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 significantly 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, confirming 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.

FIG. 1.

Effect of NG-nitroarginine methyl ester (l-NAME) on endothelial differentiation of embryonic stem cells (ESCs). (A) Gene expression time course of endothelial differentiation in the absence (Con) or presence of l-NAME (LN, 10⁻⁵ M or 10⁻⁴ M) for CD144, (B) endothelial nitric oxide synthase (eNOS), and (C) FLK1. Data normalized to 18S and expressed as relative fold change. (D) Immunoblots for endothelial markers 14 days after differentiation in the presence of l-NAME. Quantification of immunoblots for (E) eNOS and (F) FLK1 (n = 3). Statistically significant comparisons (*P < 0.05; **P < 0.01).

FIG. 2.

Purification and characterization of embryonic stem cell-derived endothelial cells (ESC-ECs). The ESCs were differentiated for 3 weeks and then purified by fluorescently activated cell sorting (FACS) based on positive expression of the mature endothelial marker, CD144. Shown are the (A) isotype negative control and (B) CD144⁺ population. After purification, the CD144⁺ cells were characterized by immunofluorescence staining for (C) CD144 or (D) endothelial nitric oxide synthase (eNOS) to confirm their endothelial phenotype. Scale bar = 25 μm. Color images available online at www.liebertonline.com/scd.

FIG. 3.

Effect of NG-nitroarginine methyl ester (l-NAME) treatment on tube-like formation of embryonic stem cell-derived endothelial cells (ESC-ECs) in Matrigel. Endothelial differentiation of ESCs was carried out in the presence of l-NAME (LN, 10⁻⁵ M or 10⁻⁴ M) for 21 days. After purification by CD144⁺ expression, the ESC-ECs were assessed for tube-like formation upon withdrawal of l-NAME for 24 h. Representative images of tube-like formation for cells having undergone chronic l-NAME or dimethyl sulfoxide (DMSO) vehicle control treatment are shown along with quantification of branch points (n = 4). Prior exposure to l-NAME (10⁻⁵ M dose only) inhibited tube formation. Statistically significant comparisons (*P < 0.05) Scale bar = 500 μm.

FIG. 4.

Time course of soluble guanylyl cyclase (sGC) and dimethylarginine dimethylaminohydrolase (DDAH) gene expression during endothelial differentiation. Embryonic stem cells (ESCs) were cultured in differentiation media for 14 days and assayed at specified time points by quantitative PCR. Data shown is normalized to 18S housekeeping gene and expressed as fold changes relative to ESC group. *Statistically significant compared to ESCs group, P < 0.05 (n = 3).

FIG. 5.

Immunofluorescence staining depicting soluble guanylyl cyclase (sGC) α₃ and sGCβ₃ expression during the course of endothelial differentiation in embryonic stem cells. Immunofluorescence staining is shown for (A) sGCα₃ and (B) sGCβ₃, with corresponding Hoechst 33342 nuclear dye. (C) After 14 days of differentiation, the cells were immunofluorescently stained for both CD144 and sGCα₃ or sGCβ₃, with corresponding Hoechst 33342 nuclear dye. Scale bar = 200 μm (A and B); 25 μm (C). Color images available online at www.liebertonline.com/scd.

FIG. 6.

Effect of endogenous or exogenous activators of nitric oxide (NO) on cyclic guanosine monophosphate (cGMP) activity. At different time points of differentiation, cell lysates were assayed for cGMP production after (A) no treatment (basal), (B) A23187, (C) SNAP, or (D) BAY41 stimulation. (E) After 14 days of differentiation in media containing NG-nitroarginine methyl ester (l-NAME) (10⁻⁵ M), cells were stimulated with activators of NO release, and lysates were assayed for cGMP. Asterisk with brackets indicates statistically significant comparisons, and asterisk without brackets indicate statistically significant when compared to embryonic stem cells (ESCs) (P < 0.05).