Publications

Simple Summary Neuroblastoma (NB), a prevalent childhood cancer, presents challenges in treatment due to its cellular diversity and the presence of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs, hindering effective therapies. We developed a stiffness-based in vitro platform simulating arterial and venous conditions to address this gap. Notably, adrenergic NB cells transdifferentiated into TECs where there was an arterial-like stiffness, while mesenchymal cells did not. This platform facilitated the identification of Globotriaosylceramide (GB3) as a novel TEC biomarker. Moreover, we harnessed Shiga toxin-functionalized nanoparticles for the specific targeting of GB3-positive cells, showing promise for future therapeutic strategies. Our study provides insights into NB heterogeneity, offers a predictive tool for assessing aggressiveness, and introduces potential targets for precision therapies.Abstract Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

JTD Keywords: Alternative vasculature, Angiogenesis, Cells, Differentiation, Gb3, Neuroblastoma, Origin, Tumor-derived endothelial cells

The problems associated with the drugs currently used to treat leishmaniasis, including resistance, toxicity, and the high cost of some formulations, call for the urgent identification of new therapeutic agents with novel modes of action. The aggregated protein dye YAT2150 has been found to be a potent antileishmanial compound, with a half-maximal inhibitory concentration (IC50) of approximately 0.5 mu M against promastigote and amastigote stages of Leishmania infantum. The encapsulation in liposomes of YAT2150 significantly improved its in vitro IC50 to 0.37 and 0.19 mu M in promastigotes and amastigotes, respectively, and increased the half-maximal cytotoxic concentration in human umbilical vein endothelial cells to >50 mu M. YAT2150 became strongly fluorescent when binding intracellular protein deposits in Leishmania cells. This fluorescence pattern aligns with the proposed mode of action of this drug in the malaria parasite Plasmodium falciparum, the inhibition of protein aggregation. In Leishmania major, YAT2150 rapidly reduced ATP levels, suggesting an alternative antileishmanial mechanism. To the best of our knowledge, this first-in-class compound is the only one described so far having significant activity against both Plasmodium and Leishmania, thus being a potential drug for the treatment of co-infections of both parasites.

JTD Keywords: Animal, Animals, Antileishmanial drugs, Antiprotozoal agent, Antiprotozoal agents, Axenic amastigotes, Colocalization, Differentiation, Discovery, Endothelial cells, Endothelium cell, Human, Humans, Identification, Leishmania, Leishmania infantum, Leishmaniasis, Parasite, Parasites, Protein aggregation, Yat2150, Yeast

A new generation of fully bioresorbable metallic Zn-based alloys could be used for stenting applications; however, the initial surface degradation delays stent re-endothelialization. Thus, this work proposes a dual strategy to control the corrosion and accelerate the endothelialization of ZnMg and ZnAg biodegradable alloys. First, a stable polycaprolactone (PCL) coating is obtained and followed by its functionalization with either linear RGD (Arg-Gly-Asp) or REDV (Arg-Glu-Asp-Val) peptides or a dual peptide-based platform combining both sequences (RGD-REDV). Scratching tests showed neither delamination nor detachment of the polymeric coating. Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) measurements confirmed the corrosion resistance after PCL coating by revealing lower current density and higher absolute impedance values. X-ray photoelectron spectroscopy (XPS) and fluorescent microscopy confirmed the correct peptide immobilization onto PCL coated Zn alloys. The functionalized samples exhibited enhanced human umbilical vein endothelial cells (HUVEC) adhesion. The higher number of adhered cells to the functionalized surfaces with the RGD-REDV platform demonstrates the synergistic effect of combining both RGD and REDV sequences. Higher corrosion resistance together with enhanced endothelialization indicates that the dual functionalization of Zn alloys with PCL and peptide-based RGD-REDV platform holds great potential to overcome the clinical limitations of current biodegradable metal stents.

JTD Keywords: Binary alloys, Biodegradable metals, Bioresorbable, Cardiovascular applications, Cell adhesive peptides, Corrosion, Corrosion resistance, Corrosion resistant alloys, Corrosion resistant coatings, Degradation, Dual peptide-based platform, Electrochemical corrosion, Electrochemical impedance spectroscopy, Endothelial cells, Endothelialization, Functionalization, Functionalizations, In-vitro, Magnesium alloys, Metallics, Mg alloy, Peptides, Polycaprolactone coating, Polymer-coatings, Rgd-functionalization, Silver alloys, Stents, Surface, X ray photoelectron spectroscopy, Zinc, Zinc alloys, Zn alloys, Zn-based alloys

