Targeted disruption of the focal adhesion kinase (FAK) gene in mice is lethal at embryonic day 8.5 (E8.5). Vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular network. We found that, although fibronectin (FN) levels were similar, its organization was less fibrillar in both FAK-/- endothelial cells and mesoderm of E8.5 FAK-/- embryos, as well as in mouse embryonic fibroblasts isolated from mutant embryos. FAK catalytic activity, proline-rich domains, and location in focal contacts were all required for proper allocation and patterning of FN matrix. Cells lacking FAK in focal adhesions fail to translocate supramolecular complexes of integrin-bound FN and focal adhesion proteins along actin filaments to form mature fibrillar adhesions. Taken together, our data suggest that proper FN allocation and organization are dependent on FAK-mediated remodeling of focal adhesions.
A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, followed by branching of a single isolated bud and its recombination with metanephric mesenchyme. Implantation of the recombined tissue results in apparent early vascularization. Thus, in principle, an unbranched epithelial tubular structure (potentially constructed from cultured cells) can be induced to form kidney tissue such that this in vitro engineered tissue is capable of being implanted in host rats and developing glomeruli with evidence of early vascularization. Optimization studies (of growth factor and matrix) indicate multiple suitable combinations and suggest both a most robust and a minimal system. A whole-genome microarray analysis suggested that recombined tissue recapitulated gene expression changes that occur in vivo during later stages of kidney development, and a functional assay demonstrated that the recombined tissue was capable of transport characteristic of the differentiating nephron. The approach includes several points where tissue can be propagated. The data also show how functional, 3D kidney tissue can assemble by means of interactions of independent modules separable in vitro, potentially facilitating systems-level analyses of kidney development. kidney development ͉ systems biology ͉ tissue engineering
Peritoneal dissemination in gastric cancer is a common fatal clinical condition with few effective therapies available. We studied the therapeutic effect of a tumor-targeting drug delivery system that uses cisplatin-encapsulated and Tf-conjugated PEG liposomes (Tf-PEG liposomes) in nude mice with peritoneal dissemination of human gastric cancer cells. Small unilamellar Tf-PEG, PEG or DSPC/CH liposomes (bare liposomes) encapsulating cisplatin were prepared by reversephase evaporation followed by extrusion. Electron microscopic studies revealed that Tf-PEG liposomes were internal- Key words: liposome; targeting; transferrin; peritoneal dissemination; gastric cancerPeritoneal dissemination is the most frequent noncurative factor and the most common type of recurrence after curative surgery in patients with gastric cancer. 1 Control of this metastasis is one of the most important challenges in treating gastric cancer. There have been few reports of effective treatment for peritoneal dissemination in patients with gastric cancer. 2,3 As a clinical locoregional chemotherapy, various anticancer drugs in solution form have been administered i.p. to expose the drug directly to peritoneal tumor cells. 4 However, small water-soluble molecules, such as cisplatin or mitomycin C, are absorbed easily through the large peritoneal surface into the circulating blood. It is difficult to maintain a high drug concentration for a long time in the peritoneal cavity. 5,6 Therefore, i.p. administration of anticancer drugs in solution form does not always produce the desired effect.Liposomes, known to be drug carriers, have various advantages. 7,8 First, they encapsulate various drugs such as water-soluble, lipid-soluble or high m.w. substances and release them in a sustained manner. Second, the antigenicity and toxicity of liposomes are very low because they consist of lipid, which is a natural component of organisms. Third, the biodistribution of liposomes can be controlled by the size or lipid component. Fourth, various materials, such as antibodies or chemical compounds, can modify the surfaces of liposomes. However, the therapeutic application of liposomes has been limited by their rapid clearance from the bloodstream and by their uptake by the RES. Recent efforts have been made to reformulate the liposome composition, to reduce its affinity to the RES. Liposomes modified with amphipathic PEG are not readily taken up by macrophages in the RES and, hence, stay in the circulation for a relatively long time. 9 -14 To increase the therapeutic effect and decrease side effects, tumor-specific targeting therapy has been advocated. Intracellular targeting using iron-saturated Tf as a ligand for receptor-mediated endocytosis has attracted attention. Tf is a glycoprotein that transports ferric ions in the body and the Tf receptor is internalized into cells by endocytosis through the binding of Tf. This receptormediated endocytosis is a normal physiologic process by which iron is delivered to cells. [15][16][17] Also, the Tf receptor concen...
S U M M A R YWe examined the distribution of cell adhesion-related molecules (CAMs) among mouse embryonic stem (ES) cells and the spatial distribution on cell surfaces before and during differentiation. The cell-cell heterogeneity of SSEA-1, PECAM-1, and ICAM-1 among the undifferentiated cells in the ES cell colonies was evident by immunohistochemistry and immuno-SEM, supporting the flow cytometry findings. In contrast, most undifferentiated ES cells strongly expressed CD9. SSEA-1 was located preferentially on the edge of low protuberances and microvilli and formed clusters or linear arrays of 3-20 particles. PECAM-1 and ICAM-1 were randomly localized on the free cell surfaces, whereas CD9 was preferentially localized on the microvilli or protuberances, especially in the cell periphery. Both the SSEA-1 ϩ fraction and the SSEA-1 Ϫ fraction of magnetic cell sorting (MACS) formed undifferentiated colonies after plating. Flow cytometry showed that these populations reverted separately again to a culture with a mixed phenotype. Differentiation induced by retinoic acid downregulated the expression of all CAMs. Immuno-SEM showed decreases of SSEA-1 in the differentiated ES cells, although some clustering still remained. Our findings help to elucidate the significance of these molecules in ES cell maintenance and differentiation and suggest that cell surface antigens may be useful for defining the phenotype of undifferentiated and differentiated ES cells.
Recently, a plasma flow has been applied to medical treatment using effects of various kinds of stimuli such as chemical species, charged particles, heat, light, shock wave and electric fields. Among them, the chemical species are known to cause an inactivation of cell viability. However, the mechanisms and key factors of this event are not yet clear. In this study, we focused on the effect of H2O2 in plasma-treated culture medium because it is generated in the culture medium and it is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance of H2O2, we assessed the differences in the effects of plasma-treated medium and H2O2-added medium against inactivation of HeLa cell viability. These two media showed comparable effects on HeLa cells in terms of the survival ratios, morphological features of damage processes, permeations of H2O2 into the cells, response to H2O2 decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H2O2 is one of the main factors responsible for inactivation of HeLa cell viability.
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