Supported cell mimetic monolayers and their interaction with blood

Surface modification using supported monolayers of phosphorylcholine containing phospholipids has been an accepted strategy for developing blood-contacting materials. We present a detailed study of the blood compatibility of the supported monolayers of phospholipid, glycolipid, and cholesterol (Chol) binary and ternary lipid combinations using in vitro techniques. The packing and orientation of these monolayers have been correlated with the blood compatibility. We have used phosphatidylcholine (PTC) for phospholipid, galactocerebroside (Gal) for glycolipid, and Chol based on the headgroup structure to represent the major lipid components of the endothelial luminal cell membrane. The interfacial behavior of various combinations of PTC, Gal, and Chol monolayers have been studied at the air/water interface and deposited on hydrophobic polycarbonate (PC) polymer substrates with the help of the Langmuir-Blodgett trough. The packing and orientation of the supported monolayers have been varied by means of changing the lipid composition rather than the deposition parameters. This approach seems to be more similar to the in vivo conditions. The different supported monolayer surfaces prepared accordingly are (1) a closely packed ordered hydrophobic surface, PC modified with the combination PTC/Chol/Gal (1:0.35:0.125), (2) a loosely packed ordered hydrophobic surface, PC modified with the combination PTC/Chol (1:0.35), and (3) a closely packed ordered hydrophilic surface, PC modified with the combination PTC/Chol (1:0.7). An optimized modified surface (PTC/Chol/Gal, 1:0.35:0.125) has been identified on the basis of the maximum transfer ratio from the air/water interface and characterized by using atomic force microscopy. The concentration of Chol has been found to be an important parameter, which influences the transfer ratio. The Gal improves the monolayer integrity under a reduced Chol concentration. The blood compatibility of these supported monolayers was studied by protein adsorption, blood cell adhesion, and calcification. The tightly packed ordered hydrophobic surface (PTC/Chol/Gal, 1:0.35:0.125), has been found to be more blood compatible because of reduced blood cell adhesion and calcification. This surface also promotes albumin adsorption and may be the reason for the reduced platelet activation, while in the case of the loosely packed ordered hydrophobic surface (PTC/Chol, 1:0.35) the protein adsorption also has been reduced along with the blood cell adhesion and calcification. When the ordered hydrophilic surface (PTC/Chol, 1: 0.7) of the monolayer has been exposed, the blood cell adhesions as well as the overall protein adsorption were significantly reduced. However, the packing of the phosphorylcholine moieties of the polar headgroup has been affecting the calcification on the surface. We have observed an increase in calcification to the surface modified with the loosely packed polar headgroup, from a relative study on chitosan and chitosan modified with the monolayer of PTC. These findings are helpful for the surface modifications for blood-contacting materials using this strategy.     

Effect of the peptide secondary structure on the peptide amphiphile supramolecular structure and interactions

Bottom-up fabrication of self-assembled nanomaterials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two different peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from α helices to β sheets. A concomitant shift in self-assembled morphology from nanoribbons to core-shell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.     

Hybrid modeling and simulation of stochastic effects on progression through the eukaryotic cell cycle

The eukaryotic cell cycle is regulated by a complicated chemical reaction network. Although many deterministic models have been proposed, stochastic models are desired to capture noise in the cell resulting from low numbers of critical species. However, converting a deterministic model into one that accurately captures stochastic effects can result in a complex model that is hard to build and expensive to simulate. In this paper, we first apply a hybrid (mixed deterministic and stochastic) simulation method to such a stochastic model. With proper partitioning of reactions between deterministic and stochastic simulation methods, the hybrid method generates the same primary characteristics and the same level of noise as Gillespie's stochastic simulation algorithm, but with better efficiency. By studying the results generated by various partitionings of reactions, we developed a new strategy for hybrid stochastic modeling of the cell cycle. The new approach is not limited to using mass-action rate laws. Numerical experiments demonstrate that our approach is consistent with characteristics of noisy cell cycle progression, and yields cell cycle statistics in accord with experimental observations.     

