In vivo remodeling of human cell-assembled extracellular matrix yarns.

Cell-assembled extracellular matrix (CAM) has been used to produce vascular grafts. While these completely biological vascular grafts performed well in clinical trials, the in vivo remodeling and inflammatory response of this truly "bio" material has not yet been investigated. In this study, human CAM yarns were implanted subcutaneously in nude rats to investigate the innate immune response to this matrix. The impact of processing steps relevant to yarn manufacturing was evaluated (devitalization, decellularization, gamma sterilization, and twisting). We observed that yarns were still present after six months, and were integrated into a non-inflamed loose connective tissue. The CAM was repopulated by fibroblastic cells and blood vessels. While other yarns caused minor peripheral inflammation at an early stage (two weeks of implantation), gamma sterilization triggered a more intense host response dominated by the presence of M1 macrophages. The inflammatory response was resolved at six months. Yarn mechanical strength was decreased two weeks after implantation except for the more compact "twisted" yarn. While the strength of other yarns was stable after initial remodeling, the gamma-sterilized yarn continued to lose mechanical strength over time and was weaker than devitalized (control) yarns at six months. This is the first study to formally demonstrate that devitalized human CAM is very long-lived in vivo and does not trigger a degradative response, but rather is very slowly remodeled. This data supports a strategy to produce human textiles from CAM yarn for regenerative medicine applications where a scaffold with low inflammation and long-term mechanical properties are critical.

Two-photon excited fluorescence (TPEF) may be useful to identify macrophage subsets based on their metabolic activity and cellular responses in atherosclerotic plaques.

BACKGROUND AND AIMS: Atherosclerosis is characterized by the formation of lipid plaques within the arterial wall. In such plaques, the massive and continuous recruitment of circulating monocyte-derived macrophages induces inflammation, leading to plaque destabilization and rupture. Plaque vulnerability is linked to the presence of (i) a large lipid core that contains necrotic, "foamy" macrophages (FMs), (ii) a thin fibrous cap that cannot limit the prothrombotic lipid core, and potentially (iii) an imbalance between inflammatory and immunoregulatory macrophages. These opposite macrophage functions rely on the use of different energy pathways (glycolysis and oxidative phosphorylation, respectively) that may lead to different levels of the auto-fluorescent cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). We hypothesized that high-resolution two-photon excited autofluorescence (TPEF) imaging of these cofactors may be used to monitor the metabolic activity and cellular responses of macrophages in atherosclerotic plaques. METHODS: Different models of human FMs were generated by exposure to acetylated or oxidized low-density lipoproteins (LDL), with/without human carotid extract (CE). Their phenotype and optical properties were compared with those of extremely polarized macrophages, inflammatory M1 (MLPS+IFNγ) and immunoregulatory M2 (MIL4+IL13). RESULTS: These FM models displayed an intermediate phenotype with low levels of M1 and M2 "specific" markers. Moreover, the NADH and FAD autofluorescence profiles of FMoxLDL ± CE cells were significantly distinct from those of M1 and M2 macrophages. CONCLUSIONS: TPEF imaging may be useful to follow the metabolic activity and cellular responses of the different macrophage subtypes present in atherosclerotic plaques in order to detect vulnerable areas.

Multimodal optical contrast agents as new tools for monitoring and tuning nanoemulsion internalisation into cancer cells. From live cell imaging to in vivo imaging of tumours.

Tailor-made NIR emitting dyes were designed as multimodal optical probes. These asymmetric amphiphilic compounds show combined intense absorption in the visible region, NIR fluorescence emission, high two-photon absorption in the NIR (with the maximum located around 1000 nm) as well as large Stokes' shift values and second-harmonic generation ability. Thanks to their structure, high loading into nanoemulsions (NEs) could be achieved leading to very high one- and two-photon brightness. These dyes were demonstrated to act as multimodal contrast agents able to generate different optical modalities of interest for bioimaging. Indeed, the uptake and carrier behaviour of the dye-loaded NEs into cancer cells could be monitored by simultaneous two-photon fluorescence and second-harmonic generation optical imaging. Multimodal imaging provided deep insight into the mechanism and kinetics of dye internalisation. Quite interestingly, the nature of the dyes was also found to influence both the kinetics of endocytosis and the internalisation pathways in glioblastoma cancer cells. By modulating the charge distribution within the dyes, the NEs can be tuned to escape lysosomes and enter the mitochondria. Moreover, surface functionalization with PEG macromolecules was realized to yield stealth NIRF-NEs which could be used for in vivo NIRF imaging of subcutaneous tumours in mice.

