Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Calcium Entry Via TRPC3 Channels Transduce Mechanical Force And Accerelate Wound Contraction
Kenichiro Kawai, M.D.,Ph.D1, Hisako Ishise, M.D.1, Barrett Larson, M.D.2, Soh Nishimoto, M.D.,Ph.D1, Toshihiro Fujiwara, M.D.,Ph.D1, Masao Kakibuchi, M.D.,Ph.D1.
1Hyogo College of Medicine, Nishinomiya, Japan, 2Stanford University, Stanford, CA, USA.

PURPOSE:
One of the major stimuli responsible for triggering pathological wound contraction is repetitive mechanical force on a lesion. However, the detailed mechanisms that account for this phenomenon are still largely unknown. We previously reported that Transient Receptor Potential Canonical Channel 3 (TRPC3) was highly expressed in contracted hypertrophic scar tissue and this channel might be a mechanotransducer that mediates stretching stimuli. In this study, we investigated the role that TRPC3 plays in wound contraction.
METHODS:
In order to investigate the role of TRPC3, TRPC3 overexpressing NIH3T3 mouse fibroblasts were created with a PMXS system. Either TRPC3 overexpressing or control-vector transfected fibroblasts were seeded on Type I collagen-coated silicon chambers (3×3 cm) at a density of 8×105 cells/chamber. Real-time calcium imaging (using the calcium indicator Fluo4) was performed during cyclic stretching of the cells at a frequency of 10 cycles/min.Next, a gel contraction assay with TRPC3 overexpressing fibroblasts was performed. Either TRPC3 overexpressing fibroblasts or vector transfected control fibroblasts were embedded in type I collagen at a final concentration of 5 × 105 cells and a collagen concentration of 1.5 mg/ml. The cells were cultured in 10% FBS for 24 hours after polymerization, followed by serum starvation with 0.5% BSA DMEM for 24 hours. Then, the TRPC3 agonist OAG, the TRPC3 specific blocker Pyr3 or a vehicle (DMSO) was added. After 72 hours of incubation, the contraction rates of the gels were recorded and analyzed.
The in vivo wound contraction rate in response to transplantation of TRPC3 overexpressing fibroblasts was then examined. Either TRPC3 overexpressing fibroblasts, control vector transfected fibroblasts (1×106 cells /mouse) or the same amount of normal saline were injected into the entire dorsal area (dermal and subdermal layers) of 8 week old female Balb/c nu/nu mice. At 10 days posttransplantation, bilateral dorsal cutaneous wounds were made and the rate of wound closure was measured.
RESULTS:
Calcium imaging demonstrated that there was increased calcium influx in response to repetitive mechanical stretching in TRPC3 overexpressing fibroblasts relative to control fibroblasts. A collagen gel assay containing TRPC3 overexpressing fibroblasts contracted more than the gels containing control fibroblasts. Moreover, adding OAG to the gels increased the contractile activities of the gels while Pyr3 attenuated the contraction of the gels. Transplantation of TRPC3 overexpressing fibroblasts resulted in a statistically significant increase in wound contraction at all time points.
CONCLUSION:
The TRPC3 channel is a potential mechanical force transducer that plays an important role in the pathogenesis of hypertrophic scar contracture. In the repetitive mechanically stretched wound, the expression of TRPC3 is induced and calcium influx through TRPC3 channel is increased. Although the mechanisms by which the transcription of TRPC3 is upregulated in stretched wounds is not clear, the findings presented here might offer new therapeutic targets for preventing hypertrophic scarring.


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