Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas

Vitronectin as a micromanager of cell response in material-driven fibronectin nanonetworks

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation

A ceramide analogue (PDMP) inhibits glycolipid synthesis in fish embryos

Glycolipids were depleted from medaka embryos using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. Embryos cultured in the presence of 20 [mu]M PDMP exhibited a dramatic decline in glycolipid synthesis and cell surface expression. Metabolic labeling of glucosylceramide declined by 87% on Days 3-6 of development and 72% on Days 7-10 (hatching occurred on Day 10). In parallel, PDMP-treated embryos exhibited a striking loss of several tissue-specific glycolipid antigens, including 9-O-acetyl GD3 from brain and retina, GT3/ GQ1C from brain, neural tube, and retina, and sulfated glycolipid from skin and gut. Despite these changes in glycolipid expression, PDMP-treated embryos were fully viable with no evidence of developmental abnormality. PDMP appears to provide a useful tool for identifying glycolipid antigens in embryos and investigating their role in development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30233/1/0000627.pd

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and αvβ3 integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer

Decreased formation of inositol trisphosphate in Madin-Darby canine kidney cells under conditions of [beta]-glucosidase inhibition

Recent work has demonstrated the enhancement of hormone-stimulated inositol trisphosphate formation in renal epithelial cells under conditions of glucosylceramide depletion. The role of glucosylceramide metabolism was explored further by exposing Madin-Darby canine kidney (MDCK) cells to the [beta]-glucosidase inhibitor conduritol B epoxide, which produced time-dependent and concentration-dependent increases in glucosylceramide levels and decreased bradykinin-stimulated inositol trisphosphate formation from isolated MDCK cell membranes. These data provide further support for an association between glucosylceramide levels and hormone-stimulated inositol trisphosphate formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30238/1/0000632.pd

Tumor-specific expression of αvβ3 integrin promotes spontaneous metastasis of breast cancer to bone

INTRODUCTION: Studies in xenograft models and experimental models of metastasis have implicated several β3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific αvβ3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear. METHODS: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of αvβ3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of αvβ3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays. RESULTS: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. αvβ3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, αvβ3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, αvβ3 increased 66cl4 tumor cell adhesion and αvβ3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro. CONCLUSION: These results demonstrate for the first time that tumor-specific αvβ3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors

The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells

The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders

External validation suggests Integrin beta 3 as prognostic biomarker in serous ovarian adenocarcinomas

<p>Abstract</p> <p>Background</p> <p>The majority of women with ovarian cancer are diagnosed in late stages, and the mortality rate is high. The use of biomarkers as prognostic factors may improve the treatment and clinical outcome of these patients. We performed an external validation of the potential biomarkers CLU, ITGB3, CAPG, and PRAME to determine if the expression levels are relevant to use as prognostic factors.</p> <p>Methods</p> <p>We analysed the gene expression of CLU, ITGB3, CAPG, and PRAME in 30 advanced staged serous adenocarcinomas with quantitative real-time polymerase chain reaction (QPCR) and the protein levels were analysed in 98 serous adenocarcinomas with western blot for semiquantitative analysis. Statistical differences in mRNA and protein expressions between tumours from survivors and tumours from deceased patients were evaluated using the Mann-Whitney U test.</p> <p>Results</p> <p>The gene and protein ITGB3 (Integrin beta 3) were significantly more expressed in tumours from survivors compared to tumours from deceased patients, which is in concordance with our previous results. However, no significant differences were detected for the other three genes or proteins CLU, CAPG, and PRAME.</p> <p>Conclusion</p> <p>The loss of ITGB3 expression in tumours from deceased patients and high expression in tumours from survivors could be used as a biomarker for patients with advanced serous tumours.</p

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis

We describe the synthesis and characterization of a 5′ conjugate between a 2′-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD–623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while ‘free’ 623 did not. However, the kinetics of luciferase expression was distinct; the RGD–623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD–623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the αvβ3 integrin