Cellular fibronectin promotes deep vein thrombosis in diet-induced obese mice

Abstract

Background: Overweight and obesity are significant risk factors for deep vein thrombosis (DVT). Cellular fibronectin containing extra domain A (Fn-EDA), an endogenous ligand for toll-like-receptor 4 (TLR4), contributes to thrombo-inflammation. The role of Fn-EDA in the modulation of DVT is not elucidated yet.

Objective: To determine whether Fn-EDA promotes DVT in the context of diet-induced obesity.

Methods: Wild-type (WT) and Fn-EDA-deficient mice were either fed control or high-fat (HF) diet for 12 weeks. DVT was induced by inferior vena cava (IVC) stenosis and evaluated after 48 hours. Cellular Fn-EDA levels in the plasma of venous thromboembolism (VTE) patients were measured by sandwich ELISA.

Results: We found that cellular Fn-EDA levels were significantly elevated in VTE patients' plasma and positively correlated with body mass index. HF diet-fed WT mice exhibited increased DVT susceptibility compared with control diet-fed WT mice. In contrast, HF diet-fed Fn-EDA-deficient mice exhibited significantly reduced thrombus weight and decreased incidence (%) of DVT compared with HF diet-fed WT mice concomitant with reduced neutrophil content and citrullinated histone H3-positive cells (a marker of NETosis) in IVC thrombus. Exogenous cellular Fn-EDA potentiated NETosis in neutrophils stimulated with thrombin-activated platelets via TLR4. Genetic deletion of TLR4 in Fn-EDA⁺ mice (constitutively express Fn-EDA in plasma and tissues), but not in Fn-EDA-deficient mice, reduced DVT compared with respective controls.

Conclusion: These results demonstrate a previously unknown role of Fn-EDA in the DVT exacerbation, which may be an essential mechanism promoting DVT in the setting of diet-induced obesity.

Keywords: NETosis; deep vein thrombosis; diet-induced obesity; fibronectin.

Figure 1:. Plasma Fn-EDA was elevated in VTE patients that positively correlated with BMI.

A, Plasma Fn-EDA levels in VTE patients and healthy controls as determined by ELISA (n=48, 100). Data are mean ± SEM. B, Correlation of plasma Fn-EDA levels with BMI in VTE patients (Pearson’s Correlation Coefficient R² 0.4845) using the data in A.

Figure 2.. Fn-EDA promotes venous thrombosis in the context of diet-induced obesity.

A, Plasma Fn-EDA levels in WT mice fed either a control diet or HF diet for 12-weeks (n=5,7, unpaired t-test). B, Thrombosis incidence (n=20,18, 20,19, chi-square test). C, Left, Representative IVC thrombus harvested 48-hour post-stenosis from each group. Right, thrombus weight (non-parametric two-way ANOVA followed by Fisher’s LSD test). Only mice that exhibited thrombosis were included to quantify the thrombus weight. Each dot represents a single mouse. Data are mean ± SEM. HF indicates high-fat.

Figure 3.. TLR4 contributes to Fn-EDA-mediated NETosis.

A, Left, Representative cross-sectional immunofluorescence image of the isolated IVC (on day-2 post-stenosis) from 12-week HF diet-fed WT and EDA⁻ mice stained for Ly6G (neutrophils, red) and anti-histone H3 (citrulline R2 + R8 + R17) (NETs, green) and DAPI (blue). Scale bar 200 μm. Right, Quantification of the % Ly6G and % CitH3 positive cells (n=7, 6, 7, 6, unpaired t-test). Data are mean ± SEM. B, NETs assay was performed by stimulating bone marrow-derived neutrophils from either WT or TLR4^−/− mice with thrombin (0.1 U/mL)-activated platelets (1X10⁷), in the presence or absence of cFn (5 μg/mL). Left, Representative microphotographs of NETs stained with PlaNET green (stains extracellular DNA, green) and counterstained with Hoechst (stains nuclei, blue). Scale bar 100 μm. Right, Quantification of the percentage of cells releasing NETs (n =5 mice/group, two-way ANOVA followed by Sidak’s multiple comparisons test). The value for each mouse represents a mean from 2 fields. cFn, cellular fibronectin, NETs, neutrophil extracellular traps.

Figure 4.. TLR4 contributes to Fn-EDA-mediated DVT.

A, Thrombosis incidence (n=11,13,12,11, chi-square test). B, Left, Representative IVC thrombus harvested 48-hour post-stenosis from each genotype. Right, Thrombus weight (non-parametric two-way ANOVA followed by Fisher’s LSD test). Only mice with thrombosis were included to quantify thrombosis weight. Data are mean ± SEM. Each dot represents a single mouse.