Shape Transformations and Self-Assembly of Hairy Particles under Confinement

Molecular dynamics simulations are used to investigate the behavior of polymer-tethered nanoparticles between two inert or attractive walls. The confinement in pores creates new possibilities for controlling the shape transformation of individual hairy particles and their self-organization. We introduce a minimalistic model of the system; only chain-wall interactions are assumed to be attractive, while the others are softly repulsive. We show how the shape of isolated particles can be controlled by changing the wall separation and the strength of the interaction with the surfaces. For attractive walls, we found two types of structures, “bridges” and “mounds”. The first structures are similar to flanged spools in which the chains are connected with both walls and form bridges between them. We observed various bridges, symmetrical and asymmetrical spools, hourglasses, and pillars. The bridge-like structures can be “nano-oscillators” in which the cores jump from one wall to the other. We also study the self-assembly of a dense fluid of hairy particles in slit-like pores and analyze how the system morphology depends on interactions with the surfaces and the wall separation. The hairy particles form layers parallel to the walls. Different ordered structures, resembling two-dimensional crystalline lattices, are reported. We demonstrate that hairy particles are a versatile soft component forming a variety of structures in the slits.     

The Cellular Choreography of Osteoblast Angiotropism in Bone Development and Homeostasis

Interaction between endothelial cells and osteoblasts is essential for bone development and homeostasis. This process is mediated in large part by osteoblast angiotropism, the migration of osteoblasts alongside blood vessels, which is crucial for the homing of osteoblasts to sites of bone formation during embryogenesis and in mature bones during remodeling and repair. Specialized bone endothelial cells that form “type H” capillaries have emerged as key interaction partners of osteoblasts, regulating osteoblast differentiation and maturation and ensuring their migration towards newly forming trabecular bone areas. Recent revolutions in high-resolution imaging methodologies for bone as well as single cell and RNA sequencing technologies have enabled the identification of some of the signaling pathways and molecular interactions that underpin this regulatory relationship. Similarly, the intercellular cross talk between endothelial cells and entombed osteocytes that is essential for bone formation, repair, and maintenance are beginning to be uncovered. This is a relatively new area of research that has, until recently, been hampered by a lack of appropriate analysis tools. Now that these tools are available, greater understanding of the molecular relationships between these key cell types is expected to facilitate identification of new drug targets for diseases of bone formation and remodeling.     

Protein Profiling of WERI-RB1 and Etoposide-Resistant WERI-ETOR Reveals New Insights into Topoisomerase Inhibitor Resistance in Retinoblastoma

Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22 higher in WERI-ETOR). Pathway analysis revealed the “retinoid metabolism and transport” pathway as an enriched metabolic pathway in WERI-ETOR cells, while the “sphingolipid de novo biosynthesis” pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of “sphingolipid de novo biosynthesis” in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB.     

Endothelial Dysfunction: An Intermediate Clinical Feature between Urolithiasis and Cardiovascular Diseases

An epidemiological relationship between urolithiasis and cardiovascular diseases has extensively been reported. Endothelial dysfunction is an early pathogenic event in cardiovascular diseases and has been associated with oxidative stress and low chronic inflammation in hypertension, coronary heart disease, stroke or the vascular complications of diabetes and obesity. The aim of this study is to summarize the current knowledge about the pathogenic mechanisms of urolithiasis in relation to the development of endothelial dysfunction and cardiovascular morbidities. Methods: A non-systematic review has been performed mixing the terms “urolithiasis”, “kidney stone” or “nephrolithiasis” with “cardiovascular disease”, “myocardial infarction”, “stroke”, or “endothelial dysfunction”. Results: Patients with nephrolithiasis develop a higher incidence of cardiovascular disease with a relative risk estimated between 1.20 and 1.24 and also develop a higher vascular disease risk scores. Analyses of subgroups have rendered inconclusive results regarding gender or age. Endothelial dysfunction has also been strongly associated with urolithiasis in clinical studies, although no systemic serum markers of endothelial dysfunction, inflammation or oxidative stress could be clearly related. Analysis of urine composition of lithiasic patients also detected a higher expression of proteins related to cardiovascular disease. Experimental models of hyperoxaluria have also found elevation of serum endothelial dysfunction markers. Conclusions: Endothelial dysfunction has been strongly associated with urolithiasis and based on the experimental evidence, should be considered as an intermediate and changeable feature between urolithiasis and cardiovascular diseases. Oxidative stress, a key pathogenic factor in the development of endothelial dysfunction has been also pointed out as an important factor of lithogenesis. Special attention must be paid to cardiovascular morbidities associated with urolithiasis in order to take advantage of pleiotropic effects of statins, angiotensin receptor blockers and allopurinol.     

