sVEGFR1 up-regulation via EGR1 impairs vascular repair in SFTSV-induced hemorrhage

Abstract

Hemorrhage is a major pathological manifestation of certain viral infections, such as severe fever with thrombocytopenia syndrome (SFTS), Ebola, Crimean-Congo hemorrhagic fever and Dengue. SFTS is an emerging viral hemorrhagic fever caused by the SFTS virus (SFTSV). Hemorrhage and angiogenesis dysfunction are key manifestations of SFTSV infection but the underlying mechanisms remain unclear. Here, we demonstrate that SFTSV infection increases soluble vascular endothelial growth factor-receptor 1 (sVEGFR1) secretion from monocytes/macrophages. Increased sVEGFR1 in the serum of SFTS patients is positively correlated with disease severity. Moreover, we show that SFTSV induces sVEGFR1 upregulation via early growth response gene 1 (EGR1), of which VEGFR1 is a downstream target. Serum from SFTS patients containing high levels of sVEGFR1 inhibit angiogenesis, which can be reversed by removal of sVEGFR1. Treatment of SFTSV-infected animals with sVEGFR1 neutralizing antibodies improves angiogenesis and prevents blood vessel leaks in vivo. In conclusion, we show that SFTSV infection induces sVEGFR1 secretion through EGR1 upregulation, thereby contributing to hemorrhage.

Keywords: EGR1; SFTSV; Vascular Dysfunction; Viral Hemorrhagic Fever; sVEGFR1.

Figure 1. SFTSV infection up-regulates sVEGFR1 expression in monocytes/macrophages.

(A) THP-1 cells were infected with SFTSV (MOI = 1) for 12, 24, 36, 48, and 60 h. SFTSV viral RNA (left) and sVEGFR1 mRNA (right) were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (SFTSV: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; sVEGFR1: ***P = 0.0002, ***P < 0.0001, ***P = 0.0001, ***P < 0.0001, ***P < 0.0001). (B) THP-1 cells were infected with SFTSV for 12, 24, 36, 48, and 60 h. Intracellular sVEGFR1 and the viral NP protein were measured by western blotting. sVEGFR1 was stained with a specific monoclonal antibody and GAPDH served as an internal control. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (ns = 0.2035, **P = 0.0043, ***P = 0.0003, ***P = 0.0002, *P = 0.0127). (C) THP-1 cells were infected with SFTSV for 12, 24, 36, 48 and 60 h. The secreted sVEGFR1 in cell supernatant was measured by capture ELISA. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (ND: Not Detected, ***P = 0.0002, **P = 0.0049, ***P < 0.0001, ***P < 0.0001). (D–H) THP-1 cells were infected with SFTSV for 12, 24, 36, 48 and 60 h at various MOIs (MOI = 0.1, 1, and 10). Viral RNA (left) and sVEGFR1 mRNA (right) were determined by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (12 h: left: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; right: *P = 0.0169, **P = 0.0087, ***P < 0.0001. 24 h: left: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; right: ***P = 0.0005, ***P < 0.0001, ***P = 0.0002. 36 h: left: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; right: ***P = 0.0004, ***P < 0.0001, ***P = 0.0002. 48 h: left: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; right: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001. 60 h: left: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; right: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001). (I–M) THP-1 cells were infected with SFTSV for 12, 24, 36, 48 and 60 h at various MOIs (MOI = 0.1, 1, and 10). sVEGFR1 in cell supernatant was measured by capture ELISA. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (12 h: ns = 0.0910, ns = 0.4550, ***P < 0.0001; 24 h: ns = 0.8551, ***P = 0.0002, ***P < 0.0001; 36 h: ns = 0.6919, ***P < 0.0001, ***P < 0.0001; 48 h: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; 60 h: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001). (N) Primary human monocyte-derived-macrophages were infected with SFTSV (MOI = 1) for 24 h. SFTSV viral RNA (left) and sVEGFR1 mRNA (right) were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (SFTSV: ***P < 0.0001; sVEGFR1: ***P = 0.0002). (O) Primary human monocyte-derived-macrophages were infected with SFTSV (MOI = 1) for 24 h. The secreted sVEGFR1 in cell supernatant was measured by capture ELISA. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0026). (P) Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were matured to macrophage and then infected with SFTSV (MOI = 1) for 24 h. BMDMs were infected with SFTSV for 24 h. Viral RNA (left) and sVEGFR1 (right) mRNA were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (SFTSV: ***P < 0.0001; sVEGFR1: *P = 0.0111). Data information: Data shown are mean ± SD of three biological replicates. (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 2. SFTSV infection up-regulates sVEGFR1 expression in hu-PBL NCG mice and in SFTS patients.

