Retromer controls epithelial cell polarity by trafficking the apical determinant crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.

Articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p <0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p <0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µm; IIB, K(d) = 8 µm; IIC, K(d) = 1.0 µm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Development and evaluation of a non-destructive adhesion test

The object of this work was to further develop the idea introduced by Muaddi et al (1981) which enables some of the disadvantages of earlier destructive adhesion test methods to be overcome. The test is non-destructive in nature but it does need to be calibrated against a destructive method. Adhesion is determined by measuring the effect of plating on internal friction. This is achieved by determining the damping of vibrations of a resonating specimen before and after plating. The level of adhesion was considered by the above authors to influence the degree of damping. In the major portion of the research work the electrodeposited metal was Watt's nickel, which is ductile in nature and is therefore suitable for peel adhesion testing. The base metals chosen were aluminium alloys S1C and HE9 as it is relatively easy to produce varying levels of adhesion between the substrate and electrodeposited coating by choosing the appropriate process sequence. S1C alloy is the commercially pure aluminium and was used to produce good adhesion. HE9 aluminium alloy is a more difficult to plate alloy and was chosen to produce poorer adhesion. The "Modal Testing" method used for studying vibrations was investigated as a possible means of evaluating adhesion but was not successful and so research was concentrated on the "Q" meter. The method based on the use of a "Q" meter involves the principle of exciting vibrations in a sample, interrupting the driving signal and counting the number of oscillations of the freely decaying vibrations between two known preselected amplitudes of oscillations. It was not possible to reconstruct a working instrument using Muaddi's thesis (1982) as it had either a serious error or the information was incomplete. Hence a modified "Q" meter had to be designed and constructed but it was then difficult to resonate non-magnetic materials, such as aluminium, therefore, a comparison before and after plating could not be made. A new "Q" meter was then developed based on an Impulse Technique. A regulated miniature hammer was used to excite the test piece at the fundamental mode instead of an electronic hammer and test pieces were supported at the two predetermined nodal points using nylon threads. This instrument developed was not very successful at detecting changes due to good and poor pretreatments given before plating, however, it was more sensitive to changes at the surface such as room temperature oxidation. Statistical analysis of test results from untreated aluminium alloys show that the instrument is not always consistent, the variation was even bigger when readings were taken on different days. Although aluminium is said to form protective oxides at room temperature there was evidence that the aluminium surface changes continuously due to film formation, growth and breakdown. Nickel plated and zinc alloy immersion coated samples also showed variation in Q with time. In order to prove that the variations in Q were mainly due to surface oxidation, aluminium samples were lacquered and anodised Such treatments enveloped the active surfaces reacting with the environment and the Q variation with time was almost eliminated especially after hard anodising. This instrument detected major differences between different untreated aluminium substrates.Also Q values decreased progressively as coating thicknesses were increased. This instrument was also able to detect changes in Q due to heat-treatment of aluminium alloys.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

A novel RGD-independent cel adhesion pathway mediated by fibronectin-bound tissue transglutaminase rescues cells from anoikis

Specific association of tissue transglutaminase (tTG) with matrix fibronectin (FN) results in the formation of an extracellular complex (tTG-FN) with distinct adhesive and pro-survival characteristics. tTG-FN supports RGD-independent cell adhesion of different cell types and the formation of distinctive RhoA-dependent focal adhesions following inhibition of integrin function by competitive RGD peptides and function blocking anti-integrin antibodies alpha5beta1. Association of tTG with its binding site on the 70-kDa amino-terminal FN fragment does not support this cell adhesion process, which seems to involve the entire FN molecule. RGD-independent cell adhesion to tTG-FN does not require transamidating activity, is mediated by the binding of tTG to cell-surface heparan sulfate chains, is dependent on the function of protein kinase Calpha, and leads to activation of the cell survival focal adhesion kinase. The tTG-FN complex can maintain cell viability of tTG-null mouse dermal fibroblasts when apoptosis is induced by inhibition of RGD-dependent adhesion (anoikis), suggesting an extracellular survival role for tTG. We propose a novel RGD-independent cell adhesion mechanism that promotes cell survival when the anti-apoptotic role mediated by RGD-dependent integrin function is reduced as in tissue injury, which is consistent with the externalization and binding of tTG to fibronectin following cell damage/stress.

