Cheung TM, Ganatra MP, Peters EB, Truskey GA. Effect of cellular senescence on the albumin permeability of blood-derived endothelial cells. Am J Physiol Heart Circ Physiol 303: H1374-H1383, 2012. First published September 28, 2012 doi:10.1152/ajpheart.00182.2012In this study, we tested the hypotheses that endothelial cells (ECs) derived from human umbilical cord blood (hCB-ECs) exhibit low permeability, which increases as hCB-ECs age and undergo senescence, and that the change in the permeability of hCB-ECs is due to changes in tight junction protein localization and the activity of exchange protein activated by cAMP (Epac)1. Albumin permeability across lowpassage hCB-EC monolayers on Transwell membranes was 10 times lower than for human aortic ECs (HAECs) (P Ͻ 0.01) but similar to in vivo values in arteries. Expression of the tight junction protein occludin and tyrosine phosphorylation of occludin were less in hCBECs than in HAECs (P Ͻ 0.05). More hCB-ECs than HAECs underwent mitosis (P Ͻ 0.01). hCB-ECs that underwent Ͼ44 population doublings since isolation had a significantly higher permeability than hCB-ECs that underwent Ͻ31 population doublings (P Ͻ 0.05). This age-related increase in hCB-EC permeability was associated with an increase in tyrosine phosphorylation of occludin (P Ͻ 0.01); permeability and occludin phosphorylation were reduced by treatment with 2 M resveratrol. Tyrosine phosphorylation of occludin and cell age influence the permeability of hCB-ECs, whereas levels of EC proliferation and expression of tight junction proteins did not explain the differences between hCB-EC and HAEC permeability. The elevated permeability in late passage hCB-ECs was reduced by 25-40% by elevation of membrane-associated cAMP and activation of the Epac1 pathway. Given the similarity to in vivo permeability to albumin and the high proliferation potential, hCB-ECs may be a suitable in vitro model to study transport-related pathologies and cell aging. vascular biology; cell aging; occludin; endothelial progenitor cell; permeability

Purpose We sought to determine the effect of stress-induced senescence on the permeability to albumin of aging endothelial progenitor cells. Methods Human umbilical cord blood derived endothelial cells (hCB-ECs) and human aortic endothelial cells (HAECs) were treated with 200 μM H2O2 and permeability to FITC-bovine serum albumin was measured. Some samples were subsequently treated with 100μM 8-pCPT-2'-O-Me-cAMP, a cAMP analog that activates the Epac1-Rap1 pathway. Cell proliferation was measured with the EdU assay. Phase contrast, and immunofluorescence images were taken to observe morphological changes in cells after exposure to H2O2. Results hCB-ECs exposed to H2O2 exhibited a significant increase in permeability, but their response differed from the HAECs. Low passage hCB-ECs had a permeability increase of about 82% (p<0.01) compared to aged cells which had a permeability increase of about 37% (p<0.05). This increase in permeability was reduced by treating the cells with 100 μM 8-pCPT-2'-O-Me-cAMP. The younger cells exhibited a significant decrease in proliferation after being subjected to various concentrations of H2O2 whereas the aged cells exhibited a more gradual decrease in the percent of cells in S-phase. These changes also correlated with changes in cell morphology and junction staining. When placed back in the original media, the morphology and permeability of the hCB-ECs returned to the control condition, while the HAECs did not. Conclusions The permeability of low and high passage hCB-ECs and HAECs initially increases in response to oxidative stress. hCB-ECs, but not HAECs, were able to recover from the stress 24 hours later. Early passage hCB-ECs were more susceptible to exogenous H2O2 than late passage hCB-ECs. The increase in permeability of hCB-ECs to H2O2 also correlated with decreased cell proliferation and changes in cell junctions.