Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Abstract

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Characterization of Endothelial and Smooth Muscle Cells From Different Canine Vessels

“…All used vessel types, the aorta, vena cava, and vena porta, were found suitable for the inversion and suturing steps necessary for EC isolation. We primarily used vessels with an approximate length of 5 cm, but the technique was also applicable for vessels as short as 1 cm (Oosterhoff et al, 2016). Canine VSMCs and CaPECs were derived from all used vessels.…”

“…Therefore, it is essential to choose an animal model where cardiovascular diseases naturally occur. Blood vessels in the canine are more similar in size and function to human vessels, and also reduce the need for genetic manipulation (Oosterhoff et al, 2016; van Steenbeek et al, 2016). Additionally, the existing overlap in commonly occurring diseases in both human and dogs such as patent ductus arteriosus and vascular stenosis is of great benefit when cells originating from canine vessels are used in disease modeling (Gittenberger-de Groot et al, 1985; den Toom et al, 2016; Laborda-Vidal et al, 2016; Van den Bossche and van Steenbeek, 2016).…”

“…Additionally, the existing overlap in commonly occurring diseases in both human and dogs such as patent ductus arteriosus and vascular stenosis is of great benefit when cells originating from canine vessels are used in disease modeling (Gittenberger-de Groot et al, 1985; den Toom et al, 2016; Laborda-Vidal et al, 2016; Van den Bossche and van Steenbeek, 2016). The novel vessel inversion technique we describe allowed us to obtain viable CaPECs and VSMCs from vessels as small as 1 cm in length (Oosterhoff et al, 2016). In addition, the isolated cells could be directly cultured, negating the need for FACS sorting.…”

Inhibition of growth of canine-derived vascular endothelial cells by non-steroidal anti-inflammatory drugs and atrial natriuretic peptide

“…This study was conducted under the Nihon University Animal Care and Use Committee approval (AP15B037). A protocol from a previous study [5, 10] was modified to isolate VECs from the arteries (aorta and bilateral carotid arteries) and veins (caudal vena cava and bilateral jugular veins) of three clinically healthy Beagles. Briefly, after one end of each blood vessel was clamped, 0.25% trypsin solution was injected into the other end using a syringe.…”

“…Previous studies have described the isolation and culture of canine VECs derived from arteries and veins using collagenase [5, 10]. In our study, trypsin was used for the intraluminal exfoliation of VECs from canine vessels because of the mRNA expression of CD31 and the positive detection of surface CD31 antigens.…”