Cell Adhesion Glycoprotein Research Articles

Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer

Background:Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and poor prognosis in several forms of cancer. In this study, we investigated the prognostic impact of PODXL expression in colorectal cancer (CRC).Methods:Using tissue microarrays and immunohistochemistry, PODXL expression was evaluated in 536 incident CRC cases from a prospective, population-based cohort study. Kaplan–Meier analysis and Cox proportional hazards modelling were used to assess the impact of PODXL expression on cancer-specific survival (CSS) and overall survival (OS).Results:High PODXL expression was significantly associated with unfavourable clinicopathological characteristics, a shorter CSS (hazard ratio (HR)=1.98; 95% confidence interval (CI) 1.38–2.84, P<0.001) and 5-year OS (HR=1.85; 95% CI 1.29–2.64, P=0.001); the latter remaining significant in multivariate analysis (HR=1.52; 95% CI 1.03–2.25, P=0.036). In addition, in curatively resected stage III (T1–4, N1–2, M0) patients (n=122) with tumours with high PODXL expression, a significant benefit from adjuvant chemotherapy was demonstrated (pinteraction =0.004 for CSS and 0.015 for 5-year OS in multivariate analysis).Conclusion:Podocalyxin-like 1 expression is an independent factor of poor prognosis in CRC. Our results also suggest that PODXL may be a useful marker to stratify patients for adjuvant chemotherapy.

P-cadherin expression as a prognostic biomarker in a 3992 case tissue microarray series of breast cancer

P-cadherin is a calcium-dependent cell–cell adhesion glycoprotein. P-cadherin expression is restricted to the myoepithelial cells in normal breast tissue, and aberrant staining has also been described in invasive tumors. Several small studies have reported P-cadherin as a marker of poor outcome in breast cancer patients but its prognostic significance in relation to other variables has not been established in a large series of breast cancers. A tissue microarray was constructed from 3992 cases of invasive breast carcinoma, and P-cadherin expression was evaluated using immunohistochemistry. Median follow-up was 12.5 years. The immunohistochemistry-based definitions of cancer subtypes were luminal (ER+ or PR+/HER2−), luminal/HER2+ (ER+ or PR+/HER2+), HER2+ (ER−/PR−/HER2+), and basal (ER−/PR−/HER2−/CK5/6+ or EGFR+). Clinical covariate and biomarker associations were assessed using contingency tables, and Pearson's χ2 or Fisher's exact test. Survival associations were assessed using Kaplan–Meier plots, logrank and Breslow tests, and Cox proportional hazards regression analysis. P-cadherin was expressed in 34.8% (1290/3710, 50% cut point) of cases. P-cadherin staining was strongly associated with HER2+ and basal carcinoma subtypes (P<0.0005). P-cadherin-positive patients showed significantly poorer short-term (0–10 years) overall survival, disease-specific survival, distant relapse-free interval, and locoregional relapse-free interval in univariable models (P<0.05). In multivariable Cox models containing standard clinical covariates and cancer subtypes, P-cadherin did not show independent prognostic value. P-cadherin expression was positively associated with histological grade, chemotherapy, Ki-67, EGFR, CK5/6, p53, YB-1, and HER2 expression (P<0.002), and negatively associated with age at diagnosis, ER, PR, and Bcl-2 expression (P<0.0005). This study shows the value of P-cadherin as a marker of poor prognosis. The large sample size of this series clarifies contradictory findings of many smaller studies. P-cadherin positivity is associated with high-grade tumor subtypes and well-established markers of poor prognosis, and may represent a promising antibody therapeutic target.

Distribution of cell adhesion glycoproteins in the human retrobulbar optic nerve

We examined the immunohistochemical distribution of six types of cell adhesion glycoproteins in the human retrobulbar optic nerve. The pial septa had laminin and entactin on the surface at the border with the optic nerve fibers, and fibronectin and vitronectin were distributed in the stroma of the septa. Some tenastin was also detectable both on the surface and in the stroma of the septa. Thrombospondin was associated with capillaries within the stroma. The surface of the arachnoid membrane, the pia mater, and the dura mater was covered by laminin and entactin. Diffuse staining with antifibronectin antibody was seen in all these meningeal tissues, but vitronectin showed a fine fibrillar appearance in these tissues. No significant staining could be found for tenastin. Thrombospondin showed only faint staining on the capillaries of the pia mater and the dura mater. These findings can provide clues for the discussion of how the extracellular matrix works and what roles it plays in disorders involving the retrobulbar optic nerve, such as optic neuritis and glaucoma.