Neuroblastoma (NB) is a highly vascularized pediatric tumor arising from undifferentiated neural crest cells early in life, exhibiting both traditional endothelial-cell-driven vasculature and an intriguing alternative vasculature. The alternative vasculature can arise from cancer cells undergoing transdifferentiation into tumor-derived endothelial cells (TEC), a trait associated with drug resistance and tumor relapse. The lack of effective treatments targeting NB vasculature primarily arises from the challenge of establishing predictive in vitro models that faithfully replicate the alternative vasculature phenomenon. In this study, we aim to recreate the intricate vascular system of NB in an in vitro context, encompassing both types of vascularization, by developing a novel neuroblastoma-on-a-chip model. We designed a collagen I/fibrin-based hydrogel closely mirroring NB's physiological composition and tumor stiffness. This biomaterial created a supportive environment for the viability of NB and endothelial cells. Implementing a physiological shear stress value, aligned with the observed range in arteries and capillaries, within the microfluidic chip facilitated the successful development of vessel-like structures and triggered transdifferentiation of NB cells into TECs. The vascularized neuroblastoma-on-a-chip model introduced here presents a promising and complementary strategy to animal-based research with a significant capacity for delving into NB tumor biology and vascular targeting therapy.

JTD Keywords: 3d tumor model, Angiogenesis, Endothelial-cells, Microfluidic device, Neuroblastoma, Origi, Transdifferentiation, Tumor, Tumor-derived endothelial cells, Tumor-on-a-chip, Vasculature

Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating 'artificial' targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating 'artificial' targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.© 2023. The Author(s).

JTD Keywords: brain endothelium, endocytic rates, ligand identification, nanoparticles, phage-display, Barrier, Brain, Brain endothelium, Brain targeting, Endocytic rates, Endothelial cells, Endothelium, Ligand identification, Peptides, Phage-display

The functionalization of 3D-printed poly-l-lactic acid (PLLA) and poly(l-lactic-co-ε-caprolactone) (PLCL) bioresorbable stents has been successfully achieved with linear RGDS and YIGSR peptides, as well as a dual platform containing both motifs within a single biomolecule.

JTD Keywords: adsorbed fibrinogen, chemistry, endothelialization, immobilization, platelets, plla, selectivity, surface, titanium, Absorbable implants, Cell adhesion, Endothelial cells, In-vitro hemocompatibility, Peptides, Polymers, Printing, three-dimensional, Stents, Tyrosyl-isoleucyl-glycyl-seryl-arginine

Sprouting angiogenesis is a core biological process critical to vascular development. Its accurate simulation, relevant to multiple facets of human health, is of broad, interdisciplinary appeal. This study presents an in-silico model replicating a microfluidic assay where endothelial cells sprout into a biomimetic extracellular matrix, specifically, a large-pore, low-concentration fibrin-based porous hydrogel, influenced by chemotactic factors. We introduce a novel approach by incorporating the extracellular matrix and chemotactic factor effects into a unified term using a single parameter, primarily focusing on modelling sprouting dynamics and morphology. This continuous model naturally describes chemotactic-induced sprouting with no need for additional rules. In addition, we extended our base model to account for matrix sensing and degradation, crucial aspects of angiogenesis. We validate our model via a hybrid in-silico experimental method, comparing the model predictions with experimental results derived from the microfluidic setup. Our results underscore the intricate relationship between the extracellular matrix structure and angiogenic sprouting, proposing a promising method for predicting the influence of the extracellular matrix on angiogenesis.Copyright © 2023 Ferre-Torres, Noguera-Monteagudo, Lopez-Canosa, Romero-Arias, Barrio, Castaño and Hernandez-Machado.

JTD Keywords: angiogenesis, biomimmetic, chemotaxis, endothelial cells, filopodia, growth, in silico model, mathematical models, mechanisms, metalloproteinase, migration, morphogenesis, phase field, pore-size, simulation, Angiogenesis, Biomimmetic, Chemotaxis, Endothelial cells, Extracellular matrix, In silico model, Mathematical models, Phase field, Tip cells

JTD Keywords: biocompatibility, bioresorbable stents, degradation, mechanical-properties, poly(epsilon-caprolactone), poly-l-lactic acid, polylactide, radiopacity, thermogel, x-ray imaging, Barium sulfate, Biocompatibility, Bioresorbable, Bioresorbable scaffolds, Bioresorbable stent, Bioresorbable stents, Blood vessels, Computerized tomography, Controlled drug delivery, Coronary heart disease, Direct-writing, Endothelial cells, Fabrication strategies, Injection molding, Lactic acid, Poly-l-lactic acid, Poly-l-lactic acids, Radiopacity, Scaffolds (biology), Solvent cast, Solvent-cast direct-writing, Solvents, Stents, Struts, Sulfur compounds, Targeted drug delivery, X-ray imaging

© 2020 Elsevier B.V. In the current work, our purpose was based on the assessment of bioactive chitosan (CS)/Poly(ethylene glycol) diacrylate (PEGDA) based scaffolds ability to stimulate in vitro angiogenesis process. The bioactivation of the scaffolds was accomplished by using organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) cues. In particular, the properties of the materials in terms of biological response promotion on human umbilical vein endothelial cells (HUVECs) were studied by using in vitro angiogenesis tests based on cell growth and proliferation. Furthermore, our interest was to examine the scaffolds capability to modulate two important steps involved in angiogenesis process: migration and tube formation of cells. Our data underlined that bioactive signals on CS/PEGDA scaffolds surface induce a desirable effect on angiogenic response concerning angiogenic marker expression (CD-31) and endothelial tissue formation (tube formation). Taken together, the results emphasized the concept that bioactive CS/PEGDA scaffolds may be novel implants for stimulating neovascularization of tissue-engineered constructs in regenerative medicine field.

