The Andes Virus Nucleocapsid Protein Directs Basal Endothelial Cell Permeability by Activating RhoA

ABSTRACTAndes virus (ANDV) predominantly infects microvascular endothelial cells (MECs) and nonlytically causes an acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every pulmonary MEC is infected, MECs are enlarged, and infection results in vascular leakage and highly lethal pulmonary edema. We observed that MECs infected with the ANDV hantavirus or expressing the ANDV nucleocapsid (N) protein showed increased size and permeability by activating the Rheb and RhoA GTPases. Expression of ANDV N in MECs increased cell size by preventing tuberous sclerosis complex (TSC) repression of Rheb-mTOR-pS6K. N selectively bound the TSC2 N terminus (1 to 1403) within a complex containing TSC2/TSC1/TBC1D7, and endogenous TSC2 reciprocally coprecipitated N protein from ANDV-infected MECs. TSCs normally restrict RhoA-induced MEC permeability, and we found that ANDV infection or N protein expression constitutively activated RhoA. This suggests that the ANDV N protein alone is sufficient to activate signaling pathways that control MEC size and permeability. Further, RhoA small interfering RNA, dominant-negative RhoA(N19), and the RhoA/Rho kinase inhibitors fasudil and Y27632 dramatically reduced the permeability of ANDV-infected MECs by 80 to 90%. Fasudil also reduced the bradykinin-directed permeability of ANDV and Hantaan virus-infected MECs to control levels. These findings demonstrate that ANDV activation of RhoA causes MEC permeability and reveal a potential edemagenic mechanism for ANDV to constitutively inhibit the basal barrier integrity of infected MECs. The central importance of RhoA activation in MEC permeability further suggests therapeutically targeting RhoA, TSCs, and Rac1 as potential means of resolving capillary leakage during hantavirus infections.IMPORTANCEHPS is hallmarked by acute pulmonary edema, hypoxia, respiratory distress, and the ubiquitous infection of pulmonary MECs that occurs without disrupting the endothelium. Mechanisms of MEC permeability and targets for resolving lethal pulmonary edema during HPS remain enigmatic. Our findings suggest a novel underlying mechanism of MEC dysfunction resulting from ANDV activation of the Rheb and RhoA GTPases that, respectively, control MEC size and permeability. Our studies show that inhibition of RhoA blocks ANDV-directed permeability and implicate RhoA as a potential therapeutic target for restoring capillary barrier function to the ANDV-infected endothelium. Since RhoA activation forms a downstream nexus for factors that cause capillary leakage, blocking RhoA activation is liable to restore basal capillary integrity and prevent edema amplified by tissue hypoxia and respiratory distress. Targeting the endothelium has the potential to resolve disease during symptomatic stages, when replication inhibitors lack efficacy, and to be broadly applicable to other hemorrhagic and edematous viral diseases.

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Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Journal of Virology ◽

10.1128/jvi.00576-10 ◽

2010 ◽

Vol 84 (14) ◽

pp. 7405-7411 ◽

Cited By ~ 53

Author(s):

Elena Gorbunova ◽

Irina N. Gavrilovskaya ◽

Erich R. Mackow

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Adherens Junctions ◽

Paracellular Permeability ◽

Cell Permeability ◽

Hantaan Virus ◽

Vascular Endothelial ◽

Vascular Endothelial Cadherin ◽

Andes Virus ◽

Endothelial Cell Permeability

ABSTRACT Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.

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Acute Pulmonary Edema after Polyethylene Glycol Intestinal Lavage in a Child

Annals of Pharmacotherapy ◽

10.1177/106002809302700905 ◽

1993 ◽

Vol 27 (9) ◽

pp. 1044-1047 ◽

Cited By ~ 14

Author(s):

Christopher M. Paap ◽

Robert Ehrlich

Keyword(s):

Polyethylene Glycol ◽

Respiratory Distress ◽

Pulmonary Edema ◽

Ventilatory Support ◽

Gastrointestinal Symptoms ◽

Acute Pulmonary Edema ◽

Severe Respiratory Distress ◽

X Ray ◽

Chest X Ray ◽

Outpatient Settings

OBJECTIVE: To report the case of an eight-year-old girl, without preexisting cardiac or renal disease, who developed acute pulmonary edema and severe respiratory distress after balanced electrolyte with polyethylene glycol (BE-PEG) intestinal lavage. CASE SUMMARY: During the nasogastric infusion of a one-liter dose of BE-PEG (OCL, Abbott), the patient experienced abdominal discomfort, gagging, vomiting, and coughing. After the nasogastric infusion, the patient again had emesis, developed tachypnea, intercostal retractions, and acute respiratory distress. She received oxygen and subsequently required intubation and ventilatory support. Physical examination revealed pulmonary congestion bilaterally but no signs of cardiac failure or sepsis. Chest X-ray revealed bilateral pulmonary edema. Ventilatory support was continued for 36 hours and the patient was extubated after two days. DISCUSSION: Enteral BE-PEG may have caused acute pulmonary edema secondary to aspiration or systemic fluid overload. Although the exact cause remains unknown, the close temporal onset of pulmonary edema after BE-PEG administration in an otherwise healthy child suggests a causal relationship. CONCLUSIONS: This case should alert clinicians to the potential for significant morbidity with BE-PEG solutions, particularly if used in outpatient settings. Patients who receive BE-PEG should be closely observed and monitored for potential aspiration, excessive infusion rates, and gastrointestinal symptoms to optimize efficacy and reduce morbidity.

