Taoufik Khalfaoui

Polycomb repressive complex 2 impedes intestinal cell terminal differentiation

Summary The crypt–villus axis constitutes the functional unit of the small intestine, where mature absorptive cells are confined to the villi, and stem cells and transit amplifying and differentiating cells are restricted to the crypts. The polycomb group (PcG) proteins repress differentiation and promote self-renewal in embryonic stem cells. PcGs prevent transcriptional activity by catalysing epigenetic modifications, such as the covalent addition of methyl groups on histone tails, through the action of the polycomb repressive complex 2 (PRC2). Although a role for PcGs in the preservation of stemness characteristics is now well established, recent evidence suggests that they may also be involved in the regulation of differentiation. Using intestinal epithelial cell models that recapitulate the enterocytic differentiation programme, we generated a RNAi-mediated stable knockdown of SUZ12, which constitutes a cornerstone for PRC2 assembly and functionality, in order to analyse intestinal cell proliferation and differentiation. Expression of SUZ12 was also investigated in human intestinal tissues, revealing the presence of SUZ12 in most proliferative epithelial cells of the crypt and an increase in its expression in colorectal cancers. Moreover, PRC2 disruption led to a significant precocious expression of a number of terminal differentiation markers in intestinal cell models. Taken together, our data identified a mechanism whereby PcG proteins participate in the repression of the enterocytic differentiation program, and suggest that a similar mechanism exists in situ to slow down terminal differentiation in the transit amplifying cell population.

Autophagy is active in normal colon mucosa.

Recently, autophagy has been found to be strongly activated in colon cancer cells, but few studies have addressed the normal colon mucosa. The aim of this study was to characterize autophagy in normal human intestinal cells. We used the expression of LC3-II and BECN1 as well as SQSTM1 as markers of autophagy activity. Using the normal human intestinal epithelial crypt (HIEC) cell experimental model, we found that autophagy was much more active in undifferentiated cells than in differentiated cells. In the normal adult colonic mucosa, BECN1 was found in the proliferative epithelial cells of the lower part of the gland while SQSTM1 was predominantly found in the differentiated cells of the upper part of the gland and surface epithelium. Interestingly, the weak punctate pattern of SQSTM1 expression in the lower gland colocalized with BECN1-labeled autophagosomes. The usefulness of SQSTM1 as an active autophagy marker was confirmed in colon cancer specimens at the protein and transcript levels. In conclusion, our results show that autophagy is active in the colonic gland and is associated with the intestinal proliferative/undifferentiated and progenitor cell populations.

Laminin Receptor 37/67LR Regulates Adhesion and Proliferation of Normal Human Intestinal Epithelial Cells

Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

Immunohistochemical analysis of cellular adhesion molecules (ICAM-1, VCAM-1) and VEGF in fibrovascular membranes of patients with proliferative diabetic retinopathy: Preliminary study

PURPOSE Diabetic fibrovascular membranes are the main pathological changes of proliferative diabetic retinopathy that can cause serious complications leading to blindness. Since the mechanism of fibrovascular membrane development is still unknown, the aim of our study was to identify potential biomarkers for this pathology. To this end, we analyzed the simultaneous expression of ICAM-1, VCAM-1 and VEGF within tissues of diabetic fibrovascular membranes. PATIENTS AND METHODS Fibrovascular membranes were taken from nine diabetic patients with proliferative diabetic retinopathy. The fibrovascular membrane specimens were analyzed by immunohistochemistry to determine ICAM-1, VCAM-1 and VEGF expression. Controls were collected on nine normal conjunctivas removed during senile cataract surgery. RESULTS Coexpression of ICAM-1, VCAM-1 and VEGF was found in most of the diabetic fibrovascular membranes studied. Thus, ICAM-1 was positive in eight of nine membranes (82%), VCAM-1 in seven of nine membranes (78%) and VEGF in all the membranes. CONCLUSIONS The substantial overexpression of adhesion molecules ICAM-1, VCAM-1 and of VEGF suggests that these molecules might contribute to the development of fibrovascular membranes in patients with proliferative diabetic retinopathy, and that they could constitute suitable markers of this pathology.