Astroglia in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is an umbrella term encompassing several neurodevelopmental disorders such as Asperger syndrome or autism. It is characterised by the occurrence of distinct deficits in social behaviour and communication and repetitive patterns of behaviour. The symptoms may be of different intensity and may vary in types. Risk factors for ASD include disturbed brain homeostasis, genetic predispositions, or inflammation during the prenatal period caused by viruses or bacteria. The number of diagnosed cases is growing, but the main cause and mechanism leading to ASD is still uncertain. Recent findings from animal models and human cases highlight the contribution of glia to the ASD pathophysiology. It is known that glia cells are not only “gluing” neurons together but are key players participating in different processes crucial for proper brain functioning, including neurogenesis, synaptogenesis, inflammation, myelination, proper glutamate processing and many others. Despite the prerequisites for the involvement of glia in the processes related to the onset of autism, there are far too little data regarding the engagement of these cells in the development of ASD.     

Involvement of Ceramide Signalling in Radiation-Induced Tumour Vascular Effects and Vascular-Targeted Therapy

Sphingolipids are well-recognized critical components in several biological processes. Ceramides constitute a class of sphingolipid metabolites that are involved in important signal transduction pathways that play key roles in determining the fate of cells to survive or die. Ceramide accumulated in cells causes apoptosis; however, ceramide metabolized to sphingosine promotes cell survival and angiogenesis. Studies suggest that vascular-targeted therapies increase endothelial cell ceramide resulting in apoptosis that leads to tumour cure. Specifically, ultrasound-stimulated microbubbles (USMB) used as vascular disrupting agents can perturb endothelial cells, eliciting acid sphingomyelinase (ASMase) activation accompanied by ceramide release. This phenomenon results in endothelial cell death and vascular collapse and is synergistic with other antitumour treatments such as radiation. In contrast, blocking the generation of ceramide using multiple approaches, including the conversion of ceramide to sphingosine-1-phosphate (S1P), abrogates this process. The ceramide-based cell survival “rheostat” between these opposing signalling metabolites is essential in the mechanotransductive vascular targeting following USMB treatment. In this review, we aim to summarize the past and latest findings on ceramide-based vascular-targeted strategies, including novel mechanotransductive methodologies.     

The Role of Beta Cell Recovery in Type 2 Diabetes Remission

Type 2 diabetes (T2D) has been considered a relentlessly worsening disease, due to the progressive deterioration of the pancreatic beta cell functional mass. Recent evidence indicates, however, that remission of T2D may occur in variable proportions of patients after specific treatments that are associated with recovery of beta cell function. Here we review the available information on the recovery of beta cells in (a) non-diabetic individuals previously exposed to metabolic stress; (b) T2D patients following low-calorie diets, pharmacological therapies or bariatric surgery; (c) human islets isolated from non-diabetic organ donors that recover from “lipo-glucotoxic” conditions; and (d) human islets isolated from T2D organ donors and exposed to specific treatments. The improvement of insulin secretion reported by these studies and the associated molecular traits unveil the possibility to promote T2D remission by directly targeting pancreatic beta cells.     

