Angiogenesis Is Differentially Modulated by Platelet-Derived Products.

Platelet-derived preparations are being used in clinic for their role in tissue repair and regenerative processes. The release of platelet-derived products such as autologous growth factors, cytokines and chemokines can trigger therapeutic angiogenesis. In this in vitro study, we evaluated and compared the ability of three platelet-derived preparations: platelet-rich-plasma (PRP), PRP-hyaluronic acid (PRP-HA) and platelet lysates (PL) at various concentrations (5-40%) to modulate human umbilical vein endothelial cells (HUVEC) biological effects on metabolism, viability, senescence, angiogenic factors secretion and angiogenic capacities in 2D (endothelial tube formation assay or EFTA) and in 3D (fibrin bead assay or FBA). HUVEC exocytosis was stimulated with PRP and PRP-HA. Cell viability was strongly increased by PRP and PRP-HA but mildly by PL. The three preparations inhibit HUVEC tube formation on Matrigel, while PRP enhanced the complexity of the network. In the fibrin bead assay (FBA), PRP and PRP-HA stimulated all steps of the angiogenic process resulting in massive sprouting of a branched microvessel network, while PL showed a weaker angiogenic response. Secretome profiling revealed modulation of 26 human angiogenic proteins upon treatment with the platelet derived preparations. These in vitro experiments suggest that PRP and PRP-HA are effective biological therapeutic tools when sustained therapeutic angiogenesis is needed.

Anti-inflammatory and Quinone Reductase-Inducing Compounds from Beilschmiedia mannii.

Previous studies on the therapeutic potential of plant species found in the diet of chimpanzees living in Taï National Park have shown that they could be potential candidates for the search of new molecules useful for humans. Based on the screening of some of these plants, the fruits of Beilschmiedia mannii, whose dichloromethane extract showed cancer chemopreventive properties, were selected. Bioactivity-guided fractionation of the extract resulted in the isolation and identification of two γ-pyrones, including desmethoxydihydromethysticin (1: ), found in a natural source for the first time, and a new congener, beilschmiediapyrone (2: ), as well as five known alkamides (3:  - 7: ). Their structures were established by using nuclear magnetic resonance spectroscopy and mass spectrometry methods. The isolated compounds were evaluated for their cancer chemopreventive potential by using quinone reductase induction and nuclear factor-kappa B inhibition tests in Hepa 1c1c7 and HEK-293/NF-κB-Luc cells, respectively. Among them, compounds 1: and 2: were the most active. The concentrations to double the quinone reductase activity were 7.5 µM for compound 1: and 6.1 µM for compound 2: . Compounds 1: and 2: inhibited nuclear factor-kappa B with IC50 values of 2.1 and 3.4 µM, respectively. These results are promising with regard to cancer chemoprevention, especially because this plant is also used for cooking by the local population around the Taï forest.

The role of γ-secretase activating protein (GSAP) and imatinib in the regulation of γ-secretase activity and amyloid-ß generation.

γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-ß production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-ß production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-ß levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-ß generation. Likewise, purified recombinant GSAP had no effect on amyloid-ß generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-ß levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-ß levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-ß generation are uncertain.

Cladribine inhibits cytokine secretion by T cells independently of deoxycytidine kinase activity.

Cladribine (2-chloro-2'-deoxyadenosine) is a purine nucleoside analogue (PNA) which causes targeted and sustained reduction of peripheral lymphocyte counts. Cladribine tablets produced significant treatment benefit for patients with relapsing-remitting multiple sclerosis in the phase 3 CLARITY study. In addition to the well-characterised cell-specific phosphorylation of PNAs responsible for lymphocyte reduction, the mode of action of cladribine may encompass distinct activities contributing to its overall effects on the immune system. Here we demonstrate that clinically relevant concentrations of cladribine also inhibit cytokine secretion by human peripheral blood T cells in vitro through mechanisms independent of the induction of lymphocyte death.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo.

We demonstrated recently that P8A-CCL2, a monomeric variant of the chemokine CCL2/MCP-1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate-mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA-sensitized mice. This anti-inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A-CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A-CCL2 and wild-type (WT) CCL2, ruling out the hypothesis that P8A-CCL2 disrupts the chemokine gradient through systemic exposure. P8A-CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A-CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A-CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP-1) from endothelial surfaces. Intravital microscopy studies indicate that P8A-CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization-deficient chemokines can exhibit anti-inflammatory properties in vivo and may represent new therapeutic modalities.

The X-ray structure of RANTES: heparin-derived disaccharides allows the rational design of chemokine inhibitors.

The biological activity of chemokines requires interactions with cell surface proteoglycans. We have determined the structure of the chemokine RANTES (regulated on activation normal T cell expressed) in the presence of heparin-derived disaccharide analogs by X-ray crystallography. These structures confirm the essential role of the BBXB motif in the interaction between the chemokine and the disaccharide. Unexpected interactions were observed in the 30s loop and at the amino terminus. Mutant RANTES molecules were designed to abrogate these interactions and their biological activity examined in vivo. The K45E mutant within the BBXB motif lost the capacity to bind heparin and the ability to elicit cellular recruitment. The Y3A mutant maintained its capacity to bind heparin but was unable to elicit cellular recruitment. Finally, a tetrasaccharide is the smallest oligosaccharide which effectively abolishes the ability of RANTES to recruit cells in vivo. These crystallographic structures provide a description of the molecular interaction of a chemokine with glycosaminoglycans.

Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines.

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1beta/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.