Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

Abstract

Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester ( l -NAME) for up to 11 days were not signifi cantly different from vehicle-treated cells in EC markers. However, by 14 days, l -NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of l -NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of l -NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by l -NAME, confi rming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.

The role of Hath6, a novel shear stress-responsive transcription factor, in endothelial differentiation and function modulation

“…NO production in response to flow-dependent shear forces applied to the surface of endothelial cells is a fundamental mechanism of the regulation of vascular tone, peripheral resistance and tissue perfusion (Balligand et al, 2009). Huang et al also reported a positive effect of NO signaling on the endothelial differentiation of ESCs (Huang et al, 2010). Accordingly, one might hypothesize that NO signaling is part of the pathway that links shear stress to endothelial differentiation -shear-stress-factor-XeNOS-NO-endothelial differentiation.…”

Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells

“…The cell-seeded scaffolds were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized in 0.1% Triton X-100 (Sigma-Aldrich), and then blocked with 1% BSA. 37, 38 The scaffolds were then incubated with primary antibodies directed against anti-human troponin-T (Abcam), anti-human connexin-43 (Sigma), and anti-human CD31 I (Dako), followed by secondary antibodies conjugated to AlexaFluor 488 or AlexaFluor 594 (both from Thermo Fisher Scientific). The samples were washed in PBS, and total nuclei were counterstained using Hoechst 33342 (Thermo Fisher Scientific) nuclear dye.…”

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

“…23,44 In brief, at 1, 3, and 5 days of differentiation, the cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized in 0.1% Triton X-100 (Sigma-Aldrich), and then blocked with 5% normal goat serum (Cell Signaling Technology). The samples were then incubated with primary antibodies mouse anti-human CD31 (Dako), sheep anti-human CD31 antibody (R&D Systems) or mouse anti-human paxillin (BD Transduction).…”