Systemic Convergent Multitarget Interactions of Plant Polyphenols Revealed by Affinity-Based Protein Profiling of Bone Cells Using C-Glucosidic Vescal(ag)in-Bearing Chemoproteomic Probes.

The ellagitannins vescalagin and vescalin, known as actin-dependent inhibitors of osteoclastic bone resorption, were mounted onto chemical probes to explore their interactions with bone cell proteins by means of affinity-based chemoproteomics and bioinformatics. The chemical reactivity of the pyrogallol units of these polyphenols toward oxidation into electrophilic ortho-quinones was exploited using NaIO4 to promote the covalent capture of target proteins, notably those expressed at lower abundance and those interacting with polyphenols at low-to-moderate levels of affinity. Different assays revealed the multitarget nature of both ellagitannins, with 100-370 statistically significant proteins captured by their corresponding probes. A much higher number of proteins were captured from osteoclasts than from osteoblasts. Bioinformatic analyses unveiled a preference for the capture of proteins having phosphorylated ligands and GTPase regulators and enabled the identification of 33 potential target proteins with systemic relevance to osteoclast differentiation and activity, as well as to the regulation of actin dynamics.

Synthesis of Flavonol-Bearing Probes for Chemoproteomic and Bioinformatic Analyses of Asteraceae Petals in Search of Novel Flavonoid Enzymes.

This study aimed at searching for the enzymes that are responsible for the higher hydroxylation of flavonols serving as UV-honey guides for pollinating insects on the petals of Asteraceae flowers. To achieve this aim, an affinity-based chemical proteomic approach was developed by relying on the use of quercetin-bearing biotinylated probes, which were thus designed and synthesized to selectively and covalently capture relevant flavonoid enzymes. Proteomic and bioinformatic analyses of proteins captured from petal microsomes of two Asteraceae species (Rudbeckia hirta and Tagetes erecta) revealed the presence of two flavonol 6-hydroxylases and several additional not fully characterized proteins as candidates for the identification of novel flavonol 8-hydroxylases, as well as relevant flavonol methyl- and glycosyltransferases. Generally speaking, this substrate-based proteome profiling methodology constitutes a powerful tool for the search for unknown (flavonoid) enzymes in plant protein extracts.

Fibrillin-1 regulates endothelial sprouting during angiogenesis.

Fibrillin-1 is an extracellular matrix protein that assembles into microfibrils which provide critical functions in large blood vessels and other tissues. Mutations in the fibrillin-1 gene are associated with cardiovascular, ocular, and skeletal abnormalities in Marfan syndrome. Here, we reveal that fibrillin-1 is critical for angiogenesis which is compromised by a typical Marfan mutation. In the mouse retina vascularization model, fibrillin-1 is present in the extracellular matrix at the angiogenic front where it colocalizes with microfibril-associated glycoprotein-1, MAGP1. In Fbn1C1041G/+ mice, a model of Marfan syndrome, MAGP1 deposition is reduced, endothelial sprouting is decreased, and tip cell identity is impaired. Cell culture experiments confirmed that fibrillin-1 deficiency alters vascular endothelial growth factor-A/Notch and Smad signaling which regulate the acquisition of endothelial tip cell/stalk cell phenotypes, and we showed that modulation of MAGP1 expression impacts these pathways. Supplying the growing vasculature of Fbn1C1041G/+ mice with a recombinant C-terminal fragment of fibrillin-1 corrects all defects. Mass spectrometry analyses showed that the fibrillin-1 fragment alters the expression of various proteins including ADAMTS1, a tip cell metalloprotease and matrix-modifying enzyme. Our data establish that fibrillin-1 is a dynamic signaling platform in the regulation of cell specification and matrix remodeling at the angiogenic front and that mutant fibrillin-1-induced defects can be rescued pharmacologically using a C-terminal fragment of the protein. These findings, identify fibrillin-1, MAGP1, and ADAMTS1 in the regulation of endothelial sprouting, and contribute to our understanding of how angiogenesis is regulated. This knowledge may have critical implications for people with Marfan syndrome.

Angiogenesis Invasion Assay to Study Endothelial Cell Invasion and Sprouting Behavior.

Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a fundamental process in developmental biology but also a pathological event that initiates or aggravates many diseases. In this complex multistep process, endothelial cells are activated by angiogenic stimuli; undergo specialization in response to VEGF/Notch signaling; degrade the basement membrane of the parent vessel; sprout, migrate, and proliferate to form capillary tubes that branch; and ultimately anastomose with adjacent vessels. Here we describe an assay that mimics the invasion step in vitro. Human microvascular endothelial cells are confronted by a VEGF-enriched basement membrane material in a three-dimensional environment that promotes endothelial cell sprouting, tube formation, and anastomosis. After a few hours, endothelial cells have become tip cells, and vascular sprouts can be observed by phase contrast, fluorescence, or time-lapse microscopy. Sprouting endothelial cells express tip cell markers, display podosomes and filopodia, and exhibit cell dynamics similar to those of angiogenic endothelial cells in vivo. This model provides a system that can be manipulated genetically to study physiological or pathological angiogenesis and that can be used to screen compounds for pro-/anti-angiogenic properties. In this chapter, we describe the key steps in setting up this assay.

The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration.

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2.

Fibrillin-1 Regulates Arteriole Integrity in the Retina.

Fibrillin-1 is an extracellular matrix protein that assembles into microfibrils that provide critical functions in large blood vessels and other tissues. Mutations in the fibrillin-1 gene are associated with cardiovascular, ocular, and skeletal abnormalities in Marfan syndrome. Fibrillin-1 is a component of the wall of large arteries but has been poorly described in other vessels. We examined the microvasculature in the retina using wild type mice and two models of Marfan syndrome, Fbn1C1041G/+ and Fbn1mgR/mgR. In the mouse retina, fibrillin-1 was detected around arterioles, in close contact with the basement membrane, where it colocalized with MAGP1. Both a mutation in fibrillin-1 or fibrillin-1 underexpression characteristically altered the microvasculature. In Fbn1C1041G/+ and Fbn1mgR/mgR mice, arterioles were enlarged with reduced MAGP1 deposition and focal loss of smooth muscle cell coverage. Losartan, which prevents aortic enlargement in Fbn1C1041G/+ mice, prevented smooth muscle cell loss and vessel leakiness when administrated in a preventive mode. Moreover, losartan also partially rescued the defects in a curative mode. Thus, fibrillin-1/MAGP1 performs essential functions in arteriolar integrity and mutant fibrillin-1-induced defects can be prevented or partially rescued pharmacologically. These new findings could have implications for people with Marfan syndrome.

miR-155 regulates physiological angiogenesis but an miR-155-rich microenvironment disrupts the process by promoting unproductive endothelial sprouting.

Angiogenesis involves cell specification orchestrated by regulatory interactions between the vascular endothelial growth factor and Notch signaling pathways. However, the role of microRNAs in these regulations remains poorly explored. Here we show that a controlled level of miR-155 is essential for proper angiogenesis. In the mouse retina angiogenesis model, antimiR-155 altered neovascularization. In vitro assays established that endogenous miR-155 is involved in podosome formation, activation of the proteolytic machinery and cell migration but not in morphogenesis. The role of miR-155 was explored using miR-155 mimics. In vivo, exposing the developing vasculature to miR-155 promoted hypersprouting, thus phenocopying defects associated with Notch deficiency. Mechanistically, miR-155 overexpression weakened Notch signaling by reducing Smad1/5 expression, leading to the formation of tip cell-like cells which did not reach full invasive capacity and became unable to undergo morphogenesis. These results identify miR-155 as a novel regulator of physiological angiogenesis and as a novel actor of pathological angiogenesis.

Erratum: Rémy et al. Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties. Cells 2019, 8, 1485.

The authors wish to make the following change to their paper [...].

Real-Time Analysis of Polyphenol-Protein Interactions by Surface Plasmon Resonance Using Surface-Bound Polyphenols.

