MAB1945 Sigma-AldrichAnti-Vitronectin Antibody, clone BV2

Neural precursor expressed, developmentally down-regulated 9 (NEDD9), a member of the Cas family of signal transduction molecules, is amplified at the genetic level in melanoma, and elevated expression levels have been shown to correlate with melanoma progression and metastasis. NEDD9 interacts with the guanine nucleotide exchange factor DOCK3 to promote Rac activation and the elongated, mesenchymal-type of tumour cell invasion, but the molecular mechanisms through which NEDD9 promotes melanoma metastasis are not fully understood. We show that signalling through increased NEDD9 levels requires integrin β3 signalling, which leads to elevated phosphorylation of integrin β3. This results in increased Src and FAK but decreased ROCK signalling to drive elongated, mesenchymal-type invasion in environments that contain vitronectin. NEDD9 overexpression does not affect ROCK signalling through activation of RhoA but decreases ROCKII signalling through Src-dependent phosphorylation of a negative regulatory site Tyr722. In NEDD9-overexpressing melanoma cells, inhibition of Src with dasatinib results in a switch from Rac-driven elongated, mesenchymal-type invasion to ROCK-dependent rounded, amoeboid invasion. These findings brings into question whether dasatinib would work as a therapeutic agent to block melanoma invasion and metastasis. On the basis of the in vitro data presented here, a combination treatment of dasatinib and a ROCK inhibitor might be a better alternative in order to inhibit both elongated, mesenchymal-type and rounded, amoeboid motility.

To examine the retinal histopathologic manifestation of aceruloplasminemia, an autosomal recessive disease caused by mutation of the ferroxidase ceruloplasmin, resulting in tissue iron overload.The morphologic features of the human aceruloplasminemic retina were studied with light and electron microscopy. Retinal iron accumulation was assessed with Perls Prussian blue staining, immunohistochemistry, and secondary ion mass spectrometry.Light and electron microscopic analysis revealed several ocular pathologic findings that resembled age-related macular degeneration, including retinal pigment epithelium (RPE) depigmentation, atrophy and hypertrophy, nodular and diffuse drusen, and lipofuscin and melanolipofuscin granules. Complement deposition was detected in drusen. The RPE cells and neural retina had increased levels of iron. Two major types of RPE cells were observed: melanosome rich and melanosome poor. Melanosome-rich cells had increased levels of iron and melanolipofuscin. The melanolipofuscin granules were observed in large aggregates, where some of the melanosomes were degrading. Melanosome-poor cells lacked melanosomes, melanolipofuscin, and lipofuscin but contained electron-dense aggregates high in iron, phosphorus, and sulfur.The findings in the aceruloplasminemic retina resemble some of those found in age-related macular degeneration. Also, they suggest that melanosomes in the RPE can be degraded via iron-mediated reactive oxygen species production.Mechanisms underlying the pathologic mechanisms found in aceruloplasminemia also may be important in age-related macular degeneration.

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/alphav integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and alphav integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and alphav integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-alphav integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/alphav integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.

Ovarian carcinomas, the most fatal gynaecological malignancies, are associated with poor prognosis predominantly because of a high recurrence rate. Ovarian cancer cells spread widely throughout the abdominal cavity leading to peritoneal metastasis. The influence of the mesothelial microenvironment on the biological mechanisms leading to cancer cell colonization of the mesothelium is poorly understood. This study aims to investigate whether mesothelial secretions affect the migration of ovarian cancer cells and focuses on the role of the adhesive molecule vitronectin and its integrin receptors. An in vitro co-culture model indicated that clusters of IGROV1 and SKOV3 cells adhere to MeT-5A mesothelial cells preferentially at intercellular sites, invade the mesothelial monolayer and alter the integrity of the mesothelium. In addition, mesothelial cell-conditioned medium (CM) induces migration of IGROV1 and SKOV3 cells in Boyden chambers and wound healing assays. Furthermore, blocking molecules directed against vitronectin or its av integrin receptor decrease mesothelial-CM-induced migration by approximately 40% and 60-70% for IGROV1 and SKOV3 ovarian cancer cells, respectively, in Boyden chamber assays. Wound healing assays that allow cell migration to be measured over 24 h periods demonstrated that blocking molecules prevent the migration of IGROV1 and SKOV3 cells. Vitronectin is present in mesothelial conditioned medium and in metastatic peritoneal tissue sections. The expression of vitronectin at the periphery of mesothelial cells and within ovarian cancer cell clusters suggests a potential role for this molecule during intraperitoneal implantation of ovarian cancer cells. Vitronectin could represent a target for the development of anti-adhesive strategies to impede ovarian cancer dissemination.

