High-dose folic acid and its effect on early stage diabetic foot ulcer wound healing.

High-dose folic acid (HDFA; vitamin B9)-5 mg, given daily, has not been evaluated as a treatment to improve early stage-diabetic foot ulcer (ES-DFU) wound healing. However, HDFA has been demonstrated to correct: (a) endothelial dysfunction and decreased nitric oxide (NO) bioavailability, associated with type-2 diabetes mellitus (T2DM); and (b) hyperhomocysteinemia (HHcy) that may promote impaired DFU-wound healing. Measures of wound area (cm2 ) reduction (wound closure; WC), over a 4-week period (4 W-WC), greater than 50% of the wound area, have been reported as a robust indicator of the potential for DFU-wound healing. By using this model, we examined the effectiveness of a wound treatment in promoting progressive healing and complete wound closure for the chronic, nonhealing DFU-wound. To investigate this possible relationship between HDFA and ES-DFU wound healing, a retrospective cohort study of medical records, between November 2018 and April 2019, was performed for Veterans with T2DM and ES-DFUs following treatment with HDFA. During the study period 29 (n = 29) Veterans with ES-DFU wounds who received HDFA treatment were identified. Medical record reviews of this retrospective cohort of ES-DFU Veterans receiving HDFA report 90% (26/29) experiencing complete DFU-wound closure during the study period. Of the 29 Veterans with ES-DFUs receiving HDFA, the medical records of nine (30%), with healed wounds, provided documentation suitable for 4 W-WC, pre- and post-HDFA treatment study comparisons. This study documents significant (P < .05) improvements comparing 4 W-WC values for standard treatment for Veterans with poorly progressing, worsening or stagnating ES-DFU-wounds to those for the same subjects following HDFA treatment. These observations suggest that chronic ES-DFUs treated with HDFA may experience significantly improved wound closure and complete healing (re-epithelialization) when compared with standard treatments without HDFA. With validation from RCTs, HDFA may be established as an effective treatment to promote wound healing and closure for nonhealing ES-DFUs.

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.

Wound nitric oxide bioactivity: a promising diagnostic indicator for diabetic foot ulcer management.

Nitric oxide (NO) is a critical molecular signal and mediator for normal wound healing. Nitric oxide deficiency has been established as an important mechanism responsible for poor healing in diabetic foot ulcer (DFU) patients. Preliminary clinical wound healing studies using wound fluid nitrate determinations, as measures of wound NO bioactivity, suggest that threshold wound fluid nitrate values may function as an effective diagnostic indicator of successful wound healing for the DFU patient. In this case, wound fluid nitrate measurements could predict the suitability of a wound for specialized wound therapies such as topical growth factors or bioengineered skin substitutes. Wound fluid nitrate determination would also identify DFU patients with wound NO deficiency-related impaired healing that may promote wound complications possibly leading to lower-extremity amputation. With clinical validation, wound fluid nitrate may qualify as a wound healing biomarker and surrogate endpoint for DFU treatment. Additionally, combined diagnostic determinations of wound NO bioactivity, bacterial load, and matrix metalloproteinase production will enhance DFU management by providing quantitative parameters for clinical wound treatment. The impact of wound fluid nitrate determination and the combined platform of wound diagnostic indicators for wound healing prediction and clinical treatment, respectively, is anticipated to provide substantial costs savings and improved outcomes for the DFU patient.

Hyperbaric oxygen therapy mediates increased nitric oxide production associated with wound healing: a preliminary study.

OBJECTIVE: The objective of this preliminary study was to document general somatic and wound nitric oxide (NO) levels during and after hyperbaric oxygen therapy (HBOT). DESIGN: The study evaluated 6 chronic wound patients that responded favorably to HBOT treatment (20 treatments; 2.0 atmosphere absolute [ATA] x 90 minutes). Successful HBOT was associated with increased wound granulation tissue formation and significantly improved wound closure. Wound fluid and fasting plasma samples were obtained for measurement of nitrate and nitrite (NOx), the stable oxidation products of NO; plasma L-arginine (L-Arg); and asymmetric dimethylarginine (ADMA). NOx measurements were obtained before treatment (baseline), after 10 and 20 treatments, and at 1 and 4 weeks after therapy. RESULTS: Wound fluid NOx levels tended to increase during treatments, were significantly elevated at 1 and 4 weeks after therapy, and correlated with reductions in wound area. Plasma L-Arg and ADMA were unchanged during and after HBOT. CONCLUSION: This preliminary study documents a significant increase in local wound NO levels (by NOx measurements) after successful HBOT and suggests that this mechanism may be an important factor in promoting enhanced wound healing and wound closure associated with this therapy.