The salient findings of this study are that (1) ESCs possess key elements of the NO-signaling machinery, but this pathway is functionally inactive in ESCs; (2) with differentiation, there is an increase in the expression of endothelial surface markers, eNOS, and subunits of sGC, in association with increasing activity of the NO-signaling machinery as assessed by cGMP production; and (3) chronic inhibition of NOS has no effect early in differentiation, but by 14 days of differentiation modestly inhibits the expression of endothelial surface markers CD144, FLK1, and eNOS, as well as the formation of endothelial tubes.
The NOS pathway is ubiquitous in the adult mammal, with endothelial, neuronal, and inducible NOS isoforms playing critical roles in every organ system, in particular cardiovascular homeostasis, thrombosis, neurotransmission, and immunity [24
]. With respect to vascular reactivity and structure, NO plays a dominant role as a vasodilation factor that also potently inhibits vascular smooth muscle proliferation, leukocyte infiltration, and platelet adherence and aggregation [25
]. Furthermore, NO is a powerful agonist of EC proliferation and migration. There is also a reciprocal reinforcing relationship between NO and vascular endothelial growth factor (VEGF), as VEGF stimulates NO synthesis, and NO increases VEGF expression [16
]. Inhibition of NOS substantially reduces growth factor-induced angiogenesis, whereas enhancing endogenous NO synthesis promotes angiogenesis [17
]. In particular, l
-NAME blocks VEGF-induced cGMP production in human umbilical venous ECs when cultured in collagen gels [27
]. Whereas l
-NAME is not metabolized by cells, asymmetrical dimethylarginine (ADMA) is an endogenous NOS inhibitor that is degraded by DDAH. Plasma ADMA is elevated in vascular diseases and associated disorders, due in part to impairment in DDAH activity [28
In view of the importance of NO in mammalian physiology and particularly its role in adult angiogenesis, it is somewhat surprising that embryonic lethality is not observed with homozygous eNOS deficiency. Even animals deficient for all 3 NOS isoforms may survive to adulthood (although hypertensive, infertile, and with nephrogenic diabetes insipidus) [31
]. Our results provide a partial explanation for this phenomenon. We observe that early in differentiation, although the key elements of the NOS pathway are present, they are functionally inactive. Accordingly, other signaling systems for endothelial growth must be primary early in development. In the adult mammal, other signaling molecules such as prostacyclin, epoxyeicosatrienoic acids, carbon monoxide, hydrogen peroxide, adrenomedullin, or C-type natriuretic peptide provide for redundancy, and become more evident when NOS is inhibited. It is possible that one or more of these signaling molecules play an important role in vascular development.
Previous studies have surveyed the differential expression of NO-signaling components during cardiac differentiation of ESCs. When murine embryoid bodies are incubated with the NOS antagonist N-monomethyl-L-arginine (l
-NMMA) or l
-nitroarginine, the number of EB-derived cardiomyocytes is not affected, but fibrillogenesis in the cardiomyocytes is reduced [32
]. This effect is mimicked by ODQ (an antagonist of soluble guanylate cyclase). These observations are consistent with those of Krumenacker et al. who showed an increase in expression of sGCα1
expression during cardiac differentiation of murine ESCs [11
]. Mujoo et al. also demonstrated an increase of sGC
subunits during the course of differentiation of human ESCs for 14 days, but the authors reported a progressive decrease in sGCβ2
mRNA from day 0 to day 14 [10
]. These observed differences may be attributed to differences in the ESC species and strains, as well as varying induction media formulations. In a recent study of human and mouse ESCs, NO donors and sGC activators could increase the mRNA expression of cardiac-specific genes myosin light chain
whereas NOS inhibitors decreased their mRNA expression [9
A limitation of the current study is the inherent heterogeneity of the population of differentiating cells, which includes ESC-ECs. Nevertheless, this mixed population can respond to pharmacological agents and provide useful biochemical and signaling cues to better understand the role of NO in ESC differentiation.
In summary, we have demonstrated that NO-signaling elements are present in ESCs, although the NOS pathway is functionally inactive until later in differentiation. Pharmacological inhibition of NOS has no observed effect early in differentiation on endothelial gene expression and surface markers, but later in differentiation causes a modest reduction of these genes and markers, as well as a modest impairment of endothelial tube-like formation. Our studies indicate that later in differentiation NO plays a role in endothelial development and function, but other signaling pathways must play a greater role early in development.