The purpose of this study was to better understand whether interleukin-13 (IL-13) and connective tissue growth factor (CTGF) are highly expressed during foreign body encapsulation of subcutaneous devices. Mock biosensors were implanted into rats for three lengths of time (7-, 21- and 48-55 days) to address different stages of the foreign body response. Using quantitative real-time PCR and immunofluorescence, the expression of IL13, CTGF, collagen 1, decorin and fibronectin were measured in this tissue. IL-13, a product of Th2 cells, was highly expressed at all time points, with greatest expression at day 21. The IL-13 expression was paralleled by increased presence of T-cells at all time points. CTGF was also found to be more highly expressed in foreign body tissue than in controls. Collagen and decorin were highly expressed at the middle and later stages. Given the increased expression of IL-13 and CTGF in foreign body tissue, and their roles in other fibrotic disorders, these cytokines may well contribute to the formation of the foreign body capsule. Since the peak gene expression of IL-13 occurred later than the previously-reported TGFbeta expression peak, IL-13 is probably not the major stimulus to TGFbeta expression during foreign body encapsulation and may contribute to fibrosis independently.

BACKGROUND: Complement factor H (CFH) polymorphism Y402H has been shown to be significantly associated with age-related macular degeneration (AMD). Furthermore, histopathological studies in AMD have implicated basal laminar deposits (BLD) in the development of choroidal neovascularization (CNV) membranes. The purpose of this study was to correlate CFH staining in BLD with the CFH genotype at the tyrosine 402 histidine (Y402H) polymorphism. PATIENTS AND METHODS: During macular translocation, 21 angiographically confirmed CNV membranes were extracted in 21 patients. The specimens were analysed histologically for BLD. The presence of CFH, complement proteins, and vitronectin was determined by immunohistochemistry. Finally, the CFH Y402H genotype was established by direct sequencing analysis. RESULTS: Histological examination demonstrated BLD in all of the excised CNV membranes. By immunostaining CFH was detected in the peripheral aspect at the inner and outer surface of BLD, which colocalized with other proteins of the complement cascade (C3, C5b-9). Similarly, vitronectin was detected in all of the BLD investigated. Four patients were noncarriers of CFH Y402H polymorphism, nine patients were heterozygous and eight patients homozygous for the CFH Y402H polymorphism. CONCLUSIONS: BLD are composed of different complement factors (factor H, C3, C5b-9) and extracellular matrix proteins such as vitronectin. The prevalence of homozygous carriers in regard to CFH Y402H polymorphism, which is suspicious for AMD, might be associated with increased secretion of vitronectin in response to dysregulation of the complement cascade.

The degree of lymphocyte infiltration is a prognostic factor in liver cancer, but to date the mechanisms by which lymphocytes infiltrate into and are retained in hepatic tumours are poorly understood. We hypothesised that the extracellular matrix glycoprotein vitronectin, a major component of the stroma of hepatic tumours, might play a role in the recruitment and retention of tumour-infiltrating lymphocytes (TIL). Thus, we investigated the ability of vitronectin to support migration and adhesion of TIL isolated from hepatocellular carcinoma and colorectal hepatic metastases. Soluble vitronectin-induced dose-dependent migration of TIL in in vitro chemotaxis and haptotaxis assays and vitronectin in tissue sections was able to support TIL adhesion to tumour stroma. Neither adhesion nor migration was inhibited by a function blocking mAb against the major vitronectin receptor alpha v beta3 and we were unable to detect alpha v beta3 on TIL in vitro or in vivo on tumour tissue. However, TIL did express high levels of urokinase-type plasminogen activator receptor (uPAR) and inhibitory antibodies and amiloride both significantly inhibited TIL adhesion to vitronectin and reduced transendothelial migration of lymphocytes across liver endothelium in vitro. Thus, we provide evidence that vitronectin in liver tumours can support the recruitment and retention of effector lymphocytes by an uPAR-dependent mechanism.

Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin alpha(v) receptor than aggregates. Blocking alpha(v) function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin alpha(v). Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.

BACKGROUND: The plasminogen activation system represents a potent mechanism of extracellular proteolysis and is an essential component of normal wound healing. It has also been implicated in the pathogenesis of chronic, nonhealing ulcers. Traditionally, urokinase-type plasminogen activator (uPA) has been associated with pericellular proteolytic activity involved in tissue remodelling processes, and tissue-type plasminogen activator (tPA) mainly with intravascular fibrinolysis. OBJECTIVES: The present study was conducted to characterize the spatial distribution of the various plasminogen activation system components in chronic ulcers and acute, well-granulating wounds. METHODS: The expression of uPA, tPA, urokinase receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and vitronectin was investigated by immunohistochemical staining, in addition to uPA, tPA and PAI-1 expression by in-situ hybridization, in samples from eight chronic venous ulcers, five decubitus ulcers, five well-granulating acute wounds and five normal skin samples. RESULTS: In chronic venous leg ulcers tPA mRNA was detected in basal and suprabasal keratinocytes at the leading wound edge, while in well-granulating wounds and in decubitus ulcers tPA mRNA was expressed only in a few keratinocytes. However, tPA was widely expressed in fibroblast- and macrophage-like cells in the stroma of well-granulating wounds, while less tPA was detected in the granulation tissue of chronic ulcers. tPA mRNA and protein were localized in the superficial granular layers in normal skin. Although no qualitative differences in expression of uPA, PAI-1 or uPAR in the wound edge keratinocytes in chronic ulcers vs. normally granulating wounds were found, their expressions were more pronounced in the granulation tissue of well-granulating wounds. CONCLUSIONS: These results suggest that in poorly healing venous leg ulcers, the pattern of tPA expression is altered in keratinocytes at the leading edge of the wound, and the patterns of tPA, uPA and PAI-1 expression are altered in the granulation tissue.

Recent studies implicate inflammation and complement mediated attack as early events in drusen biogenesis. The investigations described here sought to determine whether primary sites of complement activation could be identified within drusen substructure, and whether known inhibitors of the terminal pathway of complement are present in drusen and/or retinal pigmented epithelial (RPE) cells that lie in close proximity to drusen. Immunohistochemical examination shows two fluid phase regulators of the terminal pathway, vitronectin (Vn, S-protein) and clusterin (apolipoprotein J), to be present in drusen; Vn also accumulates in the cytoplasm of RPE cells that are closely associated with drusen. The membrane associated complement inhibitor, complement receptor 1, is also localized in drusen, but it is not detected in RPE cells immunohistochemically. In contrast, a second membrane associated complement inhibitor, membrane cofactor protein, is present in drusen associated RPE cells, as well as in small, spherical substructural elements within drusen. These previously unidentified elements also show strong immunoreactivity for proteolytic fragments of complement component C3 that are characteristically deposited at sites of complement activation. It is proposed that these structures represent residual debris from degenerating RPE cells that are the targets of complement attack. It is likely that RPE cell debris entrapped between the RPE monolayer and Bruch's membrane serves as a chronic inflammatory stimulus and a potential nucleation site for drusen formation. Thus, the process of drusen biogenesis may be envisaged as a secondary manifestation of primary RPE pathology that is exacerbated by consequences of local inflammatory processes.