Control Lung Volumes and Proteomics at Baseline
As expected, the spatial distribution of lung volumes was not uniform in the normal ventilated lung (Saline/MV group) (Figure 1A and E; Additional file 2: Figure S1a and b). Regional sVt was substantially lower in the accessory lobe R4 than other regions, particularly compared with R3 (p = 0.013), L5 (p = 0.009) and L6 (p = 0.013), whereas sEEV did not show notable regional differences. In terms of the baseline proteomic responses, the heat map showed regional heterogeneity in the protein profile (Additional file 2: Figure S2a). We used principal component analysis (PCA) to reduce the complexity of the proteomics data and to examine global trends of protein expression in the lung regions (Additional file 2: Figure S2b). In the saline group, most of the variance (63.90%) in protein expression was captured by the first three principal components. The PCA analysis suggested that L3, L6 and L1 were significantly different from other regions in relation to proteomic responses. The significant variation in baseline lung function and protein expression provided further rationale for subsequent regional analysis.
Effect of Mechanical Ventilation and Acid on Lung Volumes
As shown in Figure 2A, acid aspiration reduced global sVt by 8.2% (p = 0.047). When assessed regionally, sVt and sEEV in R4 were lower than the other regions (p < 0.001, Additional file 2: Figure S1c and d), however such variation was independent of acid exposure (p = 0.895).
We further assessed the effects of acid after 2 hours of ventilation on lung volumes. Compared to MV alone (Saline/MV), Acid/MV reduced global sVt by 9.2% (p = 0.015) (Figure 2b). MV alone had no effect on sVt (p = 0.093) or sEEV (p = 0.917) (Figure 2c), however, in the acid exposed group, global sVt decreased by 8.5% (p = 0.016) and global sEEV decreased by 6.9% (p = 0.031) (Figure 2c).
Identification of Differentially Expressed Proteins
Despite the presence of highly abundant proteins such as albumin and haemoglobin, we achieved a comparable proteome depth (Additional file 2: Figure S3) to similar studies [20]. After data filtering, 1943 proteins (FDR < 1%) were quantified across all samples relative to the control (Saline/FB) group. ON this basis, 46, 73 and 114 proteins were classified as differentially expressed in the Acid/FB, Saline/MV and Acid/MV groups respectively (Figure 3a-c) based on the q < 0.05 cut-off. Further analysis, after controlling for repeated measurements, identified 27 (up-regulation; 20: down-regulation; 7) in the Acid/FB group, 62 (up-regulation; 53: down-regulation; 9) in the Saline/MV group and 89 (up-regulation; 64: down-regulation; 25) in the Acid/MV group (Additional file 3: Table S1). Some of the differentially expressed proteins showed regional heterogeneity in expression level, including 5 in the Acid/FB group, 43 in Saline/MV and 61 in Acid/MV with 4 (3 up-regulated and 1 down-regulated) and 8 (all up-regulated) proteins showing regional differences that were influenced by treatment (Additional file 3: Table S1 a-c). Amongst the differentially expressed proteins, 42 were shared non-redundant proteins in both the Saline/MV and Acid/MV groups and 37 were uniquely present in Acid/MV group (Figure 3d).
Functional Characterization of Differentially Expressed Proteins
Functional annotation revealed that 27 of the differentially expressed proteins in the Acid/FB group are involved in transcriptional regulation process, including methylation, acetylation and RNA-binding (Additional file 3: Table S2-a). In contrast, the 62 dysregulated proteins in the Saline/MV group are involved in platelet aggregation, haemostasis, inflammation and fibrinolysis (Additional file 3: Table S2-b). Complement and coagulation cascades (CCC), comprising 8 proteins (Cfi, Cfd, Fga, Fgb, Fgg, Plg, Serpinc1, and Serpind1) was the most enriched KEGG pathway (Table 1).
