W1595 Regulation of TFF and MUC Expression By SOX2 in Gastric Epithelial Cells

Abstract

factors, but their roles in angiogenesis in general, and, specifically in angiogenesis in gastric mucosa remain unexplored. Material andMethods:We isolated gastricmucosal microvascular endothelial cells (GMMECs) from Fisher F-344 rats, 3 months of age using anti-PECAM-1 selection (Chemicon) and magnetic bead separation. PECAM-1 positive cells were separated by using a miniMACS column and grown on collagen coated dishes in endothelial cell growth media. The isolated GMMECs were characterized by immunohistochemical staining for the endothelial markers von Willebrand's factor (Factor VIII -related antigen), PECAM1 (CD31) and VEGFR2. Cells were treated with either: 100nM control siRNA, importinα1 or α3 specific siRNAs (Qiagen) for 48 or 72 hrs. Studies: 1) Importin α and VEGF mRNA expression using reverse transcription real-time PCR method with prevalidated QuantiTect assays (Qiagen, Valencia, CA), and, expression and subcellular localization of importin α and VEGF proteins using immunoflorescence studies; 2) In-vitro angiogenesis on matrigel and quantification of capillary-like endothelial tube formation using Metamorph image analysis system. Results: In control GMMECs, importin α and VEGF were strongly expressed and localized to the cell nuclei, with the former also expressed in the nuclear membrane. GMMECs plated on matrigel exhibited endothelial cell migration and capillary tube network and lumen formation at 6 hrs, reflecting in-vitro angiogenesis. Treatment of young GMMECs with importin α1 and α3 specific siRNAs significantly downregulated expression of importin α 1 and α3 (by 2.7-fold; p <0.05 and 2.0-fold; p <0.05 respectively), significantly reduced VEGF gene expression (by 3.3-fold; p <0.05 and 2.4-fold; p <0.05 respectively), and reduced in-vitro angiogenesis in GMMECs (by 1.7-fold; p<0.05 and 1.5-fold; p<0.05 respectively) vs. controls. Conclusions: 1) Knockdown of importin α1 and importin α3 significantly downregulates expression of VEGF and inhibits angiogenesis in gastric mucosal microvascular endothelial cells. 2) These data demonstrate a mechanistic role of importin α as one of the major players in gastric angiogenesis and also as a potential therapeutic target.