VEGFR2 axis that regulates EC permeability may be a central mechanism for stabilizing the endothelium and reducing the severity of HPS [127,145,175,193].
These findings suggest a plethora of targets that may regulate virally induced vascular permeability and which are already clinically approved for other indications. Moreover, targeting these responses may be broadly applicable to reducing the severity of HFRS and a wide range of viral infections that impact the endothelium and cause edematous diseases.

Future Directions and Conclusions
The endothelium plays a fundamental role in vascular disease, and stabilizing the vasculature needs to be evaluated as a means for reducing the severity and mortality of viral vascular diseases. This is especially important for viral infections that cause disease 1-2 weeks after infection, at time points when antiviral approaches are no longer viable. The ability of hantaviruses to infect LECs and alter normal fluid clearance from tissues needs to be investigated and provides a unique target and mechanism for reducing edema that has yet to be considered in HPS disease. The ability of the endothelium to regulate platelet functions, complement activation, and immune responses should also be considered as central targets for reducing the severity of viral hemorrhagic and edematous diseases.