Revisiting Virchow's triad: exploring the cellular and molecular alterations in cerebral venous congestion

Abstract

Background: Cerebral venous thrombosis (CVT) is a rare but serious condition that can lead to significant morbidity and mortality. Virchow's triad elucidates the role of blood hypercoagulability, blood flow dynamics, and endothelial damage in the pathogenesis of CVT. Cerebral venous congestion (CVC) increases the risk of cerebral venous sinus thrombosis and can lead to recurrent episodes and residual symptoms. However, the precise mechanism by which blood congestion leads to thrombosis remains unclear. Our objective was to investigate the cellular and molecular alterations linked to CVC through analysis of the pathological morphology of venous sinus endothelial cells and transcriptomic profiling.

Results: This study demonstrated a remarkable correlation between CVC and the phenotypic transformation of endothelial cells from an anticoagulant to a procoagulant state. The findings revealed that cerebral venous stasis results in tortuous dilatation of the venous sinuses, with slow blood flow and elevated pressure in the sinuses and damaged endothelial cells of the retroglenoid and internal jugular vein ligation (JVL) rat model. Mechanistically, analysis of transcriptomic results of cerebral venous sinus endothelial cells showed significant activation of platelet activation, complement and coagulation cascades pathway in the JVL rats. Furthermore, the expression of von Willebrand factor (vWF) and coagulation factor VIII (F8) in the complement and coagulation cascades and Fgg and F2 in the platelet activation was increased in the cerebral venous sinuses of JVL rats than in sham rats, suggesting that endothelial cell injury in the venous sinus induced by CVC has a prothrombotic effect. In addition, endothelial cell damage accelerates coagulation and promotes platelet activation. Significantly, the concentrations of vWF, F2 and F8 in venous sinus blood of patients with internal jugular vein stenosis were higher than in their peripheral blood.

Conclusion: Collectively, our data suggest that CVC can induce endothelial cell damage, which then exhibits a procoagulant phenotype and ultimately increases the risk of CVT. This research contributes to our understanding of the pathophysiology of CVC associated with procoagulant factors and reexamines the components of Virchow's triad in the context of CVC.

Keywords: CVT; Cerebral venous congestion; Endothelial injury; Stroke; Virchow’s triad.

Fig. 1

Cerebral venous stasis results in tortuosity and dilatation of the venous sinus, endothelial cell damage, and slow blood flow. (A) MRV of the cerebral venous sinuses of sham and JVL rats at 0 days, 1 week and 4 weeks, the white arrows indicate tortuous and dilated straight sinus (n = 5, 2 females and 3 males). (B-C) The diameters of the straight sinus (B) and SSS (C) in sham and JVL rats at 0 days, 1 week and 4 weeks (n = 5, 2 females and 3 males). (D) Pressure in the SSS of JVL rats before surgery and at 1 and 4 weeks after surgery (n = 5, 2 females and 3 males). (E) TEM images of the SSS in sham and JVL rats, the red arrows indicate tight junctions between endothelial cells (scale bars, 1 μm. n = 5, 2 females and 3 males). (F) Quantification of blood flow in cerebral venous sinuses (CVSs) in sham and JVL rats (n = 5, 2 females and 3 males). The data are representative of five independent experiments and are presented as the mean ± SD. *P < 0.05, ***P < 0.001 vs. the respective control by using an unpaired Student’s t test

Fig. 2

Transcriptome analysis demonstrated the activation of platelet activation and complement and coagulation cascades in endothelial cell injury in the cerebral venous sinus of JVL rats. (A) Schematic diagram of cerebral venous sinus isolation. (B) Volcano plot of the gene expression profiles. (C) Heatmap of DEGs (|log2FC| ≥ 1, P ≤ 0.05) in the cerebral venous sinus of sham and JVL rats. (D, E) Bubble diagram of the top 12 ranked KEGG pathways (D) and KEGG diseases (E) enrichment analysis of DEGs from the comparison between the sham and JVL group. (F, G) GSEA plot showing the enrichment of ‘platelet activation’ (F, NES: 1.82, P = 0.00) and ‘complement and coagulation cascades’ (G, NES: 1.2, P = 0.14) gene sets in the cerebral venous sinus of JVL rats

Fig. 3

Cerebral venous congestion activates the expression of the clotting factors vWF and F8 in complement and coagulation cascades. (A) Relative mRNA expression of vWF, C3, F8, Cd55 and Mbl2 in the cerebral venous sinus (CVS) of sham and JVL rats (n = 5, 2 females and 3 males). (B, C) Representative images and quantification of vWF along the SSS (lectin-green) in sham and JVL rats (scale bars, 100 μm, n = 5, 2 females and 3 males). (D, E) Representative images and quantification of vWF along the TS (lectin-green) in sham and JVL rats (scale bars, 100 μm, n = 5, 2 females and 3 males). (F, G) vWF and F8 levels in the cerebral venous sinus were determined via ELISA and were significantly greater in JVL rats than in sham control rats (n = 5, 2 females and 3 males). The data are representative of five independent experiments and are presented as the mean ± SD. **P < 0.01, ***P < 0.001 vs. the respective control by using an unpaired Student’s t test

Fig. 4

Cerebral venous congestion activates the expression of the clotting factors Fgg and F2 in the platelet activation pathway. (A) Relative mRNA expression of vWF, Fgg, Itgb1, Fcer1g, F2, Tln1, Vasp, Gp9, Mapk3, P2ry12 and Pik3cb in the cerebral venous sinus (CVS) of sham and JVL rats (n = 5, 2 females and 3 males). (B, C) Representative images and quantification of Fgg along the SSS (lectin-green) in sham and JVL rats (scale bars, 100 μm, n = 5, 2 females and 3 males). (D, E) Representative images and quantification of Fgg along the TS (lectin-green) in sham and JVL rats (scale bars, 100 μm, n = 5, 2 females and 3 males). (F) F2 levels in the cerebral venous sinus were determined via ELISA and were significantly greater in JVL rats than in sham control rats (n = 5, 2 females and 3 males). The data are representative of five independent experiments and are presented as the mean ± SD. *P < 0.05, ***P < 0.001 vs. the respective control by using an unpaired Student’s t test

Fig. 5

Cerebral venous congestion promotes platelet activation and coagulation. (A-D) JVL rats had reduced PT (A), aPTT (B) and TT (C) and elevated fibrinogen levels (D) (n = 5, 2 females and 3 males). (E-H) Cerebral venous stasis induces ATP release from platelets in the cerebral venous sinus of JVL rats (E, F) but not in peripheral blood (G, H) (n = 5, 2 females and 3 males). (I-L) Cerebral venous stasis upregulated platelet P-selectin expression in the cerebral venous sinus of JVL rats (I, J) but had no effect on platelets in peripheral blood (K, L) (n = 5, 2 females and 3 males). (M) There were greater P-selectin concentrations in the cerebral venous sinuses of JVL rats than in those of sham rats (n = 5, 2 females and 3 males). The data are representative of five independent experiments and are presented as the mean ± SD. ***P < 0.001 vs. the respective control by using an unpaired Student’s t test

Fig. 6

vWF, F2 and F8 concentrations in the peripheral blood and venous sinus blood of internal jugular vein stenosis patients. (A-C) There were greater vWF (A), F2 (B) and F8 (C) concentrations in the venous sinus blood of internal jugular vein stenosis patients than in the peripheral blood (n = 10). The data are representative of ten independent experiments and are presented as the mean ± SD. *P < 0.05 vs. the respective control by using an unpaired Student’s t test