Role and new insights of microfibrillar-associated protein 4 in fibrotic diseases.

Fibrosis is one of the most worrisome complications of chronic inflammatory diseases, leading to tissue damage, organ failure, and ultimately, death. The most notable pathological characteristic of fibrosis is the excessive accumulation of extracellular matrix (ECM) components such as collagen and fibronectin adjacent to foci of inflammation or damage. The human microfibrillar-associated protein 4 (MFAP4), an important member of the superfamily of fibrinogen-related proteins, is considered to have an extremely important role in ECM transformation of fibrogenesis. This review summarizes the structure, characteristics, and physiological functions of MFAP4 and the importance of MFAP4 in various fibrotic diseases. Meanwhile, we elaborated the underlying actions and mechanisms of MFAP4 in the development of fibrosis, suggesting that a better understand of MFAP4 broadens novel perspective for early screening, diagnosis, prognostic risk assessment, and treatment of fibrotic diseases.

The role of periostin in cardiac fibrosis.

Cardiac fibrosis, which is the buildup of proteins in the connective tissues of the heart, can lead to end-stage extracellular matrix (ECM) remodeling and ultimately heart failure. Cardiac remodeling involves changes in gene expression in cardiac cells and ECM, which significantly leads to the morbidity and mortality in heart failure. However, despite extensive research, the elusive intricacies underlying cardiac fibrosis remain unidentified. Periostin, an extracellular matrix (ECM) protein of the fasciclin superfamily, acts as a scaffold for building complex architectures in the ECM, which improves intermolecular interactions and augments the mechanical properties of connective tissues. Recent research has shown that periostin not only contributes to normal ECM homeostasis in a healthy heart but also serves as a potent inducible regulator of cellular reorganization in cardiac fibrosis. Here, we reviewed the constitutive domain of periostin and its interaction with other ECM proteins. We have also discussed the critical pathophysiological functions of periostin in cardiac remodeling mechanisms, including two distinct yet potentially intertwined mechanisms. Furthermore, we will focus on the intrinsic complexities within periostin research, particularly surrounding the contentious issues observed in experimental findings.

Unsung versatility of elastin-like polypeptide inspired spheroid fabrication: A review.

Lately, 3D cell culture technique has gained a lot of appreciation as a research model. Augmented with technological advancements, the area of 3D cell culture is growing rapidly with a diverse array of scaffolds being tested. This is especially the case for spheroid cultures. The culture of cells as spheroids provides opportunities for unanticipated vision into biological phenomena with its application to drug discovery, metabolic profiling, stem cell research as well as tumor, and disease biology. Spheroid fabrication techniques are broadly categorised into matrix-dependent and matrix-independent techniques. While there is a profusion of spheroid fabrication substrates with substantial biological relevance, an economical, modular, and bio-compatible substrate for high throughput production of spheroids is lacking. In this review, we posit the prospects of elastin-like polypeptides (ELPs) as a broad-spectrum spheroid fabrication platform. Elastin-like polypeptides are nature inspired, size-tunable genetically engineered polymers with wide applicability in various arena of biological considerations, has been employed for spheroid culture with profound utility. The technology offers a cheap, high-throughput, reproducible alternative for spheroid culture with exquisite adaptability. Here, we will brief the applicability of 3D cultures as compared to 2D cultures with spheroids being the focal point of the review. Common approaches to spheroid fabrication are discussed with existential limitations. Finally, the versatility of elastin-like polypeptide inspired substrates for spheroid culture has been discussed.

Design and Development of Extracellular Matrix Protein-Based Microcapsules as Tools for Bacteria Investigation.

