Using Systems Biology Approaches to Identify Signaling Pathways Activated During Chronic Wound Initiation. Using Systems Biology Approaches to Identify Signaling 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 hr post-wounding, to identify metabolic signaling 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 correlation network. Genes with significant Module Membership and Gene Trait Significance (p<0.05) were used to identify signaling pathways that are important for the development of chronicity. The pathway analysis using the Reactome database showed stabilization of PTEN which downregulates PI3K/AKT1, which in turn downregulates 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 downregulated 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. Overall design: For this study, we used a systems biology approach to identify metabolic signaling pathways important in initiation of wound chronicity. We chose to focus our observations on four major processes involved in wound healing: Response to Injury, Inflammation, Hypoxia and Repair Response. The window of 48hr post-wounding represents the initial stages of wound healing. Therefore, the idea of identifying the signaling pathways contributing to initiation of chronicity was what drove us to obtain the data presented in this manuscript. In addition, because patients who suffer from CW have underlying conditions such as diabetes, cardiovascular diseases etc. treatment of CW can be fine-tuned if the disrupted pathways, and thereby candidate biomarkers and therapeutic targets, can be identified. This is the gap in literature that we intend to fill with a WGCNA of chronic wound initiation. WGCNA helps identify modules significantly correlated to chronicity which puts the differentially expressed gene in the context of co-expressed and correlated genes thereby giving an insight into how the genes are regulated.