A novel quantitative model of cell cycle progression based on cyclin-dependent kinases activity and population balances

Cell cycle regulates proliferative cell capacity under normal or pathologic conditions, and in general it governs all in vivo/in vitro cell growth and proliferation processes. Mathematical simulation by means of reliable and predictive models represents an important tool to interpret experiment results, to facilitate the definition of the optimal operating conditions for in vitro cultivation, or to predict the effect of a specific drug in normal/pathologic mammalian cells. Along these lines, a novel model of cell cycle progression is proposed in this work. Specifically, it is based on a population balance (PB) approach that allows one to quantitatively describe cell cycle progression through the different phases experienced by each cell of the entire population during its own life. The transition between two consecutive cell cycle phases is simulated by taking advantage of the biochemical kinetic model developed by Gérard and Goldbeter (2009) which involves cyclin-dependent kinases (CDKs) whose regulation is achieved through a variety of mechanisms that include association with cyclins and protein inhibitors, phosphorylation–dephosphorylation, and cyclin synthesis or degradation. This biochemical model properly describes the entire cell cycle of mammalian cells by maintaining a sufficient level of detail useful to identify check point for transition and to estimate phase duration required by PB. Specific examples are discussed to illustrate the ability of the proposed model to simulate the effect of drugs for in vitro trials of interest in oncology, regenerative medicine and tissue engineering.     

Synthesis and cell cycle inhibition of the peptide enamide natural products terpeptin and the aspergillamides

Total syntheses of the peptide enamide natural products terpeptin and aspergillamides A and B are reported. An oxidative decarboxylation-elimination protocol is employed to construct the indolic enamide moiety. Unambiguous stereochemical assignment of (−)-terpeptin is accomplished by synthesis of all possible stereochemical analogues. Select compounds have been evaluated in cell cycle inhibitor assays which show that the natural amino acid configuration of terpeptin has the most potent inhibitory activity.     

Natural product-derived modulators of cell cycle progression and viral entry by enantioselective oxa Diels-Alder reactions on the solid phase

The underlying frameworks of natural product classes with multiple biological activities can be regarded as biologically selected and prevalidated starting points in vast chemical structure space in the development of compound collections for chemical biology and medicinal chemistry research. For the synthesis of natural product-derived and -inspired compound collections, the development of enantioselective transformations in a format amenable to library synthesis, e.g., on the solid support, is a major and largely unexplored goal. We report on the enantioselective solid-phase synthesis of a natural product-inspired alpha,beta-unsaturated delta-lactone collection and its investigation in cell-based screens monitoring cell cycle progression and viral entry into cells. The screens identified modulators of both biological processes at a high hit rate. The screen for inhibition of viral entry opens up avenues of research for the identification of compounds with antiviral activity.     

Subcellular topological effect of particle monolayers on cell shapes and functions

We studied topological effects of subcellular roughness displayed by a closely packed particle monolayer on adhesion and growth of endothelial cells. Poly(styrene-co-acrylamide) (SA) particles were prepared by soap-free emulsion copolymerization. Particle monolayers were prepared by Langmuir–Blodgett deposition using particles, which were 527 (SA053) and 1270 nm (SA127) in diameter. After 24-h incubation, cells tightly adhered on a tissue culture polystyrene dish and randomly spread. On the other hand, cells attached on particle monolayers were stretched into a narrow stalk-like shape. Lamellipodia spread from the leading edge of cells attached on SA053 monolayer to the top of the particles and gradually gathered to form clusters. This shows that cell–cell adhesion became stronger than cell–substrate interaction. Cells attached to SA127 monolayer extended to the reverse side of a particle monolayer and engulfed particles. They remained immobile without migration 24 h after incubation. This shows that the inhibition of extensions on SA127 monolayer could inhibit cell migration and cell proliferation. Cell growth on the particle monolayers was suppressed compared with a flat TCPS dish. The number of cells on SA053 gradually increased, whereas that on SA127 decreased with time. When the cell seeding density was increased to 200,000 cells cm⁻², some adherent cells gradually became into contact with adjacent cells. F-actin condensations were formed at the frame of adherent cells and the thin filaments grew from the edges to connect each other with time. For the cell culture on SA053 monolayer, elongated cells showed a little alignment. Cells showed not arrangement of actin stress fibers but F-actin condensation at the contact regions with neighboring cells. Interestingly, the formed cell monolayer could be readily peeled from the particle monolayer. These results indicate that endothelial cells could recognize the surface roughness displayed by particle monolayers and the response was dependent on the pitch of particle monolayers.     