The extracellular matrix (ECM) plays an immense role in the homeostasis of tissues and organs, can function as a barrier for infectious agents, but is also exploited by pathogens during infection. Therefore, the development of well-defined 3D ECM models in the form of microcapsules to elucidate the interactions between ECM components and pathogens in confinement and study disease infectivity is important, albeit challenging. Current limitations are mainly attributed to the lack of biocompatible methods for the production of protein-based microcapsules. Herein, hollow ECM-based microcapsules from laminin-111 or laminin-111/collagen IV are generated to investigate the behavior of organisms within confined 3D extracellular matrices. Microcapsules are created using water-in-oil emulsion droplets stabilized by block copolymer surfactants as templates for the charge-mediated attraction of laminin or laminin-collagen proteins to the droplets' inner periphery, allowing for the formation of modular ECM-based microcapsules with tunable biophysical and biochemical properties and organism encapsulation. The release of E. coli-laden ECM-based protein microcapsules into a physiological environment revealed differences in the dynamic behavior of E. coli depending on the constitution of the surrounding ECM protein matrix. The developed ECM-based protein microcapsules have the potential to be implemented in several biomedical applications, including the design of in vitro infection models.

ECM proteins involved in cell migration and vessel formation compromise bovine cloned placentation.

Advances in Artificial Reproductive Technologies (ARTs) in bovine embryos to produce cloned pregnancies have been developed in the last years, however high pregnancy losses rates still present. Those rates are associated to placental morphology alterations that are majorly focused on extracellular matrix (ECM) alterations and consequently placentome hyperplasia, increased trophoblast cell migration and vascular defects. Herein, we aimed to search, at protein level, pathways altered by ART that can modify the placental development harmony. For this, we used 4-month-old control (n = 3), SDS-decellularized (n = 3) and cloned (n = 3) cotyledons for proteomic analysis. Samples were grouped by condition and were washed, lysed, urea-reduced, acetone-precipitated, DTT-educed, iodoacetamide-alkylated, trypsin digested, and C-18 column purified. At the end, 3 µg protein were loaded in Orbitrap Fusion Lumos spectrometer (ThermoScientific). Generated spectra were exported to MaxQuant software (v1.6.10.43) to produce the protein list of each sample, and the LFQ intensity were statistically analyzed by Inferno software (v.1.1.6970). After this, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8. From 2577 identified protein sequences by MaxQuant software, 165 (7.1%) were filtered by selected ontologies. We found 10 proteins (B2M, COL6A6, FERMT3, LGALS3BP, NIBAN2, PDLIM5, PON1, PRP9, RASIP1 and SPARC) upregulated in clone, when compared to control condition. The ten pathways that enriched more proteins were: focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, protein digestion and absorption, amoebiasis, pathways in cancer, small cell lung cancer, platelet activation, regulation of actin cytoskeleton, and proteoglycans in cancer. Functionally, detected proteins, signaling pathways and ontologies are orchestrated to permit the binucleated trophoblastic cells migration and blood vessels modelling. In conclusion, the cloned condition presents the same mechanisms as control one, however overexpression of some specific ECM proteins could be responsible to exacerbate those mechanisms and can explain all morphophysiological alterations presented in cloned pregnancies associated to high pregnancies losses rates in this condition.

ECM proteins involved in cell migration and vessel formation compromise bovine cloned placentation

