Resolution of Deep Venous Thrombosis: Proposed Immune Paradigms

Abstract

Venous thromboembolism (VTE) is a pathology encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE) associated with high morbidity and mortality. Because patients often present after a thrombus has already formed, the mechanisms that drive DVT resolution are being investigated in search of treatment. Herein, we review the current literature, including the molecular mechanisms of fibrinolysis and collagenolysis, as well as the critical cellular roles of macrophages, neutrophils, and endothelial cells. We propose two general models for the operation of the immune system in the context of venous thrombosis. In early thrombus resolution, neutrophil influx stabilizes the tissue through NETosis. Meanwhile, macrophages and intact neutrophils recognize the extracellular DNA by the TLR9 receptor and induce fibrosis, a complimentary stabilization method. At later stages of resolution, pro-inflammatory macrophages police the thrombus for pathogens, a role supported by both T-cells and mast cells. Once they verify sterility, these macrophages transform into their pro-resolving phenotype. Endothelial cells both coat the stabilized thrombus, a necessary early step, and can undergo an endothelial-mesenchymal transition, which impedes DVT resolution. Several of these interactions hold promise for future therapy.

Keywords: NETosis; collagenolysis; deep vein thrombosis; endothelial cell; fibrinolysis; immune system; macrophage; neutrophil; stability; venous thromboembolism.

Figure 1

(Top) Histological time progression at 10× power showing venous thrombus resolution in a C57 murine IVC ligation model of DVT at 2, 4, 8, 14, and 21 days post-ligation. Formalin-fixed paraffin-embedded thrombus samples were stained using a modified Lendrum’s martius yellow, crystal scarlett, methyl blue (MSB) stain. (1) Erythrocytes are visualized in yellow, fibrin in red, and collagen in blue. As time progresses, clot composition transitions from erythrocytes to fibrin and then to collagen. (Bottom) Hematoxylin and eosin (H&E) staining at 100× power of human DVT paraffin-embedded tissue samples dated from venous stent implantation date. DVT stage was determined in accordance with the DVT pathology grading guide described by Fineschi et al. [16]. Within one week, the thrombus is mostly erythrocytes and platelets (stage 1), then by two months its composition shifts to include many infiltrating immune cells (stage 2), and finally the remaining tissue at the one year time point shows mostly collagen with sparse cells (stage 3). All photomicrographs were captured as stitched 4 picture mosaics using a Nikon E400 microscope and Nikon DS-Ri1 camera. The top row of photos were cropped horizontally.

Figure 2

Proposed mechanism of early thrombus resolution involving neutrophils (PMNs). NETosis and fibrosis are distinct mechanisms for increasing thrombus stability and may prevent embolization. NETs do not play an early role in complete stasis thrombus, but do in non-stasis models. Whether thrombus instability without NETs leads to embolization is speculative. However, it is likely that TLR9 signaling is important for later thrombus resolution, in part by driving monocyte fibrin metabolism and clearance of sterile breakdown products. Neutropenia in TLR9^-/- mice impairs both NETosis and fibrosis, resulting in an intermediate thrombus size because of instability alone. This figure also illustrates a potential mechanism of immune cell influx into the developing thrombus: neutrophils are first recruited by the chemoattractant IL8, then these neutrophils trigger an influx of monocyte-derived macrophages, partially through MCP-1 signaling.

Figure 3

Global mechanism of thrombus resolution via some of the immune system cells. The top row of cells are ambivalent or beneficial to DVT resolution, whereas the bottom row of cells impede DVT resolution. Endothelial cells can either aid in thrombus resolution by proliferating to seal off the developing thrombus, or transition into mesenchymal progenitor-like cells, which slow thrombus resolution by depositing more collagen. Both mast cells and T-cells trigger the activation of pro-inflammatory macrophages, with mast cells potentially using endothelial cells as intermediaries. T-cells secrete interferon gamma (IFNγ) which locks macrophages into the pro-inflammatory phenotype. However, with PECAM-1 from endothelial cells or hepatocyte growth factor (HGF), pro-inflammatory macrophages transition to become pro-resolving, at which point they promote fibrinolysis and collagenolysis.