Apurinic/apyrimidinic endonucelase 1 maintains adhesion of endothelial progenitor cells and reduces neointima formation.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that processes DNA-repair function and controls cellular response to oxidative stress. Endothelial progenitor cells (EPCs) are recruited to oxidative stress-rich injured vascular walls and positively contribute to vascular repair and endothelialization. We hypothesized that APE1 functions for EPCs-mediated inhibition of neointima formation in injured vasculature. EPCs isolated from bone marrow cells of C57BL6 mice (12-16 wk old) were able to survive in the presence of hydrogen peroxide (H2O2; up to 1,000 µM) due to the highly expressed reactive oxygen species (ROS) scavengers. However, adhesion capacity of EPCs was significantly inhibited by H2O2 (100 µM) even though an intracellular ROS was retained at small level. An APE1-selective inhibitor or RNA interference-mediated knockdown of endogenous APE1 in EPCs aggravated the H2O2-mediated inhibition of EPCs-adhesion. In contrast, when APE1 was overexpressed in EPCs using an adenovirus harboring the APE1 gene (APE-EPCs), adhesion was significantly improved during oxidative stress. To examine in vivo effects of APE1 in EPCs, APE-EPCs were transplanted via the tail vein after wire-mediated injury of the mouse femoral artery. The number of adherent EPCs at injured vascular walls and the vascular repair effect of EPCs were enhanced in APE-EPCs compared with control EPCs. Among the cellular functions of EPCs, adhesion is especially sensitive to oxidative stress. APE1 enhances in vivo vascular repair effects of EPCs in part through the maintenance of adhesion properties of EPCs. APE1 may be a novel and useful target gene for effective cellular transplantation therapy.

Measurement of electric current evoked by substrate transport via bi-directional H+/oligopeptide transporter over-expressed in HeLa cells: electrogenic efflux and existence of a newly observed channel-like state.

In the present study, we measured an electric current induced by substrate transport in a HeLa cell over-expressing a human intestinal di/tri-peptide transporter using the whole-cell patch-clamp technique. Gly-Sar, a typical substrate, induced an inward current associated with its uptake, which showed concentration-dependency following Michaelis-Menten-type kinetics with an apparent K(0.5) of 1.3mM as well as voltage-dependency. An outward current accompanying the efflux of Gly-Sar was also observed after washing out the cell. This outward current was voltage-dependent and was reduced by the inward proton gradient. In the case of hydrophobic dipeptides such as Gly-Phe and Gly-Leu, a distinctive current was observed: after washing out the cells, no outward current was observed, but rather, an 'inward leak' current was sustained in spite of the absence of transportable substrate. This leaky current was abolished by the perfusion of Gly-Sar and subsequent washing. It is considered that the hydrophobic substrate sticks within the substrate-binding site and causes the newly observed state, or the 'inward leak' current.