Functional Characterization of Embryonic Stem Cell-Derived Endothelial Cells

Endothelial cells (EC) derived from embryonic stem cells (ESC) require additional functional characterization before they are used as a cell therapy in order to enhance their potential for engraftment and proliferation. We explore several physiologically relevant functions of ESC-derived EC (ESCEC), such as its capacity to produce nitric oxide (NO), regulate permeability, activate and express surface molecules for the recruitment of leukocytes in response to inflammatory stimuli, migrate and grow new blood vessels, lay down extracellular matrix, and take up low-density lipoproteins. We also examined the ESC-EC ability to upregulate NO in response to shear stress and downregulate NO in response to pro-inflammatory TNF activation. Functional responses of ESCEC were compared with those of cultured mouse aortic ECs. The ESC-EC exhibit most aspects of functional endothelium, but interesting differences remain. The ESC-EC produced less NO on a per cell basis, but the same amount of NO if quantified based on the area of endothelial tissue. They also Received: May 18, 2010 Accepted after revision: January 12, 2011 Published online: May 31, 2011 Dr. Kara E. McCloskey School of Engineering University of California, Merced P.O. Box 2039, Merced, CA 95344 (USA) Tel. +1 209 228 7885, E-Mail kmccloskey @ ucmerced.edu © 2011 S. Karger AG, Basel Accessible online at: www.karger.com/jvr D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 2 :2 6: 16 P M Glaser /Gower /Lauer /Tam /Blancas /Shih / Simon /McCloskey J Vasc Res 2011;48:415–428 416 characterized these cells for a variety of endothelial specific markers using immunofluorescent labeling [2–5] . Lineage-specific markers that correlate with histological and phenotypic characteristics of somatic cells is routinely applied for the classification of many types of differentiated stem cells. Common EC markers include endothelial nitric oxide synthase (eNOS), receptors for vascular endothelial growth factors (VEGF) Flk-1 and Flt-1, vascular endothelial cadherin (VE-cadherin), CD34 and platelet EC adhesion molecule (PECAM-1). As previously published, our ESC-derived EC (ESC-EC) express these markers indicating their lineage-specific commitment, and do not contain cells expressing nondifferentiated ESC or smooth muscle cells [3, 4] . In addition to surface marker characterization, functional assays are also essential determinants of appropriate cellular maturation. Since EC or endothelial progenitor cells (EPC) differentiated in culture from stem cells are exposed to a more limited repertoire of the differentiation cues that are experienced in vivo , it is especially important to assess functional capability in addition to marker analysis. For the EC, physiologically relevant functional assays include the ability to synthesize nitric oxide (NO), regulate permeability (that is, the flux of molecules across an intact endothelium), respond to inflammatory activation by tumor necrosis factor(TNF), migrate and grow new blood vessels, and produce appropriate extracellular matrix (ECM) for developing a basal lamina. Characterization of in vitro-derived ESCEC and their response to environmental cues and inflammation is a critical step in assessing the quality of cultured cells for clinical application in cell-based therapies. EC Synthesize and Release NO EC regulate blood pressure and blood flow by releasing the vasodilators including: NO and prostacyclin, as well as vasoconstrictors including: endothelin and platelet-activating factor. Production of these molecules is critical for maintaining vascular homeostatic function [6] . Release of NO by the EC relaxes the smooth muscle cells in the walls of the arterioles, and is the principal factor that regulates dilation of the vessel wall and, in turn, blood flow. NO also inhibits the aggregation of platelets and thus keeps inappropriate clotting from interfering with blood flow. NO is synthesized within EC (among other cells) by eNOS. eNOS and NO secretion is constitutively active in EC, but is also upregulated by certain stimuli, such as laminar shear stress [7–10] . The mechanisms underlying the shear stress-induced eNOS expression include enhancing eNOS gene transcription and stabilizing eNOS mRNA. In both of the cases, the tyrosine kinase c-Src played a central role, and a complex kinase cascade including Raf, Ras, MEK1/2 and ERK1/2 seems to be involved in the signal transduction leading to eNOS transcription by shear stress [11] . In addition, TNF stimulation has been shown to downregulate eNOS mRNA, protein and activity in EC via destabilization of eNOS mRNA [12] . EC Regulate Vascular Permeability to Macromolecules and Protein In addition to regulating blood flow, endothelium plays a vital role in regulating the transport of fluids, molecules and cells between the bloodstream and surrounding tissue. An EC monolayer is relatively impermeable, ! 1% flux, to large molecules (1–100 kDa). The presence of membrane-bound receptors helps facilitate this gatekeeping role for numerous molecules including proteins, lipid-transporting particles, metabolites and hormones [6] . A number of investigators have successfully grown EC on porous supports to create in vitro models of permeability [13–15] for studying the permeability of the EC monolayer under various conditions. Transient increases in endothelial permeability do occur normally after tissue injury; however, chronic increases in permeability is abnormal and has been implicated in atherosclerosis, diabetic retinopathy and tumor growth [16] . EC Are Building Blocks for Generating New Blood Vessels It is widely accepted that the new blood vessels arise through two mechanisms during development, called vasculogenesis and angiogenesis [17] . Vasculogenesis is a process in which hemangioblasts, EC precursors from mesodermal origins, grow and organize to form vascular networks. Angiogenesis is the formation of new blood vessels by sprouting from existing vessels. Recently, an alternative view has demonstrated the presence of circulating EC in the adult [18] and their incorporation into ischemic tissue [19] , suggesting a role for vasculogenesis in the adult as well as in the embryo. It is now thought that both angiogenesis and vasculogenesis occur during postnatal life and that they are not mutually exclusive events. Both angiogenesis and vasculogenesis require EC