Increased MEOX1 or MEOX2 expression leads to increased endothelial cell senescence.

A) Representative flow cytometry showing the density of BrdU⁺ endothelial cells (upper left and right quadrants). HUVECs were transduced with N-terminal FLAG tagged MEOX1 and MEOX2 adenoviral constructs at a multiplicity of infection (MOI) of 100; 48 hours later, cells were labeled with BrdU for one hour prior to fixation. DNA was stained with 7-aminoactinomycin D (7-AAD). B) Quantification of the flow cytometry data. Expression of MEOX1, MEOX2 and MEOX2^Q235E mutant decreased cellular proliferation comparable to p53 (positive control) as assessed by BrdU incorporation into cycling cells. C) Representative images showing SA-β-gal⁺ cells (blue). Nuclei were stained with hematoxylin (brown). D) Quantification of SA-β-gal⁺ cells shows that both MEOX1 and MEOX2 expression increased the number of senescent HUVECs. In contrast, MEOX2^Q235E expression did not alter the level of endothelial cell senescence. HUVECs were transduced with N-terminal FLAG tagged MEOX1 and MEOX2 adenoviral constructs at a MOI of 250; 48 hours later cells were fixed and stained. D) MEOX proteins do not induced endothelial cell apoptosis. HUVECs were transduced with FLAG tagged MEOX1, MEOX2 and MEOX2^Q235E adenoviral constructs at a MOI of 250; 48 hours later cells were fixed and stained. Staurosporine was used as a positive control for apoptosis induction. * Indicates a statistically significant change (p<0.05) when compared to the EGFP control. ♦ Indicates a statistically significant difference (p<0.05) between MEOX1 and MEOX2.