Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 binds to atherosclerotic plaques and is pro-inflammatory. ( A ) Immunohistochemical positivity for FHR-1 (red/arrows) in necrotic/degenerated areas of an atherosclerotic plaque of a coronary artery (B) and in the media of aortas with mucoid degeneration (C) of ACVD patients but not ∆ CFHR3/1 ACVD patients. ( D ) FHR-1 co-localization with macrophages (arrows). (A–D) bars = 200 µm; each picture is representative of three patients with and without FHR-1. Binding of serum (E) FHR-1, but not (F) FHR2, to MDA-LDL (MLDL). ( G ) Detection of FHR-2 but not FHR1 by monoclonal antibody to FHR-2 mAb. gel gelatin. (H) Immobilized FHR-1 induces IL-1β secretion in whole human blood (diluted 1:10, 1:5, or 1:1), lipopolysaccharide (LPS) = control activator (I) Immobilized FHR-1 induces macrophage secretion of IL-1β IL-6 and IL-8 (BSA = bovine serum albumin) (J) upregulates expression of IL-1β, IL-8, TNFα, and CCL3 genes in neutrophils (zymosan = control activator; sFHR-1 = soluble FHR-1). Data in (E) —represent the mean ± SEM of three independent experiments with different donor cells. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by unpaired two-tailed t-tests.

Article Snippet: For imaging analysis, monoclonal FHR-1 antibodies were coupled to Alexa Fluor 647 dye (Thermo Fisher Scientific, cat no. A20502) by mixing the dye with the antibody at a 2:1 ratio and incubating the mixture for 30 min at room temperature.

Techniques: Immunohistochemical staining, Binding Assay, Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 and FHRE circulate on extracellular vesicles (EVs) in normal human serum (NHS) and normal mouse serum (MS). (A) Detection of FHR-1 in the EV fraction from NHS (EV NHS ) but not from homozygous FHR-1-deficient (EV ΔFHR-1 ) human serum by western blot analysis. FHR-1 was also absent from the supernatant fractions of both sera. Results shown are representative of three experiments. An uncropped gel is shown in . (B) Tracking of EV NHS and EV ΔFHR-1 by live-cell imaging using CLSM. EVs were stained with anti-FHR-1 antibodies conjugated with Alexa Fluor 647 (red). Bars: 10 µm. (C–F) Size distribution and number of vesicles isolated from EV FHR-1 and EV FHRE determined by DLSM using NanoSight NTA 3.2 software. Graphs in ( C,E ) represent overlays of results from 3 to 4 donors. ( (C) , SEM ± standard error, *p ≤ 0.0255 by unpaired two-tailed t-test, n = 4), and ( (E) , SEM ± standard error, *p ≤ 0.0232 by unpaired two-tailed t-test, n = 3). (G) Tracking by live-cell imaging using CLSM of EV FHR-1 and EV ΔFHR-1 stained with anti-CD9 antibody (vesicle marker) and SYTOX orange (nucleic acid marker), Bars: 10 µm. (H) Size distribution of EVs transporting CD9 and FHR-1 (EV CD9&FHR-1 ). EV FHR-1&CD9 were captured with anti-CD9-coated beads from EV FHR-1 , isolated from 1 mL NHS and analyzed by DLSM using NanoSight NTA 3.2 software. (I) Particle numbers (J) and sizes (K) in fractions obtained by size-exclusion chromatography of EV NHS measured by DLSM. (L) High FHR-1 content in fractions 13–16 determined by ELISA. FHR-1 (red) (M) and FHR-1, and vesicle marker CD9, were in close proximity (red) (N) in atherosclerotic tissues, mainly in blood vessels (stippled lines). Complexes were analyzed by proximity ligation assays using anti-FHR-1 and anti-CD9, and were detected by CLSM. EV FHR-1 carry nucleic acids (orange). Bars = 10 µm.

Article Snippet: For imaging analysis, monoclonal FHR-1 antibodies were coupled to Alexa Fluor 647 dye (Thermo Fisher Scientific, cat no. A20502) by mixing the dye with the antibody at a 2:1 ratio and incubating the mixture for 30 min at room temperature.

Techniques: Western Blot, Live Cell Imaging, Staining, Isolation, Software, Two Tailed Test, Marker, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Elevated serum FHR-1 concentrations in ACVD patients correlate with low-density lipoprotein (LDL)-cholesterol concentrations. (A) FHR-1 concentrations were significantly higher in ACVD patients than in healthy individuals, (B) whereas Factor H concentrations were similar in the two groups. (C) Equal elevation of FHR-1 concentrations in men and women with ACVD. (D–G) Correlations of FHR-1 concentrations with (D) body mass index (BMI), (E) LDL concentrations, and (F) LDL/HDL ratios, but not (G) HDL concentrations, in patients with ACVD. (H,I) Correlations of FHR1 and LDL concentrations in (H) men and (I) women with ACVD. Data in (A–C) are reported as mean ± SEM and compared by unpaired two-tailed t-tests with Welch’s correction. Data in (D–I) represent Spearman’s correlations, *p ≤ 0.05, ***p ≤ 0.001.

