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

Hypoxia increases the potential for neutrophil-mediated endothelial damage in COPD

Rationale: Chronic obstructive pulmonary disease (COPD) patients experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia, and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterised by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage assessed. Histotoxic protein levels were measured in normoxic versus hypoxic neutrophil supernatants and plasma from exacerbating COPD patients and healthy controls. Main results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from COPD patients. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia; hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of COPD patients had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive ‘hyper-secretory’ neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of 5 neutrophil degranulation and vascular injury identified in COPD patient plasma. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD

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

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