The human cancer and stem cell marker podocalyxin interacts with the glucose-3-transporter in malignant pluripotent stem cells

Podocalyxin, an integral plasma membrane cell-adhesion glycoprotein, is a marker of human pluripotent and multipotent stem cells. Podocalyxin is also a marker of many types of cancers and its expression correlates with an aggressive and poor-prognosis tumor phenotype. The function of podocalyxin in stem cells and malignant cells is unknown. Protein sequence data obtained from purified podocalyxin protein isolated from embryonal carcinoma cancer stem cells reveals peptide sequence data for the glucose-3-transporter. Protein-precipitation experiments of embryonal carcinoma protein extracts identify a podocalyxin/glucose-3-transporter protein complex. Cell imaging studies demonstrate co-localization of podocalyxin and glucose-3-transporter and confirm the interaction in vivo. Finally, siRNA podocalyxin-knockdown experiments show decreased expression levels of the glucose-3-transporter. These findings suggest a novel interaction of the glucose-3-transporter and the cell-adhesion protein podocalyxin. In pluripotent stem cells and in human cancer disease, podocalyxin may function in part to regulate and maintain the cell surface expression of the glucose-3-transporter.

Cell adhesion glycoprotein vitronectin during Xenopus laevis embryogenesis

Vitronectin (vn) is a cell-adhesive glycoprotein present in blood and extracellular matrix of all vertebrates. In the present study we reported the cDNA cloning of Xenopus laevis vitronectin and its spatial and temporal expression pattern during the embryonic development of this important model organism. The deduced amino acid sequence of Xenopus laevis vn showed 49%, 47% and 43% identity with human, chicken and zebrafish orthologs, respectively, whereas the comparison with Xenopus tropicalis vn presented 85% identity. The structural organization consisting of a somatomedin B domain and two hemopexin-like domains was similar to higher vertebrate vitronectins. The vn transcripts were detected from stage 28 onward. At tadpole stages, vn is expressed in heart, gut derivatives and in the notochord. The protein was detected in heart, liver, foregut, pronephros and notochord at stages 43 and 47 of Xenopus embryos. Our results suggest that vitronectin is developmentally regulated and could participate in embryo organogenesis.

Impact of VEGF‐dependent tumour micro‐environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice

Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs.

Crystal Structure of the LG1-3 Region of the Laminin α2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.

Platelet microparticles: a new player in malaria parasite cytoadherence to human brain endothelium

Cerebral malaria (CM) is characterized by accumulation of circulating cells within brain microvessels, among which platelets play an important role. In vitro, platelets modulate the cytoadherence of Plasmodium falciparum-parasitized red blood cells (PRBCs) to brain endothelial cells. Here we show for the first time that platelet microparticles (PMPs) are able to bind to PRBCs, thereby transferring platelet antigens to the PRBC surface. This binding is largely specific to PRBCs, because PMPs show little adherence to normal red blood cells. PMP adherence is also dependent on the P. falciparum erythrocyte membrane protein 1 variant expressed by PRBCs. PMP binding to PRBCs decreases after neutralization of PRBC surface proteins by trypsin or after treatment of PMPs with a mAb to platelet-endothelial cell adhesion molecule-1 (CD31) and glycoprotein IV (CD36). Furthermore, PMP uptake is a dynamic process that can be achieved by human brain endothelial cells (HBECs), inducing changes in the endothelial phenotype. Lastly, PMPs dramatically increase PRBC cytoadherence to HBECs. In conclusion, our study identifies several mechanisms by which PMPs may participate in CM pathogenesis while interacting with both PRBCs and HBECs. PMPs thereby provide a novel target for antagonizing interactions between vascular cells that promote microvascular sludging and blood brain barrier alteration during CM.

