Atypical Diabetic Foot Ulcer Keratinocyte Protein Signaling Correlates with Impaired Wound Healing.

Diabetes mellitus is associated with chronic diabetic foot ulcers (DFUs) and wound infections often resulting in lower extremity amputations. The protein signaling architecture of the mechanisms responsible for impaired DFU healing has not been characterized. In this preliminary clinical study, the intracellular levels of proteins involved in signal transduction networks relevant to wound healing were non-biasedly measured using reverse-phase protein arrays (RPPA) in keratinocytes isolated from DFU wound biopsies. RPPA allows for the simultaneous documentation and assessment of the signaling pathways active in each DFU. Thus, RPPA provides for the accurate mapping of wound healing pathways associated with apoptosis, proliferation, senescence, survival, and angiogenesis. From the study data, we have identified potential diagnostic, or predictive, biomarkers for DFU wound healing derived from the ratios of quantified signaling protein expressions within interconnected pathways. These biomarkers may allow physicians to personalize therapeutic strategies for DFU management on an individual basis based upon the signaling architecture present in each wound. Additionally, we have identified altered, interconnected signaling pathways within DFU keratinocytes that may help guide the development of therapeutics to modulate these dysregulated pathways, many of which parallel the therapeutic targets which are the hallmarks of molecular therapies for treating cancer.

Nitric oxide levels in wound fluid may reflect the healing trajectory.

We analyzed nitric oxide metabolites (nitrate and nitrite, NOx) and other biomarkers in human wound fluids and correlated these markers with wound healing status (progressing or worsening) based on patient's wound history. Samples were collected pre- and postcleansing from patients with wounds of various etiologies and analyzed for NOx, matrix metalloproteinase activity, and elastase activity. A laboratory method was developed to analyze NOx which can detect at least 5 µM in samples as small as 10 µL. A nitrate-free sample collection device was identified to match the sensitivity of this new assay (most "nitrate-free" products tested contained nitrate levels higher than this detection limit when extracted in such a small volume). The correlation between pre- and postcleansing biomarker values, and the diagnostic potential of the biomarkers to wound progress were analyzed. Fifty wounds provided samples that were suitable for NOx analysis. The pre- and postcleansing values for NOx showed good correlation (r = 0.72); the correlation was not very strong for matrix metalloproteinase and elastase. Data analysis showed that NOx represents the best metabolite to discriminate between worsening and progressing wounds, and suggested that a two cut point diagnostic test using NOx is better than a single cut point test to identify progressing from worsening wounds.