Development of a Transgenic Mouse Model of Age-Related Wound Healing
Parag Butala, MD, Denis Knobel, MD, James L. Crawford, BS, Caroline Szpalski, MD, Alexandre Marchac, MD, Steven M. Sultan, BA, Edward H. Davidson, MA,MBBS, Meredith T. Wetterau, MD, Pierre B. Saadeh, MD, FACS, Stephen M. Warren, MD, FACS.
NYU Langone Medical Center, New York, NY, USA.
Impaired wound healing in the elderly contributes to an increasing biomedical burden in aging populations. We introduce the Hutchinson-Gilford Progeria Syndrome Zmpste24 knockout mouse as a murine model of senescent wound healing. Furthermore, we investigate the mechanisms underlying impairment in healing and introduce pre-clinical validation of a potential therapeutic target.
Using an established wound model, 24 8-week old male Zmpste24-/- mice and 24 age-matched male C57/B6J mice underwent bilateral 6mm dorsal cutaneous wounding. Wounds were assessed with photographic analysis every other day until healed. Twelve additional 8-week male Zmpste24-/- mice were wounded and treated with the progenitor cell mobilizing agent AMD3100 (10mg/kg i.p. daily for 14 days) and wounds followed until closure. Wounds from all groups were harvested for immunohistochemistry, ELISA, and quantitative RT-PCR at day 10.
Zmpste24 mice displayed marked impairment in wound closure, with C57/B6J wild-type mice obtaining full closure at day 14 +/- 1.0 and Zmpste24 mice at day 40 +/- 2.5. Quantitative RT-PCR of wound beds demonstrated increased production of pro-apoptotic factors BAX and p53 (fold change 1.8 +/- 0.2, p<0.06 and 2.6 +/- 0.17, p<0.05) and significant decreases in VEGF (fold change 0.3 +/- 0.16, p<0.05). ELISA for VEGF and p53 corroborated RT-PCR findings (0.09pg/mL +/- 0.01 vs. 0.20pg/mL +/- 0.02, p<0.05 and 0.14pg/mL +/- 0.05 vs. 0.03pg/mL +/- 0.01, p<0.05, respectively) while CD31 immunohistochemistry supported ELISA findings and demonstrated significantly less vascularity among knockout mice. Immunohistochemistry for the proliferative marker PCNA showed decreased uptake compared to controls. Epidermal thickness was significantly decreased in knockouts compared to controls (4.9μm +/- 0.44 vs. 8.5μm +/- 0.95, p<0.04). Treatment with AMD3100 accelerated wound healing with wounds closing by day 30 +/- 4.0. RT-PCR of treated animals at day 10 demonstrated decreased p53, BAX, and PUMA expression (fold change 0.22 +/- 0.2, 0.24 +/- 0.3, and 0.29 +/- 0.3, p<0.05) and increased expression of HIF1-α, VEGF, SDF-1, and CD31. Epidermal thickness also normalized with AMD3100 treatment. Additionally, flow cytometry for quantification of circulating endothelial progenitor cells demonstrated significant increases in cEPCs after AMD3100 treatment (5.2% of lymphocytes vs. 0.12% of lymphocytes, p<0.05).
This is the first demonstration of a transgenic mouse model of senescent wound healing. We highlight the vasculogenic dysfunction in wound healing rescued with progenitor cell mobilization. The Zmpste24-/- mouse can herein serve as a novel model for the investigation of new therapies in age-related wound healing dysfunction.
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