• Jeffrey Mclean posted an update 1 week ago

    These studies revealed abnormalities in the MAT of CD patients, including the infiltration of macrophages and T cells, perivascular inflammation, fibrosis and differences in adipocytes number and size. There are currently no studies regarding apoptosis in the MAT of CD individuals, nor in the animal model of hypertrophied MAT with associated colitis. With this purpose in mind, we used TUNEL assay to evaluate apoptosis in the intestinal mucosa and in MAT, which revealed significantly fewer apoptotic cells in CD, when compared to the control groups, not only in the intestinal mucosa, but also in MAT. Intestinal barrier is maintained due to balance rates of epithelial cell proliferation and cell death. The literature has shown that healthy intestinal mucosa has high rates of cell proliferation at the base of the epithelium, with inhibition of signals to apoptosis, whereas epithelial cells that compound the intestinal villi shows cell death activation. This mechanism is not totally understood, but it seems to be a shedding cell associated to cell death. This explains the high turn-over of the intestinal epithelial cells in homeostasis conditions. In the present study, we found a low amount of TUNEL-positive epithelial cells in CD compared to controls. This may be explained by the presence of damaged mucosa consequent to inflammation, where most part of the villi are lost. In this situation, the remaining cells may be the ones that show low rate of apoptosis and high rate of proliferation in order to recover the affected area and restore function. Concerning the decreased apoptosis in lamina propria cells of CD compared to the controls, and the fact that part of these cells are immune cells, this confirms previous published results in the literature. In addition, the main novelty of our study was the correlation of the number of apoptotic cells in MAT, as evidenced by TUNEL, with reduced adipocyte size. Peyrin-Biroulet et al. previously described the morphometric features of the adipocytes from hypertrophied CD MAT relating that these cells were small in size and four times higher in number when compared to control adipose tissue. However, these authors did not correlate these findings with apoptosis. Figure 1G shows the significant correlation between the morphometric parameters of the adipose cells and the number of the apoptotic cells in the MAT of CD and controls. These findings may explain, at least in part, the intriguing features of MAT in CD: the thickening of this tissue may be due to a resistance of adipose cells to undergo apoptosis, leading to an CCF642 increased number of adipocytes that exhibit a lower perimeter and area than the control group. NF-KB activation is one of the mechanisms described that can inhibit apoptosis by inducing Bcl-2 expression. High levels of NF-KB activation are verified in CD. This factor is responsible for activate transcription of a large number of genes related to inflammation, among them, TNF-a transcription. This may explain the resistance to apoptosis in MAT of CD patients. We did not verify positivity for Ki67 in MAT from CD patients and controls. This result is in accordance with what is described in the literature concerning fat cell turn over in humans. In non-obese conditions, adipose cells are not prone to proliferation. Adipocytes proliferation occurs only in severe cases of obesity, while hypertrophy occurs across all obese states. All CD patients included in the present study, as well as the healthy controls were not obese, presenting BMI less than 25. To describe the molecular mechanisms involved with the defective apoptosis detected by TUNEL, we studied Bax and Bcl2 transcripts and also the respective encoded proteins in the intestinal mucosa and MAT of CD patients. Itoh et al. found low levels of Bax in CD lamina propria T cells, using flow cytometric analysis, when compared to UC and controls, indicating a resistance to apoptosis in CD. In the present study, we report findings of low transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the control intestinal mucosa. Moreover, we observed that the low protein levels of Bax were localized in the lamina propria, and not in the epithelium. Although there was low transcriptional expression of Bcl2 in the ICD group, no differences were observed with regard to Bcl-2 protein expression, as analyzed by immunohistochemistry in this group. These findings reinforce the data of Itoh et al. and Santaolalla et al., who associated the defective apoptosis in the lamina propria to the Baxrelated pathways. A defective apoptosis was also seen in the MAT of CD. This apoptosis correlated significantly with high levels of Bcl-2 and Caspase 3, and not with Bax protein expression. The low Bcl2 transcriptional expression observed in association with higher protein MAT levels of Bcl-2 in the ACD group could be explained by cytosine methylation, which greatly increases the stability of the Bcl2 promoter. Another possibility is that high Bcl-2 protein levels could induce a negative feedback control of Bcl2 gene transcription. The decreased expression of Caspase 3 in MAT of ACD group compared to the control confirmed an altered apoptosis in this tissue. The point of our study is to present new data that may help to explain the singular characteristics of MAT in CD patients. Given the current emphasis that has been given to the role of adipose tissue in gut homeostasis and inflammation, the defective apoptosis of MAT in CD may explain the high survival rate of these cells, which in large amount may express higher levels of proinflammatory mediators. For instance, significantly higher expression of C-reactive protein, an inflammatory marker, was detected in the MAT of CD compared to UC and controls. Moreover, a comparison of adipocyte gene expression from MAT of CD and in healthy individuals showed up-regulation of proinflammatory genes and decrease of genes involving lipid metabolism. MAT may have an important role in the maintenance of inflammation in CD, since the altered balance between proinflammatory and anti-inflammatory factors in this tissue, as well as defective autophagy, have been previously reported in the literature. Among these studies, one of them verified lower levels of adiponectin in peripheral serum and in MAT of active CD patients, revealing deficient anti-inflammatory conditions. Moreover, this tissue may be involved in the maintenance of inflammation in the late stages of the disease, and in the mechanism that leads to relapses during the course of the disease.