Inflammation-related proteomics demonstrate landscape of fracture blister fluid in patients with acute compartment syndrome

Abstract

Background: Blisters are tense vesicles or bullae that arise on swollen skin and are found in a wide range of injuries. As a complication of fracture, fracture blisters are considered soft tissue injuries, which often lead to adverse effects such as prolonged preoperative waiting time and increased risk of surgical site infection. However, our previous study found that in patients with acute compartment syndrome, fracture blisters may be a form of compartment pressure release, but the specific mechanism has not been revealed. Here, we mapped out the proteomic landscape of fracture blister fluid for the first time and compared its expression profile to cupping and burn blisters.

Methods: First, fluid samples were collected from 15 patients with fracture blisters, 7 patients with cupping blisters, and 9 patients with burn blisters. Then, the expression levels of 92 inflammatory proteins were measured using the Olink Target 96 Inflammation panel. Protein profiles were compared across the three groups using Differential Protein Expression Analysis and Principal Component Analysis (PCA).

Results: Fracture blisters had significantly higher levels of 50 proteins in comparison to cupping and 26 proteins in comparison to burn blisters. Notably, PCA showed fracture blisters closely resembled the protein expression profile of burn blisters but were distinct from the protein expression profile of cupping blisters.

Conclusion: Our study provides the first characterization of fracture blister fluid using proteomics, which provides a valuable reference for further analysis of the difference between blisters caused by fractures and those caused by other pathogenic factors. This compendium of proteomic data provides valuable insights and a rich resource to better understand fracture blisters.

Keywords: acute compartment syndrome; blisters; cytokines; fractures; inflammation; proteomics.

Figure 1

Images of blisters from fracture, burn, and cupping patients. Fracture blisters appear 24 to 48 hours after the fracture injury, and their exterior is similar to burn blisters and cupping blisters.

Figure 2

FBG, BBG, and CBG have unique protein expression profiles. (A) Venn Diagram shows the number of proteins with expression higher than the limit of detection shared and unique in FBG, BBG, and CBG. (B) PCA plot projects all 31 patients to two dimensions showing 3 groups differentiated by color. Each point represents a single patient, with patients of similar protein expression profiles positioned next to each other. Explained Variance: PCA1 = 96.93%, PCA2 = 1.45%.

Figure 3

Comparison of protein expression in the three groups. (A) Box plots show the top 5 differentially expressed proteins across all three groups as identified by ANOVA. (B) Box plots show the top 5 differentially expressed proteins between FBG and BBG as identified by t-test. (C) Box plots show the top 5 differentially expressed proteins between FBG and CBG as identified by t-test. **p<0.01; *** p<0.001; **** p<0.0001; ns not significant.

Figure 4

Pairwise comparison of differentially expressed genes. (A) Heatmap of differentially expressed proteins in FBG and BBG patients (P-value < 0.05). Proteins are manually annotated by protein family. (B) Volcano plot depicts differentially expressed genes between FBG and BBG patients. The gray dashed line indicates an exploratory cutoff of P-value < 0.05. Each red dot corresponds with a gene passing the exploratory cutoff. Genes of interest are marked with gene names. (C) Heatmap of differentially expressed proteins in FBG and CBG patients (P-value < 0.05). Proteins are manually annotated by protein family. (D)Volcano plot depicts differentially expressed genes between FBG and CBG patients. The gray dashed line indicates an exploratory cutoff of P-value < 0.05. Each red dot corresponds with a gene passing the exploratory cutoff. Genes of interest are marked with gene names.

Figure 5

Correlation and interaction of proteins in FBG. (A) Correlation Heatmap depicts pairwise Pearson correlation coefficient values between proteins in FBG. Protein modules with high intercorrelation are marked with boxes. The rows and columns are grouped by hierarchical clustering. *p<0.05; **p<0.01. (B) Protein-protein interaction network shows predicted interactions between a group of proteins. Node colors represent average protein expression in FBG and edge weights indicate confidence level (strength of data support for the interaction).

Figure 6

Validation of levels of inflammatory cytokines in blister fluid of three groups of patients. Box plots show the Validation of levels of TNF, TGF-beta-1, VEGFA and CASP-8 in FBG, BBG and CBG. *P < 0.05, **P < 0.01.