The atheroma plaque secretome stimulates the mobilization of endothelial progenitor cells ex vivo.

Endothelial progenitor cells (EPCs) constitute a promising alternative in cardiovascular regenerative medicine due to their assigned role in angiogenesis and vascular repair. In response to injury, EPCs promote vascular remodeling by replacement of damaged endothelial cells and/or by secreting angiogenic factors over the damaged tissue. Nevertheless, such mechanisms need to be further characterized. In the current approach we have evaluated the initial response of early EPCs (eEPCs) from healthy individuals after direct contact with the factors released by carotid arteries complicated with atherosclerotic plaques (AP), in order to understand the mechanisms underlying the neovascularization and remodeling properties assigned to these cells. Herein, we found that the AP secretome stimulated eEPCs proliferation and mobilization ex vivo, and such increase was accompanied by augmented permeability, cell contraction and also an increase of cell-cell adhesion in association with raised vinculin levels. Furthermore, a comparative mass spectrometry analysis of control versus stimulated eEPCs revealed a differential expression of proteins in the AP treated cells, mostly involved in cell migration, proliferation and vascular remodeling. Some of these protein changes were also detected in the eEPCs isolated from atherosclerotic patients compared to eEPCs from healthy donors. We have shown, for the first time, that the AP released factors activate eEPCs ex vivo by inducing their mobilization together with the expression of vasculogenic related markers. The present approach could be taken as a ex vivo model to study the initial activation of vascular cells in atherosclerosis and also to evaluate strategies looking to potentiate the mobilization of EPCs prior to clinical applications.

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling.

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT-Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT-Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.