Endothelial cell adhesion on polyelectrolyte multilayer films functionalised with fibronectin and collagen

The seeding of endothelial cells on biomaterial surfaces has become a major challenge to achieve better haemocompatibility of these surfaces. Multilayers of polyelectrolytes formed by the layerby-layer method are promising in this respect. In this study, the interactions of endothelial cells with multilayered polyelectrolytes films were investigated. The build-ups were prepared by selfassembled alternatively adsorbed polyanions and polycations functionalised with fibronectin and collagen. Anionic poly(sodium 4-styrenesulfonate) and cationic poly(allylamine hydrochloride) polyelectrolytes were chosen as a model system. Elaborated surfaces were characterised by electrochemical impedance spectroscopy and cyclic voltammetry. The modified electrode showed good reversible electrochemical properties and high stability in an electrolyte solution. The film ohmic resistance was highest when the film was coated with fibronectin; the parameters so determined were correlated with atomic force microscopy images. Cell colorimetric assay (WST-1) and immunofluorescence were used to quantify the cell viability and evaluate the adhesion properties. When cultured on a surface where proteins were deposited, cells adhered and proliferated better with fibronectin than with collagen. In addition, a high surface free energy was favourable to adhesion and proliferation (48.8 mJ m⁻² for fibronectin and 39.7 mJ m⁻² for collagen, respectively). Endothelial cells seeded on functionalised-polyelectrolyte multilayer films showed a good morphology and adhesion necessary for the development of a new endothelium.     

Biomimetic fibronectin/mineral and osteogenic growth peptide/mineral composites synthesized on calcium phosphate thin films

Composites of fibronectin/mineral and osteogenic growth peptide/mineral were synthesized on calcium phosphate coated substrates immersed in Dulbecco's phosphate-buffered saline solution containing biomolecules. The kinetics of coprecipitation for two biomolecules was similar, and the biomolecules participated in the formation of the crystal latticework and influenced the mineral structure and composition.     

Enhanced stability of short- and long-chain diselenide self-assembled monolayers on gold probed by electrochemistry, spectroscopy, and microscopy

Well-ordered, compact, self-assembled monolayers (SAMs) of hexyl and dodecyl diselenides have been formed on oriented (111) gold surfaces. Monolayer formation has been effected by adsorption from neat diselenides as well as millimolar solutions of diselenides in alcohol. The monolayer formation is confirmed using electrochemical quartz crystal microbalance studies. The stability and permeability of the monolayers at various temperatures have been probed using reflection absorption infrared spectroscopy (RAIRS) and electrochemistry. The RAIRS studies in the dry state show the formation of highly ordered, compact structures when adsorbed from neat compounds compared to the monolayers adsorbed in the presence of alcohol. The monolayers adsorbed from neat diselenide are quite stable as a function of temperature irrespective of the chain length. The electrochemical studies based on the blocking behavior of the monolayers toward electron transfer between a diffusing species and the electrode surface reflect the stability and the compactness of the structure. The results point out that the presence of solvent molecules during the SAM formation hinders the organization of the monolayer structure, especially in the case of short-chain diselenide monolayers.     

Expression of integrin beta1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cells (SMMC-7721) and human umbilical vein endothelial cells

To investigate expression of integrin β₁ and its roles on adhesion between different cell cycle hepatocellular carcinoma cell (HCC) and human umbilical vein endothelial cells (HUVEC), the synchronous G₁ and S phase HCC were achieved through thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Expression of integrin β₁ on hepatocellular carcinoma cells was detected with flow cytometer. Further, the adhesive force of HCC to HUVEC and the role of integrin β₁ in this adhesive course were studied by micropipette aspiration technique. The results showed that percentage of each cyclic phases of the controlled HCC (non-synchronous) are: G₂+M phase, 11%; G₁ phase, 54%; S phase, 36%; the synchronous rates of G₁ and S phase HCC amount to 74 and 98%, respectively. The expressive fluorescent intensity of integrin β₁ in G₁ phase HCC is depressed significantly than the values of S phase and controlled HCC. Accordingly, the adhesive forces of G₁ phase HCC to HUVEC was significantly lower than the value of S phase cells (P<0.01), but it has no remarkable difference when compared the adhesive force values of S phase HCC with control; the contribution of integrin β₁ was about 50% in the adhesion of HCC to HUVEC. It suggested that HCC would be synchronized preferably in G₁ and S phase with thymine-2-deoxyriboside and colchicines, the adhesive molecule integrin β₁ expressed in a high lever in HCC and presented differences in vary cell cycle, and integrin β₁ played an important roles in adhesion of HCC to HUVEC. Possibly, S phase HCC take a great action in this adhesive course.     