In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

Papaverine (PPV) is a benzylisoquinoline alkaloid isolated from Papaver somniferum that exerts antiproliferative activity. However, several questions remain regarding the biochemical pathways affected by PPV in tumourigenic cells. In this study, the influence of PPV on cell migration (light microscopy), expression of vascular endothelial growth factor (VEGF) B, VEGF R1, VEGF R2, and phosphorylated focal adhesion kinase (pFAK) were investigated using spectrophotometry in MDA-MB-231-, A549- and DU145 cell lines. The migration assay revealed that, after 48 h, PPV (100 µM) reduced cell migration to 81%, 91%, and 71% in MDA-MB-231-, A549-, and DU145 cells, respectively. VEGF B expression was reduced to 0.79-, 0.71-, and 0.73-fold after 48 h of exposure to PPV in MDA-MB-231-, A549- and DU145 cells, while PPV exposure of 48 h increased VEGF R1 expression in MDA-MB-231- and DU145 cells to 1.38 and 1.46. A fold decrease in VEGF R1 expression was observed in A549 cells to 0.90 after exposure to 150 µM. No statistically significant effects were observed on VEGF R2- and FAK expression after exposure to PPV. This study contributes to the understanding of the effects of a phytomedicinal alkaloid compound in cancer cells and may provide novel approaches to the application of non-addictive alkaloids.     

Context Dependent Sulf1/Sulf2 Functional Divergence in Endothelial Cell Activity

Signalling activities are tightly regulated to control cellular responses. Heparan sulfate proteoglycans (HSPGs) at the cell membrane and extracellular matrix regulate ligand availability and interaction with a range of key receptors. SULF1 and SULF2 enzymes modify HSPG sulfation by removing 6-O sulfates to regulate cell signalling but are considered functionally identical. Our in vitro mRNA and protein analyses of two diverse human endothelial cell lines, however, highlight their markedly distinct regulatory roles of maintaining specific HSPG sulfation patterns through feedback regulation of HS 6-O transferase (HS6ST) activities and highly divergent roles in vascular endothelial growth factor (VEGF) and Transforming growth factor β (TGFβ) cell signalling activities. Unlike Sulf2, Sulf1 over-expression in dermal microvascular HMec1 cells promotes TGFβ and VEGF cell signalling by simultaneously upregulating HS6ST1 activity. In contrast, Sulf1 over-expression in venous ea926 cells has the opposite effect as it attenuates both TGFβ and VEGF signalling while Sulf2 over-expression maintains the control phenotype. Exposure of these cells to VEGF-A, TGFβ1, and their inhibitors further highlights their endothelial cell type-specific responses and integral growth factor interactions to regulate cell signalling and selective feedback regulation of HSPG sulfation that additionally exploits alternative Sulf2 RNA-splicing to regulate net VEGF-A and TGFβ cell signalling activities.     

Circulating Exosomal miR-221 from Maternal Obesity Inhibits Angiogenesis via Targeting Angptl2

Maternal obesity disrupts both placental angiogenesis and fetus development. However, the links between adipocytes and endothelial cells in maternal obesity are not fully understood. The aim of this study was to characterize exosome-enriched miRNA from obese sow’s adipose tissue and evaluate the effect on angiogenesis of endothelial cells. Plasma exosomes were isolated and analyzed by nanoparticle tracking analysis (NTA), electron morphological analysis, and protein marker expression. The number of exosomes was increased as the gestation of the sows progressed. In addition, we found that exosomes derived from obese sows inhibited endothelial cell migration and angiogenesis. miRNA detection showed that miR-221, one of the miRNAs, was significantly enriched in exosomes from obese sows. Further study demonstrated that exosomal miR-221 inhibited the proliferation and angiogenesis of endothelial cells through repressing the expression of Angptl2 by targeting its 3′ untranslated region. In summary, miR-221 was a key component of the adipocyte-secreted exosomal vesicles that mediate angiogenesis. Our study may be a novel mechanism showing the secretion of “harmful” exosomes from obesity adipose tissues causes placental dysplasia during gestation.     

