ANDV causes hantavirus pulmonary syndrome (HPS), has a 40% mortality rate and is reportedly spread from person to person. ANDV primarily infects endothelial cells (ECs) and HPS is characterized by thrombocytopenia, hypoxia and acute pulmonary edema that leads to respiratory insufficiency. ANDV infection of ECs provides a primary means for hantaviruses to alter vascular permeability. Although ECs are not lysed by ANDV infection, ECs line capillaries of vast pulmonary alveolar beds and regulate capillary integrity. Hypoxia induces high altitude pulmonary edema through the induction of vascular endothelial growth factor (VEGF), and this response is transcriptionally directed by the hypoxia inducible factor 1? (HIF1?). ECs secrete VEGF and VEGF in turn activates VEGFR2 receptors on ECs in an autocrine and paracrine manner. VEGFR2 activation directs the disassembly of EC adherens junctions and induces capillary permeability. Interestingly, VEGF further induces transcription of HIF1?, forming a HIF1? -VEGF amplification loop that enhances EC responses to hypoxia and further increases capillary permeability. We have shown that ANDV infection of ECs dramatically enhances the permeability of ECs in response to VEGF. Consistent with this, we have recently determined that the pulmonary edema fluid of HPS patients contains high levels of VEGF. HPS patient hypoxia combined with the hypersensitivity of infected ECs to VEGF, provides a means for amplifying HIF1? -VEGF responses that contribute to pulmonary edema. We propose to study the role of hypoxia on ANDV infected ECs and define ANDV proteins and cellular responses that contribute to dramatic increases in EC permeability. Hantaviruses also cause thrombocytopenia and we have recently reported that quiescent platelets are selectively recruited to the surface of ANDV infected ECs, but not cells infected by the nonpathogenic hantavirus TULV. Platelet adherence to ANDV infected ECs is dependent on cell associated ANDV attachment to ?3 integrin receptors 3 days p.i. However, platelets are normally activated by adherence to ECs, and the mechanism by which platelets remain quiescent following binding to ANDV infected ECs remains to be determined. Interestingly, either inducing or preventing platelet activation can result in thrombocytopenia. Platelet activation is inhibited by platelet endothelial cell adherence molecule-1 (PECAM-1) and PECAM-1: 1) is activated by VEGF, 2) negatively regulates the activation of platelet integrins and 3) stimulates prostacyclin release from ECs. Curiously, hypoxia induces prostacyclin synthase in ECs and prostacyclin is a potent inhibitor of platelet activation. Further, adherence of platelets to the EC lining of alveolar capillaries could both sequester platelets and alter normal gas exchange, causing or exacerbating hypoxia. Although it is unclear whether ANDV activates PECAM-1 and induces prostacyclin, these findings are consistent with amplified HIF1? -VEGF responses of hypoxic ANDV infected ECs. Here we will investigate the mechanism by which ANDV and hypoxia induce EC responses that direct platelet adherence and quiescence. The goal of this project is to evaluate EC responses to hypoxia following ANDV infection or ANDV protein expression. A Lentivirus-puromycin selection system will be used to express ANDV proteins within primary human ECs and used to define the primary mechanisms by which ANDV and hypoxia increase permeability, alter EC adherence and induce platelet quiescence. These studies provide a principal understanding of the role of hypoxia on EC and platelet functions that contribute to ANDV induced HPS disease.

Public Health Relevance

ANDV causes hantavirus pulmonary syndrome (HPS), resulting in fluid in the lungs that causes respiratory distress and has a 40% mortality rate. ANDV infects the endothelial cell lining of capillaries which normally limits fluid leakage termed edema. We investigate the mechanism by which ANDV causes fluid leakage and the role of low oxygen levels in inducing endothelial cell responses that increase leakage. These studies are likely to identify the mechanism of leakage and provide therapeutic approaches for reducing ANDV disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Virology - A Study Section (VIRA)
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Cassetti, Cristina
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State University New York Stony Brook
Schools of Medicine
Stony Brook
United States
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Mackow, Erich R; Dalrymple, Nadine A; Cimica, Velasco et al. (2014) Hantavirus interferon regulation and virulence determinants. Virus Res 187:65-71
Gorbunova, Elena E; Gavrilovskaya, Irina N; Mackow, Erich R (2013) Slit2-Robo4 receptor responses inhibit ANDV directed permeability of human lung microvascular endothelial cells. Antiviral Res 99:108-12