Scar remodeling by alginate gel-encapsulated decoy Wnt receptor (sLRP6E1E2)-expressing adenovirus in a pig model

Abstract

An adenoviral vector (Ad) expressing a Wnt decoy receptor (sLRP6E1E2) is known to induce an anti-fibrotic effect by inhibiting Wnt signaling. We evaluated its effects in vivo using pig models and attempted to introduce an alginate gel-matrix system to prolong the effect of the Ad. Transduction efficiency was compared by fluorescent micrographs for the comparison of biological activity of Ad in different form; naked, gel-released and Ad in dissolved gel. And then, 50 days after the formation of full-thickness skin defects on the backs of Yorkshire pigs, scars were divided into five treatment groups: I, control, II, alginate gel, III, alginate gel capsulated control, IV, naked LRP6, and V, alginate gel capsulated LRP6. To evaluate its therapeutic efficacy, scar size and color index were compared, and various factors influencing scar formation and collagen rearrangement were analyzed through histology, immunohistochemistry and immunofluorescence assay. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was also analyzed for evaluation of expression levels of TGF-β1, TGF-β3, MMP-1, TIMP1, and α-SMA mRNAs. And to examine inflammatory cell infiltration within the scar tissues, the number of inflammatory cells was calculated from four serial H&E-stained tissue sections. GFP expression from gel-released Ad and Ad in dissolved gel was much higher than naked Ad. The size and color index of the scars has improved for the Ad treated group in pig model. The alginate gel encapsulating dE1-k35/sLRP6E1E2 treated scars (group V) were significantly improved in terms of scar surface size, erythema and melanin indices compared to other groups. Masson’s trichrome stain showed that premature collagen deposition was markedly decreased in group V scar tissues compared to the other groups. Picrosirius red stain also revealed that sLRP6E1E2 induces collagen rearrangement to resemble that of mature, bundle-shaped collagen fibers, and gel encapsulation enhances its efficacy. Expression levels of the major ECM components; collagen I, elastin, and fibronectin, are significantly decreased by sLRP6E1E2 overexpression, especially in the alginate gel reservoir. And the alginate gel encapsulating dE1-k35/sLRP6E1E2-treated scar tissues (group V) showed decreased TGF-β1 mRNA expression and increased TGF-β3 mRNA expression compared with other groups. The treatment also increases matrix metalloproteinase-1 mRNA expression and decreases tissue inhibitor of metalloproteinases-1 and alpha-smooth muscle actin mRNA expression in pig scar tissues. And Alginate gel encapsulating dE1-k35/sLRP6E1E2 decreases inflammatory cell counts and mast cell counts in pig scar tissues. Decoy Wnt receptor (sLRP6E1E2)-expressing adenovirus treatment improved scar quality in a pig model. Loading this construct in alginate gel allows sustained virus release into local tissues and prolongs Ad activity, thus maintaining its therapeutic effect longer in vivo. These advantages of the Ad/alginate system could markedly augment scar remodeling effects of sLRPE1E2 on scar tissue by steadily providing a high concentration of Ad with minimal toxicity.