ESC-EC are a potential inexhaustible source of functional vascular cells that exhibit the key features of a mature EC for clinical diagnostics and regenerative medicine. In this study, we examined a number of physiological responses of ESC-EC to reveal their functionality and compared these responses with MAEC. The ESC-EC constitutively synthesized and released NO into the culture medium and the amount of NO was upregulated in response to physiological shear stress. Moreover, the ESC-EC responded predictably by downregulating NO in response to the addition of TNF-α pro-inflammatory signal. Interestingly, when measuring the NO produced by a confluent layer of EC covering the 100-mm2 culture dish, the NO production of the ESC-EC was comparable to that of the MAEC per area of tissue. However, once the amount of NO produced was normalized per cell, the larger MAEC did produce significantly more NO per cell than the smaller and more densely packed ESC-EC. It is interesting to note that although MAEC produced more NO than ESC-EC, the ESC-EC are also capable of producing physiologically appropriate levels of NO per area for homeostatic tissue function.
In our assay, both ESC-EC and MAEC generated an endothelial monolayer that was relatively impermeable, <1% flux, to large molecules (70 kDa), but semi-permeable, approximately 60% flux, to small molecules (370 Da). These values are typical of normal endothelium that is resistant to the flux of large molecules, as solute permeability to dextran-70-FITC is typically less than 5% of input [44
]. A transient increase in endothelial permeability to molecules on the order of 3-kDa molecules is also possible; however, these increases in permeability are typically observed in response to acute tissue injury, whereas abnormal endothelial permeability is more persistent and has been implicated in atherosclerosis, diabetic retinopathy and tumor growth [16
Of additional interest is the increased angiogenic, and potentially tumerogenic, nature of ESC-EC when seeded on Matrigel compared to MAEC. Typically, EC create intricate spiderweb-like networks on Matrigel-coated surfaces. As expected, the MAEC formed EC cord-like structures on Matrigel. The ESC-EC also formed these same structures with increased sprouting 24 h after seeding, and continued to proliferate and develop into a cell mass with further sprouting from the center of the cell mass. This could potentially be explained by the use of Matrigel scaffolding material, as these results are not observed for EC seeded in three-dimensional collagen gels [2
]. Matrigel is derived from Engelbreth-Holm-Swarm mouse sarcoma cells [45
]. The chief components of Matrigel are structural proteins such as laminin and collagen; however, Matrigel is also highly enriched with growth factors that promote differentiation and proliferation of many cell types. The unusual behavior for our ESC-EC is not typical for mature EC, and might be indicative of the immature or embryonic-like state of our ESC-EC, especially when combined with matrix and growth factors derived from tumors. Further studies in which shear stress or inflammatory stimuli are applied to condition the ESC-EC may facilitate their ability to progress towards a more mature EC phenotype.
Endothelial synthesis of ECM is also a critical function for the generation of a basement membrane and niche for EC survival and function. Both ESC-EC and MAEC produced collagen type IV, fibronectin and laminin on tissue culture plastic. ESC-EC also produced a low level of collagen type I. Both laminin and collagen type IV are major components of the basement membrane around blood vessels [28
], and fibronectin is a key component of vasculogenesis [46
]. The synthesis of these components indicates that the ESC-EC are able to synthesize components for the generation of an appropriate niche.
Vascular inflammation is important for wound repair, clearance of bacterial and viral infections, and revascularization during ischemia. Impairment of these processes results in infection, limb loss or organ failure. Thus, it is important that endothelium derived from embryonic sources for expansion and transplant function remain appropriately responsive to the pro-inflammatory milieu in which they are introduced. At baseline the confluent monolayers of ESC-EC constitutively expressed VCAM-1 and a subpopulation expressed ICAM-1 protein. These IgG superfamily members are important for endothelial progenitor cell recruitment to ischemic tissue by providing receptor targets for their β1
-integrin (that is, VLA-4) and β2
-integrin (that is, LFA-1) as demonstrated in recovery of hind limb blood flow in mouse models [34
]. Thus, our ESC-EC may be expected to participate in such processes in the absence of additional stimuli; however, further adhesion studies in shear are warranted in order to establish their capacity to recruit monocytes and progenitor cells. The ESC-EC demonstrated the ability to increase VCAM-1 and ICAM-1 in response to TNF-α, but were more resistant to inflammation than MAEC. TNF-α increased ICAM-1 on a subset of ESC-EC constitutively expressing ICAM-1, and was also able to induce nonexpressing cells to upregulate the ICAM-1 protein in a dose-dependent manner. This suggests that inflammatory stimuli may induce the differentiation of ESC-EC into mature endothelium that would be more responsive to inflammatory and other signals.
We further explored the characterization of the EC subphenotypes. Arterial specification was characterized by Notch-1 and delta-4L while venous specification was demonstrated by the expression of the EphB4 surface protein. Our data indicate the ESC-EC are composed of significantly more venous subphenotype than MAEC. Since a venous specification appears to be the default EC subphenotype during development, it is not surprising that our ESC-EC are also predominantly venous [39
]. Interestingly, the MAEC did not indicate homogeneous arterial cell phenotype cultures even though the cells were isolated from the abdominal aorta. It seems that some of the MAEC have lost their arterial specification in culture, possibly due to the lack of shear stress signaling in the static culture dishes.
Unfortunately, it is difficult to compare ESC-EC with EPC isolated from blood because EPC lack a unifying phenotype. The most recent literature discusses many different types of EPC generated using different culture methods [48
]. The three categories of EPC include: (1) colony-forming unit-Hill cells, (2) circulating angiogenic cells, and (3) endothelial colony-forming cells (ECFC). These cells are described by the distinct derivation methods used to generate the EPC. The colony-forming unit-Hill method plates peripheral blood mononuclear cells on fibronectin for 5 days and then replates the nonadherent peripheral blood mononuclear cells that subsequently grow into clusters of cells with sprouts at the peripheries. The circulating angiogenic cells are isolated from culturing the adherent cells with the nonadherent cells, and the ECFC are the expanded late colony outgrowths after the nonadherent cells are removed from cultures. Of these 3 types of EPC, all populations express VEGFR-2, CD34, vWF, eNOS and VE-cadherin; however, only ECFC late colony outgrowths form new blood vessel in vivo. Most early outgrowth cell cultures seem to be derivatives of hematopoietic progenitors expressing CD45, CD14 or CD115, which retain the phagocytic ability to ingest bacteria [48
]. We suspect that our ESC-EC might be most closely related to the ECFC type of EPC due to their ability to form vascular networks with lumens in vitro and expression of VEGFR-2, CD34, eNOS, VE-cadherin and respond to TNF-α, but direct comparisons between phenotypes would be more informative [2
In the effort to culture ESC-EC for cell transplantation or as substrates for pharmacological testing, it is important that these stem cell-derived EC function in predictable ways following incorporation into ECM or in the inflammatory milieu in which they will be placed. Our data indicate that the ESC-EC exhibit most aspects of functional endothelium, but interesting differences remain, such as a more robust proliferative profile and a less inflammatory phenotype of the ESC-EC as compared to mature aortic EC. In addition, our ESC-EC are more consistent with a venous EC subphenotype, and we suspect that physiological shear and inflammatory stimuli may play a role in the induction of these ESC-EC to behave more like mature arterial EC.