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Endothelial lesions induce migration and proliferation of neighboring cells in vivo and in vitro, aimed at closing the damage as quickly as possible. Thereby, different endothelial cell populations (tip- and stalk cells) play a specific role.
In horses it has been known for a long time that thrombosis evolves frequently in both, arterial and venous localizations. In contrast, this phenomenon is not often seen in other domestic animals. To examine the role of migration of venous and arterial endothelial cells in equine thrombosis, a scratch assay was established. As a control group, different human endothelial cell lines were used.
MATERIALS AND METHODS
Human endothelial cells of the skin (HDMEC), the umbilical vein (HUVEC) and the abdominal aorta were bought from different manufactures and cultivated in endothelial cell growth medium (EGM-2 MV, Lonza). Equine endothelial cells were isolated from the Arteria carotis communis and the Vena jugularis externa via enzymatic digestion. Identification and sorting of these cells was performed by flow cytometry with the endothelial marker CD31. Additionally, equine endothelial cells were identified by labelling immunohistochemically with anti-von Willebrand factor and Weibel-Palade-bodies, i.e., endothelial marker organelles were visualized using transmission electron microscopy. After the emergence of a complete monolayer, a lesion was set using a sterile Eppendorf pipette tip (10μl). The cellular migration was investigated over a period up to 60 hours using an air-conditioned Life Science Microscope (Olympus IX81). For documentation, the shooting interval was set to one picture per 30 minutes.
Human endothelial cells
All of the three cell lines examined displayed continuous migration and proliferation towards the middle of the scratch. Tip-cells could be observed at the monolayer edges consistently. The joining stalk-cells were following permanently. The scratch area was closed by the migrating cells after about 16 hours and a complete monolayer was reconstructed. Endothelial cells presented the typical cobblestone pattern and a slight reduction of the cell density, as well as a hypertrophy of single cells could be observed at the periphery of the field of view.
Equine endothelial cells
Immunohistochemical and electron microscopic examinations confirmed endothelial identity of the isolated equine cells.
The induced lesion in the arterial endothelial cell monolayer was closed after 33 hours. Migration of these cells towards the middle of the scratch was not continuous but showed focal sprouting of tip- and stalk-cells. In addition, single cells (especially tip-cells) displayed a specific migration pattern. They detached from the cell complex a few times and migrated up to 20 hours in the denuded scratch area. Neither reduction of the cell density nor hypertrophy of single cells could be monitored. The cells displayed an elongated shape but also in a cobblestone pattern.
The equine endothelial cells from the Vena jugularis externa displayed an increased migration compared to the arterial equine endothelial cells. A complete monolayer was rebuild after around 23 hours. The migration pattern was equate to that of the arterial equine endothelial cells.
A fast and uniform migration of human endothelial cells to close the wound in scratch assays is described in many studies (e.g. Liang et al., 2007). In our study, we were able to confirm this. For the equine endothelial cells this was not true. Wound closure in equine cells was not uniform and also slower than in the human cultures. The solitary migrating cells might be a special subpopulation of tip-cells.