JTD Keywords: angiogenesis, bmp-2 peptide, chitosan/pegda based scaffolds, human umbilical vein endothelial cells huvecs, Angiogenesis, Bmp-2 peptide, Chitosan/pegda based scaffolds, Human umbilical vein endothelial cells huvecs, Osteogenesis

The main drawbacks of cardiovascular bare-metal stents (BMS) are in-stent restenosis and stent thrombosis as a result of an incomplete endothelialization after stent implantation. Nano- and microscale modification of implant surfaces is a strategy to recover the functionality of the artery by stimulating and guiding molecular and biological processes at the implant/tissue interface. In this study, cobalt-chromium (CoCr) alloy surfaces are modified via direct laser interference patterning (DLIP) in order to create linear patterning onto CoCr surfaces with different periodicities (≈3, 10, 20, and 32 μm) and depths (≈20 and 800 nm). Changes in surface topography, chemistry, and wettability are thoroughly characterized before and after modification. Human umbilical vein endothelial cells' adhesion and spreading are similar for all patterned and plain CoCr surfaces. Moreover, high-depth series induce cell elongation, alignment, and migration along the patterned lines. Platelet adhesion and aggregation decrease in all patterned surfaces compared to CoCr control, which is associated with changes in wettability and oxide layer characteristics. Cellular studies provide evidence of the potential of DLIP topographies to foster endothelialization without enhancement of platelet adhesion, which will be of high importance when designing new BMS in the future.

JTD Keywords: CoCr, Direct laser interference patterning, Endothelial cells, Linear surface pattern, Platelets

Here we report on the development of a new type of hybrid fibrinogen-polylactic acid (FBG-PLA) nanofibres (NFs) with improved stiffness, combining the good mechanical properties of PLA with the excellent cell recognition properties of native FBG. We were particularly interested in the dorsal and ventral cell response to the nanofibres' organization (random or aligned), using human umbilical endothelial cells (HUVECs) as a model system. Upon ventral contact with random NFs, the cells developed a stellate-like morphology with multiple projections. The well-developed focal adhesion complexes suggested a successful cellular interaction. However, time-lapse analysis shows significantly lowered cell movements, resulting in the cells traversing a relatively short distance in multiple directions. Conversely, an elongated cell shape and significantly increased cell mobility were observed in aligned NFs. To follow the dorsal cell response, artificial wounds were created on confluent cell layers previously grown on glass slides and covered with either random or aligned NFs. Time-lapse analysis showed significantly faster wound coverage (within 12 h) of HUVECs on aligned samples vs. almost absent directional migration on random ones. However, nitric oxide (NO) release shows that endothelial cells possess lowered functionality on aligned NFs compared to random ones, where significantly higher NO production was found. Collectively, our studies show that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization.

JTD Keywords: Electrospun nanofibers, Endothelial cells, Fibrinogen, Guided cellular behavior, Polylactic acid, Vascular tissue engineering

Apart from the paradigm that cell-biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)-a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self-assembled monolayers (SAMs), a positively charged - NH(2), and negatively charged -COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network-like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC-labeled Col IV was quantified and showed about twice more protein on NH(2) substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both alpha(1) and alpha(2) integrins on positively charged NH(2) substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH(2) and Col IV functionalization may support endothelization of cardiovascular implants.

JTD Keywords: Collagen type IV, SAMs, AFM, Surface-induced protein assembly, Endothelial cells, Vascular grafts

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

JTD Keywords: Contraction, Human umbilical vein endothelial cells, Permeability, Traction force, Cell-cell contact, Cell-substrate contact, Substrate stiffness, Rho kinase, Vascular endothelial cadherin, Thrombin

A technique for producing micrometer-scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three-dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one-micrometer-tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).

JTD Keywords: Biocompatible Materials/ chemistry, Cell Adhesion, Cell Culture Techniques/ methods, Cell Proliferation, Cells, Cultured, Chitosan/ chemistry, Crystallization/methods, Endothelial Cells/ cytology/ physiology, Humans, Materials Testing, Nanostructures/ chemistry/ ultrastructure, Nanotechnology/methods, Particle Size, Surface Properties, Tissue Engineering/methods

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.

JTD Keywords: Cell Line, Cell Nucleus/ physiology, Cell Proliferation, Cell Size, Computer Simulation, Endothelial Cells/ cytology/ physiology, G1 Phase/ physiology, Humans, Mechanotransduction, Cellular/ physiology, Models, Biological, Statistics as Topic