Induction of endothelial cell proliferation by recombinant and microparticle-tissue factor involves β1-integrin and extracellular signal regulated kinase activation

Objective: Increased levels of circulating tissue factor (TF) in the form of microparticles increase the risk of thrombosis. However, any direct influence of microparticle-associated TF on vascular endothelial cell proliferation is not known. In this study, the influence of recombinant and microparticle- associated TF on endothelial cell proliferation and mitogen-activated protein kinase signaling mechanisms was examined. Methods and Results: Incubation of human coronary artery endothelial cells with lipidated recombinant full-length TF, or TF-containing microparticles (50 to 200 pmol/L TF), increased the rate of cell proliferation and induced phosphorylation of extracellular signal regulated kinase 1 in a TF-dependent manner. Inhibition of extracellular signal regulated kinase 1/2 using PD98059 or extracellular signal regulated kinase 1/2 antisense oligonucleotides or inhibition of c-Jun N-terminal kinase reduced recombinant TF-mediated cell proliferation. PD98059 also reduced cell proliferation in response to TF-containing microparticles. Inclusion of FVIIa (5 nmol/L) and FXa (10 nmol/L) or preincubation of cells with an inhibitory anti-FVIIa antibody had no additional influence on TF-mediated cell proliferation. However, preincubation of exogenous TF with a β1-integrin peptide (amino acids 579 to 799) reduced TF-mediated proliferation. Conclusion: High concentrations of recombinant or microparticle-associated TF stimulate endothelial cell proliferation through activation of the extracellular signal regulated kinase 1/2 pathway, mediated through a novel mechanism requiring the interaction of exogenous TF with cell surface β1-integrin and independent of FVIIa. © 2010 American Heart Association, Inc

Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90 % suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation

Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA–mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cell, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53

Novel food grade dispersants : review of recent progress

Many foreseen advances in the design of food structures, suitable for ever demanding nutrient delivery systems, tailored controlled release, microencapsulation and protection of active ingredients, require a generation of superior dispersants than those currently provided by proteins. While the most efficient structure for such dispersants is relatively easy to specify, in foods they cannot simply be synthetically manufactured. The review highlights several possible strategies for realising more efficient food colloid stabilisers and summarises the key recent progress for each approach, both experimentally and theoretically. The emphasis is on those methods that lead to macromolecularly adsorbed layers. Practical aspects apart, we also discuss a number of interesting fundamental questions that each approach raises

On the structural polydispersity of random copolymers adsorbed at interfaces : comparison of surface and bulk distributions

Synthesis of random copolymers leads to a structurally polydispersed distribution of polymer chains, where one of the constituent monomers prefer residing on the interface, while the others have a tendency for remaining in the bulk. Previous studies have demonstrated the very strong dependence of the level of adsorption with the degree of blockiness and number of adsorbing residues of the chains. Using self-consistent field (SCF) calculations, we obtain the distribution of the adsorbed copolymers and compare this with the bulk distribution of such chains. In our study, the whole range of structurally polydisperse chains in the distribution derived for a given random copolymer, are simultaneously present and can compete with each other for adsorption. We show that the distribution of chains on the surface is grossly different to that in the bulk and is largely dominated by those rare chains at the tail end of the latter distribution

Improved enzymatic accessibility of peanut protein isolate pre-treated using thermosonication

Thermosonication pre-treatment was used to enhance the pancreatin-induced proteolysis of peanut protein isolate (PPI). Response surface methodology was applied to optimize the thermosonication conditions (including power-output and temperature), and the highest degree of hydrolysis (7.16%) was obtained at 475.0 W, 72 °C. SDS-PAGE analysis showed that at this optimized condition, the enzymatic accessibility of the major constitutive protein arachin in thermosonicated PPI (TS-PPI) was substantially improved compared to that in untreated PPI or sonicated PPI (475 W, 30°C; S-PPI), resulting in a remarkable increase in protein solubility for the hydrolysates. Protein denaturation and conformation profiles of untreated PPI, S-PPI and TS-PPI were investigated using differential scanning calorimetry, intrinsic fluorescence emission spectroscopy, Fourier transform infra-red spectroscopy and thioflavin-T (ThT) fluorescence assay. It was found that heat could present a markedly additive effect to ultrasound on denaturing peanut proteins, leading to significant changes in protein conformation. TS-PPI was characterized by the appearance of high proportion of parallel intermolecular β-sheets and a strong fluorescence enhancement upon binding to ThT, suggesting that the protein unfolding and aggregation induced by thermosonication probably resulted in the formation of fibril protein aggregates in TS-PPI rather than spherical protein aggregates formed in S-PPI. As a result, the protein conformation of TS-PPI appeared to be more unfolded and flexible than that of untreated PPI or S-PPI, and therefore was more easily accessible to protease. This study shows that thermosonication pre-treatment could be a highly effective and feasible technique to improve the enzymatic accessibility of globular proteins, producing prominent functional benefits for the protein hydrolysates