(A) hu-PBL NCG mice were infected with SFTSV (n = 5) at 1 × 10³ TCID₅₀ or mock-infected (n = 5), and blood samples were collected after 14 days. SFTSV viral RNA (left) and sVEGFR1 mRNA (right) in blood cells were determined by qPCR. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (SFTSV, ***P < 0.0001; sVEGFR1, ***P = 0.0001). (B) The serum level of sVEGFR1 protein was measured by capture ELISA. Each point represents an individual. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P = 0.0008). (C) The infected mice (n = 5) were euthanized, and spleens were collected. sVEGFR1 and the viral NP protein in spleens were measured by western blotting (left). Numeric label indicates individual mice. The sVEGFR1 protein expression relative to GAPDH was shown (right). n = 3 biological replicates for Mock; n = 5 biological replicates for SFTSV. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0074). (D) Detection of sVEGFR1 by immunofluorescence in the spleens after SFTSV infection (left). SFTSV-Gn (red), sVEGFR1 (green) and nuclei (DAPI, blue) were visualized using either antibodies specific for Gn or sVEGFR1, or DAPI for nuclei. A merged image of red, green and blue channels is shown (merge). The quantifications of relative sVEGFR1 intensities were determined by ImageJ software (right). Scale bars, 25 µm, respectively. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P < 0.0001). (E) Blood samples were obtained from 60 SFTS patients (grouped into severe and mild based upon clinical criteria) and 30 healthy individuals as controls. The serum level of sVEGFR1 was measured by capture ELISA. n = 30 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (Mild, ***P < 0.0001; Severe, ***P < 0.0001). Data information: Data shown are mean ± SD of at least three biological replicates with each data point representing a biological experiment (**P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 3. SFTSV-upregulated sVEGFR1 induces angiogenesis dysfunction in vitro.

(A) HUVECs were introduced into the Matrigel in the presence of serum. Each serum collected from SFTS patients of either mild symptom or severe symptom was placed under UV light for 30 min for residual virus inactivation. Tube formation of HUVECs was examined at 12 h after treatment. Representative capillary tubule structures were shown (top). Red arrowheads indicate tube elongation. Bar graph (bottom) represent the fold change of tubule formation. Scale bar = 50 μm. n = 3 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (Mild, ***P < 0.0001; Severe, ***P < 0.0001). (B) Representative images (top) of mouse aortic rings from which microvessels sprouted after treatment with sera collected from either mild patients or severe patients. Each serum was placed under UV light for 30 min for residual virus inactivation. Red arrowheads indicate micro-vascular sprouts. Bar graph (bottom) represent the fold change of microvessels outgrowth from aortic rings. Scale bar = 50 μm. n = 3 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (Day 3: *P = 0.0281, *P = 0.0281; Day 5: ***P < 0.0001, ***P < 0.0001). (C) Sera from healthy individuals and SFTS patients were placed under UV light for 30 min to inactivate the virus, and then pre-treated with a sVEGFR1 neutralizing antibody-conjugated or control antibody-conjugated magnetic beads to remove sVEGFR1. HUVECs were introduced into the Matrigel in the presence of pre-treated-serum from either healthy individuals or SFTS patients, and tube formation of HUVECs was examined at 12 h after treatment. Representative capillary tubule structures were shown. Red arrowheads indicate tube elongation. Scale bar = 50 μm. Bar graph on the right represent the fold change of tubule formation. n = 3 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (Control, **P = 0.0065; SFTS, *P = 0.0177). (D) Representative images of the tube formation following incubation with pre-treated sera from either healthy individuals or SFTS patients added with exogenous recombinant sVEGFR1 (10 ng/ml) or VEGFA (20 ng/ml) protein, respectively. Red arrowheads indicate tube elongation. Scale bar = 50 μm. Bar graph on the right represent the fold change of tubule formation. n = 3 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (12 h: **P = 0.0041, **P = 0.0021; 24 h: **P = 0.0030, **P = 0.0022). Data information: Data shown are mean ± SD of three biological replicates. (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 4. SFTSV infection up-regulates EGR1.