Oligosaccharyltransferase-Subunit Mutations in Nonsyndromic Mental Retardation

Mental retardation (MR) is the most frequent handicap among children and young adults. Although a large proportion of X-linked MR genes have been identified, only four genes responsible for autosomal-recessive nonsyndromic MR (AR-NSMR) have been described so far. Here, we report on two genes involved in autosomal-recessive and X-linked NSMR. First, autozygosity mapping in two sibs born to first-cousin French parents led to the identification of a region on 8p22-p23.1. This interval encompasses the gene N33/TUSC3 encoding one subunit of the oligosaccharyltransferase (OTase) complex, which catalyzes the transfer of an oligosaccharide chain on nascent proteins, the key step of N-glycosylation. Sequencing N33/TUSC3 identified a 1 bp insertion, c.787_788insC, resulting in a premature stop codon, p.N263fsX300, and leading to mRNA decay. Surprisingly, glycosylation analyses of patient fibroblasts showed normal N-glycan synthesis and transfer, suggesting that normal N-glycosylation observed in patient fibroblasts may be due to functional compensation. Subsequently, screening of the X-linked N33/TUSC3 paralog, the IAP gene, identified a missense mutation (c.932T-->G, p.V311G) in a family with X-linked NSMR. Recent studies of fucosylation and polysialic-acid modification of neuronal cell-adhesion glycoproteins have shown the critical role of glycosylation in synaptic plasticity. However, our data provide the first demonstration that a defect in N-glycosylation can result in NSMR. Together, our results demonstrate that fine regulation of OTase activity is essential for normal cognitive-function development, providing therefore further insights to understand the pathophysiological bases of MR.

Expression Analysis of Kidney-specific Cadherin in a Wide Spectrum of Traditional and Newly Recognized Renal Epithelial Neoplasms: Diagnostic and Histogenetic Implications

Kidney-specific cadherin (Ksp-cad) is a membrane-associated cell adhesion glycoprotein expressed by the distal nephron tubular cells in its later developmental stages. Chromophobe renal cell carcinoma and renal oncocytoma are reported to be variably positive for Ksp-cad with some studies suggesting a discriminatory role for Ksp-cad. Immunoreactivity in other tumors with granular eosinophilic cytoplasm including clear cell and papillary renal cell carcinomas needs to be clearly elucidated and its expression in emerging novel and other unusual renal epithelial neoplasm subtypes including tumors with uncertain histogenesis is not yet known. In this study, we performed a detailed immunohistochemical analysis for Ksp-cad in a broad range of 136 renal epithelial neoplasms. Reactivity with Ksp-cad was observed in the following tumors: chromophobe renal cell carcinoma [23/25 (92%), diffuse (>50% of tumor cells)] positivity and membranous characteristically accentuating the "plant cell-like" histomorphology of the typical (clear) type, renal oncocytoma [15/20 (75%), usually diffuse staining with predominantly membranous accentuation], papillary renal cell carcinoma [5/17 (29%) all focal to moderate, eosinophilic type or type 2-3/7 (43%), basophilic type or type 1-2/10 (20%)], Xp11 translocation carcinoma [1/4 (25%), diffuse positivity] and clear cell renal cell carcinoma [6/36 (17%) all focal, clear cell renal cell carcinoma with prominent eosinophilic cells 1/7 (14%)]. Immunoreactivity was higher when evaluating whole histologic sections than with tissue microarrays for both chromophobe renal cell carcinoma (100% vs. 60%) and renal oncocytoma (100% vs. 55%). No immunoreactivity was observed in mucinous tubular and spindle cell carcinomas (0/23), high-grade collecting duct carcinomas (of Bellini) (0/3), renal medullary carcinomas (0/2), and urothelial carcinomas (0/6). Our study documents the immunoreactivity of Ksp-cad in the range of contemporarily classified renal epithelial neoplasms. The findings argue against the use of Ksp-cad in differentiating chromophobe renal cell carcinoma and renal oncocytomas and further support their relationship to the distal nephron. Ksp-cad may be helpful in distinguishing these two tumor types from clear cell renal cell carcinoma with prominent eosinophilic cells particularly in cases with limited tissue samples (ie, needle core biopsy). In the similar diagnostic setting, caution must be exercised, however, in differentiating chromophobe renal cell carcinoma and renal oncocytoma from the eosinophilic variant of papillary renal cell carcinoma as moderate expression of Ksp-cad may be observed in papillary renal cell carcinoma. The histogenesis of mucinous tubular and spindle cell carcinoma remains debatable as this tumor does not express Ksp-cad, which is highly expressed normally in the thick ascending loop of Henle and the distal convoluted tubules. In conclusion, Ksp-cad is a useful tumor type associated marker for distinguishing chromophobe renal cell carcinoma and renal oncocytoma from the wide range of nonintercalated cell-related adult renal epithelial neoplasms; addition of this marker to a panel comprised of other histologic subtype-associated markers may greatly facilitate histologic subclassification of adult renal epithelial neoplasms.

Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central beta-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to alpha-helix F (alphaHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k(lim)) of RCL insertion for reactions with beta-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k(lim) for tPA but decreased the k(lim) for beta-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of alphaHF, decreased k(lim) and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for beta-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k(lim) but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to alphaHF, controls bifurcation between the inhibitory and the substrate pathways.

