When Activator and Inhibitor of PPARα Do the Same: Consequence for Differentiation of Human Intestinal Cells.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-dependent transcription factor that plays a role in various processes including differentiation of several cell types. We investigated the role of PPARα in the differentiation of intestinal cells using HT-29 and Caco2 cell lines as a model as well as human normal colon and colorectal carcinoma tissues. We detected a significant increase in PPARα expression in differentiated HT-29 cells as well as in normal surface colon epithelium where differentiated cells are localised. Thus, it seems that PPARα may play a role in differentiation of intestinal cells. Interestingly, we found that both PPARα activators (fenofibrate and WY-14643) as well as its inhibitor (GW6471) regulated proliferation and differentiation of HT-29 cells in vitro in the same way. Both compounds led to a decrease in proliferation accompanied by a significant increase in expression of villin, intestinal alkaline phosphatase (differentiation markers). Moreover, the same trend in villin expression was observed in Caco2 cells. Furthermore, villin expression was independent of subcellular localisation of PPARα. In addition, we found similar levels of PPARα expression in colorectal carcinomas in comparison to adjacent normal epithelium. All these findings support the hypothesis that differentiation of intestinal epithelium is PPARα-independent.

Comparative Analysis of Immunohistochemical Staining Intensity Determined by Light Microscopy, ImageJ and QuPath in Placental Hofbauer Cells.

Software based analyses of immunohistochemical staining are designed for obtaining quantitative, reproducible, and objective data. However, often times only a certain type of positive cells or structures need to be quantified thus whole image analysis cannot be performed. Such an example is Hofbauer placental cells, which show positivity of some antigens together with trophoblast, but only Hofbauer cells represent the regions of interest (ROIs). Two independent observers evaluated the immunohistochemical staining intensity of Hofbauer cells in placenta samples stained for cytoplasmic antigens by ImageJ, QuPath and light microscopy. Thus, the precise manual determination of ROIs, i.e. Hofbauer cells, was necessary. We detected low inter-observer variability in staining intensity. Almost perfect agreement between observers was reached for ImageJ and QuPath whilst substantial agreement was reached for light microscopy evaluation. As for the comparison of ImageJ, QuPath and light microscopy, the agreement of all three methods (identical immunohistochemical intensity) was achieved for 38.1% samples. The almost perfect agreement of staining intensities was reached between ImageJ and QuPath, and moderate agreement for comparison of the light microscopy to both software. Software analyses are much more time-consuming, thus their utilization is at least questionable to evaluate ROIs with selection.

Morphometric analysis of Hofbauer cells in normal placenta and chorioamnionitis in humans.

Hofbauer cells are macrophages residing in the stroma of placental villi and play a number of roles during normal pregnancy, as well as pathological conditions. A morphometric analysis of Hofbauer cells, in particular to investigate the number of cells, their size and shape in samples of normal human placenta from 1st trimester, term and with chorioamnionitis was performed. Tissue samples were immunostained for CD206 antigen and evaluated using ImageJ software. We detected significant changes in number and morphology of HBCs between normal placenta and placenta with chorioamnionitis samples. In chorioamnionitis, the cells were unevenly distributed within the villi, generally present in higher numbers, larger and more elongated than those in normal 1st trimester and term placenta.

Soluble Epoxide Hydrolase as an Important Player in Intestinal Cell Differentiation.

There is growing evidence that soluble epoxide hydrolase (sEH) may play a role in cell differentiation. sEH metabolizes biologically highly active and generally cytoprotective epoxyeicosatrienoic acids (EETs), generated from arachidonic acid metabolism by CYP epoxygenases (CYP2C and CYP2J subfamilies), to less active corresponding diols. We investigated the effect of sEH inhibitor (TPPU) on the expression of villin, CYP2C8, CYP2C9, CYP2J2, and sEH in undifferentiated and in vitro differentiated HT-29 and Caco2 cell lines. The administration of 10 µM TPPU on differentiated HT-29 and Caco2 cells resulted in a significant decrease in expression of villin, a marker for intestinal cell differentiation. It was accompanied by a disruption of the brush border when microvilli appeared sparse and short in atomic force microscope scans of HT-29 cells. Although inhibition of sEH in differentiated HT-29 and Caco2 cells led to an increase in sEH expression in both cell lines, this treatment had an opposite effect on CYP2J2 expression in HT-29 and Caco2 cells. In addition, tissue samples of colorectal carcinoma and adjacent normal tissues from 45 patients were immunostained for sEH and villin. We detected a significant decrease in the expression of both proteins in colorectal carcinoma in comparison to adjacent normal tissue, and the decrease in both sEH and villin expression revealed a moderate positive association. Taken together, our results showed that sEH is an important player in intestinal cell differentiation.