Temporal and differential proteomic profile of molecular mediators associated with chronic and acute wound healing.

The underlying pathophysiology of nonhealing chronic wounds is poorly understood due to the changes occurring at the gene level and the complexity arising in their proteomic profile. Here, we elucidated the temporal and differential profile of the normal and diabetic wound-healing mediators along with their interactions and associated pathways. Skin tissues corresponding to normal and diabetic wounds were isolated at Days 0, 3, 6, and 9 representing different healing phases. Temporal gene expression was analyzed by quantitative real-time PCR. Concurrently, differential protein patterns in the wound tissues were identified by Nano LC-ESI-TOF mass spectrometry and later confirmed by Western blot analysis. Gene ontology annotation, protein-protein interaction, and protein pathway analysis were performed using DAVID, PANTHER, and STRING bioinformatics resources. Uniquely identified proteins (complement C3, amyloid beta precursor protein, and cytoplasmic linker associated protein 2) in the diabetic wound tissue implied that these proteins are involved in the pathogenesis of diabetic wound. They exhibit enhanced catalytic activity, trigger pathways linked with inflammation, and negatively regulate wound healing. However, in the normal wound tissue, axin 1, chondroitin sulfate proteoglycan 4, and sphingosine-1-phosphate receptor were identified, which are involved in proliferation, angiogenesis, and remodeling. Our findings demonstrate the correlation between elevated gene expression of tumor necrosis factor-α, interleukin (IL)-1β, and identified mediators: aryl hydrocarbon receptor nuclear translocator, 5'-aminolevulinate synthase 2, and CXC-family, that inflicted an inflammatory response by activating downstream MAPK, JAK-STAT, and NF-κB pathways. Similarly, in normal wound tissue, the upregulated IL-4 and hepatocyte growth factor levels in conjunction with the identified proteins, serine/threonine-protein kinase mTOR and peroxisome proliferator-activated receptor gamma, played a significant role in the cellular response to platelet-derived growth factor stimulus, dermal epithelialization, and cell proliferation, processes associated with the repair mechanism. Furthermore, Western blot analysis indicated elevated levels of inflammatory markers and reduced levels of proliferative and angiogenic factors in the diabetic wound.