Aggregation of a peptide antibiotic alamethicin at the air-water interface and its influence on the viscoelasticity of phospholipid monolayers

The aggregation properties of an antibiotic membrane-active peptide alamethicin at the air-water interface have been studied using interfacial rheology and fluorescence microscopy techniques. Fluorescence microscopy of alamethicin monolayers revealed a coexistence of liquid expanded (LE) and solid phases at the surface concentrations studied. Interfacial oscillatory shear measurements on alamethicin monolayers indicate that its viscoelastic properties are determined by the area fraction of the solid domains. The role of zwitterionic phospholipids dioleoylphosphatidyl choline (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE) on the peptide aggregation behavior was also investigated. Fluorescence microscopy of alamethicin/phospholipid monolayers revealed an intermediate phase (I) in addition to the solid and LE phase. In mixed monolayers of phospholipid (L)/alamethicin (P), with increase in L/P, the monolayer transforms from a viscoelastic to a viscous fluid with the increase in area fraction of the intermediate phase. Further, a homogeneous mixing of alamethicin/lipid molecules is observed at L/P > 4. Our studies also confirm that the viscoelasticity of alamethicin/phospholipid monolayers is closely related to the alamethicin/phospholipid interactions at the air-water interface.     

Hierarchical assembly of model cell surfaces: synthesis of mucin mimetic polymers and their display on supported bilayers

Molecular level analysis of cell-surface phenomena could benefit from model systems comprising structurally defined components. Here we present the first step toward bottom-up assembly of model cell surfaces-the synthesis of mucin mimetics and their incorporation into artificial membranes. Natural mucins are densely glycosylated O-linked glycoproteins that serve numerous functions on cell surfaces. Their large size and extensive glycosylation makes the synthesis of these biopolymers impractical. We designed synthetically tractable glycosylated polymers that possess rodlike extended conformations similar to natural mucins. The glycosylated polymers were end-functionalized with lipid groups and embedded into supported lipid bilayers where they interact with protein receptors in a structure-dependent manner. Furthermore, their dynamic behavior in synthetic membranes mirrored that of natural biomolecules. This system provides a unique framework with which to study the behavior of mucin-like macromolecules in a controlled, cell surface-mimetic environment.     

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27Kip1 ubiquitination pathway in hepatocellular carcinoma

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27^Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27^Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27^Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27^Kip1 for degradation by the 26S proteasome, leading to accumulation of p27^Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.     

Effective isolation of magnesium lithospermate B and its inhibition of aldose reductase and fibronectin on mesangial cell line

We developed an effective isolation method of magnesium lithospermate B from Salviae miltiorrhizae Radix and found for the first time that magnesium lithospermate B shows strong in vitro inhibition (IC50=0.04 microM) of aldose reductase (AR), 2.5 times than that of clinically used epalrestat (IC50=0.1 microM) and accumulation of fibronectin dose dependently.     

Effects of lidocaine-HCl salt and benzocaine on the expansion of lipid monolayers employed as bio-mimicking cell membrane

Effects of lidocaine-HCl salt and benzocaine on the expansion of lipid monolayers employed as bio-mimicking cell membrane were investigated using Langmuir-Blodgett film balance to figure out the molecular mechanism for anesthesia by these local anesthetics. Lidocaine-HCl salt in subphase expanded the monolayer of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE). Benzocaine was not mixed with lipids in the monolayer, but the monolayer of lipids on the surface of water saturated with benzocaine was expanded same as the case of lidocaine-HCl salt. Even though this study can not explain the whole molecular mechanism for anesthesia by lidocaine-HCl salt and benzocaine, it can be asserted from the results of this study that the expansion of cell membrane by lidocaine-HCl salt and benzocaine contribute, at least partially, to the generation of anesthesia.     

