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Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells
Tamara B Wills1, Allison M Heaney, K Jane Wardrop
1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7060, USA. twills@vetmed.wsu.edu
Insights
Immunomagnetic isolation effectively enumerates circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in canine blood. This method provides a reliable way to count these cells for potential disease biomarker studies in dogs.
Area of Science:
- Veterinary Hematology
- Cellular Biology
- Biomarker Discovery
Background:
- Elevated circulating endothelial cells (CECs) indicate vascular injury in various human diseases.
- Immunomagnetic isolation is a key technique for enumerating CECs, even at low concentrations.
Purpose of the Study:
- To validate a standard immunomagnetic isolation protocol for enumerating CECs and endothelial progenitor cells (EPCs) in canine whole blood.
- To assess the feasibility of using this method in canine research.
Main Methods:
- Canine aortic endothelial cells were characterized using von Willebrand factor.
- Anti-CD146 magnetic beads were used for immunomagnetic isolation of CECs/EPCs from cultured cells and canine blood.
- Endothelial origin was confirmed with Ulex europaeus agglutinin 1 (UEA-1) staining; cells were counted via fluorescent microscopy.
Main Results:
- Anti-CD146 beads successfully bound cultured canine endothelial cells, with >86% recovery.
- Immunomagnetic isolation yielded 43.4 ± 15.6 CECs/mL from canine whole blood.
- A high percentage of cultured cells were UEA-1 positive, confirming endothelial markers.
Conclusions:
- Immunomagnetic isolation is a viable method for enumerating canine CECs/EPCs in whole blood.
- Further research is needed to explore the clinical relevance of CEC/EPC counts in canine pathologies.
Background:
Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells.
Objectives:
The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood.
Methods:
Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti-CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146-positive cells were stained with fluorescein-conjugated Ulex europaeus agglutinin 1 (UEA-1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA-anticoagulated whole blood samples from 10 healthy client-owned dogs.
Results:
The anti-CD146-coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA-1-positive cells were obtained from whole blood, while >85-90% of cultured canine aortic endothelial cells were UEA-1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10-40 microm in size. Using immunomagnetic isolation, 43.4 +/- 15.6 CECs/mL (range 24-70/mL) were isolated from canine whole blood samples.
Conclusions:
Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.
