Visualization of new vessel formation during venous thrombus organization using genetic fate mapping.

BACKGROUND

Endothelial cell (EC) dysfunction contributes to venous thrombosis, and disrupting endothelial angiogenic signaling delays venous thrombus resolution. However, the time course of EC appearance during venous thrombus organization and the identity of cells lining the intrathrombotic vessels are largely unknown.

OBJECTIVES

We used a genetic fate mapping approach to visualize tamoxifen-inducible endothelial receptor tyrosine kinase (TIE2) reporter ECs during venous thrombus resolution and human pulmonary thrombi of different organizational stages to validate our findings.

METHODS

We induced stenosis and deep vein thrombosis by ligating the inferior vena cava in mice expressing membrane targeted green fluorescent protein in postembryonic TIE2 lineage cells. TIE2 reporter ECs started to appear on day 7 and lined the newly formed vessel-like structures within thrombi on day 21.

RESULTS

Immunolabeling identified endomucin as most specific marker of angiogenic ECs in venous thrombi. Intercellular adhesion molecule 2, a marker of EC activation, was expressed predominantly on ECs lining the adjacent aorta, not the inferior vena cava. Cells expressing the hypoxia surrogate marker carboanhydrase IX, stromal cell-derived factor 1 alpha, or CD34 peaked on day 7, whereas endothelial protein C receptor-expressing cells appeared later. Platelet-derived growth factor beta lineage-derived pericytes and fibrosis were detected from day 14 onwards. Histologic analysis of thrombus material from patients with acute pulmonary embolism or chronic thromboembolic pulmonary hypertension confirmed the presence of endomucin-, CD34-, and endothelial protein C receptor-positive cells in organized pulmonary thrombus material, whereas vessel-like structures were only seen in chronic thromboembolic pulmonary hypertension thrombi.

CONCLUSION

TIE2 reporter ECs appear in venous thrombi early and prior to measurable reductions in thrombus size, whereas pericytes appear later and possibly contribute to vessel stabilization.