(A) RNA-seq was performed in SFTSV-infected or mock-infected HUVECs. The heatmap showed that the 81 genes relating to angiogenesis were among 422 genes with significant differences (based on an adjusted P value ≤ 0.05) in expression after SFTSV infection. (B) Comparison of gene expression of SFTSV-infected or mock-infected HUVECs. Red dots represent genes with higher expression in SFTSV-infected cells and blue dots represent genes with higher expression in mock-infected cells (based on an adjusted P value ≤ 0.05). (C) HeLa cells were infected with SFTSV for 4, 6, 12, 24 and 36 h. Intracellular EGR1 protein was analyzed by western blotting (left). The EGR1 expression relative to GAPDH was shown (right). n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P = 0.0010, ***P = 0.0006, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001). (D) are the same experiments as in (C), but evaluated in THP-1 cells. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (*P = 0.0281, **P = 0.0053, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001). (E) Primary human monocyte-derived-macrophages were infected with SFTSV (MOI = 1) for 24 h. EGR1 mRNA was measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0046). (F) BMDMs from C57BL/6J mice were matured to macrophage and then infected with SFTSV (MOI = 1) for 24 h. BMDMs were infected with SFTSV for 24 h. EGR1 mRNA was measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P < 0.0001). (G) hu-PBL NCG mice were infected with SFTSV (n = 5) at 1 × 10³ TCID₅₀ or mock-infected (n = 5), and blood samples were collected. EGR1 mRNA in blood cells was determined by qPCR. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (*P = 0.0152). (H) Infected mice (n = 5) were euthanized and spleens were collected. The indicated proteins in spleens were measured by western blotting (left). Numeric label indicates individual mice. The EGR1 protein expression relative to GAPDH was shown (right). n = 3 biological replicates for Mock; n = 5 biological replicates for SFTSV. Statistical significance was determined by two-tailed unpaired Student t test. (*P = 0.049). (I) Detection of EGR1 by immunohistochemistry in the spleen sections after SFTSV infection (left). Shown on the right are subset images of the regions outlined by rectangles on the left. Scale bar = 50 μm. The quantifications of relative EGR1 intensities were determined by ImageJ software (right). n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P = 0.0002). Data information: Data shown are mean ± SD of at least three biological replicates with each data point representing a biological experiment. (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 5. EGR1 positively regulates the expression of sVEGFR1.

(A) HeLa cells were transiently transfected with a plasmid expressing EGR1 or an empty vector (EV-his). After 36 h post transfection, the relative EGR1 mRNA (left) and sVEGFR1 mRNA (right) in HeLa cells were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (EGR1: ***P < 0.0001; sVEGFR1: ***P < 0.0001). (B) The expression of sVEGFR1 was measured by western blotting (left) and quantified by density of the WB bands (right). n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P = 0.0008). (C) sVEGFR1 protein level in HeLa cell culture supernatant was measured by ELISA. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0013). (D) HEK-293T cells were infected with a recombinant lentivirus vector (pLV-Puro-EGR1) and screened with puromycin for cell clones with stable EGR1 overexpression (EGR1-293T cells). EGR1-293T cells, along with HEK-293T cells infected with pLV-Puro vector (Con-293T cells), were tested for the mRNA levels of EGR1 and sVEGFR1 by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (EGR1: ***P < 0.0001; sVEGFR1: **P = 0.0050). (E) The protein levels of EGR1 and sVEGFR1 in EGR1-293T cells and Con-293T cells were measured by western blotting (left) and quantified by density of the WB bands (right). n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P = 0.0003). (F) sVEGFR1 protein in cell culture supernatant was measured by ELISA. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (***P < 0.0001). (G) Cells transfected with siNC or siEGR1 (siEGR1-1, siEGR1-2 and siEGR1-3) were harvested at 36 hpt to evaluate EGR1 protein knock-down efficiency and sVEGFR1 protein levels by western blotting. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (EGR1: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001; sVEGFR1: ***P < 0.0001, ***P < 0.0001, ***P < 0.0001). Data information: Data shown are mean ± SD of three biological replicates. (**P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 6. sVEGFR1 neutralizing antibodies restore angiogenesis in a Matrigel plug embedded in SFTSV-infected mice in vivo.