Investigation of Wall Shear Stress in Cardiovascular Research and in Clinical Practice—From Bench to Bedside

In the 1900s, researchers established animal models experimentally to induce atherosclerosis by feeding them with a cholesterol-rich diet. It is now accepted that high circulating cholesterol is one of the main causes of atherosclerosis; however, plaque localization cannot be explained solely by hyperlipidemia. A tremendous amount of studies has demonstrated that hemodynamic forces modify endothelial athero-susceptibility phenotypes. Endothelial cells possess mechanosensors on the apical surface to detect a blood stream-induced force on the vessel wall, known as “wall shear stress (WSS)”, and induce cellular and molecular responses. Investigations to elucidate the mechanisms of this process are on-going: on the one hand, hemodynamics in complex vessel systems have been described in detail, owing to the recent progress in imaging and computational techniques. On the other hand, investigations using unique in vitro chamber systems with various flow applications have enhanced the understanding of WSS-induced changes in endothelial cell function and the involvement of the glycocalyx, the apical surface layer of endothelial cells, in this process. In the clinical setting, attempts have been made to measure WSS and/or glycocalyx degradation non-invasively, for the purpose of their diagnostic utilization. An increasing body of evidence shows that WSS, as well as serum glycocalyx components, can serve as a predicting factor for atherosclerosis development and, most importantly, for the rupture of plaques in patients with high risk of coronary heart disease.     

Laser Direct Writing via Two-Photon Polymerization of 3D Hierarchical Structures with Cells-Antiadhesive Properties

We report the design and fabrication by laser direct writing via two photons polymerization of innovative hierarchical structures with cell-repellency capability. The structures were designed in the shape of “mushrooms”, consisting of an underside (mushroom’s leg) acting as a support structure and a top side (mushroom’s hat) decorated with micro- and nanostructures. A ripple-like pattern was created on top of the mushrooms, over length scales ranging from several µm (microstructured mushroom-like pillars, MMP) to tens of nm (nanostructured mushroom-like pillars, NMP). The MMP and NMP structures were hydrophobic, with contact angles of (127 ± 2)° and (128 ± 4)°, respectively, whereas flat polymer surfaces were hydrophilic, with a contact angle of (43 ± 1)°. The cell attachment on NMP structures was reduced by 55% as compared to the controls, whereas for the MMP, a reduction of only 21% was observed. Moreover, the MMP structures preserved the native spindle-like with phyllopodia cellular shape, whereas the cells from NMP structures showed a round shape and absence of phyllopodia. Overall, the NMP structures were more effective in impeding the cellular attachment and affected the cell shape to a greater extent than the MMP structures. The influence of the wettability on cell adhesion and shape was less important, the cellular behavior being mainly governed by structures’ topography.     

Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

Resistin is a novel hormone that is secreted by human adipocytes and mononuclear cells and is associated with obesity, insulin resistance and inflammation. Recently, resistin has been postulated to play a role in angiogenesis. Here, we investigated the hypothesis that resistin regulates ovary carcinoma production of vascular endothelial growth factor (VEGF) and the angiogenic processes. We found that in human ovarian epithelial carcinoma cells (HO-8910), resistin (10–150 ng/mL) enhanced both VEGF protein and mRNA expression in a time- and concentration-dependent manner, as well as promoter activity. Furthermore, resistin enhanced DNA-binding activity of Sp1 with VEGF promoter in a PI3K/Akt-dependent manner. PI3K/Akt activated by resistin led to increasing interaction with Sp1, triggering a progressive phosphorylation of Sp1 on Thr453 and Thr739, resulting in the upregulation of VEGF expression. In an in vitro angiogenesis system for endothelial cells (EA.hy926) co-cultured with HO-8910 cells, we observed that the addition of resistin stimulated endothelial cell tube formation, which could be abolished by VEGF neutralizing antibody. Our findings suggest that the PI3K/Akt-Sp1 pathway is involved in resistin-induced VEGF expression in HO-8910 cells and indicates that antiangiogenesis therapy may be beneficial treatment against ovarian epithelial carcinoma, especially in obese patients.     

