Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells.

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.

A technique for in vitro culture of canine valvular interstitial cells.

OBJECTIVE: To develop a method for in vitro culture of canine valvular interstitial cells (VICs). ANIMALS, MATERIALS AND METHODS: Canine VICs were isolated from the distal third of the anterior mitral valve leaflet using an explant technique and maintained in cell culture. Molecular phenotyping of the cultured cells was performed using reverse transcription polymerase chain reaction and immunocytochemistry. RESULTS: Cells resembling fibroblasts migrated from canine mitral valve explants and were maintained in culture for up to eight passages. Establishment of the valve explant required collagen but once established, subsequent passages grew on non-coated plastic plates. At confluence the cultured cells exhibited the characteristic whorled pattern of fibroblasts in culture. The isolated valve cells expressed vimentin but not platelet endothelial cell adhesion molecule or von Willebrand's factor, consistent with the molecular phenotype of VICs. CONCLUSIONS: VICs can be readily isolated from canine mitral valve leaflets and successfully maintained in culture using standard culture techniques. The described techniques permit the study of bioactive VICs in a controlled environment and may be a useful in vitro model for investigation of cellular and molecular alterations associated with canine chronic degenerative valve disease.

Endovascular retrieval of embolized jugular catheter fragments in three dogs using a nitinol gooseneck snare.

Two dogs were referred to the Kansas State University Veterinary Medical Teaching Hospital for pulmonary arterial embolization and one to Washington State University Veterinary Teaching Hospital for right heart embolization of jugular catheter fragments. Endovascular retrieval of foreign bodies was accomplished under general anesthesia in all three cases. Although the incidence of, and complications associated with catheter embolization in dogs are unknown, it appears that they can be safely and easily retrieved with a nitinol gooseneck snare.

Electrocardiographic and echocardiographic evidence of atrial dissociation.

A 4-year-old Boxer dog with a history of ventricular premature contractions was evaluated for anorexia. An electrocardiogram revealed atrial dissociation and an echocardiogram supported simultaneous mechanical atrial systole with the ectopic atrial electrical activity. This case and the supporting images provide further credence to the existence of atrial dissociation.