A selection of bioactive polyphenols of different structural classes, such as the ellagitannins vescalagin and vescalin, the flavanoids catechin, epicatechin, epigallocatechin gallate (EGCG), and procyanidin B2, and the stilbenoids resveratrol and piceatannol, were chemically modified to bear a biotin unit for enabling their immobilization on streptavidin-coated sensor chips. These sensor chips were used to evaluate in real time by surface plasmon resonance (SPR) the interactions of three different surface-bound polyphenolic ligands per sensor chip with various protein analytes, including human DNA topoisomerase IIα, flavonoid leucoanthocyanidin dioxygenase, B-cell lymphoma 2 apoptosis regulator protein, and bovine serum albumin. The types and levels of SPR responses unveiled major differences in the association, or lack thereof, and dissociation between a given protein analyte and different polyphenolic ligands. Thus, this multi-analysis SPR technique is a valuable methodology to rapidly screen and qualitatively compare various polyphenol-protein interactions.

Intersection of TKS5 and FGD1/CDC42 signaling cascades directs the formation of invadopodia.

Tumor cells exposed to a physiological matrix of type I collagen fibers form elongated collagenolytic invadopodia, which differ from dotty-like invadopodia forming on the gelatin substratum model. The related scaffold proteins, TKS5 and TKS4, are key components of the mechanism of invadopodia assembly. The molecular events through which TKS proteins direct collagenolytic invadopodia formation are poorly defined. Using coimmunoprecipitation experiments, identification of bound proteins by mass spectrometry, and in vitro pull-down experiments, we found an interaction between TKS5 and FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42, which is known for its role in the assembly of invadopodial actin core structure. A novel cell polarity network is uncovered comprising TKS5, FGD1, and CDC42, directing invadopodia formation and the polarization of MT1-MMP recycling compartments, required for invadopodia activity and invasion in a 3D collagen matrix. Additionally, our data unveil distinct signaling pathways involved in collagenolytic invadopodia formation downstream of TKS4 or TKS5 in breast cancer cells.

Regulation of podosome formation in aortic endothelial cells vessels by physiological extracellular cues.

Invadosomes are specialised actin-based dynamic microdomains of the plasma membrane. Their occurrence has been associated with cell adhesion, matrix degrading and mechanosensory functions that make them crucial regulators of cell migration and invasion. Monocytic, cancer cell and Src-transformed cell invadosomes have been extensively described. Less well defined are the structures which form in other cell types, i.e., non-haematopoietic and non-transformed cells, exposed to specific stimuli. We herein describe the specificities of podosomes induced in aortic endothelial cells stimulated with TGFß in vitro and in conditions that more closely resemble the in vivo situation. These podosomes display the typical architecture of monocytic podosomes. They organise into large rosette-shape superstructures where they exhibit collective dynamic behavior consisting in cycles of formation and regression. At the ultrastructural level, microfilament arrangements in individual podosomes were revealed. Oxygen levels and hemodynamic forces, which are key players in endothelial cell biology, both influence the process. In 3D environment, podosomes appear as globular structures along cellular extensions. A better characterization of endothelial podosomes has far-reaching implications in the understanding and, possibly, in the treatment of some vascular diseases.

BMP-SMAD1/5 Signaling Regulates Retinal Vascular Development.

Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of Smad1 and Smad5. We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.

Podosomes in endothelial cell--microenvironment interactions.

PURPOSE OF REVIEW: The discovery of podosomes in endothelial cells during the process of angiogenesis in vivo opens a new era in vascular biology. Podosomes are actin-based microdomains located at the plasma membrane that have been extensively described but in vitro and in other cells. This review focuses on podosomes in endothelial cells and aims to rise hypotheses about when and how these structures mediate cell--microenvironment interactions. RECENT FINDINGS: A wealth of new information regarding podosome organization and functioning has been collected in simple 2D models. Characterization of their modular architecture has unravelled their mechanics. However, context matters and podosome characteristics and functioning are shaped by the microenvironment. Although matrix degradation was seen as the typical function of podosomes, mechanosensing now appears equally prominent and involved in setting of the proteolytic machinery. Endothelial podosomes breach the basement membrane, and are thus, involved in vascular remodelling. SUMMARY: In endothelial cells, podosomes are involved in breaking up the basement membrane, giving the cells the opportunity to invade adjacent tissues and to engage in new cell--cell interactions. Such functions are particularly relevant to vascular biology and the exploration of podosomes in in vivo settings should bring clues to many unanswered questions.

Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties.

: Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O2] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.

Cell Migration in Microfluidic Devices: Invadosomes Formation in Confined Environments.