Cells, surfaces and adhesion

This thesis concerns cell adhesion to polymer surfaces with an experimental emphasis on hydrogels. The thesis begins with a review of the literature and a synthesis of recent evidence to describe the process of cell adhesion in a given situation. The importance of understanding integrin-adhesion protein interactions and adhesion protein-surface interactions is emphasised. The experimental chapters describe three areas of investigation. Firstly, in vitro cell culture techniques are used to explore a variety of surfaces including polyethylene glycol methacrylate (PEGMA) substituted hydrogels, sequence distribution modified hydrogels and worn contact lenses. Cell adhesion to PEGMA substituted gels is found to decrease with increases in polyethylene oxide chain length and correlations are made between sequence distribution and adhesion. Worn contact lenses are investigated for their cell adhesion properties in the presence of antibodies to specific adhesion proteins, demonstrating the presence of vitronectin and fibronectin on the lenses. The second experimental chapter addresses divalent cation regulation of integrin mediated cell adhesion. Several cell types and various cations are used. Zinc, previously not regarded as an important cation in the process, is found to inhibit 3T3 cell adhesion to vitronectin that is promoted by other divalent cations. The final experimental chapter concerns cell adhesion and growth on macroporous hydrogels. A variety of freeze-thaw formed porous gels are investiated and found generally to promote cell growth rate.Interpenetrating networkbased gels (IPN) are made porous by elution of dextrin particles of varying size and loading density. These materials provide the basis for synthetic cartilage. Cartilage cells (chondrocytes) plated onto the surface of the porous IPN materials maintain a rounded shape and hence phenotypic function when a critical pore size and density is achieved. In this way, a prospective implant, made porous at the perpendicular edges contacting natural cartilage can be both mechanically stabilised and encourage the maintenance of normal matrix production at the tissue interface.

The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.

Phospholipid chlorohydrin induces leukocyte adhesion to Apoe(-/-) mouse arteries via upregulation of P-selectin

HOCl-modified low-density lipoprotein (LDL) has proinflammatory effects, including induction of inflammatory cytokine production, leukocyte adhesion, and ROS generation, but the components responsible for these effects are not completely understood. HOCl and the myeloperoxidase-H2O2-halide system can modify both protein and lipid moieties of LDL and react with unsaturated phospholipids to form chlorohydrins. We investigated the proinflammatory effects of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine (SOPC) chlorohydrin on artery segments and spleen-derived leukocytes from ApoE-/- and C57 Bl/6 mice. Treatment of ApoE-/- artery segments with SOPC chlorohydrin, but not unmodified SOPC, caused increased leukocyte-arterial adhesion in a time- and concentration-dependent manner. This could be prevented by pretreatment of the artery with P-selectin or ICAM-1-blocking antibodies, but not anti-VCAM-1 antibody, and immunohistochemistry showed that P-selectin expression was upregulated. However, chlorohydrin treatment of leukocytes did not increase expression of adhesion molecules LFA-1 or PSGL-1, but caused increased release of ROS from PMA-stimulated leukocytes by a CD36-dependent mechanism. The SOPC chlorohydrin-induced adhesion and ROS generation could be abrogated by pretreatment of the ApoE-/- mice with pravastatin or a nitrated derivative, NCX 6550. These findings suggest that phospholipid chlorohydrins formed in HOCl-treated LDL could contribute to the proinflammatory effects observed for this modified lipoprotein in vitro.

Importance of syndecan-4 and syndecan -2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex

Tissue transglutaminase (TG2) has been identified as an important extracellular crosslinking enzyme involved in matrix turnover and in bone differentiation. Here we report a novel cell adhesion/survival mechanism in human osteoblasts (HOB) which requires association of FN bound TG2 with the cell surface heparan sulphates in a transamidase independent manner. This novel pathway not only enhances cell adhesion on FN but also mediates cell adhesion and survival in the presence of integrin competing RGD peptides. We investigate the involvement of cell surface receptors and their intracellular signalling molecules to further explore the pathway mediated by this novel TG-FN heterocomplex. We demonstrate by siRNA silencing the crucial importance of the cell surface heparan sulphate proteoglycans syndecan-2 and syndecan-4 in regulating the compensatory effect of TG-FN on osteoblast cell adhesion and actin cytoskeletal formation in the presence of RGD peptides. By use of immunoprecipitation and inhibitory peptides we show that syndecan-4 interacts with TG2 and demonstrate that syndecan-2 and the a5ß1 integrins, but not a4ß1 function as downstream modulators in this pathway. Using function blocking antibodies, we show activation of a5ß1 occurs by an inside out signalling mechanism involving activation and binding of protein kinase PKCa and phosphorylation of focal adhesion kinase (FAK) at Tyr861 and activation of ERK1/2.

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

Electrolytic processes on surfaces:contributions to eletrolytic polishing, anodising, adhesion, colour, lithography and electronic applications

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.