The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker

Abstract

Aims/hypothesis Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. Methods Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n=17) and non-healing (NH, n=11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. Results Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in nondiabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. Conclusions/interpretation We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.

NETosis Delays Diabetic Wound Healing in Mice and Humans

“…We previously developed and validated a proteomic platform for the identification of biomarkers and new molecular pathways related to diabetic wound healing (14). This platform required the proteomic characterization of tissue lysates obtained from wound biopsies in patients ("discovery cohort") with wound outcomes clearly defined as RH (n = 17) or NH (n = 11).…”

“…This platform required the proteomic characterization of tissue lysates obtained from wound biopsies in patients ("discovery cohort") with wound outcomes clearly defined as RH (n = 17) or NH (n = 11). The details of this protocol were previously published (14). Here, we reanalyzed the data to evaluate the differential abundance of proteins associated with NETs in NH versus RH wounds.…”

“…We recently established a platform for the discovery and validation of novel biomarkers for tissue healing in patients with DFUs (14). We identified several proteins differentially expressed in RH versus NH diabetic wounds from a biopsy of the wound margin using tissue digestion, protein extraction, and proteomic analysis (14).…”

“…We identified several proteins differentially expressed in RH versus NH diabetic wounds from a biopsy of the wound margin using tissue digestion, protein extraction, and proteomic analysis (14). The DAVID bioinformatic resource for functional annotation of genes/ proteins revealed that proteins enriched in NH versus RH wounds belonged to "cell death," "protease activity," and "defense response" pathways (14). NETosis is a type of cell death that is associated with protease activation and triggered by infection.…”

“…Therefore, we set out to validate these preclinical findings using a novel mouse model and to establish the effect of NETosis on the delayed wound healing in patients with diabetes. We took advantage of our platform developed for the identification of new candidate biomarkers and therapeutic targets in diabetic wound healing (14). This platform was based on a proteomic analysis of wound biopsies obtained from a "discovery" cohort of patients with DFUs who were unequivocally categorized as rapidly healing (RH) or nonhealing (NH).…”