Characterization of a Cell-Assembled extracellular Matrix and the effect of the devitalization process.

We have previously shown that the Cell-Assembled extracellular Matrix (CAM) synthesized by normal, human, skin fibroblasts in vitro can be assembled in a completely biological vascular graft that was successfully tested in the clinic. The goal of this study was to perform a detailed analysis of the composition and the organization of this truly bio-material. In addition, we investigated whether the devitalization process (dehydration) used to store the CAM, and thus, make the material available "off-the-shelf," could negatively affect its organization and mechanical properties. We demonstrated that neither the thickness nor the mechanical strength of CAM sheets were significantly changed by the dehydration/freezing/rehydration cycle. The identification of over 50 extracellular matrix proteins highlighted the complex composition of the CAM. Histology showed intense collagen and glycosaminoglycan staining throughout the CAM sheet. The distribution of collagen I, collagen VI, thrombospondin-1, fibronectin-1, fibrillin-1, biglycan, decorin, lumican and versican showed various patterns that were not affected by the devitalization process. Transmission electron microscopy analysis revealed that the remarkably dense collagen network was oriented in the plane of the sheet and that neither fibril density nor diameter was changed by devitalization. Second harmonic generation microscopy revealed an intricate, multi-scale, native-like collagen fiber orientation. In conclusion, this bio-material displayed many tissue-like properties that could support normal cell-ECM interactions and allow implantation without triggering degradative responses from the host's innate immune system. This is consistent with its success in vivo. In addition, the CAM can be devitalized without affecting its mechanical or unique biological architecture. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) defines biological function and mechanical properties of tissues and organs. A number of promising tissue engineering approaches have used processed ECM from cadaver/animal tissues or cell-assembled ECM in vitro combined with scaffolds. We have shown the clinical potential of a scaffold-free approach based on an entirely biological material produced by human cells in culture without chemical processing. Here, we perform a comprehensive analysis of the properties of what can truly be called a bio-material. We also demonstrate that this material can be stored dried without losing its remarkable biological architecture.

Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso and Trigonella foenum-graecum L. on liver fat accumulation in C57BL/6J mice with high-fat diet-induced type 2 diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: Centaurium erythraea Rafn (CE), Artemisia herba-alba Asso (AHA) and Trigonella foenum-graecum L. (TFG) are traditionally used to treat type 2 diabetes in Algeria, previous studies have found that extracts of these plants were effective to treat or prevent experimental diabetes induced by high-fat diet (HFD). AIM OF THE STUDY: Describe the additional effects of these extracts on lipid tissue deposition in HFD. MATERIALS AND METHODS: Male C57BL/6J mice were fed with HFD to induce type 2 Diabetes. Groups of mice were given plant extracts orally at 2g/kg/bodyweight daily for 20 weeks during establishment of diabetes, or for 18 weeks after confirmation of diabetes at the 17th week. Liver and other tissue samples were stained with Oil Red O. RESULTS: Liver steatosis was confirmed with HFD. CE, AHA and TFG extracts improved liver steatosis by the end of the preventive (20 weeks) and curative periods (35 weeks). This was most marked for CE extract (p<0.05), less so with TFG and AHA. No steatosis was found in other tissues. CONCLUSION: CE extract had a clear hepatoprotective effect in this mouse model of diet-induced type 2 diabetes. AHA and TFG had a minimal or no significant effect on steatosis. Beyond its effect as an antidiabetic agent, CE may also be promising to prevent or treat non-alcoholic liver steatosis.

Glutamate-induced AMPA receptor desensitization increases their mobility and modulates short-term plasticity through unbinding from Stargazin.

