Tissue Transglutaminase Enhances Fibrin-Dependent Angiogenesis and Extracellular Matrix Formation by Altering Gene Expression during Wound Healing.

Fibrin deposition triggers an injury response that involves the migration of inflammatory cells formation of new blood vessels and the synthesis of extracellular matrix (ECM). Tissue transglutaminase (TTG) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TTG is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGF beta-1) and expressed during wound healing response. In this study, we investigated how TTG modulated fibrin-dependent wound healing and the associated angiogenic response. We used an animal model consisting of fibrin Z-chambers (F-ZC, dual porous plexiglass chambers containing fibrin), implanted into the subcutaneous tissue of rats and harvested subsequently for quantitative assessment of granulation tissue formation (wound healing) and microvessel density (angiogenesis). We found that local administration of recombinant TTG into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p<0.001), an effect similar in magnitude to 25 ng/ml of TGFbeta1 administered in the F-ZC. The pro-healing effect of TTG was associated with a 2-fold increase in microvessel density in granulation tissue at day 6 of wound healing response (p<0.001). As a negative control, inactive recombinant TTG mutant did not exhibit increased wound healing response or pro-angiogenic effect. The data suggested that TTG enhanced the transition from the inflammatory stage of wound healing to proliferation stage. The two areas where TTG enhanced wound healing were 1) angiogenesis and 2) deposition of matrix. To investigate TTG-induced gene expression, total RNAs were isolated from control- and TTG-treated F-ZCs (at Day 6) using Trizol reagent (Invitrogen, CA). Biotin-labeled cDNA probes were synthesized, and hybridized to nylon membranes containing angiogenesis-related gene arrays (Superarray, MD). The signals were detected using streptavidin-peroxidase and quantitated using Superarray’s software. We identified increased expression of VEGF receptors Flk-1, Flt1 and neuropilin, suggesting increased responsiveness to the potent angiogenic factor VEGF. In addition, increased levels of angiopoietin-1 and ephrin B2 were observed which are involved in vascular development and stabilization. For matrix enhancing effects, considerably decreased levels (5-fold) of matrix metalloproteinases (MMPs) coupled with increased TGFbeta receptors and connective tissue growth factor (CTGF) were observed. The gene expression profile suggests that TTG alters the balance between matrix production and destruction in favor of production resulting in increased deposition of ECM in granulation tissue. In conclusion, we have identified that TTG 1) enhances fibrin-dependent wound healing response, 2) increases angiogenesis through enhanced VEGF receptors, angiopoietin-1 and ephrin B2 expression, and 3) promoted matrix deposition by simultaneously reducing MMPs and increasing CTGF and TGFbeta receptors expression.