Advances in Artificial Reproductive Technologies (ARTs) in bovine embryos to produce cloned pregnancies have been developed in the last years, however high pregnancy losses rates still present. Those rates are associated to placental morphology alterations that are majorly focused on extracellular matrix (ECM) alterations and consequently placentome hyperplasia, increased trophoblast cell migration and vascular defects. Herein, we aimed to search, at protein level, pathways altered by ART that can modify the placental development harmony. For this, we used 4-month-old control (n = 3), SDS-decellularized (n = 3) and cloned (n = 3) cotyledons for proteomic analysis. Samples were grouped by condition and were washed, lysed, urea-reduced, acetone-precipitated, DTT-educed, iodoacetamide-alkylated, trypsin digested, and C-18 column purified. At the end, 3 μg protein were loaded in Orbitrap Fusion Lumos spectrometer (ThermoScientific). Generated spectra were exported to MaxQuant software (v1.6.10.43) to produce the protein list of each sample, and the LFQ intensity were statistically analyzed by Inferno software (v.1.1.6970). After this, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8. From 2577 identified protein sequences by MaxQuant software, 165 (7.1%) were filtered by selected ontologies. We found 10 proteins (B2M, COL6A6, FERMT3, LGALS3BP, NIBAN2, PDLIM5, PON1, PRP9, RASIP1 and SPARC) upregulated in clone, when compared to control condition. The ten pathways that enriched more proteins were: focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, protein digestion and absorption, amoebiasis, pathways in cancer, small cell lung cancer, platelet activation, regulation of actin cytoskeleton, and proteoglycans in cancer. Functionally, detected proteins, signaling pathways and ontologies are orchestrated to permit the binucleated trophoblastic cells migration and blood vessels modelling. In conclusion, the cloned condition presents the same mechanisms as control one, however overexpression of some specific ECM proteins could be responsible to exacerbate those mechanisms and can explain all morphophysiological alterations presented in cloned pregnancies associated to high pregnancies losses rates in this condition.

The anti-ageing effects of a natural peptide discovered by artificial intelligence.

OBJECTIVE: As skin ages, impaired extracellular matrix (ECM) protein synthesis and increased action of degradative enzymes manifest as atrophy, wrinkling and laxity. There is mounting evidence for the functional role of exogenous peptides across many areas, including in offsetting the effects of cutaneous ageing. Here, using an artificial intelligence (AI) approach, we identified peptide RTE62G (pep_RTE62G), a naturally occurring, unmodified peptide with ECM stimulatory properties. The AI-predicted anti-ageing properties of pep_RTE62G were then validated through in vitro, ex vivo and proof of concept clinical testing. METHODS: A deep learning approach was applied to unlock pep_RTE62G from a plant source, Pisum sativum (pea). Cell culture assays of human dermal fibroblasts (HDFs) and keratinocytes (HaCaTs) were subsequently used to evaluate the in vitro effect of pep_RTE62G. Distinct activities such as cell proliferation and ECM protein production properties were determined by ELISA assays. Cell migration was assessed using a wound healing assay, while ECM protein synthesis and gene expression were analysed, respectively, by immunofluorescence microscopy and PCR. Immunohistochemistry of human skin explants was employed to further investigate the induction of ECM proteins by pep_RTE62G ex vivo. Finally, the clinical effect of pep_RTE626 was evaluated in a proof of concept 28-day pilot study. RESULTS: In vitro testing confirmed that pep_RTE62G is an effective multi-functional anti-ageing ingredient. In HaCaTs, pep_RTE62G treatment significantly increases both cellular proliferation and migration. Similarly, in HDFs, pep_RTE62G consistently induced the neosynthesis of ECM protein elastin and collagen, effects that are upheld in human skin explants. Lastly, in our proof of concept clinical study, application of pep_RTE626 over 28 days demonstrated anti-wrinkle and collagen stimulatory potential. CONCLUSION: pep_RTE62G represents a natural, unmodified peptide with AI-predicted and experimentally validated anti-ageing properties. Our results affirm the utility of AI in the discovery of novel, functional topical ingredients.

