Human peripheral blood endothelial progenitor cells synthesize and express functionally active tissue factor.
BACKGROUND
Endothelial progenitor cells are circulating cells able to home to sites of vascular damage and to contribute to the revascularization of ischemic areas. We evaluated whether endothelial progenitor cells synthesize tissue factor, a procoagulant protein also involved in angiogenesis.


METHODS
Endothelial progenitor cells were obtained from the peripheral blood mononuclear fraction of normal donors and cultured in endothelial medium supplemented with specific growth factors. The procoagulant activity expressed by cells disrupted by freeze-thaw cycles was assessed by a one stage clotting assay. Tissue factor mRNA expression was evaluated by RT-PCR.


RESULTS
Endothelial progenitor cells do not express procoagulant activity in baseline conditions. However, lipopolysaccharide induces the expression of procoagulant activity. The effect is dose-dependent and reaches statistical significance at 100 ng/mL lipopolysaccharide. Inhibition with an anti-tissue factor antibody and amplification of cDNA with primers based on the tissue factor sequence confirm the identity of this activity with tissue factor. The kinetics of tissue factor expression by endothelial progenitor cells is identical to that of human umbilical vein endothelial cells showing maximal activity within 4 hours, and then decreasing; in contrast, tissue factor expression by mononuclear cells lasts for longer times. Both 5,6-dichloro-beta D-ribofuranosyl-benzimidazole and cycloheximide prevented the expression of procoagulant activity. Stimulation of endothelial progenitor cells with tumor necrosis factor-alpha did not elicit any detectable procoagulant activity.


CONCLUSIONS
Endothelial progenitor cells can be stimulated by lipopolysaccharide to synthesize tissue factor. This protein might be involved in thrombotic phenomena and might contribute to endothelial progenitor cells related neovascularization.
Di Stefano
Rossella
R
Cardiovascular Research Laboratory, Cardiac, Thoracic and Vascular Department, University of Pisa, Pisa, Italy. r.distefano@ao-pisa.toscana.it
Barsotti
Maria Chiara
MC
Armani
Chiara
C
Santoni
Tatiana
T
Lorenzet
Roberto
R
Balbarini
Alberto
A
Celi
Alessandro
A
D053687:Adult Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D018919:Neovascularization, Physiologic; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics*
D008070:Lipopolysaccharides; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D004004:Dichlororibofuranosylbenzimidazole; D013925:Thromboplastin; D003513:Cycloheximide