Table 1: Pathway analysis of the differentially expressed proteins in different groups
KEGG Pathway
|
Count (%)
|
p
|
Benjamini (p)
|
Proteins involved
|
Proteins identified from comparison of Saline/MV and Saline/FB
|
|
|
|
|
Complement and coagulation cascades
|
8 (12.9%)
|
0.000
|
0.000
|
Cfi, Cfd, Fga, Fgb, Fgg, Plg, Serpinc1, Serpind1
|
Staphylococcus aureus infection
|
6 (9.7%)
|
0.000
|
0.000
|
Cfi, Cfd, Fgg, H2-Ea-ps, Itgb2, Plg
|
Phagosome
|
7 (11.3%)
|
0.000
|
0.002
|
Coro1a, H2-Ea-ps, Itgb2, Itgb3, Mpo, Thbs1, Tubb1
|
Platelet activation
|
6 (9.7%)
|
0.000
|
0.005
|
Fermt3, Fga, Fgb, Fgg, Itga2b, Itgb3
|
Shared proteins in Saline/MV and Acid/MV groups
|
|
|
|
|
Complement and coagulation cascades
|
7 (16.7)
|
0.000
|
0.000
|
Cfi, Fga, Fgb, Fgg, Plg, Serpinc1, Serpind1
|
Staphylococcus aureus infection
|
4 (9.5%)
|
0.000
|
0.013
|
Cfi, Fgg, Itgb2, Plg
|
Platelet activation
|
5 (11.9%)
|
0.001
|
0.012
|
Fermt3, Fga, Fgb, Fgg, Itgb3
|
Unique proteins in Acid/MV group
|
|
|
|
|
Complement and coagulation cascades
|
6 (16.2%)
|
0.000
|
0.000
|
C3, C4b, Cfh, Cfb, Kng1, Serpinf2
|
Parkinson's disease, Huntington's disease
|
7 (18.9%)
|
0.000
|
0.000
|
Ndufa9, Cox5a, Cyc1, Uqcrc2, Uqcrc1, Vdac1, Vdac2
|
Staphylococcus aureus infection
|
4 (10.8%)
|
0.001
|
0.008
|
C3, C4b, Cfh, Cfb
|
Oxidative phosphorylation
|
5 (13.5%)
|
0.001
|
0.011
|
Ndufa9, Cox5a, Cyc1, Uqcrc2, Uqcrc1
|
Cardiac muscle contraction
|
4 (10.8%)
|
0.002
|
0.015
|
Cox5a, Cyc1, Uqcrc2, Uqcrc1
|
MV: mechanical ventilation; FB: free-breathing.
Mapping of the 42 overlapping proteins dysregulated in Saline/MV confirmed that these CCC proteins were also present in the double hit model of acid aspiration and MV. Analysis of 37 unique proteins in Acid/MV group identified an additional 6 proteins relating to the enriched CCC pathway (C3, C4b, Cfh, Cfb, Kng1, Serpinf2). Other pathways identified from the set of unique proteins, consisted of 7 down-regulated proteins that have been linked to Parkinson's disease, Huntington's disease and cardiac muscle contraction. On further examination, these proteins belong to mitochondrial respiratory chain components (MRC) including Ndufa9 (Complex I), Cyc1, Uqcrc2, Uqcrc1 (Complex III), Cox5a (Complex IV), and Vdac1, Vdac2 (mitochondrial porin family). The details for the functional annotation of these overlapping and unique proteins can be found in Additional file 3: Table S2 c-d.
A regulatory network was established using the differentially expressed proteins (89) in the Acid/MV group (Additional file 2: Figure S4). Confirming our pathway analysis above, the network comprised of two clear clusters: 1) proteins associating with complement, coagulation, and fibrinolysis as hubs and 2) proteins involving the mitochondrial respiratory chain.
Association between Identified Protein Sets and Lung Volumes
Due to functional relevance and strong correlation of the proteins belonging to the same pathway (Additional file 2: Figure S5, p < 0.001 for all associations), we employed principal component analysis (PCA) to group these variables. Using this approach, we identified a principal component (PCA 1) for overlapping CCC proteins accounting for 61.07% of the total variance, as well as a PCA 1 for unique CCC proteins (78.60%), and a PCA 1 for unique MRC proteins (80.16%).
PCA 1 for both shared and unique CCC proteins was negatively associated with regional sVt (p < 0.05) (Figure 4a), whereas PCA 1 for the MRC proteins was positively correlated with regional sEEV (r = 0.381, p < 0.001) (Figure 4b).
Comparison of PCA 1 scores amongst the four groups showed that, for overlapping CCC proteins, both Saline/MV and Acid/MV had higher PCA 1 scores than the Saline/FB (control) group (Figure 4c). While, for the unique CCC and MRC proteins, the significant differences of PCA 1 scores were only found between the Acid/MV and Saline/FB groups (Figure 4d and e).