Are Natural Killer Cells Distributed in Relationship to Nerve Fibers in the Pregnant Mouse Uterus?

Specialized lymphocytes, called uterine Natural Killer (uNK) cells, appear in human and rodent uteri and become abundant at implantation sites during decidualization and early pregnancy. The hallmark of human uNK cells is intense expression of CD56, a neural cell adhesion glycoprotein (NCAM-1) while mature (granulated) mouse uNK cells express asialoGM1, a brain ganglioside. Murine uNK cells initiate the normal structural changes induced in maternal spiral arteries by pregnancy but regulation of their recruitment, localization and activation is incompletely understood. To address whether uNK cell distribution is co-localized with nerve fiber distribution, sections of gestation day (gd) 6-12 implantation sites from C57BL/6 (B6) mice were studied. Nerve fibers reactive with antibodies to pan neurofilament 150 kD or with tyrosine hydroxylase, an enzyme restricted to sympathetic fibers, were present the walls of branches from the uterine artery in the mesentery. Reactivity was lost as the vessels crossed the myometrium and entered endometrium/decidua. Periodic Acid Schiffs reactive uNK cells were absent from the mesentery and enriched in decidua basalis where they transcribed NCAM-1 and associated with non-innervated segments of the uterine arteries, including spiral arteries. These data suggest that the localization and activation of mature uNK cells are unlikely to be neurotransmitter regulated.

The vitronectin gene in rainbow trout: Cloning, expression and phylogenetic analysis

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout ( Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon–intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1–Ig6), five fibronectin-type III (FN1–FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5–6 (L1/Ig5–6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5–6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5–6 was produced for four days in 2 L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5–6 were immobilized on poly- d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.

Signal Transduction Pathways of Inflammatory Gene Expressions and Therapeutic Implications

Intercellular adhesion molecule-1 (ICAM-1), an inducible cell adhesion glycoprotein of the immunoglobulin supergene family and cyclooxygenase-2 (COX-2), an inducible prostaglandin G/H synthase, are overexpressed by proinflammatory mediators in a wide variety of cell types. These stimuli increase ICAM-1 or COX-2 expression primarily through activation of ICAM-1 or COX-2 gene transcription. The architecture of the ICAM-1 or COX-2 promoter is complex, containing a large number of binding site for inducible transcription factors, the most important of which is NF-kB. NF-kB acts in concert with other transcription factors or transcriptional coactivators which facilitate the assembly of distinct stereospecific transcription complexes on the ICAM-1 or COX-2 promoter. These transcription complexes presumably mediate the induction of ICAM-1 or COX-2 expression in different cell types and in response to different stimuli. In this review, I summarize the current understanding of ICAM-1 and COX-2 gene regulation with a particular emphasis on the transcription factors or coactivators, and signal transduction pathways critical for their expression. A PKC-dependent c-Src pathway activating NF-kB or GAS to enhance ICAM-1 or COX-2 gene expression is discussed. Furthermore, natural products and novel agents targeting on the transcription factor with potential anti-inflammation and anti-tumor activity are also discussed.

Approaches to improving the biocompatibility of porous perfluoropolyethers for ophthalmic applications

Porous perfluoropolyether (PFPE) membranes for ophthalmic applications were prepared with a zwitterion monomer, 3-[[2-(methacryloxy) ethyl]( N, N-dimethyl)ammonio]-propane-1-sulphonate, copolymerized in weight ratios of 0–10%. The polymer samples were assessed for a range of physical properties, including equilibrium water content, bovine serum albumin permeability, transparency, refractive index and the ability to support corneal epithelial cell and tissue attachment, growth and migration. In vitro assessment of the polymers using bovine corneal epithelial cells and tissue showed that a zwitterion incorporation level of between 0% and 6% in the PFPE membranes supported the migration of an intact sheet of epithelial tissue without compromising epithelial cell attachment and growth, with 4–6% being the optimal level for these properties. Binding patterns of the cell adhesion glycoprotein fibronectin were also found to reflect the cell and tissue response. Effective nutrient permeability, refractive index and optical transparency were also maintained by the porous PFPE polymers containing this concentration of zwitterionic monomer. The presence of amounts of zwitterion greater than 6% was inhibitory to both tissue migration and cell growth and was associated with increased optical haze. These results demonstrated that it is possible to achieve the potential for increased biocompatibility in zwitterion-containing PFPE polymers without compromising existing beneficial characteristics.