The last 20 years have seen the blooming of microfluidics technologies applied to biological sciences. Microfluidics provides effective tools for biological analysis, allowing the experimentalists to extend their playground to single cells and single molecules, with high throughput and resolution which were inconceivable few decades ago. In particular, microfluidic devices are profoundly changing the conventional way of studying the cell motility and cell migratory dynamics. In this chapter we will furnish a comprehensive view of the advancements made in the research domain of confinement-induced cell migration, thanks to the use of microfluidic devices. The chapter is subdivided in three parts. Each section will be addressing one of the fundamental questions that the microfluidic technology is contributing to unravel: (i) where cell migration takes place, (ii) why cells migrate and, (iii) how the cells migrate. The first introductory part is devoted to a thumbnail, and partially historical, description of microfluidics and its impact in biological sciences. Stress will be put on two aspects of the devices fabrication process, which are crucial for biological applications: materials used and coating methods. The second paragraph concerns the cell migration induced by environmental cues: chemical, leading to chemotaxis, mechanical, at the basis of mechanotaxis, and electrical, which induces electrotaxis. Each of them will be addressed separately, highlighting the fundamental role of microfluidics in providing the well-controlled experimental conditions where cell migration can be induced, investigated and ultimately understood. The third part of the chapter is entirely dedicated to how the cells move in confined environments. Invadosomes (the joint name for podosomes and invadopodia) are cell protrusion that contribute actively to cell migration or invasion. The formation of invadosomes under confinement is a research topic that only recently has caught the attention of the scientific community: microfluidic design is helping shaping the future direction of this emerging field of research.

Variations on the theme of podosomes: A matter of context.

Extensive in vitro studies have described podosomes as actin-based structures at the plasma membrane, connecting the cell with its extracellular matrix and endowed with multiple capabilities. Contractile actin-myosin cables assemble them into a network that constitutes a multifaceted cellular superstructure taking different forms - with common characteristics - but manifesting different properties depending on the context of study. Their morphology and their role in cell functioning and behavior are therefore now apprehended in in vivo or in vitro situations relevant to physiological processes. We focus here on three of them, namely: macrophage migration, antigen presentation by dendritic cells and endothelial cell sprouting during angiogenesis to highlight the characteristics of podosomes and their functioning shaped by the microenvironment.

Anti-osteoclastic effects of C-glucosidic ellagitannins mediated by actin perturbation.

Actin subunits assemble into actin filaments whose dynamics and three-dimensional architectures are further regulated by a variety of cellular factors to establish the functional actin cytoskeleton. The C-glucosidic ellagitannin vescalagin and its simpler analogue vescalin, affect both the dynamics and the ultrastructure of the actin cytoskeleton by directly binding to F-actin. Herein, we show that in vitro, the two compounds induce the formation of distinct F-actin networks characterized by different superstructures and dynamics. In living mature osteoclasts, highly specialized bone-degrading cells that constantly remodel their cytoskeleton, vescalagin and vescalin alter actin dynamics at podosomes and compromise the integrity of the podosome belt that forms the bone-degrading apparatus. Both compounds target the bone-resorbing activity at concentrations that preserve osteoclastic maturation and survival and with no detectable impact on the behaviour of bone-forming osteoblastic cells. This anti-osteoclastic activity of vescalagin and vescalin reveals the potential of targeting actin dynamics as a new therapeutic opportunity and, in this case, as a plausible approach for the local treatment of osteoporosis.

[Variations on the theme of podosomes, context matters]. / Variations sur le thème des podosomes, une affaire de contexte.

Podosomes are actin-based microdomains connecting the cell with its extracellular matrix. Contractile actin-myosin cables assemble them into a network that constitutes a versatile cellular superstructure. Discovered and extensively described in in vitro conditions, podosomes now appear as major actors of specific physiological processes. They share common characteristics but their morphology and their effect on cell functioning can only be apprehended in specific cellular contexts. We focus here on three cellular processes involving podosomes and discuss their properties in context.

Targeting Cx40 (Connexin40) Expression or Function Reduces Angiogenesis in the Developing Mouse Retina.

OBJECTIVE: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown. APPROACH AND RESULTS: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice. CONCLUSIONS: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis.