SummaryCoeliac disease is characterized by immunoglobulin-A (IgA)-class autoantibodies targeted against transglutaminase 2 (TG2), a multi-functional protein also with a role in angiogenesis. These antibodies are present in patient serum but are also found bound to TG2 below the epithelial basement membrane and around capillaries in the small intestinal mucosa. Based on these facts and the information that the mucosal vasculature of coeliac patients on a gluten-containing diet is disorganized, we studied whether the coeliac diseasespecific autoantibodies targeted against TG2 would disturb angiogenesis. The effects of coeliac disease-specific autoantibodies on in vitro angiogenesis were studied in angiogenic cell cultures. The binding of the antibodies to cells, endothelial sprouting, migration of both endothelial and vascular mesenchymal cells, the integrity of the actin cytoskeleton in both cell types and the differentiation of vascular mesenchymal cells were recorded. In vitro, IgA derived from coeliac disease patients on a gluten-containing diet binds to surface TG2 on endothelial and vascular mesenchymal cells and this binding can be inhibited by the removal of TG2. In addition, coeliac disease-specific autoantibodies targeting TG2 disturb several steps of angiogenesis: endothelial sprouting and the migration of both endothelial and vascular mesenchymal cells. Furthermore, the autoantibodies cause disorganization of the actin cytoskeleton in both capillary cell types that account most probably for the defective cellular migration. We conclude that coeliac disease-specific autoantibodies recognizing TG2 inhibit angiogenesis in vitro. This disturbance of the angiogenic process could lead in vivo to the disruption of the mucosal vasculature seen in coeliac disease patients on a gluten-containing diet.

Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients' serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a "gatekeeper" in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.

SummaryIn coeliac disease, the intake of dietary gluten induces small-bowel mucosal damage and the production of immunoglobulin (Ig)A class autoantibodies against transglutaminase 2 (TG2). We examined the effect of coeliac patient IgA on the apical-to-basal passage of gluten-derived gliadin peptides p31-43 and p57-68 in intestinal epithelial cells. We demonstrate that coeliac IgA enhances the passage of gliadin peptides, which could be abolished by inhibition of TG2 enzymatic activity. Moreover, we also found that both the apical and the basal cell culture media containing the immunogenic gliadin peptides were able to induce the proliferation of deamidation-dependent coeliac patient-derived T cells even in the absence of exogenous TG2. Our results suggest that coeliac patient IgA could play a role in the transepithelial passage of gliadin peptides, a process during which they might be deamidated.

In coeliac disease, ingestion of gluten leads to altered appearance of small-bowel mucosal microvasculature. It is thus conceivable that the small-bowel mucosal vascular biology might be involved in the pathogenesis of coeliac disease.

Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies.

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