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Updated: Mar 8, 2026
Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins
Published on: November 18, 2016
Endothelial protein C receptor-dependent antichemotactic effects of canine protein C
Insights
Canine neutrophils express the endothelial protein C receptor (EPCR), which mediates the anti-migratory effects of protein C on these cells. This suggests EPCR signaling could be a therapeutic target in canine inflammatory diseases.
Area of Science:
- Immunology
- Hematology
- Veterinary Medicine
Background:
- Neutrophil migration is crucial in inflammatory responses.
- Protein C is a key regulator of coagulation and inflammation.
- The role of canine protein C (CnPC) and its receptor in neutrophil function is not well understood.
Purpose of the Study:
- To investigate the antichemotactic effects of CnPC on canine neutrophils.
- To determine the expression of endothelial protein C receptor (EPCR) on canine neutrophils.
- To elucidate the role of EPCR in canine neutrophil chemotaxis.
Main Methods:
- Neutrophils were isolated from healthy and sick dogs.
- EPCR mRNA and protein expression were analyzed using RT-PCR and flow cytometry.
- Neutrophil migration assays were performed using CnPC or activated CnAPC, with or without an anti-EPCR antibody (RCR-379).
- Chemotaxis index (Chx) was calculated based on fluorescence measurements.
Main Results:
- Canine neutrophils express EPCR mRNA and protein.
- The anti-chemotactic effects of CnPC and activated CnAPC were dependent on EPCR.
- Inhibition of EPCR with RCR-379 normalized neutrophil migration, indicating EPCR's role in regulating chemotaxis.
- These effects were independent of protein C concentration or activation status.
Conclusions:
- Canine neutrophils express EPCR, mediating the inhibitory effects of protein C on their migration.
- EPCR signaling is a critical regulator of canine neutrophil chemotaxis.
- Targeting EPCR may offer a novel therapeutic strategy for inflammatory conditions in dogs.
Abstract:
OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram. PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti-human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed. RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C. CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.