proliferation, migration and three-dimensional organization [20] . The ability of EC to form lines and tube-like formations in vitro has been demonstrated to be a good measure of vasculogenic and angiogenic potential in tissue. A variety of culture conditions for generating these vascular structures have been employed, including culturing cells on the surface of collagen or fibrin gels, on a D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 2 :2 6: 16 P M ESC-Derived Endothelial Cells J Vasc Res 2011;48:415–428 417 thin layer of fibronectin, using a Matrigel invasion assay, collagen or fibrin gel sandwich assay, or by directly embedding EC in three-dimensional gels [for review, see 2 ]. One of the most common (and controversial) assays involves seeding EC directly on Matrigel. Although a common assay for EC function, recent evidence suggests that the EC may also be responding to tension forces of cellular traction due to the low rigidity of the adhesive support [21–23] , rather than undergoing true vasculogenesis. Angiogenesis, on the other hand, can be observed using the simple Matrigel assay and observing evidence of new cell proliferation and sprout formations. EC Produce ECM Vascular endothelium is tightly associated with a basal lamina that consists of multiple ECM molecules arranged in a sheet-like structure that supports the endothelium [24] . The ECM of EC is also vital to cell signaling processes and structural developments [25] in order to stabilize their microenvironment and to provide a niche for the vascular tissue. EC lining the lumen of blood vessels produce their own ECM material components during the formation of a basement membrane, which separates endothelial layer from the underlying smooth muscle cells. This basement membrane is composed of a variety of ECM materials including collagen type IV, laminin and fibronectin. Collagen type IV is a network-forming collagen that is the most abundant factor in the ECM-surrounding blood vessels providing structural stability [26] . Laminin is another major component of the basement membrane around blood vessels. Laminin-8 and laminin-10 are specific isoforms produced by EC [27] . Laminin interacts with collagen type IV in the basement membrane and helps stimulate growth and differentiation of vascular cells [28] . Laminin is also specifically necessary for maturation of vascular smooth muscle cells in the blood vessel [38] . Previous studies have shown laminin to be more actively produced in subconfluent EC cultures, with a decrease in laminin production once cells become more densely packed [29] . Fibronectin is another insoluble protein component of the ECM and is found as a soluble disulfide-linked dimer in the plasma. Recent studies suggest that fibronectin is a key component of vasculogenesis [30, 31] , and that unlike laminin, fibronectin production by EC is independent of cell density [29] . Inflammation and EC Activation During inflammation the endothelium alters its adhesive properties supporting leukocyte recruitment by sequential binding of selectins, chemokines and 2-integrins that function cooperatively to elicit rolling, arrest and transmigration. In vitro, endothelium can be ‘activated’ using acute inflammatory cytokines such as TNF. This, in turn, activates a mitogen-activated protein kinase signaling pathway that leads to the transcription and upregulation of cell adhesion molecules including E-selectin, ICAM-1 and VCAM-1 as well as chemokines which guide recruitment of leukocytes [32, 33] . The process also facilitates the homing of highly proliferative endothelial progenitor cells and thus is important in both neovascularization in regions of ischemia or re-endothelialization of areas where the endothelium is denuded [34, 35] . Low-Density Lipoprotein Uptake Normal EC are able to take up low-density lipoprotein (LDL), a lipoprotein that carries cholesterol through the bloodstream, and these functions transport cholesterol to the arteries. Not only is LDL uptake an important EC function, it is one of the most commonly used assessments for identification of an EC. Studies have shown that in areas of low and disturbed flows, normally active atheroprotective genes are suppressed and the pro-atherogenic genes are upregulated, leading to an increase in the engagement and synthesis of LDL cholesterol by EC and an increase in the permeability of the endothelium to LDL. This ultimately promotes the subendothelial accumulation of LDL [36] . Oxidative stress is also increased by promoting the production of reactive oxygen species and oxidation of LDL [36] . Vascular Diversification at the Cellular Level The vascular system is a complex network of arteries that transport oxygen-rich blood to tissues and veins that carry the oxygen-depleted blood back to the heart. Arterial endothelia are surrounded by a thick layer of smooth muscle cells embedded in collagen, whereas venous endothelia lack smooth muscle cells. Due to these anatomical and physiological differences, one expects that the endothelium for these structures would have distinct molecular signatures. Recent discoveries of molecular markers for arterial, venous and lymphatic EC now allow a more sophisticated characterization of endothelial diversity [37, 38] . Arterial specification, promoted by Notch signaling, is characterized by ephrinB2, delta-like (Dll)-4, Notch1 and 4, Jagged-1, and connexin-40 expression. Venous endothelium, potentially a default pathway of EC differentiation [39] , is characterized by EphB4 and COUP-TFII. Committed lymphatic EC, differentiated from venous EC, express Prox-1 as the most specific lymphatic EC marker. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 2 :2 6: 16 P M Glaser /Gower /Lauer /Tam /Blancas /Shih / Simon /McCloskey J Vasc Res 2011;48:415–428 418 For the EC that might be used clinically for cell therapies, especially if derived in vitro from stem cells, it is important that these cells function similar to those in vivo counterparts. Here, we examined the capacity of in vitro stem cell-derived EC to produce NO, regulate permeability, activate and express surface molecules for the recruitment of leukocytes in response to inflammatory cues, migrate and grow new blood vessels, lay down appropriate ECM for the basal lamina, and to take up LDL. We then compared the responses of ESC-EC within cultured mouse aortic ECs (MAEC). These assays reflect some of the most important known functions of a mature EC.