OBJECTIF: À mesure que la peau vieillit, une altération de la synthèse des protéines de la matrice extracellulaire (ECM) et une action accrue des enzymes dégradantes se manifestent par une atrophie, des rides et un laxisme. Il existe de plus en plus de preuves du rôle fonctionnel des peptides exogènes dans de nombreux domaines, y compris pour compenser les effets du vieillissement cutané. Ici, en utilisant une approche d'intelligence artificielle (AI), nous avons identifié le peptide RTE62G (pep_RTE62G), un peptide naturel non modifié avec des propriétés de stimulation ECM. Les propriétés anti-âge prédites par l'IA de pep_RTE62G ont ensuite été validées par des tests cliniques in vitro, ex vivo et de validation de principe. LES MÉTHODES: Une approche d'apprentissage en profondeur a été appliquée pour déverrouiller pep_RTE62G à partir d'une source végétale, Pisum sativum (pois). Des tests de culture cellulaire de fibroblastes dermiques humains (HDF) et de kératinocytes (HaCaTs) ont ensuite été utilisés pour évaluer l'effet in vitro de pep_RTE62G. Des activités distinctes telles que la prolifération cellulaire et les propriétés de production de protéines ECM ont été déterminées par des tests ELISA. La migration cellulaire a été évaluée à l'aide d'un test de cicatrisation des plaies, tandis que la synthèse des protéines ECM et l'expression des gènes ont été analysées, respectivement, par microscopie à immunofluorescence et PCR. L'immunohistochimie des explants de peau humaine a été utilisée pour approfondir l'induction des protéines ECM par pep_RTE62G ex vivo. Enfin, l'effet clinique de pep_RTE626 a été évalué dans une étude pilote de 28 jours de validation de principe. RÉSULTATS: Les tests in vitro ont confirmé que pep_RTE62G est un ingrédient anti-âge multifonctionnel efficace. Dans HaCaTs, le traitement pep_RTE62G augmente de manière significative à la fois la prolifération et la migration cellulaire. De même, dans les HDF, pep_RTE62G a induit de manière cohérente la néosynthèse de la protéine ECM élastine et collagène, effets qui sont maintenus dans les explants de peau humaine. Enfin, dans notre étude clinique de preuve de concept, l'application de pep_RTE626 sur 28 jours a démontré un potentiel stimulant anti-rides et collagène. CONCLUSION: pep_RTE62G représente un peptide naturel, non modifié avec des propriétés anti-âge prédites par l'IA et validées expérimentalement. Nos résultats confirment l'utilité de l'IA dans la découverte de nouveaux ingrédients topiques fonctionnels.

Production of a Recombinant Non-Hydroxylated Gelatin Mimetic in Pichia pastoris for Biomedical Applications.

Proteins derived from the natural extracellular matrix like collagen or gelatin are common in clinical research, where they are prized for their biocompatibility and bioactivity. Cells are able to adhere, grow and remodel scaffolds based on these materials. Usually, collagen and gelatin are sourced from animal material, risking pathogenic transmission and inconsistent batch-to-batch product quality. A recombinant production in yeast circumvents these disadvantages by ensuring production with a reproducible quality in animal-component-free media. A gelatin mimetic protein, based on the alpha chain of human collagen I, was cloned in Pichia pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. A producing clone was selected and cultivated at the 30 L scale. The protein was secreted into the cultivation medium and the final yield was 3.4 g·L-1. Purification of the target was performed directly from the cell-free medium by size exclusion chromatography. The gelatin mimetic protein was tested in cell culture for biocompatibility and for promoting cell adhesion. It supported cell growth and its performance was indistinguishable from animal-derived gelatin. The gelatin-mimetic protein represents a swift strategy to produce recombinant and human-based extracellular matrix proteins for various biomedical applications.

Tenascin C regulates multiple microglial functions involving TLR4 signaling and HDAC1.

Tenascin C (Tnc) is an extracellular matrix glycoprotein, expressed in the CNS during development, as well as in the setting of inflammation, fibrosis and cancer, which operates as an activator of Toll-like receptor 4 (TLR4). Although TLR4 is highly expressed in microglia, the effect of Tnc on microglia has not been elucidated to date. Herein, we demonstrate that Tnc regulates microglial phagocytic activity at an early postnatal age (P4), and that this process is partially dependent on microglial TLR4 expression. We further show that Tnc regulates proinflammatory cytokine/chemokine production, chemotaxis and phagocytosis in primary microglia in a TLR4-dependent fashion. Moreover, Tnc induces histone-deacetylase 1 (HDAC1) expression in microglia, such that HDAC1 inhibition by MS-275 decreases Tnc-induced microglial IL-6 and TNF-α production. Finally, Tnc-/- cortical microglia have reduced HDAC1 expression levels at P4. Taken together, these findings establish Tnc as a regulator of microglia function during early postnatal development.

