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Abstract
To understand the molecular mechanism of hemorrhagic tendency represented in hemorrhagic fever with renal syndrome (HFRS), the effect of Hantaan virus (HTNV) on the von Willebrand factor (vWF) was observed in human umbilical vein endothelial cells (HuVECs). An immunofluorescence assay (IFA) showed a significant reduction of the vWF in the cytoplasm of HTNV-infected HuVECs. The amount of vWF protein in HTNV-infected HuVECs was reduced to 86, 49, 67, and 42% of those in control HuVECs at 1st, 3rd, 5th, and 7th days of post infection (d.p.i.), respectively. However, there were no significant differences in the vWF mRNA expression in both groups at all time courses by reverse transcriptase polymerase chain reaction (RT-PCR). The amounts of secreted vWF in the culture supernatants of the HTNV-infected HuVECs were 79, 87, 83, and 82% of those in control HuVECs at 1st, 3rd, 5th, and 7th d.p.i., respectively. These results indicated that the reduction of vWF by HTNV was regulated at post-transcriptional level and this might delay the coagulation process on the site of HTNV infection, thus leading to hemorrhage in HFRS.
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Figure 1.
Expression of von Willebrand factor (vWF) in HuVECs. The vWF is shown in red, HTNV in green, and the cell nuclei in blue. The vWF and HTNV were double immunostained with rabbit polyclonal vWF antibody and HFRS patient serum as primary antibodies, followed by incubation with donkey anti-rabbit IgG coupled to Cy3, as well as goat anti-human IgG coupled to FITC, both of which were used as secondary antibodies in the control HuVEC (A) and HTNV-infected HuVEC (B, C). Nuclei were counterstained with Hoechest 33258 (C).
Figure 2.
HTNV reduces vWF protein levels in the HuVECs. The control and HTNV-infected HuVECs were harvested at the indicated times (1, 3, 5, and 7 d.p.i.). (A) The cells were lysed in Laemmli's sample buffer, and the proteins were separated by SDS-PAGE and detected via immunoblotting using anti-vWF and anti-tubulin antibodies. (B) Total RNA was extracted from both the control and HTNV-infected HuVECs, and the levels of HTNV mRNA expression were assessed via RT-PCR.
Figure 3.
The expression of vWF mRNA was not altered by HTNV infection in HuVECs. HuVECs were either infected with HTNV or not infected, and the cells were harvested at the indicated times (1, 3, 5, and 7 d.p.i.). Total RNA was extracted from both the control and HTNV-infected HuVECs, and the levels of mRNA expression were assessed via RT-PCR.
Figure 4.
HTNV reduced the levels of vWF secretion. The culture supernatants were harvested at the indicated times (1, 3, 5, and 7 d.p.i.) from both the control and HTNV-infected HuVECs, and the amounts of vWF were quantified by ELISA. Each value represents mean ± SD of three independent experiments.
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