Two-dimensional patterning of thin coatings for the control of tissue outgrowth

Control of the precise location and extent of cellular attachment and proliferation, and of tissue outgrowth is important in a number of biomedical applications, including biomaterials and tissue engineered medical devices. Here we describe a method to control and direct the location and define boundaries of tissue growth on surfaces in two dimensions. The method relies on the generation of a spatially defined surface chemistry comprising protein adsorbing and non-adsorbing areas that allow control over the adsorption of cell-adhesive glycoproteins. Surface modification was carried out by deposition of thin acetaldehyde and allylamine plasma polymer coatings on silicon wafer and FEP substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved by masking during plasma polymerization. XPS and AFM were used to provide evidence of successful surface modifications. Adsorption of the extracellular matrix protein collagen I followed by tissue outgrowth experiments with bovine corneal epithelial tissue for up to 21 days showed that two-dimensional control over tissue outgrowth is achievable with our patterning method over extended time frames. The method promises to be an effective tool for use in a number of in vitro and in vivo applications.

Additivity in Effects of Vitronectin and Monoclonal Antibodies against α-Helix F of Plasminogen Activator Inhibitor-1 on Its Reactions with Target Proteinases

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.

Nicotine and fibronectin expression in lung fibroblasts: implications for tobacco-related lung tissue remodeling.

Tobacco-related lung diseases are associated with alterations in tissue remodeling and are characterized by increased matrix deposition. Among the matrix molecules found to be highly expressed in tobacco-related lung diseases is fibronectin, a cell adhesive glycoprotein implicated in tissue injury and repair. We hypothesize that nicotine, a component of tobacco, stimulates the expression of fibronectin in lung fibroblasts via the activation of intracellular signals that lead to increased fibronectin gene transcription. In support of this, we found that nicotine stimulated the expression of fibronectin in lung fibroblasts and that its stimulatory effect was associated with activation of protein kinase C and mitogen-activated protein kinases, increased levels of intracellular cAMP, and phosphorylation and DNA binding of the transcription factor CREB. Increased transcription of the gene was dependent on cAMP-response elements (CREs) present on the 5' end of its gene promoter. The stimulatory effect of nicotine on fibronectin expression was abolished by alpha-bungarotoxin, an inhibitor of alpha7 nicotinic acetylcholine receptors (alpha7 AChRs). Of note, nicotine increased the expression of alpha7 nAChRs on fibroblasts. Our data suggest that nicotine induces lung fibroblasts to produce fibronectin by stimulating alpha7 nAChR-dependent signals that regulate the transcription of the fibronectin gene.

Prostate cancer invasion is influenced more by expression of a CD44 isoform including variant 9 than by Muc18

The standard form of cell adhesion glycoprotein CD44 is a metastasis suppressor in prostate cancer. However, we previously showed by RT-PCR and Western blotting that cancer overexpresses unique CD44 variant v7–v10 isoforms. Muc18 is another cell adhesion marker reportedly overexpressed by prostate cancer. Matched frozen section-confirmed tumor and benign tissues were harvested from 10 prostatectomy specimens and tumor was microdissected from two lymph node metastases. Tissues were homogenized for RNA preparations, and RT-PCR was performed for the CD44v7–v10 sequence. In cultured prostate cancer cells, we caused RNA interference against CD44v9 and/or Muc18. We used PC3M cells and a derivative cell line called Gsα, that constitutively expresses this G-protein and is more invasive. Lipofection was performed for a green fluorescent protein plasmid and for two 22-mer DNA fragments, cloned into a plasmid expression vector to generate hairpin, interfering dsRNA. Assays for invasion into Matrigel, a basement membrane matrix, were performed in 4–5 experiments. RT-PCR demonstrated expression of a 608 bp band representing CD44v7–v10 or a 638 bp band of CD44v6–v10 in prostate cancer tissues and metastases but not benign tissue. Cultured Gsα cells overexpressed CD44v9 by comparison with PC3M cells. At 90 h after 6-hour lipofection, protein silencing was evident by Western blots. Silencing the CD44v9 expression reduced invasiveness into Matrigel to 21.6±7.0% in PC3M cells (P<0.001) and 31.2±18.3% in Gsα cells (P=0.001), compared to cells exposed to transfection vehicle alone. Silencing Muc18 expression reduced invasiveness to 76.9±13.5% of the control value in PC3M cells (P<0.05) and 84.8±29.9% in Gsα cells (P=0.18). Prostate cancer invasion is facilitated more by its overexpression of CD44 variant 9 than by Muc18. Its relative overexpression by Gsα cells is a novel finding, suggesting a link between signal transduction and cell adhesion marker expression.