Abstract
Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate in vitro models resembling native vasculature are of great importance. Unfortunately, existing in vitro models do not sufficiently reflect their in vivo counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.
Highlights
Two essential cell types in blood vessels are endothelial cells (ECs), which line the inside of all blood vessels, and vascular smooth muscle cells (VSMCs), which regulate vessel stability
After Canine primary ECs (CaPECs) isolation, the vessel was minced into small pieces and a 4-h digestion with collagenase type II (0.09 U/mL) in Dulbecco’s Modified Eagle’s Medium (DMEM) GlutaMAX was performed at 37◦C, allowing collection of the VSMC fraction
We primarily used vessels with an approximate length of 5 cm, but the technique was applicable for vessels as short as 1 cm (Oosterhoff et al, 2016)
Summary
Two essential cell types in blood vessels are endothelial cells (ECs), which line the inside of all blood vessels, and vascular smooth muscle cells (VSMCs), which regulate vessel stability. The main function of ECs is to provide a barrier between the blood and the rest of the body, they maintain vascular homeostasis by orchestrating the selective exchange of nutrients, oxygen and immune cells between tissues or organs, and act as key players in angiogenesis and vasculogenesis, aiding the formation of new microvasculature to create an organized network (Jain, 2003). Human umbilical vein ECs (HUVECs) and endothelial progenitor cells (EPCs) are widely used to model ECs in vitro. Isolation of these two cell types does not require surgery or even death of the donor, as HUVECs are derived from the umbilical cord and EPCs are derived from blood samples (Melero-Martin et al, 2007, 2008; Emontzpohl et al, 2017). Using HUVECs and EPCs to model adult blood vessels in vitro remain challenging due to molecular and functional differences between ECs (Hauser et al, 2017)
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