Elastin-Derived Peptides Are New Regulators of Thrombosis

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The Innate Immune Response in Eisenia Fetida to Microbial Challenges

The common earthworm, Eisenia fetida, exhibits a rudimentary immune system. The earthworm needs cellular and chemical responses against a constant microbial exposure from its natural environment. Some cellular and chemical responses are found in the coelomic fluid and have been shown to demonstrate anti-microbial characteristics. This project uses microscopy and modified staining techniques to differentiate and categorize the cellular components found in the coelomic fluid. Following a microbial challenge by Klebsiella pneumoniae, an inflammatory response was initiated. Six groups of earthworms were injected with 0.05 ml of 1.0 x 106 cfu /ml K. pneumoniae on day one and tested over a period of five days. A group of three worms was shocked each day for the next five days to cause the coelomic fluid and cells to pass through the body wall. The coelomic fluid was placed directly on glass slides, dried and stained with a modified Wright’s stain using a wash buffer solution with a pH of 6.3. The stained cells were differentiated into four categories. Total cell counts were determined. The data indicated a marked proliferation in total cell counts in comparison to the control worms. This trend of increasing total cell counts continued over the five days. The percent ages of the four types of coelomic cells from the differential remained constant. Cells were photographed and documented for comparisons. Additional studies are ongoing to determine how long the Eisenia fetida take to remove Klebsiella pneumoniae from the coelomic cavity

Elastin-derived peptides potentiate atherosclerosis through the immune Neu1-PI3Kγ pathway

Aims Elastin is degraded during vascular ageing and its products, elastin-derived peptides (EP), are present in the human blood circulation. EP binds to the elastin receptor complex (ERC) at the cell surface, composed of elastin-binding protein (EBP), a cathepsin A and a neuraminidase 1. Some in vitro functions have clearly been attributed to this binding, but the in vivo implications for arterial diseases have never been clearly investigated. Methods and results Here, we demonstrate that chronic doses of EP injected into mouse models of atherosclerosis increase atherosclerotic plaque size formation. Similar effects were observed following an injection of a VGVAPG peptide, suggesting that the ERC mediates these effects. The absence of phosphoinositide 3-kinase γ (PI3Kγ) in bone marrow-derived cells prevented EP-induced atherosclerosis development, demonstrating that PI3Kγ drive EP-induced arterial lesions. Accordingly, in vitro studies showed that PI3Kγ was required for EP-induced monocyte migration and ROS production and that this effect was dependent upon neuraminidase activity. Finally, we showed that degradation of elastic lamellae in LDLR−/− mice fed an atherogenic diet correlated with atherosclerotic plaque formation. At the same time, the absence of the cathepsin A-neuraminidase 1 complex in cells of the haematopoietic lineage abolished atheroma plaque size progression and decreased leucocytes infiltration, clearly demonstrating the role of this complex in atherogenesis and suggesting the involvement of endogenous EP. Conclusion Altogether, this work identifies EP as an enhancer of atherogenesis and defines the Neuraminidase 1/PI3Kγ signalling pathway as a key mediator of this function in vitro and in viv

Elastin Peptides Signaling Relies on Neuraminidase-1-Dependent Lactosylceramide Generation

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM3 level decreases while lactosylceramide (LacCer) content increases consistently with a GM3/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM3 blocking antibody shows that GM3 is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM3/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest

Etude des voies de signalisation activées par les peptides d'élastine dans les fibrolastes dermiques humains (application à la régulation de la production de la pro-MMP-1 dans le cadre de la réaction stromale)

L'élastine est la protéine de la matrice extracellulaire responsable de la résilience des tissus. Cependant, son rôle n'est pas restreint à cette fonction : la dégradation de l'élastine lors de la progression tumorale conduit à la genèse de peptides d'élastine doués d'activités biologiques. Ces peptides induisent ainsi l'expression de pro-MMP-1 par les fibroblastes dermiques, métalloprotéinase matricielle impliquée dans l'invasion tumorale. Nous montrons par l'utilisation d'approches pharmacologiques et génétiques que les peptides d'élastine induisent l'expression de la pro-MMP-1 via la voie ERK1/2. Les facteurs de transcription de la famille AP-1 sont activés par cette voie. L'activation de ERK1/2 met en jeu deux voies complémentaires : protéine G0/Gi / p110g / Raf-1 / MEK1/2 et AMPc / PKA / B-Raf / MEK1/2. p110g est activée par l'hétérodimère bg dérivé de la protéine G. Ras n'est pas impliqué dans l'activation de la voie ERK1/2. L'analyse préliminaire du mode de fonctionnement du complexe récepteur de l'élastine suggère que son activité neuraminidase stimulée par les peptides d'élastine est le point d'initiation de la transduction du signal. Une analyse par microscopie confocale montre que le récepteur est localisé dans les radeaux lipidiques de la membrane plasmique. La destruction de ces microdomaines par déplétion du cholestérol qu'ils contiennent met en évidence leur rôle crucial dans la transduction du signal du récepteur. Enfin, la stimulation des cellules par les peptides d'élastine induit la production de lactosylcéramide, second messager pouvant être généré par l'action de la neuraminidase et qui pourrait être responsable de l'activation de la voie ERK1/2Elastin is the extracellular matrix molecule responsible for resilience of tissues. However, its role is not restricted to this function. During cancer progression, elastin is degraded leading to the production of elastin peptides which exhibit biological activities. These peptides induce pro-MMP-1 production by dermal fibroblasts, a matrix metalloproteinase involved in tumour development. Using pharmacological and genetic approaches, we show that elastin peptides induce pro-MMP-1 production via the ERK1/2 pathway. AP-1 transcription factors are activated via these kinases. ERK1/2 activation is achieved by the complementary involvement of two signalling modules G0/Gi protein / p110g / Raf-1 / MEK1/2 and cAMP / PKA / B-Raf / MEK1/2. p110g is activated by bg subunits derived from the G protein. Ras does not participate to ERK1/2 pathway activation. Preliminary analysis of the elastin receptor operational mechanism suggests that its elastin peptides-induced neuraminidase activity is the initiating point of its signalling. Confocal microscopy analysis shows that the receptor colocalize with plasma membrane lipids rafts. Destruction of rafts by cholesterol depletion demonstrate the key role of these microdomains in the elastin receptor signalling. Finally, we show that cell stimulation with elastin peptides induce the production of the second messenger lactosylceramide. This molecule could be generated through neuraminidase activity and could be responsible for ERK1/2 pathway activationREIMS-BU Sciences (514542101) / SudocSudocFranceF

Influence des peptides d élastine sur l apoptose des fibroblastes dermiques humains de sujets jeunes, matures et âgés

REIMS-BU Sciences (514542101) / SudocSudocFranceF

From elastin peptides to neuraminidase-1-dependent lactosylceramide generation

International audienc