Isolation of equine endothelial cells and life cell angiogenesis assay

Abstract

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis.

Endothelial cells and angiogenesis in the horse in health and disease—A review

“…Tip and stalk cells (Figure 4) were documented in equine endothelial cell cultures by Dietze et al. (2014) and Rieger et al. (2018).…”

“…For horses, as in many other species, immunohistochemical staining of the coagulation co‐factor von Willebrand factor (vWF) (Figure 3) has been found to be a useful marker of endothelial cells (Bochsler, Slauson, Chandler, & Suyemoto, 1989; Bosch, Moleman, Barneveld, van Weeren, & van Schie, 2011; Chiam et al., 2006; Dietze et al., 2014; Johnson et al., 1996; MacEachern, Smith, & Nolan, 1997; Rieger et al., 2018; Rosenberg, Greengard, & Montgomery, 2000). The quantitative expression of this marker seems to vary in the horse.…”

“…The aortic ring model has been used to assess the effects of cortisol in equine vessels (Morgan et al., 2018). The development of capillary‐like structures was observed in isolated equine endothelial cells in vitro (Dietze et al., 2014). In vitro tubule formation on basement membrane matrix has been done on both equine endothelial progenitor cells (Salter et al., 2015; Seeto et al., 2017; Sharpe et al., 2016) and equine endothelial cells (Bussche & Van de Walle, 2014).…”

“…Briefly, angiogenic stimuli cause increased endothelial cell permeability, invasiveness and proliferation; basement membrane components are proteolysed to facilitate invasion of endothelial cells into the stroma of the neighbouring tissue; lumen formation occurs, as the sprout forms a multicellular structure and meets up with an adjacent sprout; the new capillary is stabilized with a basement membrane and pericytes are recruited (Potente & Carmeliet, 2017; Yoo & Kwon, 2013). The angiogenic cascade of endothelial cells in vitro has been classified and quantified for several species, including the horse (Dietze et al., 2014). In splitting or intussusceptive angiogenesis, a single vessel is split in two.…”

Subcellular Interactions during Vascular Morphogenesis in 3D Cocultures between Endothelial Cells and Fibroblasts

“…We hypothesize that these structures are microvesicles that were released by the cells, serving as vehicles for the transfer of angiogenic factors over longer distances [ 29 ]. Microparticles were found to be released in monocultures of ECs in an earlier study at the time of their integration from the monolayer into three-dimensional capillary-like structures [ 30 ]. It is only recently that the role of microvesicles in intercellular interactions has become a focus of research interest.…”