1Universitätsmedizin der Johannes Gutenberg-Universität Mainz Kardiologie 1, Zentrum für Kardiologie Mainz, Deutschland
Background and Aim: Endothelial cells play a central role for venous thrombosis, and work from us and others has shown that experimental disruption of endothelial angiogenic signaling delays venous thrombus resolution. Transdifferentiation of hematopoietic to endothelial cell lineages (and the reverse) also has been described during new vessel formation, but its significance for thrombus revascularization is unknown. The aim of this study was to visualize the sequence of cellular events occurring during venous thrombus formation and resolution and to genetically fate-map the origin of cells revascularizing venous thrombi.
Methods: Reporter mice (Rosa26-mTmG) with enhanced membrane-tethered green fluorescent protein (mEGFP) expression specifically in either erythrocytes (EpoR.Cre), endothelial cells (Tie2.CreERT2) or pericytes (PDGFRB.CreERT2) were subjected to inferior Vena cava ligation to induce stasis and deep vein thrombosis. Non-invasive ultrasound was performed, and veins containing thrombi were isolated at different time points (at 2, 7, 14, 21 and 28 days) after surgery. Serial longitudinal sections were cut from cryo-preserved thrombi and subjected to Carstairs staining to visualize erythrocytes, platelets and fibrin, as well as to immunofluorescent staining using antibodies against general endothelial cell markers, such as platelet endothelial cell adhesion molecule-1 (PECAM1 or CD31) and isolectin B4 (IB4), and endothelial cell markers with specific functions, such as endomucin (EMCN) and intracellular adhesion molecule-2 (ICAM2).
Results: Our analysis revealed mEGFP-labeled erythrocyte accumulation near the vein wall and immediately attached to the inner vein endothelial cell layer on day 2, whereas unlabeled nucleated cells invaded this area and translocated erythrocyte signals towards the center of the thrombus at later time points. mEGFP-labeled endothelial cells started to occur at day 7 and to form vessel-like structures within thrombi, primarily at the outer layers of the thrombus, starting at day 14, and their numbers increased over the time. mEGFP-labeled pericytes were detected from day 14 onwards. Although RBC-mEGFP and CD31 signals were found to co-localize in 2-days-old thrombi, nuclear staining was negative suggesting activated platelets as its main origin. Endothelial mEGFP and CD31 double-positive cells were present from day 7 onwards, whereas pericyte mEGFP expressing cells did not stain positive for CD31. mEGF-CD31-labeled endothelial cells were also observed in the tissue surrounding veins at all time points, whereas isolectin B4 labeled cells were detected in thrombi at day 2 post-surgery, but not in the surrounding, injured vein. The venous endothelial cell marker endomucin was expressed in injured veins at all times, and endomucin-positive endothelial cells formed vessels-like structures inside the thrombi at day 21 post-surgery. Interestingly, expression of ICAM2, a marker of endothelial cell activation, was observed only in the endothelial cells lining the neighbouring aorta, but not in those lining the IVC.
Conclusions: Our analyses visualize the time-dependent repopulation of venous thrombi with diverse types of cells, focusing on RBC’s, endothelial cells and pericytes.