Advanced Glycation End Products Increase Transglutaminase Activity in Primary Porcine Tenocytes

Abstract

Objectives: Tendon abnormalities, such as increased stiffness, thickness, and excess calcification, occur commonly in patients with diabetes mellitus and cause considerable disability. These changes are frequently attributed to increased cross-linking of extracellular matrix components by advanced glycation end-products (AGEs). However, cellular effects of AGEs, such as increased activity of the cross-linking transglutaminase (Tgase) enzymes, could also contribute to altered tissue biomechanics and calcification in diabetic tendons. We determined the effect of AGE-modified protein on tenocyte Tgase activity. Research Design and Methods: Primary porcine tenocytes were exposed to Nε- carboxymethyl-lysine (CML)-modified type I collagen in high or normal glucose media. Protein and mRNA levels of the Tgase enzymes and Tgase activity levels were measured, as were markers of apoptosis. We also determined the effect of antioxidants on CML-collagen mediated Tgase activity. Results: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes 2.3- to 5.6-fold over unmodified collagen controls in both normal and high glucose media, without altering enzyme protein levels. Anti-oxidant treatment reduced the effect of CML-collagen on Tgase activity. Deoxyribonucleic acid laddering and annexin V protein levels were not altered by CML-collagen exposure. Conclusions: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes, likely posttranslationally. Increased levels of Tgase-mediated cross-links may contribute to the excess calcification and biomechanical pathology seen in diabetic tendons.

Nicotine reduced MMP‐9 expression in the primary porcine tenocytes exposed to cyclic stretch

“…Primary porcine tenocytes were isolated from rotator cuff tendons. The procedure for the isolation was based on the method previously described by Rosenthal et al11 and Pauly et al12 The infraspinatus tendons were scraped free of any adherent synovium or fat. The tenocytes were enzymatically released from the surrounding matrix using 0.25% trypsin for 1 h, followed by 1 mg/ml collagenase P (Roche, Indianapolis, IN) in Dulbecco's modified Eagle medium (DMEM: Invitrogen, Carlsbad, CA) for 3 h. Isolated cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin (P/S: Invitrogen); that is, growth medium.…”

Tendinopathy in diabetes mellitus patients—Epidemiology, pathogenesis, and management

“…Matrix stiffness is known to regulate the differentiation of mesenchymal stem cells (MSCs) . AGEs were reported to increase transglutaminase activity in tenocytes in vitro . Transglutaminase 2 is a cross‐linking enzyme of ECM proteins and it also facilitates cell‐ECM interaction through integrins.…”

“…99 AGEs were reported to increase transglutaminase activity in tenocytes in vitro. 100 Transglutaminase 2 is a cross-linking enzyme of ECM proteins and it also facilitates cell-ECM interaction through integrins. The increase in matrix stiffness as a result of AGE formation and transglutaminase 2 activation may modify the niche and hence promote nontenocyte differentiation of stem cells in tendons.…”

Site-specific AGE modifications in the extracellular matrix: a role for glyoxal in protein damage in diabetes

“…Cells grown on glucose-modified collagen expressed less of MMP-2 and more of endogenous collagen IV and MMP inhibitor TIMP-1 compared to cells grown on unmodified collagen (38, 46). Exposure of tenocytes to CML-collagen I increased intracellular activity of transglutaminase, enzyme that catalyzes formation of non-AGE cross-links in ECM (47). In addition, non cross-link GO-derived modifications such as CML, G-H1 and CMA may contribute to the lower digestibility of diabetic ECM because the consensus cleavage sites of many MMPs contain lysine and arginine residues (48).…”