Xanthatin mediates G2/M cell cycle arrest,autophagy and apoptosis via ROS/XIAP signaling in human colon cancer cells

Abstract

Xanthatin is a natural plant bicyclic sesquiterpene lactone extracted from Xanthium plants (Asteraceae). In the present study, we demonstrated for the first time that Xanthatin inhibited cell proliferation and mediated G₂/M phase arrest in human colon cancer cells. Xanthatin also activated caspase and mediated apoptosis in these cells. Concomitantly, Xanthatin triggered cell autophagic response. We found down-regulation of X-linked inhibitor of apoptosis protein (XIAP) contribute to the induction of apoptosis and autophagy. Moreover, reactive oxygen species (ROS) production was triggered upon exposure to Xanthatin in colon cancer cells. ROS inhibitor N-acetylcysteine (NAC) significantly reversed Xanthatin-mediated XIAP down-regulation, G₂/M phase arrest, apoptosis and autophagosome accumulation. In summary, our findings demonstrated that Xanthatin caused G₂/M phase arrest and mediated apoptosis and autophagy through ROS/XIAP in human colon cancer cells. We provided molecular bases for developing Xanthatin as a promising antitumor candidate for colon cancer therapy. Abbreviations ROS reactive oxygen species

DMSO dimethyl sulfoxide

5-FU 5-Fluorouracil

3-MA 3-Methyladenine

DCFH-DA 2’7’-dichlorfluorescein-diacetate

NAC N-acetylcysteine

XIAP X-linked inhibitor of apoptosis protein

     

Fabrication and hemocompatibility of cell outer membrane mimetic surfaces on chitosan by layer by layer assembly with polyanion bearing phosphorylcholine groups

Three random copolymers poly(2-methacryloyloxyethyl phosphorylcholine-co-methacrylic acid) (PMAs) were synthesized by free radical polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) and methacrylic acid (MA) with different monomer ratios under monomer-starved conditions. The synthesized PMA polyanions were assembled on chitosan (CS) film surfaces via electrostatic interactions. Using layer by layer (LbL) assembly with PMA polyanion and chitosan polycation, PMA/CS multilayer thin films with phosphorylcholine groups on the outer surfaces were fabricated. The modified surfaces were characterized by dynamic contact angle (DCA), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Hemocompatibility of the surfaces was estimated by protein adsorption and platelet adhesion measurements. The results indicated that cell outer membrane mimetic structures were formed on the modified surfaces with PMA as the outermost layer, and the hemocompatibility of the modified surfaces was significantly improved. This facile method of fabricating cell outer membrane mimetic surfaces may have potential applications in the fields of hemocompatible coatings, drug delivery, and tissue engineering.     

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.     

Expression of integrin β1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cells (SMMC-7721) and human umbilical vein endothelial cells

To investigate expression of integrin β₁ and its roles on adhesion between different cell cycle hepatocellular carcinoma cell (HCC) and human umbilical vein endothelial cells (HUVEC), the synchronous G₁ and S phase HCC were achieved through thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Expression of integrin β₁ on hepatocellular carcinoma cells was detected with flow cytometer. Further, the adhesive force of HCC to HUVEC and the role of integrin β₁ in this adhesive course were studied by micropipette aspiration technique. The results showed that percentage of each cyclic phases of the controlled HCC (non-synchronous) are: G₂+M phase, 11%; G₁ phase, 54%; S phase, 36%; the synchronous rates of G₁ and S phase HCC amount to 74 and 98%, respectively. The expressive fluorescent intensity of integrin β₁ in G₁ phase HCC is depressed significantly than the values of S phase and controlled HCC. Accordingly, the adhesive forces of G₁ phase HCC to HUVEC was significantly lower than the value of S phase cells (P<0.01), but it has no remarkable difference when compared the adhesive force values of S phase HCC with control; the contribution of integrin β₁ was about 50% in the adhesion of HCC to HUVEC. It suggested that HCC would be synchronized preferably in G₁ and S phase with thymine-2-deoxyriboside and colchicines, the adhesive molecule integrin β₁ expressed in a high lever in HCC and presented differences in vary cell cycle, and integrin β₁ played an important roles in adhesion of HCC to HUVEC. Possibly, S phase HCC take a great action in this adhesive course.