Using systems biology approaches to identify signalling pathways activated during chronic wound initiation.

Chronic wounds are a significant health problem worldwide. However, nothing is known about how chronic wounds initiate and develop. Here we use a chronic wound model in diabetic mice and a Systems Biology Approach using nanoString nCounter technology and weighted gene correlation network analysis (WGCNA), with tissues collected at 6, 12, 24 and 48 h post-wounding, to identify metabolic signalling pathways involved in initiation of chronicity. Normalized counts obtained from the nanoString nCounter Mouse Metabolic Panel were used for the WGCNA, which groups genes into co-expression modules to visualize the correlation network. Genes with significant module membership and gene trait significance (p < 0.05) were used to identify signalling pathways that are important for the development of chronicity. The pathway analysis using the Reactome database showed stabilization of PTEN, which down-regulates PI3K/AKT1, which in turn down-regulates Nrf2, as shown by ELISA, thus disabling antioxidant production, resulting in high oxidative stress levels. We find that pathways involved in inflammation, including those that generate pro-inflammatory lipids derived from arachidonic acid metabolism, IFNγ and catecholamines, occur. Moreover, HIF3α is over-expressed, potentially blocking Hif1α and preventing activation of growth factors and cytokines that promote granulation tissue formation. We also find that FGF1 is under-expressed, while thrombospondin-1 is over-expressed, resulting in decreased angiogenesis, a process that is critical for healing. Finally, enzymes involved in glycolysis are down-regulated, resulting in decreased production of pyruvate, a molecule critical for ATP production, leading to extensive cell death and wound paralysis. These findings offer new avenues of study that may lead to the development of novel treatments of CW to be administered right after debridement.