Submission Number: 10345. Tuesday, 10 October 2006, 2:00 PM to 2:15 PM, Presentation 2:04 PM

Continuous Live Imaging of TGF-Beta Activity Using a Novel Transgenic Mouse Wound-Healing Model

Thomas Satterwhite, BS, Alphonsus K. Chong, MD, Jian Luo, PhD, Hung Pham, BS, Melinda Costa, MD, Michael T. Longaker, MD, MBA, Tony Wyss-Coray, PhD, James Chang, MD

INTRODUCTION: Scar formation is a fundamental plastic surgery problem. The pathogenesis of scarring is unclear, and the treatment options are time-consuming, expensive, and consistently unsuccessful ([1], [2]). Increased transforming growth factor beta (TGF-Beta) expression correlates with scarring and excess collagen deposition ([3], [4], [5]); however, the spatial and temporal expression patterns of TGF-Betaduring normal and pathological wound repair remain unresolved. We used a novel transgenic mouse system that allows real-time continuous in vivo tracking of TGF-Beta1 activity by measuring bioluminescence after skin wounding. . .

METHOD: The transgenic mouse model was generated using a Smad2/3-responsive luciferase (SBE-luc) construct ([6]). In vivo: Two dorsal excisional skin wounds were made using a skin biopsy punch in SBE-luc mice (n=8). Bioluminescence was detected with the In Vivo Imaging System (Xenogen) at fixed time points following surgery (Figure 1). Mice were euthanized, and wounds were harvested for immunohistochemical staining.

In vitro: A TGF-Beta1 dose response was conducted on dermal fibroblasts harvested from SBE-luc mice (n=6), with maximal luciferase induction occurring at 0.1 ng/ml. Fibroblasts were then co-treated with TGF-Beta1 and increasing doses of either neutralizing antibody to TGF-Beta (NA-TGFBeta) or SB-431542, a novel inhibitor of the TGF-Beta receptor ([7]). Bioluminescence was measured with an automatic luminometer. SBE-luc fibroblasts underwent Western blot analysis for collagen type I production. Statistical analysis was performed with Student’s t-test and ANOVA.

RESULTS: In vivo: Bioluminescence peaked on Day 17 and was 4-fold higher than baseline (p<0.05) (Figure 1). The number of hair follicles increased around the wound edge, and the base of the hair follicles stained intensely for phosphorylated Smad2/3 (Figure 3A and 3B), suggesting that increased Smad2/3 activity in the hair follicles of wounded SBE-luc skin increases the bioluminescence seen on in vivo imaging.

Figure 1: SBE-luc mouse with dorsal skin

wounds (left) with corresponding image after

bioluminescent detection and photon quantification

of wounds (right, circled).

Figure 2: In vivo bioluminescent activity at various time points post-wounding in SBE-luc mice. Bioluminescence was expressed as fold increase in activity over baseline at Day 0. Bioluminescence peaked on Day 17 and was 4-fold higher than baseline (p<0.05).

Figure 3: Immunohistochemical staining for phosphorylated Smad2/3 in (A) unwounded skin and (B) wounded skin 17 days post-wounding (100X magnification). Wounded skin had an increased number of hair follicles and increased staining for phosphorylated Smad2/3 in the base of the hair follicles.

In vitro: NA-TGFBeta inhibited luciferase activity in a dose-dependent fashion in cultured SBE-luc fibroblasts, with complete inhibition achieved by 0.1 ug/ml (p<0.05) (Figure 4A). SB-431542 inhibited luciferase activity in a dose-dependent fashion, with complete inhibition achieved by 1 uM (p<0.05) (Figure 4B). SB-431542 inhibited collagen type I production in a dose-dependent fashion in SBE-luc fibroblasts (Figure 5).

A B

Figure 4: Effect of (A) NA-TGFBeta and (B) SB-431542 on luciferase activity in SBE-luc fibroblasts in vitro. Bioluminescence was expressed as fold increase in activity over non-treated control. NA-TGFBeta and SB-431542 inhibited luciferase activity in a dose-dependent fashion with complete inhibition achieved at a concentration of 0.1 ug/ml and 1 uM, respectively (p<0.05). Error bars represent standard deviation, n=3.

Figure 5: Western blot of the effect of SB-431542 on collagen I production in SBE-luc fibroblasts. SB-431542 inhibited collagen I production in a dose-dependent fashion, with inhibition observed at 1 uM and 5 uM.

CONCLUSION: The in vivo data suggest that bioluminescence after wounding can be measured through real-time imaging, and it correlates with Smad2/3 activation. The in vitro data suggest that the bioluminescence from dermal SBE-luc fibroblasts is responsive to TGF-Beta1, it can be inhibited, and it correlates with the synthesis of collagen—a key component in wound repair. Taken together, the in vivo and in vitro results validate the SBE-luc mouse as a novel wound-healing model. The use of biophotonics and continuous real-time imaging will translate to a more thorough understanding of the healing process, leading to better anti-scarring treatments.


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