Spatiotemporal Analyses of Cellular Tractions Describe Subcellular Effect of Substrate Stiffness and Coating.

Cells interplay with their environment through mechanical and chemical interactions. To characterize this interplay, endothelial cells were cultured on polyacrylamide hydrogels of varying stiffness, coated with either fibronectin or collagen. We developed a novel analysis technique, complementary to traction force microscopy, to characterize the spatiotemporal evolution of cellular tractions: We identified subpopulations of tractions, termed traction foci, and tracked their magnitude and lifetime. Each focus consists of tractions associated with a local single peak of maximal traction. Individual foci were spread over a larger area in cells cultured on collagen relative to those on fibronectin and exerted higher tractions on stiffer hydrogels. We found that the trends with which forces increased with increasing hydrogel stiffness were different for foci and whole-cell measurements. These differences were explained by the number of foci and their average strength. While on fibronectin multiple short-lived weak foci contributed up to 30% to the total traction on hydrogels with intermediate stiffness, short-lived foci in such a number were not observed on collagen despite the higher tractions. Our approach allows for the use of existing traction force microscopy data to gain insight at the subcellular scale without molecular probes or spatial constraining of cellular tractions.

Substrate stiffness affects neural network activity in an extracellular matrix proteins dependent manner.

Neuronal growth, differentiation, extension, branching and neural network activity are strongly influenced by the mechanical property of extracellular matrix (ECM). However, the mechanism by which substrate stiffness regulates a neural network activity, and the importance of ECM composition in conferring substrate stiffness sensing have not been explored. To address this question, the hippocampal neurons were seeded on the polydimethylsiloxane (PDMS) substrate with different stiffness, which were coated with fibronectin and laminin respectively. Our results show that voltage-gated Ca2+ channel currents are greater in neurons on the stiff substrate than on the soft substrate. In addition, the neurons exhibit a greater increase of Ca2+ currents on laminin-coated stiff substrate than on those coated with fibronectin, indicating that the composition of ECM can modulate responses to substrate stiffness of neurons. Paired patch-clamp recordings have shown that upregulation of neural effective synaptic connectivity is greater on the laminin-coated stiff substrate than on the fibronectin-coated ones. Consistently, laminin-coated stiff substrate enhances Ca2+ oscillations of neurons is greater that compared with the fibronectin-coated ones. Our study demonstrates that a direct role for substrate stiffness in regulating neuronal network activity and indicate that this modulation is dependent on a specific type of ECM protein, which should be taken into account for the design of biomaterials for neuronal tissue engineering.

Transforming growth factor ß2 (TGFß2) signaling plays a key role in glucocorticoid-induced ocular hypertension.

Elevation of intraocular pressure (IOP) is a serious adverse effect of glucocorticoid (GC) therapy. Increased extracellular matrix (ECM) accumulation and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) is associated with GC-induced IOP elevation. However, the molecular mechanisms by which GCs induce ECM accumulation and ER stress in the TM have not been determined. Here, we show that a potent GC, dexamethasone (Dex), activates transforming growth factor ß (TGFß) signaling, leading to GC-induced ECM accumulation, ER stress, and IOP elevation. Dex increased both the precursor and bioactive forms of TGFß2 in conditioned medium and activated TGFß-induced SMAD signaling in primary human TM cells. Dex also activated TGFß2 in the aqueous humor and TM of a mouse model of Dex-induced ocular hypertension. We further show that Smad3-/- mice are protected from Dex-induced ocular hypertension, ER stress, and ECM accumulation. Moreover, treating WT mice with a selective TGFß receptor kinase I inhibitor, LY364947, significantly decreased Dex-induced ocular hypertension. Of note, knockdown of the ER stress-induced activating transcription factor 4 (ATF4), or C/EBP homologous protein (CHOP), completely prevented Dex-induced TGFß2 activation and ECM accumulation in TM cells. These observations suggested that chronic ER stress promotes Dex-induced ocular hypertension via TGFß signaling. Our results indicate that TGFß2 signaling plays a central role in GC-induced ocular hypertension and provides therapeutic targets for GC-induced ocular hypertension.

Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction.

The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.

Effect of stem cell niche elasticity/ECM protein on the self-beating cardiomyocyte differentiation of induced pluripotent stem (iPS) cells at different stages.

Stem cell-based myocardial regeneration therapies have emerged as alternative strategies to heart transplantation for serious heart diseases, but autologous beating mature cardiomyocytes are not available. Here we investigated the effect of culture substrates on the cardiomyocyte differentiation of induced pluripotent stem cells (iPSs) in vitro by separately evaluating the following continuous three steps: (1) cardiac marker gene expression, (2) contractile gene expression and self-beating, and (3) beating duration. To this end, we used iPS cells to study the cardiac differentiation, and neonatal rat cardiomyocytes (NCMs) to study beating behavior. These cells were cultured on substrates with different natures, i.e., an elastic substrate (Es) with the modulus of 9, 20, or 180 kPa, and hard tissue culture polystyrene dishes (TCPS) coated with collagen type I (Col), gelatin (Gel), or fibronectin (FN). The results revealed that the effective niches in each step were very different. The cardiac marker gene (GATA4, Tbx5, MEF2C) expression of iPSs at the 1st step was very high on the TCPS coated with FN or Gel, whereas on the FN-coated Es (especially with the 9 kPa modulus), the undifferentiated marker gene (Nanog) expression of iPSs was maintained. The expression of the contractile genes α-MHC, TnC1, and TnT2 and the self-beating (the 2nd step) of the NCMs were high on FN-coated TCPS and Col-coated Es. The 3rd step (beating duration) of the NCMs was effective on the Es, and at 21 days both the iPSs and NCMs stopped beating on the TCPS but were still beating on the Es. Overall, cardiac differentiation 'preferred' ECM-rigid culture substrates, and beating-behavior 'preferred' Col-soft culture substrates. These results are important for understanding and designing cardiac differentiation niches for regenerative medicine, and they suggest that a single culture substrate is not suitable for preparing self-beating cardiomyocytes. STATEMENT OF SIGNIFICANCE: The transplantation of beating cardiomyocytes (BCMs) is expected to be made more effective for serious heart diseases. The identification of the appropriate engineering processes and suitable culture substrates for inducing stem cell differentiation into BCMs is thus indispensable. The differentiation can be divided into three major processes, the cardiac differentiation step, the beating-induction step and the beating-duration step. A protocol with the higher efficiency in all of the steps must be useful. In this study, we separately evaluated the effect of culture substrates at each three step. We clarified that the biological and the physical properties of the culture substrates required at these steps were different. We found useful criteria for effective cardiac cell niche systems design.

Quantitative Analyses of Dynamic Features of Fibroblasts on Different Protein-Coated Compliant Substrates.

Cell response to substrate rigidity, closely related to extracellular matrix protein composition, requires actomyosin-generated contractility. By introducing coefficients describing cell spreading and traction dynamics, and a revised high-resolution traction force microscopy, we analyzed the static and dynamic features of fibroblasts on fibronectin- or collagen- coated stiff or soft substrates. Large cell spreading area and branchlike morphology were more favorable on fibronectin than collagen. Cell spreading on fibronectin-coated substrates was more sensitive to rigidity compared with collagen. Low concentration fibronectin-coated substrate induced more dynamic lamellipodia movement than other conditions. Interestingly, the static average cell traction on high concentration fibronectin-coated stiff and soft substrates showed no difference. However, the lamellipodium traction dynamics was sensitive to rigidity on fibronectin. Particularly, lamellipodia on fibronectin-coated soft substrate performed much higher local traction dynamics compared with other groups. Together, dynamics of cell adhesion and traction are regulated by extracellular matrix protein composition, coupled with substrate rigidity.