Article Snippet: For imaging analysis, monoclonal FHR-1 antibodies were coupled to Alexa Fluor 647 dye (Thermo Fisher Scientific, cat no. A20502) by mixing the dye with the antibody at a 2:1 ratio and incubating the mixture for 30 min at room temperature.

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Elevated serum FHR-1 concentrations in ACVD patients correlate with inflammation markers. (A) C-reactive protein (CRP) concentrations were significantly higher in ACVD patients than in healthy controls (mean ± SEM, unpaired two-tailed t-test). (B) Correlations of FHR-1 concentrations with CRP concentrations and of (C) SAA concentrations with CRP concentrations. (H) ACVD patients with high FHR-1 levels (> 80 µg mL −1 ) show elevated SAA concentrations (mean ± SEM, unpaired two-tailed t-test), which correlate with (I) neopterin and (J) IL-6 concentrations. Data in (B,C,I,J) represent Spearman’s correlations.

Article Snippet: For imaging analysis, monoclonal FHR-1 antibodies were coupled to Alexa Fluor 647 dye (Thermo Fisher Scientific, cat no. A20502) by mixing the dye with the antibody at a 2:1 ratio and incubating the mixture for 30 min at room temperature.

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 activates the coagulation pathway. (A) Correlation between serum FHR-1 concentrations and numbers of thrombocytes and (B) Quick test in atherosclerosis patients (Spearman’s correlation). (C) FHR-1 induction of the expression of genes encoding proteins in the coagulation pathway in monocytes (n = 4 donors). (D) FHR-1-treated monocytes express tissue factor on their surfaces. Representative dot plots from one of three donors are shown. VD viability dye. Quantitative analysis of TF expression following inhibition of PLC and NF-κB (right panel). (E) FHR-1-treated monocytes show cleavage of Factor VIIa substrate, which is blocked by inhibitors of PLC and NFκB or (F) EMR2. (G) EMR2 inhibition blocks FHR1-induced, but not LPS-induced, TNFα secretion by monocytes. Data in (D–G) represent the mean ± SEM of three independent experiments with cells from different donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (unpaired two-tailed t-test).

Article Snippet: For imaging analysis, monoclonal FHR-1 antibodies were coupled to Alexa Fluor 647 dye (Thermo Fisher Scientific, cat no. A20502) by mixing the dye with the antibody at a 2:1 ratio and incubating the mixture for 30 min at room temperature.

Techniques: Coagulation, Expressing, Inhibition, Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Model of FHR-1-induced inflammation. Extracellular vesicles that transport FHR-1 (EV FHR1 ) bind to oxLDL in necrotic cores of atherosclerotic plaques and activate neutrophils and monocytes. These monocytes and neutrophils release inflammatory cytokines, which attract more immune cells. FHR-1 also induces monocytes to express tissue factor on their surfaces, which can trigger the coagulation pathway and thrombosis, the ultimate complication of atherosclerosis.

Article Snippet: For imaging analysis, monoclonal FHR-1 antibodies were coupled to Alexa Fluor 647 dye (Thermo Fisher Scientific, cat no. A20502) by mixing the dye with the antibody at a 2:1 ratio and incubating the mixture for 30 min at room temperature.

Techniques: Coagulation

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Homozygous CFHR1 deficiency in patients and healthy individuals.

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 binds to atherosclerotic plaques and is pro-inflammatory. ( A ) Immunohistochemical positivity for FHR-1 (red/arrows) in necrotic/degenerated areas of an atherosclerotic plaque of a coronary artery (B) and in the media of aortas with mucoid degeneration (C) of ACVD patients but not ∆ CFHR3/1 ACVD patients. ( D ) FHR-1 co-localization with macrophages (arrows). (A–D) bars = 200 µm; each picture is representative of three patients with and without FHR-1. Binding of serum (E) FHR-1, but not (F) FHR2, to MDA-LDL (MLDL). ( G ) Detection of FHR-2 but not FHR1 by monoclonal antibody to FHR-2 mAb. gel gelatin. (H) Immobilized FHR-1 induces IL-1β secretion in whole human blood (diluted 1:10, 1:5, or 1:1), lipopolysaccharide (LPS) = control activator (I) Immobilized FHR-1 induces macrophage secretion of IL-1β IL-6 and IL-8 (BSA = bovine serum albumin) (J) upregulates expression of IL-1β, IL-8, TNFα, and CCL3 genes in neutrophils (zymosan = control activator; sFHR-1 = soluble FHR-1). Data in (E) —represent the mean ± SEM of three independent experiments with different donor cells. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by unpaired two-tailed t-tests.