Laser Direct Writing of Dual-Scale 3D Structures for Cell Repelling at High Cellular Density

The fabrication of complex, reproducible, and accurate micro-and nanostructured interfaces that impede the interaction between material’s surface and different cell types represents an important objective in the development of medical devices. This can be achieved by topographical means such as dual-scale structures, mainly represented by microstructures with surface nanopatterning. Fabrication via laser irradiation of materials seems promising. However, laser-assisted fabrication of dual-scale structures, i.e., ripples relies on stochastic processes deriving from laser–matter interaction, limiting the control over the structures’ topography. In this paper, we report on laser fabrication of cell-repellent dual-scale 3D structures with fully reproducible and high spatial accuracy topographies. Structures were designed as micrometric “mushrooms” decorated with fingerprint-like nanometric features with heights and periodicities close to those of the calamistrum, i.e., 200–300 nm. They were fabricated by Laser Direct Writing via Two-Photon Polymerization of IP-Dip photoresist. Design and laser writing parameters were optimized for conferring cell-repellent properties to the structures, even for high cellular densities in the culture medium. The structures were most efficient in repelling the cells when the fingerprint-like features had periodicities and heights of ≅200 nm, fairly close to the repellent surfaces of the calamistrum. Laser power was the most important parameter for the optimization protocol.     

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs’ barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.     

Critical Role of LSEC in Post-Hepatectomy Liver Regeneration and Failure

Liver sinusoids are lined by liver sinusoidal endothelial cells (LSEC), which represent approximately 15 to 20% of the liver cells, but only 3% of the total liver volume. LSEC have unique functions, such as fluid filtration, blood vessel tone modulation, blood clotting, inflammatory cell recruitment, and metabolite and hormone trafficking. Different subtypes of liver endothelial cells are also known to control liver zonation and hepatocyte function. Here, we have reviewed the origin of LSEC, the different subtypes identified in the liver, as well as their renewal during homeostasis. The liver has the exceptional ability to regenerate from small remnants. The past decades have seen increasing awareness in the role of non-parenchymal cells in liver regeneration despite not being the most represented population. While a lot of knowledge has emerged, clarification is needed regarding the role of LSEC in sensing shear stress and on their participation in the inductive phase of regeneration by priming the hepatocytes and delivering mitogenic factors. It is also unclear if bone marrow-derived LSEC participate in the proliferative phase of liver regeneration. Similarly, data are scarce as to LSEC having a role in the termination phase of the regeneration process. Here, we review what is known about the interaction between LSEC and other liver cells during the different phases of liver regeneration. We next explain extended hepatectomy and small liver transplantation, which lead to “small for size syndrome” (SFSS), a lethal liver failure. SFSS is linked to endothelial denudation, necrosis, and lobular disturbance. Using the knowledge learned from partial hepatectomy studies on LSEC, we expose several techniques that are, or could be, used to avoid the “small for size syndrome” after extended hepatectomy or small liver transplantation.     

Dynamic “Molecular Portraits” of Biomembranes Drawn by Their Lateral Nanoscale Inhomogeneities

To date, it has been reliably shown that the lipid bilayer/water interface can be thoroughly characterized by a sophisticated so-called “dynamic molecular portrait”. The latter reflects a combination of time-dependent surface distributions of various physicochemical properties, inherent in both model lipid bilayers and natural multi-component cell membranes. One of the most important features of biomembranes is their mosaicity, which is expressed in the constant presence of lateral inhomogeneities, the sizes and lifetimes of which vary in a wide range—from 1 to 10³ nm and from 0.1 ns to milliseconds. In addition to the relatively well-studied macroscopic domains (so-called “rafts”), the analysis of micro- and nanoclusters (or domains) that form an instantaneous picture of the distribution of structural, dynamic, hydrophobic, electrical, etc., properties at the membrane-water interface is attracting increasing interest. This is because such nanodomains (NDs) have been proven to be crucial for the proper membrane functioning in cells. Therefore, an understanding with atomistic details the phenomena associated with NDs is required. The present mini-review describes the recent results of experimental and in silico studies of spontaneously formed NDs in lipid membranes. The main attention is paid to the methods of ND detection, characterization of their spatiotemporal parameters, the elucidation of the molecular mechanisms of their formation. Biological role of NDs in cell membranes is briefly discussed. Understanding such effects creates the basis for rational design of new prospective drugs, therapeutic approaches, and artificial membrane materials with specified properties.