(A) Schematic representation of experimental design. The Matrigel plug assays were performed by subcutaneous injection of Matrigel containing sVEGFR1 Ab or Control Ab into hu-PBL NCG mice followed with or without SFTSV infection. Seven days after infection, the Matrigel plugs were removed and photographed. (B) Images of Matrigel plugs containing control Ab (10 µg/ml) or sVEGFR1 Ab (10 µg/ml) recovered from mice dorsum after the mice were infected or uninfected with SFTSV. (C) Representative cross-sections of H&E-staining of the Matrigel plugs, harvested as described in (B). Shown on the right are subset images of the regions outlined by rectangles on the left. Scale bars, 250 µm (left) and 50 µm (right), respectively. n = 3 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (Mock, ***P < 0.0001; SFTSV, ***P = 0.0006). (D) After the plugs were embedded in wax and sectioned, the sections were immune-stained with CD31 antibodies. Representative images of immunostaining are shown. Shown on the right are subset images of the regions outlined by rectangles on the left. Scale bars, 250 µm (left) and 50 µm (right), respectively. n = 3 biological replicates for each group. Statistical significance was determined by two-tailed unpaired Student t test. (Mock, ***P = 0.0007; SFTSV, **P = 0.0039). Data information: Data shown are mean ± SD of three biological replicates. (**P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 7. sVEGFR1 expression is reduced in EGR1 KO mice during SFTSV infection, alleviating angiogenesis dysfunction.

(A) PCR (left) and q-PCR (right) were used to detect the genotype and KO efficiency, respectively (n = 3 biologically independent WT mice and KO mice). ND, Not Detected. (B) Experimental scheme of the SFTSV-infected mouse model. WT and EGR1-KO mice were infected with SFTSV at 1 × 10⁵ TCID₅₀, respectively. Liver, spleen, lung and kidney were isolated from two groups for analysis. (C, D) The percent of body weight and survival changes were monitored for 7 days. n = 5 biological replicates for each group. Statistical significance was determined by Log-rank test. ((D): **, P = 0.0048) (E) sVEGFR1 (left) and SFTSV viral RNA mRNA (right) in blood cells from the WT and KO mice (n = 5) were determined by qPCR. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (sVEGFR1: **P = 0.0087; SFTSV: *P = 0.0481) (F) The serum level of sVEGFR1 protein was measured by capture ELISA. Each point represents an individual. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (*P = 0.0193). (G) Histopathological examinations of the tissues collected from the WT and KO mice. Tissues were collected from mice on day 4. The representative photographs of H&E were shown. The black arrows indicate hyperemia. Bars, 100 μm. (H) The infected mice were euthanized, and spleens were collected. sVEGFR1 mRNA in the spleens from the WT and KO mice were determined by qPCR. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (*P = 0.0235). (I) sVEGFR1 and the viral NP protein in spleens were measured by western blotting (left). The sVEGFR1 and SFTSV-NP protein expression relative to GAPDH was shown (right), respectively. n = 3 biological replicates for Mock; n = 5 biological replicates for SFTSV. Statistical significance was determined by two-tailed unpaired Student t test. (sVEGFR1: **P = 0.0054; NP: **P = 0.0018). (J) Detection of sVEGFR1 and viral Gn protein by immunofluorescence in the spleens after SFTSV infection (left). SFTSV-Gn (green), sVEGFR1 (red) and nuclei (DAPI, blue) were visualized using either antibodies specific for Gn or sVEGFR1, or DAPI for nuclei. A merged image of green, red and blue channels is shown (merge). The quantifications of relative SFTSV-Gn and sVEGFR1 intensities were determined by ImageJ software (right). Scale bars, 25 µm. n = 5 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (Gn: ***P < 0.0001; sVEGFR1: ***P < 0.0001). Data information: Data shown are mean ± SD of at least three biological replicates with each data point representing a biological experiment. (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are available online for this figure.

Figure 8. Graphical illustration showing the mechanism of hemorrhage.

The mechanism of hemorrhage that SFTSV-induced sVEGFR1 release through upregulation of EGR1 contributes to hemorrhage, suggesting that the EGR1/sVEGFR1/hemorrhage axis may be an effective target for the development of novel anti-hemorrhagic therapies during SFTSV infection.

Figure EV1. SFTSV infection up-regulates total VEGFR1 and membrane-bound VEGFR1 expression.

