Reduced proliferation of endothelial colony-forming cells in unprovoked venous thromboembolic disease as a consequence of endothelial dysfunction.

BACKGROUND: Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). METHODS AND RESULTS: Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20-50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated ß-galactosidase (SA-ß-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). CONCLUSIONS: As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events.

Molecular characterization and tissue expression profile of porcine Ephrin-B2.

Ephrin-B2 (EFNB2) is a signaling molecule that plays an important role in cell adhesion, proliferation, and migration in humans. However, little is known about this molecule in pigs. In order to investigate whether EFNB2 is associated with the skeletal muscle in pigs, we cloned the full-length cDNA of EFNB2 (GenBank accession No. KF500033) from the longissimus dorsi muscle of Yorkshire pigs by rapid amplification of cDNA ends. The results indicated that its full-length cDNA comprises 1991 bp, with an open reading frame of 1002 bp, a 5' end of 88 bp, and a 3' end of 901 bp. We analyzed the homology of porcine EFNB2 with sequences from other species, and the phylogenetic tree showed that pig EFNB2 was most closely related to that from sheep, followed by domestic cats and wolf, with mackerel being the most distantly related. Porcine EFNB2 is a water-soluble protein with a theoretical molecular weight of 36,928.1 Da, an isoelectric point of 8.98, and a hydrophilic transmembrane-spanning region. It contains 19 glycosylation sites and eight phosphorylation sites. The tertiary structure of the EFNB2 protein showed a forniciform helix structure. The porcine EFNB2 gene was expressed in ten different tissues from 25-day-old Shaziling and Yorkshire piglets, with the highest expression observed in the longissimus dorsi. These results lay the foundation for further study on the EFNB2 gene in pigs.