Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Differentiation potential of pediatric adipose tissue-derived stem cells for future applications in tissue reconstruction.

Background and aims: Stem cells derived from adipose tissue are a potential important source for autologous cell-based therapies, including treatment of birth-related defects. Critical for their use in therapeutic applications is to fully understand their differentiation potential and its consistency or variability among individuals, information still lacking, particularly in children. Therefore, the aim of this study was to better understand the differentiation potential of human adipose-derived stem cells (ADSCs) obtained from different pediatric patients with craniofacial defects, for future applications in autologous craniofacial reconstruction as a new approach to tissue repair in children. To address these issues, I have investigated ADSCs phenotype and analysed expression levels of some differentiation markers and a molecule possibly involved in ADSCs differentiation, PAD3, an enzyme known to play a role in calcium-dependent processes. PAD enzymes citrullinate (change an arginine to citrulline) proteins and increased citrullination has been associated with several human diseases including neural or cartilage disorders, such as rheumatoid arthritis. A crucial point of my research was to define the potentiality of the single cells, focusing my study on the clonal lines analysis, to assess whether cell lines derived from a single cell display a multilineage differentiation potential or whether each phenotype arises from a subset of committed progenitor cells that exist within a heterogeneous population. Methods: I have cultured ADSCs, isolated from liposuction aspirates, both on plastic and on Matrigel or poly-L-lysin/laminin-coated plates to promote cell adhesion, migration, growth and differentiation. Cells differentiated in culture media containing lineage-specific induction factors were compared to cells grown in control medium, without differentiation factors. I have assessed the expression of some lineage-specific differentiation markers at the RNA and protein level using RT-qPCR and immunocytochemistry techniques after two, three and four weeks of differentiation. I have also analysed morphological differences between induced and non-induced cells using different microscopy techniques. Results: ADSCs responded to chondrogenic, adipogenic, osteogenic, neural and epithelial induction by clearly changing their morphology in comparison with control cells which show a fibroblast-like cell shape and by up-regulating tissue-specific differentiation markers, independently from patient of origin. Specifically, for chondrogenic-induced cells results of RT-qPCR analysis reveal an up-regulation of chondrogenic markers Aggrecan and Col II in 2 and 3 weeks induced ADSCs of patients H21 and H23. Results of the analysis at clonal level of neurally-induced cells, show a consistent up-regulation of the Schwann cell marker P0, both at 2 and 4 weeks after neural induction, while mRNA levels of neuron-specific enoalse (NSE) remain unchanged between control and induced cells. Immunocytochemistry reveals a clearly different expression pattern of NF-200 in non-induced and neural-induced cells: in neural-induced cells, this protein is localized in filamentous structures both after 2 and 4 weeks after induction. Following epithelial induction, mRNA up-regulation of the epithelial marker cytokeratin-18 (Ck-18) in epithelial-induced cells is observed at both 3 and 4 weeks after induction, whereas the mRNA levels of the tight junction protein, zonula occludens-1 (ZO-1) remain unchanged between non-induced and induced cells. However, translocation of the ZO-1 protein from the cytoplasm to the cell margin at points of cell-cell contact in the epithelial-induced cells, consistent with tight junction formation typical of epithelial cells, is observed. In addition, immunocytochemistry also demonstrates the induction of filamentous cytokeratin expression found in epithelial cells. PAD3 mRNA is significantly up-regulated in neural-induced cells, showing results consistent among different clones. Also in chondrogenic-induced cells PAD3 expression levels are higher than in control cells. Summary and conclusions: This study supports the hypothesis that ADSC cultures contain pluripotent adult stem cell and are not solely a mixed population of unipotent progenitor cells, extending this finding to non-mesenchymal lineages and pediatric ADSCs, that had not been previously studied at the clonal level. Together, the gene and protein expression results confirm that ADSCs can differentiate into cell lineages of mesodermal and non-mesodermal origin, demonstrating for the first time that ADSCs can undergo epithelial differentiation at clonal level. In addition, this study shows up-regulation of PAD3 upon differentiation suggesting a role for this citrullination in this process that will require further investigation

