Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia

Abstract

Summary Venous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1-/- and TLR4-/- mice underwent treatment with saline or LPS (10 mg/kg i. p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4-/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1-/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated V T.

Genome-wide association analysis of venous thromboembolism identifies new risk loci and genetic overlap with arterial vascular disease

“…Therefore, in the interest of humane and responsible animal use, wild type C57BL/6 mice (WT) were utilized as controls. Animals underwent a wellcharacterized DVT model, stasis inferior vena cava (IVC) thrombosis, at 8-10 weeks of age and 20-25 grams body weight 24,25,[45][46][47] . Isoflurane 2% was administered as inhaled anesthetic.…”

Immune Factors in Deep Vein Thrombosis Initiation

“…In addition to cell recruitment, activated endothelium enhances blood coagulation and exerts suppressed anticoagulant function, thus contributing to thrombus formation [40] . Upregulation of the endothelial surface adhesion receptor ICAM-1 underlies augmented DVT under the endotoxemic conditions [41] . Clinically, endothelial activation has been reported in patients with VTE and thrombosis of superficial veins 42 , 43 .…”

Resolution of Deep Venous Thrombosis: Proposed Immune Paradigms

“…Resolvin D4, an anti-inflammatory lipid mediator, reduces PMN infiltration and antagonizes NET formation, in addition to recruiting pro-resolving monocytes and reducing the thrombus size [70]. Furthermore, both toll-like receptor 4 (TLR4), an immune receptor, and intercellular adhesion molecule 1 (ICAM-1) respond to lipopolysaccharide binding, but reducing PMN counts increases thrombosis and increases plasminogen activator inhibitor-1 (PAI-1), increases circulating Factor XIII, and decreases uPA observed in endotoxemia [71]. This contradicts a prior result showing no neutropenic change on thrombus size in mice.…”

“…Thrombus resolution also involves endothelial cells, which assume many roles within the developing thrombus, similar to macrophages. Some of the effects of proteins secreted by endothelial cells can be understood by their linkage to the aforementioned parts of the immune system, such as ICAM-1 which is also expressed by PMNs [71] and pro-inflammatory Mo/MΦ cells [94], and Vascular Endothelial Growth Factor (VEGF) which triggers pro-resolving Mo/MΦ activation [89,101]. As noted above, endothelial cells can act as intermediaries between mast cells and infiltrating immune cells [141].…”