Wound Healing Society

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Protease-activated Receptor-2 Knockdown Attenuates The Fibrotic Phenotype In Post-burn Hypertrophic Scar Fibroblasts
Jayson W. Jay, Anesh Prasai, Amina El Ayadi, David N. Herndon, Celeste C. Finnerty.
University of Texas Medical Branch, Galveston, TX, USA.

BACKGROUND: Hypertrophic scarring (HTS) following major burn injury remains a critical morbidity with limited treatment options that ultimately diminishes quality of life for burn victims. We and others have found that cutaneous mast cells (MC) are increased significantly in burn wounds and HTS. Tryptase, released by the stimulated MC, activates the protease-activated receptor-2 (PAR2) and is thought to play an important role in post-burn HTS pathophysiology. Here, we examined serum tryptase concentrations in a pediatric burn population. Additionally, in an in vitro model, we investigated the anti-fibrotic effects of PAR2 knockdown in primary post-burn HTS fibroblast cultures. METHODS: Serum was collected from age-matched non-burned and burned pediatric patients with greater than 20% of total body surface area burned in this IRB-approved study. Tryptase-β2 was measured by ELISA. In vitro, primary HTS fibroblasts were treated with siRNA to PAR2 or scrambled siRNA for 72 hours prior to 1-hour treatment with PAR2 activating peptide SLIGKV. After treatment, mRNA targets of PAR2 activation and subsequent fibrotic signaling were quantified by RT-qPCR in three independent experiments. RESULTS: Following severe burn injury, serum tryptase increased significantly during acute ICU admission (n=5, 6.104 ng/mL ± 0.93, p=0.0002) and remained elevated for at least 6 months post-burn (n=5, 5.105 ng/mL ± 1.793, p=0.0009) compared to non-burned controls (n=6, 0.7083 ng/mL). Furthermore, PAR2 siRNA knockdown significantly reduces mRNA transcript expression of fibrotic genes, including Collagens-1, and -3, MMPs-2, and -9, and α-SMA (p<0.05) compared to control-treated cells. Moreover, stimulation with SLIGKV confirmed complete PAR2 knockdown as there was no increase in the expression of the fibrotic genes. CONCLUSION: Together, these data show PAR2 activation intensifies the HTS fibrotic phenotype in burn HTS fibroblasts and further suggests that targeted PAR2 antagonism at wound sites may decrease MC tryptase’s proliferative effect and potentially limit detrimental fibrosis in post-burn HTS over time.


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