Angiogenesis-associated pathways play critical roles in neonatal sepsis outcomes

Abstract

Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here provide the mechanistic evidence in support of the relevance for these observations. Angiopoetin-1 (Ang-1), which promotes vascular integrity, was decreased in blood plasma of human and murine septic newborns. In preclinical models, administration of Ang-1 provided prophylactic protection from septic death. Arachidonic acid metabolism appears to be functionally connected to Ang-1 via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Strengthening this intersection via oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death while also increasing plasma Ang-1 levels among septic newborns. Our data highlight that targeting angiogenesis-associated pathways with interventions that increase Ang-1 activity directly or indirectly through ROS/eNOS provide promising avenues to prevent and/or treat severe neonatal sepsis.

Figure 1

Ang/TIE signaling is associated with neonatal sepsis. (A) Plasma Ang1 (left panel) and Ang2 (right panel) levels in human infants with LOS (sPLA-2/Ang sepsis cohort; n = 22 infants with n = 31 samplings) or with no LOS (n = 40 infants with n = 43 samples) as measured by ELISA (Wilcoxon rank-sum with median and 95% CI). (B) Plasma Ang1/Ang2 ratio in human infants with LOS vs without LOS, as measured by ELISA (Wilcoxon rank-sum). (C) Overall survival of cecal slurry (CS)-challenged mice after prophylactic administration of exogenous Ang1 or Ang2 (n = 21 per group, derived from two independent experiments; p = 0.0019, log-rank). (D) Overall survival of CS-challenged mice after prophylactic administration of anti-Ang2 blocking antibody (n = 11–15 mice per group, derived from two independent experiments; p = 0.0077, log-rank). (E) Overall survival of CS-challenged mice treated with either saline (CS) or Ang1 plus Ang2 (Ang1 + Ang2) one hour prior to CS challenge (n = 19–20 per group, derived from two independent experiments; p = 0.493, log rank). Violin plots use a combination a boxplot and a kernel density curve to show the distribution of data. The upper and lower bounds of the boxplot indicate the first and third quartiles, respectively, with the median displayed by a horizontal line; whiskers extend no further than 1.5 × the interquartile range from the hinge.

Figure 2

Nitric oxide regulates functionality of the Ang/TIE signaling axis. (A) Overall survival of CS-challenged mice with or without concurrent administration of the universal NOS inhibitor L-NAME (n = 14 CS, 22 L-NAME mice per group, derived from two independent experiments; p = 0.0013; log rank test). (B) Fold change over control uninfected mice of serum Ang1 and Ang2 levels in CS-challenged mice with or without concurrent administration of L-NAME (paired Wilcoxon rank-sum; n = 4–5 mice per group derived from 3 independent experiments). (C) Accumulation of ROS (O₂⁻) as measured by dihydroethidium staining in the liver of CS-challenged mice with or without concurrent administration of L-NAME as measured by dihydroethidium staining (paired Wilcoxon rank-sum; n = 8 control, 6 CS, 6 L-NAME, all pooled across 3 independent experiments). (D) Overall survival of CS-challenged mice (at LD50) with or without administration of L-NAME 4–6 h prior to challenge (n = 18–21 mice per group, derived from two independent experiments; p < 0.0001; log rank). (E) Survival of CS-challenged mice (at LD90) treated with saline only, L-Arg, Ang1 or combined L-Arg and Ang1 1-h post challenge (n = 15–21 mice per group, derived from two independent experiments; p < 0.0001; log rank test). Boxplots indicate medians with first and third quartiles (25–75%) at the lower and upper bounds of the box; whiskers extend no further than 1.5 × interquartile range from the hinge.

