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Toll-like Receptor Agonism Upregulates Endothelial Populations And Supports Healing Of Diabetic Wound
Shawn Jeffrey Loder, MD, Fuat Baris Bengur, MD, Wayne Vincent Nerone, BS, Phoebe Lee, BS, David Guerrero, BS, Lauren Kokai, PhD.
University of Pittsburgh, Pittsburgh, PA, USA.

PURPOSE: The diabetic wound microenvironment has several pathologic modifications which maintains its chronicity. The endothelium acts as a primary gatekeeper of the wound environment, which in its disease-related dysregulated state, engenders hypoxia and allows for a milieu of inflammatory markers and cells to accumulate and delay wound closure. Thymalfasin is an FDA approved synthetic analog of the Toll-like Receptor (TLR) -2/9 agonist Thymosin alpha-1; it is primarily used to enhance cell-mediated immunity in Hepatitis and melanoma, but also promotes endothelial migration and acute wound closure. Given that diabetic wounds display vascular deficiency and chronically elevated inflammation, we hypothesized that Thymalfasin could be used to more effectively treat chronic diabetic wounds. To test this hypothesis, we explored systemic use of Thymalfasin in our novel murine diabetic non-healing wound model.
METHODS: Male obese diabetic (B6.Cg-Lepob) mice received bilateral edge-inverted wounds mimicking the clinical architecture of epibole. Wounds were allowed to mature for 4-weeks to develop a stable granulation base. All wounds were measured photographically and dressed with tegaderm. Thereafter, mice received daily intraperitoneal Thymalfasin injections for 1 week at 250ug/kg. Dressings were then taken down and all wounds were re-measured. Closure fraction and wound areas were determined and then redressed. In parallel, a second cohort of Thymalfasin treated mice were used to collect granulation tissue for flow cytometric analysis using antibodies against CD31, VEGFR2, CD34, and CD45. The fraction of mature endothelial cells in the wound was determined by enrichment of (CD31+VEGFR2+CD34-CD45-) cells.
RESULTS: Prior to drug-therapy, there was no statistical difference in chronic wound area between the treatment and vehicle-control groups (0.41+/-0.32 vs. 0.42+/-0.26 cm2). After 1 week of intraperitoneal Thymalfasin therapy, chronic wounds demonstrated a post-treatment area of 0.06+/-0.13 cm2 with only 40% of wounds remaining open. Conversely, time-matched vehicle-treated wounds demonstrated area of 0.32+/-0.19 cm2 in area and no closure post-injury week 5. This was a statistically significant decrease in the Thymalfasin-treated group (91.58+/-16.48 % [Treatment] vs. 8.19 +/- 23.41% [Control]; p=0.0002). Tissues collected from the wound site were significantly enriched for mature endothelial (CD31+VEGFR2+CD34-CD45-) cells in the Thymalfasin-treated group (13.1 % [Treatment] vs. 6.8% [Control]; p<0.0001).
CONCLUSION: In pre-formed chronic diabetic wounds in mice, Thymalfasin-therapy was sufficient to trigger early closure and to significantly decrease overall wound area vs. vehicle-treated control. This was associated with significant enrichment in mature endothelial population. Mechanistically, Thymalfasin acts as a TLR 2/9 agonist with possible activity at TLR4 suggesting the potential for TLR agonism to enhance the vascular niche and speed healing in diabetic chronic wounds.


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