Plasma proteomics reveals gestational age-specific responses to mechanical ventilation and identifies the mechanistic pathways that initiate preterm lung injury

Abstract

The preterm lung is particularly vulnerable to ventilator-induced lung injury (VILI) as a result of mechanical ventilation. However the developmental and pathological cellular mechanisms influencing the changing patterns of VILI have not been comprehensively delineated, preventing the advancement of targeted lung protective therapies. This study aimed to use SWATH-MS to comprehensively map the plasma proteome alterations associated with the initiation of VILI following 60 minutes of standardized mechanical ventilation from birth in three distinctly different developmental lung states; the extremely preterm, preterm and term lung using the ventilated lamb model. Across these gestations, 34 proteins were differentially altered in matched plasma samples taken at birth and 60 minutes. Multivariate analysis of the plasma proteomes confirmed a gestation-specific response to mechanical ventilation with 79% of differentially-expressed proteins altered in a single gestation group only. Six cellular and molecular functions and two physiological functions were uniquely enriched in either the extremely preterm or preterm group. Correlation analysis supported gestation-specific protein-function associations within each group. In identifying the gestation-specific proteome and functional responses to ventilation we provide the founding evidence required for the potential development of individualized respiratory support approaches tailored to both the developmental and pathological state of the lung.

Figure 1

Multi-variate analysis confirmed GA-specificity of the plasma proteome response to ventilation. Multi-variate analysis of SWATH-MS data included (A) principle component analysis (PCA) and (B) partial least squares discriminant analysis (PLS-DA). Venn diagrams depicting the number of proteins expressing increased (C) or decreased (D) differential expression in response to ventilation as determined by paired t-test, p < 0.05. N = 7–8/group.

Figure 2

Normalized MS-SWATH HBB values (A) and ELISA-validation of plasma HBB concentration (B) in extremely preterm (open circle), preterm (grey square) and term (closed triangle) lambs during 60 minutes of ventilation. All data are expressed as mean ± SD. ^***Differs from 0 minute concentration in extremely preterm group, P < 0.001 (one-way ANOVA with Tukey’s post tests), ^†††Differs from 0 minute concentration in preterm group, P < 0.001 (one-way ANOVA with Tukey’s post tests), ^‡‡‡Differs from 0 minute concentration in term group, P < 0.001 (one-way ANOVA with Tukey’s post tests). N = 5–7/group.

Figure 3

Network mapping of differential protein expression was GA-specific. Log₂ fold changes and the GA-specific IPA-derived network pathways associated with differentially-expressed proteins in (A) extremely preterm, (B) preterm and (C) term ventilated lambs (P < 0.05, paired t-test pre-ventilatoin vs. 60 minute post-ventilation). Red and green nodes/text represent increased and decreased protein expressionat 60 minutes ventilation, relative to matched pre-ventilation samples. Proteins shown by white nodes were not identified in plasma samples. Solid lines indicate direct interactions or relationships, while dashed lines indicate indirect effects mediated by additional proteins. Proteins known to be influenced by antenatal betamethasone are shown in italics.

Figure 4

Alterations in protein expression predominantly mapped to the coagulation-complement cascade. Protein alterations observed in the extremely preterm (red), preterm (green) or term (blue) groups mapped to the coagulation-complement cascades. Grey boxes indicate identified proteins which were not altered by ventilation and black boxes represent proteins that were not identified in plasma samples. N = 7–8/group.

Figure 5

Unique protein-function associations were identified within each GA-group. CIM map representation of significant associations between changes in protein level measured prior to ventilation and at 60 minutes ventilation and functional outcomes in extremely preterm (A), preterm (B) or term (C) lambs. Red squares represent positive Pearson correlations (r ≥ 0.7) and blue squares represent negative Pearson correlations (r ≤ −0.7), with color intensity indicative of strength of correlation. Significance of correlations was calculated using a Mantel test with 1000 permutations. Non-significant results with r values of −0.69 to 0.69 are show in in grey. Functional outcomes have been grouped according to the colour key displayed. N = 7–8/group.