Dual sources of vitronectin in the human lower urinary tract: synthesis by urothelium vs. extravasation from the bloodstream.

Vitronectin (VN), secreted into the bloodstream by liver hepatocytes, is known to anchor epithelial cells to basement membranes through interactions with cell surface integrin receptors. We report here that VN is also synthesized by urothelial cells of urothelium in vivo and in vitro. In situ hybridization, dideoxy sequencing, immunohistochemistry, and ELISA of urothelial cell mRNA, cDNA, tissue, and protein extracts demonstrated that the VN gene is active in vivo and in vitro. The expression of VN by urothelium is hypothesized to constitute one of several pathways that anchor basal cells to an underlying substratum and explains why urothelial cells adhere to glass and propagate under serum-free conditions. Therefore, two sources of VN in the human urinary bladder are recognized: 1) localized synthesis by urothelial cells and 2) extravasation of liver VN through fenestrated capillaries. When human plasma was fractionated by denaturing heparin affinity chromatography, VN was isolated in a biologically active form that supported rapid spreading of urothelial cells in vitro under serum-free conditions. This activity was inhibited by the matricellular protein SPARC via direct binding of VN to SPARC through a Ca(+2)-dependent mechanism. A novel form of VN, isolated from the same heparin affinity chromatography column and designated as the VN(c) chromatomer, also supported cell spreading but failed to interact with SPARC. Therefore, the steady-state balance among urothelial cells, their extracellular milieu, and matricellular proteins constitutes a principal mechanism by which urothelia are anchored to an underlying substrata in the face of constant bladder cycling.

Fibroblast growth factor-10 signals development of von Brunn's nests in the exstrophic bladder.

von Brunn's nests have long been recognized as precursors of benign lesions of the urinary bladder mucosa. We report here that von Brunn's nests are especially prevalent in the exstrophic bladder, a birth defect that predisposes the patient to formation of bladder cancer. Cells of von Brunn's nest were found to coalesce into a stratified, polarized epithelium which surrounds itself with a capsule-like structure rich in types I, III, and IV collagen. Histocytochemical analysis and keratin profiling demonstrated that nested cells exhibited a phenotype similar, but not identical, to that of urothelial cells of transitional epithelium. Immunostaining and in situ hybridization analysis of exstrophic tissue demonstrated that the FGF-10 receptor is synthesized and retained by cells of von Brunn's nest. In contrast, FGF-10 is synthesized and secreted by mesenchymal fibroblasts via a paracrine pathway that targets basal epithelial cells of von Brunn's nests. Small clusters of 10pRp cells, positive for both FGF-10 and its receptor, were observed both proximal to and inside blood vessels in the lamina propria. The collective evidence points to a mechanism where von Brunn's nests develop under the control of the FGF-10 signal transduction system and suggests that 10pRp cells may be the original source of nested cells.

The adaptor-related protein complex 2, alpha 2 subunit (AP2α2) gene is a peroxisome proliferator-activated receptor cardiac target gene.

A peroxisome proliferator-actived receptor (PPAR) response element (RE) in the promoter region of the adaptor-related protein complex 2, alpha 2 subunit (AP2α2) of mouse heart has been identified. The steroid hormone nuclear PPARs and the retinoid X receptors (RXRs) are important transcriptional factors that regulate gene expression, cell differentiation and lipid metabolism. They form homo- (RXR) and hetero- (PPAR-RXR) dimers that bind DNA at various REs. The AP2α2 gene is part of complex and process that transports lipids and proteins from the plasma membrane to the endosomal system. A PPAR activator (Wy14643) and DMSO (vehicle) was introduced into control and δ337T thyroid hormone receptor (TRß1) transgenic mice. Heart tissue was extracted and AP2α2 gene expression was compared using Affymetrix expression arrays and qRT PCR among four groups [control, control with Wy14643, δ337T TRß1 and δ337T TRß1 with Wy14643]. The gene expression of AP2α2 in the Wy14643 control and transgenic mouse groups was significantly up regulated over the vehicle mouse groups in both the array (p < 0.01) and qRT PCR (p < 0.01) studies. Duplex oligo DNAs containing the PPAR/RXR motif (AGGTCA/TCCAGT) from the AP2α2 promoter were used in EMSA to verify binding of the PPAR and RXR receptors to their REs. pGL4.0 [Luc] constructs of the AP2α2 promoter with and without the PPAR/RXR motifs were co-transfected with mouse PPARα, ß or γ1 into HepG2 cells and used in lucerifase assays to verify gene activation. In conclusion our study revealed that PPARα regulates the mouse cardiac AP2α2 gene in both the control and transgenic mouse.

Differential affinity of vitronectin versus collagen for synthetic biodegradable scaffolds for urethroplastic applications.

Cell-seeded synthetic polymer scaffolds constitute an emerging technology for urethroplastic applications. The study goal was to identify urethral proteins appropriate for cell attachment and optimize their adsorption onto two types of scaffolds: porous poly(ester urethane) with a poly(caprolactone) soft segment (PEU-PCL) and poly-(96% L/4% D)-lactic acid (P96L/4DLA). Specimens from eight men undergoing urethral reconstruction for stricture diseases were subjected to immunohistochemical analysis. Type I collagen, type IV collagen and vitronectin were detected at the interface between the epithelium and its basement membrane. Electrophoresis confirmed that polypeptide chains in the starting material were also present in fractions eluted from adsorbed scaffolds. Over a 4 week incubation assay, only vitronectin exhibited 100% retention levels for all scaffolds. The saturation point for each protein on each scaffold type was determined by titration and ELISA. The collective evidence indicates the concept that vitronectin > type IV collagen > type I collagen are preferred adsorption proteins for PEU-PCL and P96L/4DLA.