"Investigative Study on Nitric Oxide Production In Human Dermal Fibroblast Cells Under Normal And High Glucose Conditions"

Background: Despite the advances in wound care, the healing of diabetic foot ulcers (DFU) continues to present a clinical challenge.  Altered nitric oxide (NO) production in macrophages has been identified as a factor in the dysregulation of diabetic wound healing, but it has not been established if other cells contribute to the NO needs of healing tissue. Due to the short half-life of NO, it is difficult to measure the actual impairment of cellular NO production. Previous studies have used nitrite and nitrate measurements to estimate NO production. Using a novel measurement system developed in our lab with proven reliability and validity, we are able to accurately measure real-time NO produced by cells grown in established tissue culturing protocols.

Purpose: In this study, we measured the actual NO levels released by human dermal fibroblasts under normal and high glucose conditions.

Results: Our results showed that NO production was significantly higher in normal glucose compared to high glucose conditions. The real-time NO was compared to the nitrite levels using the triiodide assay.  We found a direct correlation in normal glucose conditions. However, real-time NO and nitrite measurement did not correlate under high glucose conditions. We used Western blot analysis to measure the inducible nitric oxide synthase (iNOS) enzyme responsible for NO production; iNOS was upregulated in both normal and high glucose conditions. The difference in proliferation rate of fibroblasts was not statistically significant between all groups. Our results show that using nitrite to assess NO production is not an accurate method to measure NO present in the wound bed of patients with diabetes.

Conclusions: The results will be utilized with further studies to develop a NO-releasing/absorbing dressing that can be tuned to the needs of the wound at each stage of healing.