Human blood outgrowth endothelial progenitor cells (BOECs), derived in culture from peripheral blood mononuclear cells, possess high proliferative capacity, and endothelial protein expression profiles (i.e. CD34, 31, 133, 144, 105, 146, KDR, Ac-LDL and vWF). As such they hold potential for autologous cell seeding applications in cardiovascular biomaterials or as prognostic indicators of endothelial mediated diseases. Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular homeostasis and loss of eNOS activity is a hallmark of endothelial dysfunction. We therefore examined the expression of eNOS in BOECs finding markedly lower protein (0.34 ± 0.13, Western blot) as well as activity levels (0.49 ± 0.18, Nitrite analysis) when compared to human umbilical vein endothelial cells (HUVECs). BOEC eNOS protein levels initially decreased with both length of time in culture and passage, but the levels stabilized between Passage 5 to Passage 8. To further examine the role of eNOS expression on BOEC functional activity we performed transfection studies with eNOS minicircle vectors (double stranded DNA of reduced size and are devoid of bacterial sequences). Two promoters were tested for expression efficiency in BOECs, the CMV promoter (pMini-CMV-eNOS) and the EF1α promoter (pMini-EF1α-eNOS). Transfection with pMini-CMV-eNOS achieved 24.8 ± 5.1 times more eNOS expression when compared to null transfected cells at 24 hours, a marked improvement over that achieved with conventional PVAX plasmid (10.2 ± 4.7 fold increase) or pMini-EF1α-eNOS (8.2 ± 1.2 fold increase both compared to null transfected control). pMini-CMV-eNOS also produced more sustained eNOS expression (3.1 ± 0.7 fold increase at 72 hours). Boyden chamber and tube formation assays showed that pMini-CMV-eNOS mediated overexpression can both improve cell migration (24.9 ± 4.2 versus 13.8 ± 3.7 cells/field) and angiogenesis (0.92 ± 0.13% versus 0.58 ± 0.19% of total pixels per high power field both for eNOS transfected versus null). These results suggest that minicircle vectors can be used to markedly enhance eNOS expression in BOECs and as such enhance their angiogenic and regenerative potential.
Extracellular Matrix Differentially Regulates Endothelial Nitric Oxide Synthase Production in HUVECs and Human Blood Outgrowth Endothelial Progenitor Cells
