Tissue Transglutaminase Enhances Fibrin-Dependent Angiogenesis and Extracellular Matrix Formation During Tissue Repair by Altering Gene Expression and Is Inhibited by Aspirin.

Abstract 3055

Poster Board II-1031

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 (TGM2) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TGM2 is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGFbeta-1) and expressed during tissue injury. In this study, we investigated how TGM2 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 TGM2 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 TGM2 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 C277A/TGM2 mutant did not exhibit increased wound healing response or proangiogenic effect. The data suggested that TGM2 enhanced the transition from the inflammatory stage of wound healing to proliferation stage. The two areas where TGM2 enhanced wound healing were 1) angiogenesis and 2) deposition of ECM. To investigate TGM2-induced angiogenesis-related gene expression, total RNAs were isolated from control- and TGM2-treated F-ZCs (at Day 6). 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. We identified increased expression of VEGF receptors Flk-1 (2-fold), Flt1 and neuropilin (1.4-fold), angiopoietin-1 (2-fold) and ephrin B2 (1.8-fold). There were decreased levels (5-fold) of matrix metalloproteinases (MMPs) and increased TGFbeta-1 receptors (1.5-fold) and connective tissue growth factor (CTGF)(1.4-fold) levels. The gene expression profile suggests that TGM2 promotes angiogenesis and enhances deposition of ECM.

We then investigated whether Aspirin (Acetylsalicylic Acid, ASA) a potent anti-inflammatory agent would inhibit TGM2. ASA and another chemical acetylating agent, sulfosuccinimidyl acetate (SNA), were used to investigate whether acetylation would alter the crosslinking activity of TGM2. We found acetylation by either SNA or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys⁴⁶⁸ and Lys⁶⁶³. Molecular modeling indicates that these Lys residues play an important role in the conformation change that occurs in TGM2 from a closed-to-open shape, i.e. inactive-to-active, transitions. In conclusion, we show that TGM2-fibrin crosslinking accelerates angiogenesis and promotes ECM deposition. This suggests that TGM2-fibrin interactions mediates outside-in signaling events that aides wound healing. Furthermore aspirin can acetylate and inhibit critical residues in TGM2 that regulate TGM-2 function.