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques: Immunohistochemical staining, Binding Assay, Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 and FHRE circulate on extracellular vesicles (EVs) in normal human serum (NHS) and normal mouse serum (MS). (A) Detection of FHR-1 in the EV fraction from NHS (EV NHS ) but not from homozygous FHR-1-deficient (EV ΔFHR-1 ) human serum by western blot analysis. FHR-1 was also absent from the supernatant fractions of both sera. Results shown are representative of three experiments. An uncropped gel is shown in . (B) Tracking of EV NHS and EV ΔFHR-1 by live-cell imaging using CLSM. EVs were stained with anti-FHR-1 antibodies conjugated with Alexa Fluor 647 (red). Bars: 10 µm. (C–F) Size distribution and number of vesicles isolated from EV FHR-1 and EV FHRE determined by DLSM using NanoSight NTA 3.2 software. Graphs in ( C,E ) represent overlays of results from 3 to 4 donors. ( (C) , SEM ± standard error, *p ≤ 0.0255 by unpaired two-tailed t-test, n = 4), and ( (E) , SEM ± standard error, *p ≤ 0.0232 by unpaired two-tailed t-test, n = 3). (G) Tracking by live-cell imaging using CLSM of EV FHR-1 and EV ΔFHR-1 stained with anti-CD9 antibody (vesicle marker) and SYTOX orange (nucleic acid marker), Bars: 10 µm. (H) Size distribution of EVs transporting CD9 and FHR-1 (EV CD9&FHR-1 ). EV FHR-1&CD9 were captured with anti-CD9-coated beads from EV FHR-1 , isolated from 1 mL NHS and analyzed by DLSM using NanoSight NTA 3.2 software. (I) Particle numbers (J) and sizes (K) in fractions obtained by size-exclusion chromatography of EV NHS measured by DLSM. (L) High FHR-1 content in fractions 13–16 determined by ELISA. FHR-1 (red) (M) and FHR-1, and vesicle marker CD9, were in close proximity (red) (N) in atherosclerotic tissues, mainly in blood vessels (stippled lines). Complexes were analyzed by proximity ligation assays using anti-FHR-1 and anti-CD9, and were detected by CLSM. EV FHR-1 carry nucleic acids (orange). Bars = 10 µm.

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques: Western Blot, Live Cell Imaging, Staining, Isolation, Software, Two Tailed Test, Marker, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Elevated serum FHR-1 concentrations in ACVD patients correlate with low-density lipoprotein (LDL)-cholesterol concentrations. (A) FHR-1 concentrations were significantly higher in ACVD patients than in healthy individuals, (B) whereas Factor H concentrations were similar in the two groups. (C) Equal elevation of FHR-1 concentrations in men and women with ACVD. (D–G) Correlations of FHR-1 concentrations with (D) body mass index (BMI), (E) LDL concentrations, and (F) LDL/HDL ratios, but not (G) HDL concentrations, in patients with ACVD. (H,I) Correlations of FHR1 and LDL concentrations in (H) men and (I) women with ACVD. Data in (A–C) are reported as mean ± SEM and compared by unpaired two-tailed t-tests with Welch’s correction. Data in (D–I) represent Spearman’s correlations, *p ≤ 0.05, ***p ≤ 0.001.

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Elevated serum FHR-1 concentrations in ACVD patients correlate with inflammation markers. (A) C-reactive protein (CRP) concentrations were significantly higher in ACVD patients than in healthy controls (mean ± SEM, unpaired two-tailed t-test). (B) Correlations of FHR-1 concentrations with CRP concentrations and of (C) SAA concentrations with CRP concentrations. (H) ACVD patients with high FHR-1 levels (> 80 µg mL −1 ) show elevated SAA concentrations (mean ± SEM, unpaired two-tailed t-test), which correlate with (I) neopterin and (J) IL-6 concentrations. Data in (B,C,I,J) represent Spearman’s correlations.

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 activates the coagulation pathway. (A) Correlation between serum FHR-1 concentrations and numbers of thrombocytes and (B) Quick test in atherosclerosis patients (Spearman’s correlation). (C) FHR-1 induction of the expression of genes encoding proteins in the coagulation pathway in monocytes (n = 4 donors). (D) FHR-1-treated monocytes express tissue factor on their surfaces. Representative dot plots from one of three donors are shown. VD viability dye. Quantitative analysis of TF expression following inhibition of PLC and NF-κB (right panel). (E) FHR-1-treated monocytes show cleavage of Factor VIIa substrate, which is blocked by inhibitors of PLC and NFκB or (F) EMR2. (G) EMR2 inhibition blocks FHR1-induced, but not LPS-induced, TNFα secretion by monocytes. Data in (D–G) represent the mean ± SEM of three independent experiments with cells from different donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (unpaired two-tailed t-test).

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques: Coagulation, Expressing, Inhibition, Two Tailed Test

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: Model of FHR-1-induced inflammation. Extracellular vesicles that transport FHR-1 (EV FHR1 ) bind to oxLDL in necrotic cores of atherosclerotic plaques and activate neutrophils and monocytes. These monocytes and neutrophils release inflammatory cytokines, which attract more immune cells. FHR-1 also induces monocytes to express tissue factor on their surfaces, which can trigger the coagulation pathway and thrombosis, the ultimate complication of atherosclerosis.

Article Snippet: FHR-1 concentrations were measured in size-exclusion chromatography fractions using Human FHR-1 ELISA kits (RayBiotech, cat no. ELH-CFHR1) according to the manufacturer’s instructions.

Techniques: Coagulation