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Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA

10.1101/2021.03.05.434186 ◽

2021 ◽

Author(s):

Elena E. Gorbunova ◽

Erich R. Mackow

Keyword(s):

Endothelial Cells ◽

Protein Kinase ◽

Pulmonary Edema ◽

Regulatory Protein ◽

Underlying Mechanism ◽

N Protein ◽

Inactive State ◽

C Terminus ◽

Andes Virus ◽

Protein Kinase Cα

AbstractAndes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs) causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients virtually every PMEC is infected, however the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase, RhoA, in primary human PMECs causing VE-Cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA, but co-precipitates RhoGDI (Rho GDP-dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C-terminus (69-204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N-terminus (1-69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and VEGF increase RhoA induced PMEC permeability by directing Protein Kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing Protein Kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV induced capillary permeability, edema and HPS.ImportanceHPS causing hantaviruses infect pulmonary endothelial cells causing vascular leakage, pulmonary edema and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses don’t lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34 phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC phosphorylated RhoGDI, synergistically enhancing hypoxia directed RhoA activation and PMEC permeability. Our data suggests inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.

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ACUTE PULMONARY EDEMA AND ELEVATED TROPONIN: WHAT IS THEIR SIGNIFICANCE? CLINICAL REVIEW

Brazilian Journal of Medicine and Human Health ◽

10.17267/2317-3386bjmhh.v4i2.923 ◽

2016 ◽

Vol 4 (2) ◽

Author(s):

Maria Das Neves Dantas da Silveira Barros ◽

Vander Weyden Batista De Sousa ◽

Isabelle Adjanine Borges De Lima ◽

Cecília Raquel Bezerra Marinho Nóbrega ◽

Isabelle Conceição Albuquerque Machado Moreira ◽

...

Keyword(s):

Heart Failure ◽

Respiratory Distress ◽

Pulmonary Edema ◽

Clinical Signs ◽

Myocardial Necrosis ◽

Acute Pulmonary Edema ◽

Cardiac Damage ◽

Atypical Symptoms ◽

Coronary Syndrome ◽

Coronary Events

Acute pulmonary edema (APE) is a clinical condition characterized by severe acute respiratory distress, frequently accompanied by crackling lung sounds and sudoresis. One classification system divides APE into cardiogenic or non-cardiogenic adult respiratory distress syndrome – ARDS. This study reviews cardiogenic APE, which is the most severe clinical presentation of heart failure (ADHF), and its relationship with the increase of troponin along with other factors such as abnormalities in the electrocardiogram (ECG) that may be mistaken for acute coronary syndrome (ACS). Atypical symptoms could occur in the presentation of ACS in 8,4% of cases, as has been shown in the Global Registry of Acute Coronary Events (GRACE): dyspnea (49%), sudoresis (26%), nauseas or vomiting (24%) and syncope (19%). The CK-MB enzyme was replaced by cardiac troponin (cTn) as the chosen marker of myocardial necrosis in the diagnosis of AMI in the late 90s. The cTn is a marker of cardiac damage, not just cardiac ischemia, and this may pose questions as whether there is a myocardium infarction or not. The positive results of a cTn should be interpreted considering the clinical signs of myocardial ischemia. The challenge remains and further studies are needed to aid in accurate diagnosis of both conditions (APE and ACS) as well as cases in which one results from the other because the role of coronary artery disease in acute heart failure has not been well studied in clinical trials.

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Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA

Journal of Virology ◽

10.1128/jvi.00396-21 ◽

2021 ◽

Author(s):

Elena E. Gorbunova ◽

Erich R. Mackow

Keyword(s):

Endothelial Cells ◽

Protein Kinase ◽

Pulmonary Edema ◽

Regulatory Protein ◽

Underlying Mechanism ◽

N Protein ◽

Inactive State ◽

C Terminus ◽

Andes Virus ◽

Protein Kinase Cα

Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs) causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients virtually every PMEC is infected, however the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase, RhoA, in primary human PMECs causing VE-Cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA, but co-precipitates RhoGDI (Rho GDP-dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C-terminus (69-204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N-terminus (1-69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and VEGF increase RhoA induced PMEC permeability by directing Protein Kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing Protein Kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV induced capillary permeability, edema and HPS. Importance HPS causing hantaviruses infect pulmonary endothelial cells causing vascular leakage, pulmonary edema and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses don't lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34 phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC phosphorylated RhoGDI, synergistically enhancing hypoxia directed RhoA activation and PMEC permeability. Our data suggests inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.

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