Short-term plasticity of AMPAR currents during high-frequency stimulation depends not only on presynaptic transmitter release and postsynaptic AMPAR recovery from desensitization, but also on fast AMPAR diffusion. How AMPAR diffusion within the synapse regulates synaptic transmission on the millisecond scale remains mysterious. Using single-molecule tracking, we found that, upon glutamate binding, synaptic AMPAR diffuse faster. Using AMPAR stabilized in different conformational states by point mutations and pharmacology, we show that desensitized receptors bind less stargazin and are less stabilized at the synapse than receptors in opened or closed-resting states. AMPAR mobility-mediated regulation of short-term plasticity is abrogated when the glutamate-dependent loss in AMPAR-stargazin interaction is prevented. We propose that transition from the activated to the desensitized state leads to partial loss in AMPAR-stargazin interaction that increases AMPAR mobility and allows faster recovery from desensitization-mediated synaptic depression, without affecting the overall nano-organization of AMPAR in synapses.

Astrocytosis in parkinsonism: considering tripartite striatal synapses in physiopathology?

The current concept of basal ganglia organization and function in physiological and pathophysiological conditions excludes the most numerous cells in the brain, i.e., the astrocytes, present with a ratio of 10:1 neuron. Their role in neurodegenerative condition such as Parkinson's disease (PD) remains to be elucidated. Before embarking into physiological investigations of the yet-to-be-identified "tripartite" synapses in the basal ganglia in general and the striatum in particular, we therefore characterized anatomically the PD-related modifications in astrocytic morphology, the changes in astrocytic network connections and the consequences on the spatial relationship between astrocytic processes and asymmetric synapses in normal and PD-like conditions in experimental and human PD. Our results unravel a dramatic regulation of striatal astrocytosis supporting the hypothesis of a key role in (dys) regulating corticostriatal transmission. Astrocytes and their various properties might thus represent a therapeutic target in PD.

Cascade reactions in multicompartmentalized polymersomes.

Enzyme-filled polystyrene-b-poly(3-(isocyano-L-alanyl-aminoethyl)thiophene) (PS-b-PIAT) nanoreactors are encapsulated together with free enzymes and substrates in a larger polybutadiene-b-poly(ethylene oxide) (PB-b-PEO) polymersome, forming a multicompartmentalized structure, which shows structural resemblance to the cell and its organelles. An original cofactor-dependent three-enzyme cascade reaction is performed, using either compatible or incompatible enzymes, which takes place across multiple compartments.

Chromogranin A induces the biogenesis of granules with calcium- and actin-dependent dynamics and exocytosis in constitutively secreting cells.

Chromogranins are a family of acidic glycoproteins that play an active role in hormone and neuropeptide secretion through their crucial role in secretory granule biogenesis in neuroendocrine cells. However, the molecular mechanisms underlying their granulogenic activity are still not fully understood. Because we previously demonstrated that the expression of the major component of secretory granules, chromogranin A (CgA), is able to induce the formation of secretory granules in nonendocrine COS-7 cells, we decided to use this model to dissect the mechanisms triggered by CgA leading to the biogenesis and trafficking of such granules. Using quantitative live cell imaging, we first show that CgA-induced organelles exhibit a Ca(2+)-dependent trafficking, in contrast to native vesicle stomatitis virus G protein-containing constitutive vesicles. To identify the proteins that confer such properties to the newly formed granules, we developed CgA-stably-expressing COS-7 cells, purified their CgA-containing granules by subcellular fractionation, and analyzed the granule proteome by liquid chromatography-tandem mass spectrometry. This analysis revealed the association of several cytosolic proteins to the granule membrane, including GTPases, cytoskeleton-based molecular motors, and other proteins with actin- and/or Ca(2+)-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca(2+)-evoked exocytosis of the CgA-containing granules, indicating that these granules interact with microtubules and cortical actin for the regulated release of their content. These data demonstrate for the first time that the neuroendocrine factor CgA induces the recruitment of cytoskeleton-, GTP-, and Ca(2+)-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroendocrine secretory granules.