Social Pharmacy

Il progetto di Social Pharmacy si basa su un concetto di farmacia che si configura sulla nuova realtà multiculturale e cosmopolita delle città odierne. L’indicatore principale di questa nuova realtà è il cambiamento della popolazione in termini di numero, varietà, nonché i fenomeni di grande mobilità internazionale e migrazioni. Questo cambiamento sociale porta con sé la necessità di affrontare nuovi e differenti scenari, volti a soddisfare le esigenze di vari profili di utenti, attraverso un’offerta variegata di prodotti e servizi. In termini di approccio al problema risulta centrale il concetto di diversification strategy, ovvero un approccio all’offerta della farmacia basato sulla varietà e la personalizzazione, proseguendo con un consequenziale sviluppo di nuove pratiche e servizi, al fine di realizzare una dimensione “senza confini né barriere”, con percorsi accessibili secondo la disponibilità di tempo dell’utenza. Secondo un dato Federfarma, le funzioni sociali e sanitarie attribuite alla farmacia sono quelle prevalenti. La farmacia va quindi considerata un’impresa professionale che opera a tutela della salute pubblica. La varietà di offerta e servizio si sviluppa attraverso la contaminazione con le culture d’altrove al fine di realizzare un sistema a servizio del benessere totale, all’interno del quale il farmacista riveste il ruolo di councelor e l’utente si inserisce in un ambiente accogliente e accessibile. La farmacia diventa un luogo della comunità, locale ed internazionale, che offre allo stesso tempo servizi all’avanguardia basati sulla tecnologia e sulla praticità, nonché servizi di supporto, orientamento ed informazione in loco. Attraverso una strategia di diversificazione, a cavallo tra tradizione ed innovazione, si punta ad un sistema di Pharmaceutical Care focalizzato sull’empowerment delle figure del farmacista e dell’utente. Affinché essa corrisponda ad un modello Social virtuoso, è necessario massimizzare l’attenzione alla collettività e, allo stesso tempo in modo più mirato, al “singolo”, in un sistema di fruizione Responsive User Experience

Programmable zwitterionic droplets as biomolecular sorters and model of membraneless organelles

It is emerging that cells can regulate biochemical functions by generating open compartments with well-defined composition. One important mechanism underlying this control is simple coacervation driven by disordered proteins that encode multivalent interactions. Inspired by these observations, here we develop programmable droplets based on simple coacervation of synthetic responsive polymers that mimic the architecture of these biological disordered proteins. We have adopted a bottom-up approach starting from zwitterionic polymers, demonstrating that they can form liquid droplets that exclude most molecules. Starting from this reference material we have progressively introduced in the polymer architecture an increasing number of different intermolecular interactions. With this strategy, we can independently control multiple properties of the droplets, such as stimulus responsiveness, polarity, selective uptake of client molecules and miscibility. We demonstrate that these droplets can separate and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation and in diagnostics

Programmable Zwitterionic Droplets as Biomolecular Sorters and Model of Membraneless Organelles

Increasing evidence indicates that cells can regulate biochemical functions in time and space by generating membraneless compartments with well-defined mesoscopic properties. One important mechanism underlying this control is simple coacervation driven by associative disordered proteins that encode multivalent interactions. Inspired by these observations, programmable droplets based on simple coacervation of responsive synthetic polymers that mimic the "stickers-and-spacers" architecture of biological disordered proteins are developed. Zwitterionic polymers that undergo an enthalpy-driven liquid-liquid phase separation process and form liquid droplets that remarkably exclude most molecules are developed. Starting from this reference material, different functional groups in the zwitterionic polymer are progressively added to encode an increasing number of different intermolecular interactions. This strategy allowed the multiple emerging properties of the droplets to be controlled independently, such as stimulus-responsiveness, polarity, selective uptake of client molecules, fusion times, and miscibility. By exploiting this high programmability, a model of cellular compartmentalization is reproduced and droplets capable of confining different molecules in space without physical barriers are generated. Moreover, these biomolecular sorters are demonstrated to be able to localize, separate, and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation, and diagnostics.ISSN:0935-9648ISSN:1521-409

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.ISSN:2041-6520ISSN:2041-653

Dynamics of Synthetic Membraneless Organelles in Microfluidic Droplets

ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

The interface of condensates of the hnRNPA1 low-complexity domain promotes formation of amyloid fibrils

The maturation of liquid-like protein condensates into amyloid fibrils has been associated with several neurodegenerative diseases. However, the molecular mechanisms underlying this liquid-to-solid transition have remained largely unclear. Here we analyse the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in amyotrophic lateral sclerosis. We show that phase separation and fibrillization are connected but distinct processes that are modulated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. We further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results reveal that the interface of biomolecular condensates of hnRNPA1 promotes fibril formation, therefore suggesting interfaces as a potential novel therapeutic target against the formation of aberrant amyloids mediated by condensation.ISSN:1755-4349ISSN:1755-433