VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability

Abstract

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ∼70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.

FIG. 1.

Src knockdown inhibits ANDV-induced EC permeability. (A) Endothelial cells were transfected with Src or scrambled siRNA, and Src mRNAs were analyzed by qRT-PCR (69). mRNA levels were quantitated and standardized to GAPDH mRNA levels and presented as a percentage of controls. (B) Endothelial cells were plated on Transwell inserts and infected at an MOI of 0.5 in triplicate with ANDV. One day postinfection (p.i.), cells were transfected with control or Src-specific siRNAs using SureFECT siRNA transfection reagent. Three days p.i., VEGF (100 ng/ml) and FITC-dextran were added to medium in the upper chamber, and the presence of FITC-dextran in the lower chamber was quantitated at indicated times (26, 27, 31). The percent change in FITC-dextran over controls is presented as a measure of EC monolayer permeability (27).

FIG. 2.

IC₅₀s of compounds that block ANDV-induced EC permeability. The concentrations of VEGFR2 and SFK inhibitors required to inhibit ANDV-induced endothelial cell permeability by 50% (IC₅₀s) were determined. Endothelial cells were ANDV infected, and 3 days postinfection the permeability of cells in response to VEGF addition was determined (27) in the presence or absence of increasing amounts of kinase inhibitor. Endothelial cell permeability was determined as described for Fig. 1, and the permeability of ANDV- versus mock-infected controls was determined to be 100%. The effect of inhibitors is presented as the percentage of ANDV-induced permeability of inhibitor-treated monolayers 3 days postinfection and 30 min post-VEGF and FITC-dextran addition (27). Data are derived from at least three different experiments for each condition.

FIG. 3.

VEGFR2-Src inhibitors block ANDV-induced permeability. Endothelial cells were plated on vitronectin-coated Transwell inserts and infected at an MOI of 0.5 in triplicate with ANDV. Three days postinfection, the permeability of ANDV- and mock-infected endothelial cell monolayers was determined as described for Fig. 1 (27) at indicated times in the presence or absence of the kinase inhibitors pazopanib (A), dasatinib (B), bosutinib (C), PP1 (D), and Src inhibitor 1 (E). The percent change in FITC-dextran over controls is presented as a measure of EC monolayer permeability (27). Data are derived from two independent experiments performed in triplicate with comparable results.

FIG. 4.

Dasatinib and pazopanib block VE-cadherin internalization in ANDV-infected HUVECs. Human endothelial cells were mock infected or infected with pathogenic ANDV at an MOI of 0.5. Three days postinfection, endothelial cells were pretreated with kinase inhibitors dasatinib (1 nM/liter) and pazopanib (100 nM/liter) or left untreated for 15 min prior to VEGF addition (100 ng/ml, 2 h). Cells were incubated with an antibody to the extracellular domain of VE-cadherin (BV9; Clonetics) at 4°C for 1 h (23, 26, 31). After antibody removal and washing, cells were incubated at 37°C in 5% CO₂ (31) (1 h) to permit intracellular trafficking of antibody-bound VE-cadherin (23). Cells were subsequently washed with a mild acid solution (2 mM PBS-glycine, pH 2.0; three times for 5 min each) in order to remove VE-cadherin antibody that was not internalized or were left untreated (23, 26, 31). Cells were paraformaldehyde fixed and Triton X-100 permeabilized prior to incubation with an FITC-tagged anti-mouse secondary antibody and examined on an Olympus IX51 microscope. Data are presented as a percentage of cells with internalized VE-cadherin in ANDV-infected ECs pretreated only with VEGF (control) versus mock-treated infected cells (n = 500; P < 0.001) (31). Data represent the results from two independent experiments.