Localization and functional characterization of glycosaminoglycan domains in the normal human kidney as revealed by phage display-derived single chain antibodies

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy.     

Bone tissue engineering on amorphous carbonated apatite and crystalline octacalcium phosphate-coated titanium discs

Poor fixation of bone replacement implants, e.g. the artificial hip, in implantation sites with inferior bone quality and quantity may be overcome by the use of implants coated with a cultured living bone equivalent. In this study, we tested, respectively, amorphous carbonated apatite (CA)- and crystalline octacalcium phosphate (OCP)-coated discs for their use in bone tissue engineering. Subcultured rat bone marrow cells were seeded on the substrates and after 7 days of culture, the implants were subcutaneously implanted in nude mice for 4 weeks. After 7 days of culture, the cells had formed a continuous multi-layer that covered the entire surface of the substrates. The amount of cells was visually higher on the crystalline OCP-coated discs compared to the amorphous CA-coated discs. Furthermore, the amorphous CA-coated discs exhibited a visually higher amount of mineralized extracellular matrix compared to the crystalline OCP-coated discs. After 4 weeks of implantation, clear de novo bone formation was observed on all discs with cultured cells. The newly formed bone on the crystalline OCP-coated discs was more organized and revealed a significantly higher volume compared to the amorphous CA-coated discs. The percentage of bone contact with the discs was also significantly higher on the OCP-coated discs. Overall, the results suggest that a crystalline OCP coating is more suitable for bone tissue engineering than an amorphous CA coating.     

Effect of second- and third-generation oral contraceptives on the protein C system in the absence or presence of the factor VLeiden mutation: a randomized trial

A plausible mechanism to explain thrombotic risk differences associated with the use of second- and third-generation oral contraceptives (OCs), particularly in carriers of factor V(Leiden), is still lacking. In a double-blind trial, 51 women without and 35 women with factor V(Leiden) were randomized to either a second- (30 microg ethinylestradiol/150 microg levonorgestrel) or third- (30 microg ethinylestradiol/150 microg desogestrel) generation OC. After 2 cycles of use and a wash-out of 2 cycles, the participants continued with the corresponding progestagen-only preparation. Hemostatic variables that probe the activity of the anticoagulant protein C system were determined. Compared with levonorgestrel, desogestrel-containing OCs significantly decreased protein S and increased activated protein C (APC) resistance in both groups. OCs with desogestrel had the most pronounced effects in carriers of factor V(Leiden). Progestagen-only preparations caused changes of anticoagulant parameters opposite to those of combined OCs, which in a number of cases were more pronounced with levonorgestrel. Our data show that progestagens in combined OCs counteract the thrombotic effect of the estrogen component. The higher thrombotic risk associated with third-generation OCs compared with second-generation OCs may be explained by the fact that desogestrel appeared less antithrombotic than levonorgestrel, especially in women with factor V(Leiden).     

Proteomic analysis of exosomes secreted by human mesothelioma cells

Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.     

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.