An in vitro evaluation of inflammation response of titanium functionalized with heparin/fibronectin complex

Immobilization of biomolecules with a variety of biological functions has been a promising method to improve the biocompatibility of biomaterials. However, little is known about their inflammatory property and cytotoxicity, which are both key aspects to most biomaterials designed for tissue engineering applications and in vivo implantation. In this in vitro study, heparin/fibronectin complex (Hep/Fn) was coimmobilized onto titanium surface (HF-Ti), which had been proven to have the properties of both anticoagulation and endothelialization in our previous study. Fourier transform infrared (FTIR) spectroscopy and water contact angle measurement were utilized to determine the surface chemical compositions and physical properties. Toluidine Blue O (TBO) and immunochemistry methods were performed to quantify the surface-immobilized heparin and fibronectin. The early inflammatory responses elicited by pristine Ti and HF-Ti were investigated by proinflammatory cytokine secretion of tumor necrosis factor-alpha (TNF-α) released by attached peritoneal macrophages, monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β (IL-1β) released by attached human umbilical vein endothelial cells (ECs), respectively. Scanning electronic microscopy (SEM) and immunofluorescence were employed to investigate the changes in macrophages and ECs morphologies. The incubation period for both cells was 24 h and the results showed that HF-Ti revealed a weaker inflammatory response than pristine Ti, which provoked a stronger inflammatory response and higher activation of macrophages. Our data suggest that Hep/Fn coimmobilized biomaterials surface may develop to be a new generation of biomaterials with both biocompatibility and anti-inflammatory properties, especially for used as cardiovascular implants and in tissue engineering applications.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Multiple Ligands of von Willebrand Factor-binding Protein (vWbp) Promote Staphylococcus aureus Clot Formation in Human Plasma

Staphylococcus aureus secretes coagulase (Coa) and von Willebrand factor-binding protein (vWbp) to activate host prothrombin and form fibrin cables, thereby promoting the establishment of infectious lesions. The D1-D2 domains of Coa and vWbp associate with, and non-proteolytically activate prothrombin. Moreover, Coa encompasses C-terminal tandem repeats for binding to fibrinogen, whereas vWbp has been reported to associate with von Willebrand factor and fibrinogen. Here we used affinity chromatography with non-catalytic Coa and vWbp to identify the ligands for these virulence factors in human plasma. vWbp bound to prothrombin, fibrinogen, fibronectin, and factor XIII, whereas Coa co-purified with prothrombin and fibrinogen. vWbp association with fibrinogen and factor XIII, but not fibronectin, required prothrombin and triggered the non-proteolytic activation of FXIII in vitro. Staphylococcus aureus coagulation of human plasma was associated with the recruitment of prothrombin, FXIII, and fibronectin as well as the formation of cross-linked fibrin. FXIII activity in staphylococcal clots could be attributed to thrombin-dependent proteolytic activation as well as vWbp-mediated non-proteolytic activation of FXIII zymogen.     

Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils

Growing interest and research efforts have recently been focused on elucidating the molecular mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was observed when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS–PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.     

Essential roles of fibronectin in the development of the left-right embryonic body plan

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left–right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5β1, is also required for the development of the left–right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left–right axis of asymmetry in vertebrates.     

Domain swapping and amyloid fibril conformation

For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins β₂-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.     

PMEL: a pigment cell‐specific model for functional amyloid formation

PMEL is a pigment cell‐specific protein responsible for the formation of fibrillar sheets within the pigment organelle, the melanosome. The fibrillar sheets serve as a template upon which melanins polymerize as they are synthesized. The PMEL fibrils are required for optimal pigment cell function, as animals that either lack PMEL expression or express mutant PMEL variants show varying degrees of hypopigmentation and pigment cell inviability. The PMEL fibrils have biophysical properties of amyloid, a protein fold that is frequently associated with neurodegenerative and other diseases. However, PMEL is one of a growing number of non‐pathogenic amyloid proteins that contribute to the function of the cell and/or organism that produces them. Understanding how PMEL generates amyloid in a non‐pathogenic manner might provide insights into how to avoid toxicity due to pathological amyloid formation. In this review, we summarize and reconcile data concerning the fate of PMEL from its site of synthesis in the endoplasmic reticulum to newly formed melanosomes and the role of distinct PMEL subdomains in trafficking and amyloid fibril formation. We then discuss how its progression through the secretory pathway into the endosomal system might allow for the regulated and non‐toxic conversion of PMEL into an ordered amyloid polymer.     

The anti-Parkinsonian drug selegiline delays the nucleation phase of α-synuclein aggregation leading to the formation of nontoxic species

Parkinson's disease (PD) is a movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of intraneuronal inclusions called Lewy bodies, which are composed mainly of α-synuclein (α-syn). Selegiline (Sel) is a noncompetitive monoamino oxidase B inhibitor that has neuroprotective effects and has been administered to PD patients as monotherapy or in combination with l-dopa. Besides its known effect of increasing the level of dopamine (DA) by monoamino oxidase B inhibition, Sel induces other effects that contribute to its action against PD. We evaluated the effects of Sel on the in vitro aggregation of A30P and wild-type α-syn. Sel delays fibril formation by extending the lag phase of aggregation. In the presence of Sel, electron microscopy reveals amorphous heterogeneous aggregates, including large annular species, which are innocuous to a primary culture enriched in dopaminergic neurons, while their age-matched counterparts are toxic. The inhibitory effect displayed by Sel is abolished when seeds (small fibril pieces) are added to the aggregation reaction, reinforcing the hypothesis that Sel interferes with early nuclei formation and, to a lesser extent, with fibril elongation. NMR experiments indicate that Sel does not interact with monomeric α-syn. Interestingly, when added in combination with DA (which favors the formation of toxic protofibrils), Sel overrides the inhibitory effect of DA and favors fibrillation. Additionally, Sel blocks the formation of smaller toxic aggregates by perturbing DA-dependent fibril disaggregation. These effects might be beneficial for PD patients, since the sequestration of protofibrils into fibrils or the inhibition of fibril dissociation could alleviate the toxic effects of protofibrils on dopaminergic neurons. In nondopaminergic neurons, Sel might slow the fibrillation, giving rise to the formation of large nontoxic aggregates.