Atomic Force Microscopy Protocol for Measurement of Membrane Plasticity and Extracellular Interactions in Single Neurons in Epilepsy.

Physiological interactions between extracellular matrix (ECM) proteins and membrane integrin receptors play a crucial role in neuroplasticity in the hippocampus, a key region involved in epilepsy. The atomic force microscopy (AFM) is a cutting-edge technique to study structural and functional measurements at nanometer resolution between the AFM probe and cell surface under liquid. AFM has been incrementally employed in living cells including the nervous system. AFM is a unique technique that directly measures functional information at a nanoscale resolution. In addition to its ability to acquire detailed 3D imaging, the AFM probe permits quantitative measurements on the structure and function of the intracellular components such as cytoskeleton, adhesion force and binding probability between membrane receptors and ligands coated in the AFM probe, as well as the cell stiffness. Here we describe an optimized AFM protocol and its application for analysis of membrane plasticity and mechanical dynamics of individual hippocampus neurons in mice with chronic epilepsy. The unbinding force and binding probability between ECM, fibronectin-coated AFM probe and membrane integrin were strikingly lower in dentate gyrus granule cells in epilepsy. Cell elasticity, which represents changes in cytoskeletal reorganization, was significantly increased in epilepsy. The fibronectin-integrin binding probability was prevented by anti-α5ß1 integrin. Thus, AFM is a unique nanotechnique that allows progressive functional changes in neuronal membrane plasticity and mechanotransduction in epilepsy and related brain disorders.

Differential deposition of fibronectin by asthmatic bronchial epithelial cells.

Altered ECM protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or nonasthmatic HBECs and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)-ß1-regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol 3 kinase, and activin-like kinase 5 (ALK5). Asthmatic HBECs deposited higher levels of Fn in the ECM than nonasthmatic cells under basal conditions, whereas cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-ß1 increased mRNA levels and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in nonasthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells after TGF-ß1 stimulation persisted compared with nonasthmatic cells. Inhibition of ALK5 completely prevented TGF-ß1-induced Fn deposition. Importantly, ECM Fn increased HBEC proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM, is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represents a novel, therapeutic target.

PLAP-1/Asporin Regulates TLR2- and TLR4-induced Inflammatory Responses.

Periodontal ligament-associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2- and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-κB activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, IκB kinase α degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2- and TLR4-induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.

Effectiveness of an acellular synthetic matrix in the treatment of hard-to-heal leg ulcers.

Hard-to-heal leg ulcers are a major cause of morbidity in the elderly population. Despite improvements in wound care, some wounds will not heal and they present a significant challenge for patients and health care providers. A multi-centre cohort study was conducted to evaluate the effectiveness and safety of a synthetic, extracellular matrix protein as an adjunct to standard care in the treatment of hard-to-heal venous or mixed leg ulcers. Primary effectiveness criteria were (i) reduction in wound size evaluated by percentage change in wound area and (ii) healing assessed by number of patients healed by end of the 12 week study. Pain reduction was assessed as a secondary effectiveness criteria using VAS. A total of 45 patients completed the study and no difference was observed between cohorts for treatment frequency. Healing was achieved in 35·6% and wound size decreased in 93·3% of patients. Median wound area percentage reduction was 70·8%. Over 50% of patients reported pain on first visit and 87·0% of these reported no pain at the end of the study. Median time to first reporting of no pain was 14 days after treatment initiation. The authors consider the extracellular synthetic matrix protein an effective and safe adjunct to standard care in the treatment of hard-to-heal leg ulcers.