Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

Tissue transglutaminase and the progression of human renal scarring

ABSTRACT. Experimental renal scarring indicates that tissue transglutaminase (tTg) may be associated with the accumulation of extracellular matrix (ECM), both indirectly via TGF-β1 activation and directly by the formation of ε(γ-glutamyl) lysine dipeptide bonds within the ECM. The latter potentially accelerates deposition and confers the ECM with resistance to proteolytic digestion. Studied were 136 human renal biopsy samples from a range of chronic renal diseases (CRD) to determine changes in tTg and ε(γ-glutamyl) lysine crosslinking. Immunofluorescence for insoluble tTg showed a 14-fold increase in the kidneys of CRD patients (5.3 ± 0.5 versus 76 ± 54 mV/cm2), which was shown to be active by a similar 11-fold increase in the ε(γ-glutamyl) lysine crosslink (1.8 ± 0.2 versus 19.3 ± 14.2 mV/cm2). Correlations were obtained with renal function for tTg and crosslink. In situ hybridization for tTg mRNA showed that tubular epithelial cells were the major source of tTg; however, both mesangial and interstitial cells also contributed to elevated levels in CRD. This mRNA pattern was consistent with immunohistochemistry for soluble tTg. Changes in renal tTg and its product, the ε(γ-glutamyl) lysine crosslink, occur in progressive renal scarring in humans independently of the original etiology and in a similar manner to experimental models. tTg may therefore play a role in the pathogenesis of renal scarring and fibrosis in patients with CRD and can therefore be considered a potential therapeutic target. E-mail: T.Johnson@sheffield.ac.uk

Increases in renal ε-(γ-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy:Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, e-(γ-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney e-(γ-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p <0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p <0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular e-(γ-glutamyl) lysine (+ 361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular e-(γ-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

TG2: a credible therapeutic target in fibrotic diseasedue to its multifunctional roles

The role of TG2 in fibrosis is reported to be related to two important effects. The first in mediating the deposition and accumulation of the fibrotic extracellular matrix (ECM) via its cross-linking of proteins like fibronectin, collagen I and collagen III; and the second, the activation of latent matrix bound TGFβ1. We report here that the role of TG2 in fibrosis progression can be much more complex. We also report a new family of TG2-specific inhibitors that can not only inhibit protein cross-linking, but also regulate other functions of TG2, thus increasing their potency which can be demonstrated by their effectiveness in inhibiting fibrosis in two different fibrotic in vivo models.

Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function:implications for age-related macular degeneration (AMD)

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D. Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test. Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure. Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.