OBJECTIVE To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses.
SAMPLE Jugular venous blood samples from 24 adult horses.
PROCEDURES Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates.
RESULTS Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry.
CONCLUSIONS AND CLINICAL RELEVANCE ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.
Objective—To determine significant molecular and
cellular factors responsible for differences in secondintention
healing in thoracic and metacarpal wounds
Animals—6 adult mixed-breed horses.
Procedure—A full-thickness skin wound on the
metacarpus and another such wound on the pectoral
region were created, photographed, and measured,
and tissue was harvested from these sites weekly for
4 weeks. Gene expression of type-I collagen, transforming
growth factor (TGF)-β1, matrix metalloproteinase
(MMP)-1, and tissue inhibitor of metalloproteinase
(TIMP)-1 were determined by quantitative in
situ hybridization. Myofibroblasts were detected by
immunohistochemical labeling with α-smooth muscle
actin (α-SMA). Collagen accumulation was detected
by use of picrosirius red staining. Tissue morphology
was examined by use of H&E staining.
Results—Unlike thoracic wounds, forelimb wounds
enlarged during the first 2 weeks. Myofibroblasts,
detected by week 1, remained abundant with superior
organization in thoracic wounds. Type-I collagen
mRNA accumulated progressively in both wounds.
More type-I collagen and TGF-β1 mRNA were seen in
forelimb wounds. Volume of MMP-1 mRNA
decreased from day 0 in both wounds. By week 3,
TIMP-1 mRNA concentration was greater in thoracic
Conclusions and Clinical Relevance—Greater collagen
synthesis in metacarpal than thoracic wounds
was documented by increased concentrations of
myofibroblasts, type-I collagen mRNA, TGF-β1 mRNA,
and decreased collagen degradation (ie, MMP-1).
Imbalanced collagen synthesis and degradation likely
correlate with development of exuberant granulation
tissue, delaying healing in wounds of the distal portions
of the limbs. Factors that inhibit collagen synthesis
or stimulate collagenase may provide treatment
options for horses with exuberant granulation
tissue. (Am J Vet Res 2002;63:1564–1570)