Figure 1.
Formation of capillary-like vessels in vitro.
HUVEC and PaSMC (A & C) or HUVEC and hMSC (B & D) were plated and co-cultured for 5 days to form in vitro capillaries. Cells were fixed and processed for scanning electron microscopy. (A) Capillary tubes were long and branched. Size bar = 10 µm. (B) EC tubes (arrow) were stabilized and wrapped by additional cells that were presumably hMSC-differentiated cells (asterick). Size bar = 10 µm. (C) Cells were fixed and stained with antibodies specific for SMA (brown) and COL18A1 (blue) or (D) vWF (red) and COL18A1 (green).
Figure 2.
Hantavirus infection of in vitro capillaries.
HUVEC and PaSMC were plated and co-cultured for 3 days to form in vitro capillaries. Samples were then mock-infected or infected with HTNV or ANDV. (A) Cells were fixed and stained with antibodies specific for vWF (red), hantavirus N (green), and nuclei (DAPI). Arrows indicate hantavirus-infected HUVEC and arrowheads indicate hantavirus-infected PaSMC (10×). (B) PaSMC were mock-infected or infected with HTNV or ANDV for 3 days. Cells were fixed and stained with antibodies to SMA (red), hantavirus N (green), and nuclei (DAPI) (20×). (C) PaSMC were mock-infected or infected with HTNV and ANDV for 4 days. Whole cell lysates were harvested on days 1, 2, 3, and 4 and prepared for immunoblotting. Separated proteins were transferred to PVDF membranes and probed with antibodies to detect hantavirus N or GAPDH.
Figure 3.
Hantavirus infection of in vitro capillaries does not alter VE-cadherin or VEGF levels.
HUVEC and PaSMC were plated and co-cultured for 3 days to form in vitro capillaries then mock-infected or infected with HTNV or ANDV. (A) Cells were lysed and prepared for immunoblotting. Separated proteins were transferred to a PVDF membrane and probed with antibodies that detect VE-cadherin, hantavirus N, or GAPDH. (B) Supernatants were collected and analyzed by ELISA to measure concentrations of VEGF. Data are represented as mean +/− SEM, n = 4.
Figure 4.
Liberation of bradykinin on hantavirus-infected in vitro capillaries, PaSMC, and HUVEC.
Capillaries, PaSMC, and HUVEC were plated separately and mock-infected or infected with HTNV or ANDV for 3 days. Cells were treated with 1 µM HOE 140 and 5 µM Trandolapril and incubated with 50 nM of FXII, PK, and HK in buffer for 1 h at 37°C. Supernatants were collected and analyzed using a bradykinin enzyme immunoassay. As controls, FXII, PK, and HK were also tested individually for non-specific binding in the immunoassay. Data are represented as mean +/− SEM, n = 6. ***p≤0.001, ****p≤0.0001.
Figure 5.
Cleavage of HK on hantavirus-infected HUVEC.
HUVEC were plated and mock- infected or infected with HTNV or ANDV for 4 days. 50 nM of HK in buffer was added to the monolayer and incubated for 1 h at 37°C. The cells were incubated an additional 1 h with (A) 50 nM FXII and 50 nM PK in the absence or presence of 1 µM CTI or (B) 50 nM PK. To examine cleavage of HK, cells were lysed and prepared for immunoblotting. Full length, heavy, and light chain fragments of HK, hantavirus N, or GAPDH antibodies were used to detect protein. Levels of full length HK were analyzed by densitometery. (C) Samples were examined by immunoblotting to assess the levels of PRCP and HSP90 in mock-, HTNV-, and ANDV-infected HUVEC.
Figure 6.
Enzymatic activity of FXIIa and KAL on hantavirus-infected HUVEC.
HUVEC cells were plated and mock-infected or infected with HTNV or ANDV for 4 days. 20 nM of HK in buffer was added to the monolayer and incubated for 1 h at 37°C. To measure enzyme activity cells were washed and incubated with 20 nM FXII, 20 nM PK, and 0.8 mM S2302 for 1 h at 37°C. The liberation of paranitroanilide (pNA) from substrate (S2302) was measured at 405 nm. As controls, FXII, PK, and HK were also added individually to wells. Data are represented as mean +/− SEM, n = 12. ****p≤0.0001.
Figure 7.
Kallikrein-kinin system activation and changes in permeability of hantavirus-infected HUVEC.
Mock-, HTNV-, or ANDV-infected HUVEC were trypsinized, seeded onto ECIS chamberslides, and cultured until confluent. Media were removed and replaced with phenol red free EBM containing Zn2+. After an equilibration, 0.2 ml of phenol red free EBM containing Zn2+, FXII, PK, and HK (100 nM each) were added to cells (time zero) (black lines) (A). To measure inhibition of activation, some samples were treated with CTI (1 µM) (blue line), PKSI-527 (5 µM) (red line), or HOE 140 (1 µM) (green line) simultaneously with factors (B, C, & D). Real time measurements frequency measurements were taken throughout the assay.
Figure 8.
FXII binding and autoactivation on hantavirus-infected HUVEC.
HUVEC were plated and mock-infected or infected with HTNV or ANDV for 4 days. (A) 100 nM of FXII in buffer was added to trypsinized cells in suspension and incubated for 1 h at 37°C. To examine FXII protein levels, cells were lysed and prepared for immunoblotting. FXII, hantavirus N, or GAPDH antibodies were used to detect protein. (B) To measure conversion of FXII to FXIIa, cells were washed and incubated with 25 nM FXII in the absence or presence of 25 nM PK and 0.8 mM S2302 for 2 h at 37°C. The liberation of paranitroanilide (pNA) from substrate (S2302) was measured at 405 nm. Data are represented as mean +/− SEM, n = 6. ****p≤0.0001.
Figure 9.
Kallikrein-kinin system and BK inhibitors.
A schematic illustration of three inhibitors that impact BK-mediated increase in endothelial cell permeability. CTI (corn trypsin inhibitor) inhibits FXIIa; PKSI-527 (plasma kallikrein specific inhibitor) inhibits KAL activity; Icatibant (HOE 140) is a peptidomimetic drug consisting of ten amino acids, which is a selective and specific antagonist of BKB2 receptors and is used in humans under orphan drug status for symptomatic treatment of hereditary angioedema.