The episomal constructs eNOS pGL3 −1193/+109, vWF pGL3 −487/+247, and VE-cadherin pGL3 −1912/+1 were transiently transfected into the EC human coronary artery endothelial cells (HCAECs), human pulmonary artery endothelial cells (HPAECs), HUVECs, and non-EC types HeLa, HEK293, and HuAoVSMCs. Shown are the fold increases in luciferase expression of eNOS pGL3 −1193/+109, vWF pGL3 −487/+247, and VE-cadherin pGL3 −1912/+1 constructs. Studies are controlled for transfection efficiency across cell types, as previously demonstrated.⁷  Steady-state levels of eNOS, VE-cadherin, and vWF mRNAs were not detectable in HuAoVSMCs as determined by reverse transcriptase (RT)-qPCR (supplemental Figure 1), in contrast to the observed in vitro activity of episomal EC-enriched promoter/reporter constructs. The eNOS pGL3 −1193/+109 episomal construct was active in the non-EC types HeLa, HEK293, and HuAoVSMCs, as previously described.⁷  Similar to eNOS, the VE-cadherin pGL3 −1912/+1 construct demonstrated a 5.17 ± 0.45- and 6.95 ± 0.56-fold increase in expression when transiently transfected into HEK293 and HeLa cell types, respectively. Robust expression was also demonstrated in all EC types. Interestingly, the vWF pGL3 −487/+247 construct also demonstrated expression when transfected into the non-ECs HeLa, HEK293, and HuAoVSMCs (1.49 ± 0.15, 2.72 ± 0.29, and 1.02 ± 0.09, respectively), as well as in the 3 EC types assayed.

Data expressed as luciferase units, relative to empty vector for each cell type. Shown is the mean ± standard error of the mean (n = 3).