Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors.

Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage

Effect of pharmacological inhibition of RIP2 and NOD1 siRNA mediated knockdown on responses of hESC-EC to <i>Haemophilus influenzae</i> (HIN) and C12-iE-DAP.

<p>(A) Relative expression (vs. GAPDH) of NOD1 following 48 hour incubation with NOD1 siRNA normalized to non-targeting siRNA; n = 6. (B) CXCL8 release from hESC-EC following 48 hour pre-incubation with non-targeting siRNA (open bars) or NOD1-siRNA (filled bars) and 24 hour treatment +/− C12-iE-DAP (10 µg/ml) or <i>Haemophilus influenzae (HIN)</i> (10⁷–10⁸ CFU/ml); n = 7–8. (C) Effect of GSK'214 (300 nM; RIP2 inhibitor) or GSK'217 (300 nM; NOD1 inhibitor), given 30 minutes before a 24 hour treatment with HIN (10⁷ CFU/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release; n = 4. It should be noted that GSK drugs increased CXCL8 release under basal conditions; for each experiment this was subtracted from treatment groups. For panel A, statistical significance was determined by one-sample t-test. For panel B statistical significance within siRNA groups was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05), and between groups by two-way ANOVA followed by Bonferroni's post-test (+p<0.05). For panel C statistical significance for the effects of inhibitor of C12-iE-DAP or HIN induced CXCL8 was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05).</p

MSD analysis of cytokine (pg/ml) release from hESC-EC.

<p>Data mean are ± SEM for n = 3. hESC-EC were treated for 24 hours with vehicle, LPS (1 µg/ml), or C12-iE-DAP (10 µg/ml). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05). ND = non-detectable.</p

Effect of <i>in vivo</i> ‘conditioning’ on TLR4 and NOD1 expression.

<p>TLR4 and NOD1 expression in (A) hESC-EC and (B) HUVEC before (pre-implant; open bars) and 21 days after (post-implant; filled bars) implantation <i>in vivo</i> (‘conditioning’). Data are mean ± SEM and are normalized at unity (1) to gene levels in pre-implant cells. HUVEC; NOD1 pre-implant n = 8, post implant n = 4: HUVEC; TLR4 pre-implant n = 10, post implant n = 3. hESC-ECs; NOD1 pre-implant n = 6, post implant n = 5: hESC-ECs; TLR4 pre-implant n = 10, post implant n = 6. Data was obtained from 2 independent experiments (using up to 12 rats per group). Statistical significance was determined by one-sample t-test where results were compared to a theoretical control of 1 (*p<0.05). ND = none detectable.</p

Responses of hESC-EC and HUVEC to 24 hour infection with <i>Heamophilus influenzae.</i>

<p>(A) Effect of LPS (1 µg/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release from hESC-EC and HUVEC after 24 hours. (B) Effect of <i>Haemophilus influenzae</i> (HIN) (10⁵–10⁸ CFU/ml) on CXCL8 release from hESC-EC (solid line) or HUVEC (dashed line) after 24 hours. Data are mean ± SEM; n = 3 representative of 6 hESC-EC isolations. Statistical significance for responses to drugs or bacteria was determined by one-way ANOVA followed by Dunnett's multiple comparison test (p<0.05).</p

MSD analysis of cytokine (pg/ml) release from HUVEC.

<p>Data are mean ± SEM for n = 3. HUVEC were treated for 24 hours with vehicle, LPS (1 µg/ml), or C12-iE-DAP (10 µg/ml). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05).</p