Figure 3

Whole blood, liver and spleen transcriptomics comparing likely survivors and non-survivors of murine neonatal sepsis. (A) Unsupervised clustering of normalized RNA-seq data using principal component analyses of transcriptomic data collected from blood , liver (middle) and spleen (bottom); (n = 7 Control, 9 CS-Survive, 10 (blood) or 13 (liver, spleen) CS-Die; derived from 5 independent experiments). (B) Selected immune response and metabolic pathways from top enriched GO terms (by q-value) from differentially expressed genes in predicted survivors and non-survivors in the blood, liver, and spleen. (C) Network of clustered enriched GO biological process terms from differentially expressed genes in predicted survivor vs non-survivor mice in the liver and whole blood. The size of the node reflects the number of genes in the term, while the colour represents the adjusted p-value. Each functional cluster is represented by a different color. Edges connect terms that have shared genes (similarity > 0.2), and the length of the edge is inversely proportional to term similarity.

Figure 4

Arachidonic acid signaling modulates Ang1/Ang2 ratio and is relevant in murine and human neonatal sepsis. (A) Overall survival of CS-challenged mice with or without prophylactic AA treatment 4–6 h prior to challenge (n = 16 CS, 29 AA mice, derived from two independent experiments; p = 0.003, log rank). (B) ROS (O₂⁻) accumulation in the liver of CS-challenged mice 8 h post challenge as determined by relative fluorescence intensity of dihydroethidium normalized to untreated controls (p < 0.001, paired Wilcoxon rank-sum test, n = 22 controls, 19 cs, and 10 AA treated mice (CS + AA) derived from 3 independent experiments). (C) Serum Ang1 and Ang2 levels in control, challenged (CS) and challenged / AA treated mice (CS + AA) (paired Wilcoxon rank-sum; n = 5 control, 10 cs, and 10 AA treated mice (CS + AA) derived from 3 independent experiments). (D) qPCR analysis of fold changes between ALOX15 (left), PTGS2 (middle) or CYP2J2 (right) to reference gene TUBB in a cohort of septic neonates from Malawi (n = 14 cases and 16 controls; Wilcoxon rank-sum). (E) Plasma sPLA-2 levels in human infants with any LOS vs. no LOS, as measured by ELISA (Wilcoxon rank-sum). (F) Spearman correlation showing relationship between plasma sPLA-2 levels and plasma ANG1 levels (left-panel), plasma ANG2 levels (middle panel) and plasma ANG1/ANG2 ratio (right panel) for all neonates (22 cases and 40 controls) within our cohort (Spearman correlation). Boxplots indicate medians with first and third quartiles (25–75%) indicated by the lower and upper bounds of the box; whiskers extend no further than 1.5 × interquartile range from the hinge. Violin plots display a kernel density curve overlaying a boxplot.

Figure 5

Proposed model of enhancing vascular resilience in neonatal sepsis via 3 complementary strategies. During the steady state, vascular integrity is maintained by sequestration of Ang2 within intracellular vesicles. eNOS remains coupled to the cellular membrane, and converts L-Arg into NO. During the septic inflammatory response, eNOS is uncoupled from the cellular membrane and converts L-Arg into ROS. ROS releases Ang2 from within the endothelial cell which outcompetes Ang1 from binding to the TIE2 receptor. The Ang2:TIE2 complex triggers the release of tight junctions and causes vascular instability. Vascular resilience was enhanced through three avenues: (1) L-Arg supplementation ensured that eNOS could access sufficient substrate to generate NO instead of ROS, improving survival outcomes. (2) Exogenous Ang1 ensures a more favorable Ang1:Ang2 ratio which led to decreased levels of ROS improving survival. (3) Oral supplementation with ARA prior to challenge resulted in decreased levels of ROS in the liver during sepsis (via a still unknown mechanism), while increasing levels of plasma Ang1 and improving survival outcomes. L-Arg L-arginine, NO nitric oxide, eNOS endothelial nitric oxide synthase, Ang1 angiopoetin-1, Ang2 angiopoetin-2, ROS reactive oxygen species, ARA arachidonic acid. This image was created using Biorender.