Clément Faye

Isolation, Characterization and Biological Evaluation of Jellyfish Collagen for Use in Biomedical Applications

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2–10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications.

Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (KD = ∼1.8 × 10−8 m, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (KD = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

The First Draft of the Endostatin Interaction Network

Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds to heparin/heparan sulfate and to a number of proteins, but its molecular mechanisms of action are not fully elucidated. We have used surface plasmon resonance (SPR) arrays to identify new partners of endostatin, and to give further insights on its molecular mechanism of action. New partners of endostatin include glycosaminoglycans (chondroitin and dermatan sulfate), matricellular proteins (thrombospondin-1 and SPARC), collagens (I, IV, and VI), the amyloid peptide Aβ-(1–42), and transglutaminase-2. The biological functions of the endostatin network involve a number of extracellular proteins containing epidermal growth factor and epidermal growth factor-like domains, and able to bind calcium. Depending on the trigger event, and on the availability of its members in a given tissue at a given time, the endostatin network might be involved either in the control of angiogenesis, and tumor growth, or in neurogenesis and neurodegenerative diseases.

Transglutaminase-2: a new endostatin partner in the extracellular matrix of endothelial cells.

Endostatin, a C-terminal fragment of collagen XVIII, binds to TG-2 (transglutaminase-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (Kd=6.8 nM). Enzymatic assays indicated that, in contrast with other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, Arg27 and Arg139, are crucial for its binding to TG-2. They are also involved in the binding to heparin [Sasaki, Larsson, Kreuger, Salmivirta, Claesson-Welsh, Lindahl, Hohenester and Timpl (1999) EMBO J. 18, 6240-6248], and to alpha5beta1 and alphavbeta3 integrins [Faye, Moreau, Chautard, Jetne, Fukai, Ruggiero, Humphries, Olsen and Ricard-Blum (2009) J. Biol. Chem. 284, 22029-22040], suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the hypothesis that the GTP-binding site of TG-2 is a potential binding site for endostatin. Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells under hypoxia, which stimulates angiogenesis. This interaction, occurring in a cellular context, might participate in the concerted regulation of angiogenesis and tumorigenesis by the two proteins.

Extended interaction network of procollagen C-proteinase enhancer-1 in the extracellular matrix

PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases such as BMP-1 (bone morphogenetic protein 1). PCPE-1 interacts with several proteins in addition to procollagens and BMP-1, suggesting that it could be involved in biological processes other than collagen maturation. We thus searched for additional partners of PCPE-1 in the extracellular matrix, which could provide new insights into its biological roles. We identified 17 new partners of PCPE-1 by SPR (surface plasmon resonance) imaging. PCPE-1 forms a transient complex with the β-amyloid peptide, whereas it forms high or very high affinity complexes with laminin-111 (KD=58.8 pM), collagen VI (KD=9.5 nM), TSP-1 (thrombospondin-1) (KD1=19.9 pM, KD2=14.5 nM), collagen IV (KD=49.4 nM) and endostatin, a fragment of collagen XVIII (KD1=0.30 nM, KD2=1.1 nM). Endostatin binds to the NTR (netrin-like) domain of PCPE-1 and decreases the degree of superstimulation of PCPE-1 enhancing activity by heparin. The analysis of the PCPE-1 interaction network based on Gene Ontology terms suggests that, besides its role in collagen deposition, PCPE-1 might be involved in tumour growth, neurodegenerative diseases and angiogenesis. In vitro assays have indeed shown that the CUB1CUB2 (where CUB is complement protein subcomponents C1r/C1s, urchin embryonic growth factor and BMP-1) fragment of PCPE-1 inhibits angiogenesis.

In vivo evidence for a bridging role of a collagen V subtype at the epidermis-dermis interface.

Collagen V is the defective product in most cases of classical Ehlers-Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V)(2)α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V)(3) minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V)(3) homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V)(3)-containing fibrils leads to ultrastructural modifications at the epidermis-dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal-dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.

Antibody-based surfaces: rapid characterization using two complementary colorimetric assays.

Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A(2)HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.

ANGPTL4-αvβ3 interaction counteracts hypoxia-induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2.

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia‐induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvβ3. In vitro and in vivo functional assays with Angptl4‐deficient mice demonstrate that ANGPTL4–αvβ3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4–αvβ3 interaction enhances Src recruitment to integrin αvβ3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia‐induced breakdown of VEGFR2–VE‐cadherin and VEGFR2–αvβ3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia‐driven vascular permeability through integrin αvβ3 binding, modulation of VEGFR2–Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4‐deficient mice show increased vascular leakage in vivo in a model of laser‐induced choroidal neovascularization, indicating that this newly identified ANGPTL4–αvβ3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright

On-line capillary electrophoresis-based enzymatic methodology for the study of polymer-drug conjugates ☆

This work aims at studying the potentialities of an on-line capillary electrophoresis (CE)-based digestion methodology for evaluating polymer-drug conjugates degradability in the presence of free trypsin (in-solution digestion). A sandwich plugs injection scheme with transverse diffusion of laminar profile (TDLFP) mode was used to achieve on-line digestions. Electrophoretic separation conditions were established using poly-l-Lysine (PLL) as reference substrate. Comparison with off-line digestion was carried out to demonstrate the feasibility of the proposed methodology. The applicability of the on-line CE-based digestion methodology was evaluated for two PLL-drug conjugates and for the four first generations of dendrigraft of lysine (DGL). Different electrophoretic profiles presenting the formation of di, tri, and tetralysine were observed for PLL-drug and DGL. These findings are in good agreement with the nature of the linker used to link the drug to PLL structure and the predicted degradability of DGL. The present on-line methodology applicability was also successfully proven for protein conjugates hydrolysis. In summary, the described methodology provides a powerful tool for the rapid study of biodegradable polymers.

Irradiation effects on antibody performance in the frame of biochip-based instruments development for space exploration

Several instruments based on immunoassay techniques have been proposed for life-detection experiments in the framework of planetary exploration but few experiments have been conducted so far to test the resistance of antibodies against cosmic ray particles. We present several irradiation experiments carried out on both grafted and free antibodies for different types of incident particles (protons, neutrons, electrons and 12C) at different energies (between 9 MeV and 50 MeV) and different fluences. No loss of antibodies activity was detected for the whole set of experiments except when considering protons with energy between 20 and 30 MeV (on free and grafted antibodies) and fluences much greater than expected for a typical planetary mission to Mars for instance. Our results on grafted antibodies suggest that biochip-based instruments must be carefully designed according to the expected radiation environment for a given mission. In particular, a surface density of antibodies much larger than the expected proton fluence would prevent significant loss of antibodies activity and thus assuring a successful detection.