(A) THP-1 cells were infected with SFTSV for 12, 24, 36, 48, and 60 h. Total VEGFR1 mRNA was measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (ns = 0.0548, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001) (B) THP-1 cells were infected with SFTSV for 12, 24, 36, 48, and 60 h. Then the cells were incubated with anti-VEGFR1 for 1 h at 4 °C and VEGFR1 level on the cell membrane was determined by flow cytometry. X-axes show anti-VEGFR1 antibody (logarithm of fluorescence), Y axes depict the cell count. (C, D) THP-1 cells were induced to differentiate into macrophage-like cells by 150 nM phorbol-12-myristate-13-acetate (PMA) and then infected with SFTSV. Total VEGFR1 (C) and sVEGFR1 (D) mRNAs were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. ((C): ***P < 0.0001; (D): ***P < 0.0001). (E) Primary human monocyte-derived-macrophages were infected with SFTSV (MOI = 1) for 24 h. Total VEGFR1 mRNA was measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (*P = 0.0198). Data information: Data shown are mean ± SD of three biological replicates. (ns, P > 0.05; *P < 0.05; ***P < 0.001).

Figure EV2. sVEGFR1 is mainly produced by M2 macrophages.

(A, B) THP-1 cells were induced to differentiate into M1 macrophage-like cells by 24 h incubation with 150 nM PMA, followed by IFN-γ (20 ng/ml) and LPS (10 pg/ml), or THP-1 cells were induced to differentiate into M2 macrophage-like cells by 24 h incubation with 150 nM PMA, followed by interleukin 4 (20 ng/ml) and interleukin 13 (20 ng/ml) for 72 h. Total VEGFR1 (A) and sVEGFR1 (B) mRNAs were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. ((A): ***P < 0.0001; (B): ***P < 0.0001). (C, D) Primary human monocyte-derived-macrophages were differentiated into M1 macrophages by culture in the presence of IFN-γ (20 ng/ml) and LPS (20 ng/ml) for 24 h, or were differentiated into M2 macrophages by culture in the presence of interleukin 4 (20 ng/ml) and interleukin 13 (20 ng/ml) for 72 h. Total VEGFR1 (C) and sVEGFR1 (D) were measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. ((C): **P = 0.0036; (D): ***P < 0.0001). (E) Primary human monocyte-derived-macrophages were differentiated into M1 macrophages or M2 macrophages. The secreted sVEGFR1 in cell supernatant was measured by capture ELISA. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0011). (F) Primary human monocyte-derived-macrophages were differentiated into M1 macrophages or M2 macrophages. EGR1 mRNA was measured by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0018). Data information: Data shown are mean ± SD of three biological replicates. (**P < 0.01; ***P < 0.001).

Figure EV3. SFTSV infection up-regulates EGR1 expression in SFTS patients.

(A) PBMCs were isolated from 10 SFTS patients and 10 healthy individuals as controls. EGR1 expression in PBMCs among SFTS patients were measured by qPCR. n = 10 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. (**P = 0.0085). Data information: Data shown are mean ± SD of ten biological replicates with each data point representing a biological experiment (**P < 0.01).

Figure EV4. Overexpression of EGR1 activates the VEGFR1 promoter.

(A, B) Luciferase reporter assay. For the VEGFR1 promoter luciferase reporter assay, the predicted binding sites of VEGFR1 and EGR1 were obtained from the eukaryotic promoter database (EPD). The corresponding promoter constructs in the promoter region of VEGFR1 was synthesized and then inserted into the pGL4.19-basic firefly luciferase reporter vector (PPL), named pGL4.19-FLT1 promoter. These vectors were co-transfected into HeLa or HEK-293T cells with EGR1 construct or empty vector. Measurement of luciferase activity was conducted at 48 h post-transfection. Representative results were from three independent experiments. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. ((A): ***P = 0.0001; (B): ***P < 0.0001). Data information: Data shown are mean ± SD of three biological replicates (***P < 0.001).

Figure EV5. Knock-down of EGR1 reduces sVEGFR1 expression.

(A, B) THP-1 cells transfected with siNC or siEGR1 (siEGR1-2 in Fig. 5G) were harvested at 36 hpt to evaluate EGR1 mRNA (A) knock-down efficiency and sVEGFR1 mRNA (B) levels by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student t test. ((A): ***P = 0.0003; (B): *P = 0.0243). Data information: Data shown are mean ± SD of three biological replicates (*P < 0.05; ***P < 0.001).