NFAT5 directs hyperosmotic stress-induced fibrin deposition and macrophage infiltration via PAI-1 in endothelium

Abstract

Although stress can significantly promote atherosclerosis, the underlying mechanisms are still not completely understood. Here we successfully unveiled that high salt-induced nuclear factor of activated T cells 5 (NFAT5) control the endothelial-dependent fibrinolytic activity and the inflammatory adhesion-related molecules expression through regulation of plasminogen activator inhibitor-1 (PAI-1). We first observed that high salt diets instigated the expression of NFAT5 and PAI-1 in the endothelium which brought about the fibrin deposition and macrophage infiltration in the atherosclerotic arteries of ApoE^-/- mice. Overexpression of NFAT5 increased PAI-1-mediated antifibrinolytic activity and activated inflammatory adhesion-related genes in endothelial cells. Knockdown of NFAT5 by siRNA inhibited the expression of PAI-1, antifibrinolytic and adhesive molecules. Moreover, chromatin immunoprecipitation assay demonstrated that high salt intake significantly promoted the binding of NFAT5 to PAI-1 promoter (TGGAATTATTT) in endothelial cells. Our study identified that NFAT5 has great potential to activate the PAI-1-mediated fibrinolytic dysfunction and inflammatory cell adhesion, thus promoting high salt-induced atherosclerosis disease.

Keywords: NFAT5; PAI-1; atherosclerosis; endothelial cells; high salt.

Figure 1

High-salt intake predisposes fibrin deposition, macrophage infiltration and atherosclerosis formation in aortas of ApoE^-/- mice. (A) The schematic diagram shows the process of fibrin deposition, macrophage infiltration and atherosclerosis formation. (B, C) Representative sections of the AA region in ApoE^-/- mice stained for fibrin and macrophage marker F4/80 (n = 7). Nuclei, hematoxylin staining. Fibrin and F4/80-positive macrophages were marked respectively by arrow heads. (D) Oil Red O staining of the artery and quantification of percentage lesion areas in the thoracic aorta (TA) and aortic arch (AA) of ApoE^-/- mice (n = 10) fed with a normal or high-salt diet for 12 weeks. (E) En face immunofluorescent staining of PAI-1 (red) in ECs of TA and AA regions of ApoE^-/- mice in normal and high salt groups after 4 weeks feeding. Nuclei were stained by DAPI. All data were presented as mean ± SEM, N≥7. *p < 0.05 versus control group.

Figure 2

High-salt intake induces endothelial fibrinolytic dysfunction and thrombi. (A–C) Protein levels of PAI-1, active plasmin and D-Dimer in plasma of mice in normal and high salt groups after 4 weeks feeding. (D) Representative sections of the livers in ApoE^-/- mice stained for fibrin. Nuclei, hematoxylin staining. Microthrombi were marked by arrowheads. (E, F) mRNA and protein expression of PAI-1 in HUVECs that cultured with different hyper-osmotic media (270, 290, 310, 330 and 350 mosmol/kg) for two or three days. 270 mosmol/kg was as the control. (G, H) mRNA and protein level of plasmin in HUVECs that cultured with different hyper-osmotic media for two or three days. 270 mosmol/kg was as the control. (I) Representative immunofluorescent staining of PAI-1 (green) in HUVECs that exposed to different hyper-osmotic media for three days. Nuclei were stained by DAPI. All data were presented as mean ± SEM, N≥3. *p < 0.05 versus control group.

Figure 3

High-salt promotes adhesion molecules expression and inflammatory cells infiltration in ECs via PAI-1. (A–D) mRNA expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1, and MCP-1) in TA and AA regions of ApoE^-/- mice in normal and high salt groups after 4 weeks feeding. (E–H) mRNA expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1, and MCP-1) in HUVECs that were treated with Ctr siRNA or PAI-1 siRNA under high-salt condition for two days. (I) Representative images of adherent monocytes to HUVECs that were treated with Ctr siRNA or PAI-1 siRNA under high-salt condition. (J) Representative images of infiltrated monocytes into HUVECs that were treated with Ctr siRNA or PAI-1 siRNA under high-salt condition. All data were presented as mean ± SEM, N≥3. *p < 0.05 versus control group.

Figure 4

High-salt intake induces NFAT5 nuclear translocation in ECs. (A) En face immunofluorescent staining of NFAT5 (red) in ECs of TA and AA regions of ApoE^-/- mice in normal and high salt groups after 4 weeks feeding. Nuclei were stained by DAPI. NFAT5 nuclear translocation in ECs was marked by circles. (B) Protein expression of NFAT5 in HUVECs that exposed to different hyper-osmotic media for three days. All data were presented as mean ± SEM, N≥3. *p < 0.05 versus control group.

Figure 5

High-salt induces the dysfunction of PAI-1-dependent fibrinolysis in ECs via NFAT5. (A–D) mRNA and protein expression of fibrinolysis genes (PLAT, PLAU and PLG) in HUVECs that treated by Adenovirus-null (Ad-null) or Adenovirus-NFAT5 (Ad-NFAT5). (E–H) mRNA and protein expression of fibrinolysis genes (PLAT, PLAU and PLG) in HUVECs that transfected with Ctr siRNA or NFAT5 siRNA under high-salt condition. All data were presented as mean ± SEM, N≥3. *p < 0.05 versus control group.

Figure 6

High-salt induces the dysfunction of monocytes adhesion and infiltration in ECs via NFAT5. (A–D) mRNA expression of adhesive molecules (E-selectin, ICAM-1, VCAM-1, and MCP-1) in HUVECs that treated by Ad-null or Ad-NFAT5. (E–H) mRNA expression of adhesive molecules (E-selectin, ICAM-1, VCAM-1, and MCP-1) in HUVECs that transfected by Ctr siRNA or NFAT5 siRNA under high-salt condition. All data were presented as mean ± SEM, N≥3. *p < 0.05 versus control group.

Figure 7

NFAT5 directly regulates PAI-1 transcription in ECs. (A, B) mRNA and protein expression of PAI-1 in HUVECs that treated by Ad-null or Ad-NFAT5. (C, D) High-salt increases binding of NFAT5 to PAI-1 promoter. Diagram showing the region of the NFAT5 binding site upstream of the transcription start site (TSS) of PAI-1, and the regions that were used to analyze NFAT5 binding by ChIP. ChIP results were relative to 270 mosmol/kg. (E, F) mRNA and protein expression of PAI-1 in HUVECs that transfected with Ctr siRNA or NFAT5 siRNA under high-salt condition. All data were presented as mean ± SEM, N≥3. *p < 0.05 versus control group.

Figure 8

The schematic diagram shows the process of fibrin deposition, macrophage infiltration and atherosclerosis formation. Stage I: Hypertonicity → NFAT5-dependent PAI-1 gene transcription → PAI-1 secretion. Stage II: PAI-1 secretion → Antifibrinolytic activation/adhesive molecules → Fibrin deposition/monocytes adhesion and infiltration. Stage III: Endothelial dysfunction leads to fibrin deposition, macrophage-driven foam cells and phenotype conversion of smooth muscle cells, contributing to the formation of atherosclerotic plaque.