On the Origin of Seemingly Non-Surface Active Particles Partitioning between Phase Separated Solutions of Incompatible Non-Adsorbing Polymers and Their Adsorption at the Phase Boundary.

We have computed the free energy per unit area (i.e., interfacial tension) between a solid surface and two co-existing polymer solutions, where there is no specific interaction between the particles and either polymer, via self-consistent field calculations. Several different systems have been studied, including those where the two polymers differ in molecular weight (Mw) by a factor of ~ 2, or where the polymers have the same Mw but one set of chains is branched with the other being linear. In the absence of any enthalpic contribution resulting from adsorption on the particle, the differences in free energy per unit area resulting from the polymer depleted regions around the particles in the two co-exiting phases are found to be ~ 1 μN m-1. Although this value may seem rather small, this difference is more than capable of inducing the partitioning of particles of 100 nm in size (or larger) into the phase with the lower interfacial free energy at a solid surface. By examining the density profile variation of the polymers close to the solid surface, we can also infer information about the wettability and contact angle (Θ) of the solid particle at the interface between the two co-existing phases. This leads to the conclusion that for all systems of this type, when the incompatibility between the two polymers is sufficiently large, will be close to 90°

Early warning signals in plant disease outbreaks

Infectious disease outbreaks in plants threaten ecosystems, agricultural crops and food trade. Currently, several fungal diseases are affecting forests worldwide, posing a major risk to tree species, habitats and consequently ecosystem decay. Prediction and control of disease spread are difficult, mainly due to the complexity of the interaction between individual components involved. In this work, we introduce a lattice-based epidemic model coupled with a stochastic process that mimics, in a very simplified way, the interaction between the hosts and pathogen. We studied the disease spread by measuring the propagation velocity of the pathogen on the susceptible hosts. Our quantitative results indicate the occurrence of a critical transition between two stable phases: local confinement and an extended epiphytotic outbreak that depends on the density of the susceptible individuals. Quantitative predictions of epiphytotics are performed using the framework early-warning indicators for impending regime shifts, widely applied on dynamical systems. These signals forecast successfully the outcome of the critical shift between the two stable phases before the system enters the epiphytotic regime. Our study demonstrates that early-warning indicators could be useful for the prediction of forest disease epidemics through mathematical and computational models suited to more specific pathogen–host-environmental interactions. Our results may also be useful to identify a suitable planting density to slow down disease spread and in the future, design highly resilient forests

Biomarkers of coronary endothelial health: correlation with invasive measures of collateral function, flow and resistance in chronically occluded coronary arteries and the effect of recanalization

Objectives: In the presence of a chronically occluded coronary artery, the collateral circulation matures by a process of arteriogenesis; however, there is considerable variation between individuals in the functional capacity of that collateral network. This could be explained by differences in endothelial health and function. We aimed to examine the relationship between the functional extent of collateralization and levels of biomarkers that have been shown to relate to endothelial health. Methods: We measured four potential biomarkers of endothelial health in 34 patients with mature collateral networks who underwent a successful percutaneous coronary intervention (PCI) for a chronic total coronary occlusion (CTO) before PCI and 6-8 weeks after PCI, and examined the relationship of biomarker levels with physiological measures of collateralization. Results: We did not find a significant change in the systemic levels of sICAM-1, sE-selectin, microparticles or tissue factor 6-8 weeks after PCI. We did find an association between estimated retrograde collateral flow before CTO recanalization and lower levels of sICAM-1 (r=0.39, P=0.026), sE-selectin (r=0.48, P=0.005) and microparticles (r=0.38, P=0.03). Conclusion: Recanalization of a CTO and resultant regression of a mature collateral circulation do not alter systemic levels of sICAM-1, sE-selectin, microparticles or tissue factor. The identified relationship of retrograde collateral flow with sICAM-1, sE-selectin and microparticles is likely to represent an association with an ability to develop collaterals rather than their presence and extent