Apoptotic factors (Bcl-2 and Bax) and diabetic retinopathy in type 2 diabetes

The expression of apoptotic factors Bcl-2 and Bax were studied in the conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination including ocular fundus and retinal fluorescein angiography. The indirect immunoperoxidase method was performed on 15 normal conjunctiva taken during cataract surgery (group 1), on 40 eyes of 40 patients with type 2 diabetes without diabetic retinopathy (group 2) and 13 eyes of 13 patients with diabetic retinopathy (group 3). In normal human conjunctiva, Bax showed positive expression in epithelial, vascular and stromal cells whereas Bcl-2 staining was negative. In the conjunctiva of diabetic patients without diabetic retinopathy, Bax was widely, and strongly, expressed in epithelial cells, vascular endothelial cells, fibroblasts and infiltrating cells such as macrophages. For patients with diabetic retinopathy, Bax was consistently strong to very strong. Bcl-2 protein expression became weak to negative for diabetic patients both with and without diabetic retinopathy. Immunoreactivity was not correlated between Bcl-2 and Bax in the conjunctiva of diabetic patients. Bax was always localized in tissues characterized by a high rate of apoptosis, whereas, Bcl-2 was absent. Our results suggest that diabetic human conjunctiva, with its inflammatory phenomena, is considered as a privileged target for programmed cell death.

Immunohistochemical study of vascular endothelial growth factor (VEGF), tumor suppressor protein (p53) and intercellular adhesion molecule (ICAM-1) in the conjunctiva of diabetic patients

SummaryThe expression pattern of VEGF, p53 and ICAM-1 was studied in conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination, including retinal fluorescein angiography. Indirect immunoperoxidase method was performed on 20 eyes of 20 patients with type II diabetes without DR and on 5 eyes of 5 patients with PDR. A control study was performed on 6 normal conjunctiva undertaken during cataract surgery. Immunoreactivity of VEGF, p53 and ICAM-1 was found in epithelial, fibroblast and vascular endothelial cells. For the same duration of diabetes, a strong to moderate or weak immunoreactivity was observed in the conjunctiva of patients without retinopathy. In patients with PDR, the expression was strong for all these proteins. The immunoreactivity was correlated between VEGF, p53 and ICAM-1. In the normal conjunctiva, a weak to negative immunostaining was observed. The presence of these proteins in the conjunctiva of diabetic patients without retinopathy may add new data in the pathogenesis of diabetic retinopathy. Further studies are needed to confirm this hypothesis.

INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor.

Understanding β cell–extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic β cell survival and function, even enhancing glucose‐stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin‐mediated interactions, while much less attention has been paid on non‐integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor‐ligand interaction through the adhesion of INS‐1 cells (a rat insulinoma cell line) to CDPGYIGSR‐, GRGDSPC‐ or CDPGYIGSR + GRGDSPC‐covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose‐stimulated insulin secretion were investigated. Culture of the INS‐1 cells on the bioactive surfaces was improved compared to the low‐fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose‐stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose‐stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion. Copyright

Expression de la molécule d’adhésion intercellulaire de type 1 au niveau de la conjonctive des patients diabétiques: Une étude préliminaire

PURPOSE Recent research has incriminated adhesion molecules in the pathogenesis of diabetic retinopathy. These molecules have been found to be expressed in many cells participating in inflammatory processes and neovascularization. The purpose of our investigation was to study the expression of intercellular adhesion molecule type 1 (ICAM-1) in the conjunctiva of diabetic patients without retinopathy in comparison with normal human conjunctiva. PATIENTS AND METHODS Fifteen conjunctival biopsies were obtained from diabetic patients without retinopathy. The ocular fundus examination and retinal fluorescein angiography were normal. The normal human conjunctiva were taken from five patients undergoing senile cataract surgery. Immunohistochemical analysis consisted of indirect immunoperoxidase using the monoclonal antibody ICAM-1. RESULTS The adhesion molecule ICAM-1 was immunolocalized in epithelial, vascular endothelial, and inflammatory cells. The expression of this molecule was different in diabetic patients for the same duration. In the normal human conjunctiva, the expression of ICAM-1 was very low. CONCLUSION This preliminary study shows that ICAM-1 is present in the conjunctiva of diabetic patients without retinopathy and thus may add new insights into the pathogenesis of diabetic retinopathy.