Endothelial Cells Promote Osteogenesis by Establishing a Functional and Metabolic Coupling With Human Mesenchymal Stem Cells

Bone formation involves a complex crosstalk between endothelial cells (EC) and osteodifferentiating stem cells. This functional interplay is greatly mediated by the paracrine and autocrine action of soluble factors released at the vasculature-bone interface. This study elucidates the molecular and functional responses triggered by this intimate interaction. In this study, we showed that human dermal microvascular endothelial cells (HMEC) induced the expression of pro-angiogenic factors in stem cells from human exfoliated deciduous teeth (SHED) and sustain their osteo-differentiation at the same time. In contrast, osteodifferentiating SHED increased EC recruitment and promoted the formation of complex vascular networks. Moreover, HMEC enhanced anaerobic glycolysis in proliferating SHED without compromising their ability to undergo the oxidative metabolic shift required for adequate osteo-differentiation. Taken together, these findings provide novel insights into the molecular mechanism underlying the synergistic cooperation between EC and stem cells during bone tissue renewal

Quatsomes Loaded with Squaraine Dye as an Effective Photosensitizer for Photodynamic Therapy

Photodynamic therapy is a non-invasive therapeutic strategy that combines external light with a photosensitizer (PS) to destroy abnormal cells. Despite the great progress in the development of new photosensitizers with improved efficacy, the PS’s photosensitivity, high hydrophobicity, and tumor target avidity still represent the main challenges. Herein, newly synthesized brominated squaraine, exhibiting intense absorption in the red/near-infrared region, has been successfully incorporated into Quatsome (QS) nanovesicles at different loadings. The formulations under study have been characterized and interrogated in vitro for cytotoxicity, cellular uptake, and PDT efficiency in a breast cancer cell line. The nanoencapsulation of brominated squaraine into QS overcomes the non-water solubility limitation of the brominated squaraine without compromising its ability to generate ROS rapidly. In addition, PDT effectiveness is maximized due to the highly localized PS loadings in the QS. This strategy allows using a therapeutic squaraine concentration that is 100 times lower than the concentration of free squaraine usually employed in PDT. Taken together, our results reveal the benefits of the incorporation of brominated squaraine into QS to optimize their photoactive properties and support their applicability as photosensitizer agents for PDT

Mechanical and Biological Characterization of PMMA/Al2O3 Composites for Dental Implant Abutments

The mechanical and biological behaviors of PMMA/Al2O3 composites incorporating 30 wt.%, 40 wt.%, and 50 wt.% of Al2O3 were thoroughly characterized as regards to their possible application in implant-supported prostheses. The Al2O3 particles accounted for an increase in the flexural modulus of PMMA. The highest value was recorded for the composite containing 40 wt.% Al2O3 (4.50 GPa), which was about 18% higher than that of its unfilled counterpart (3.86 GPa). The Al2O3 particles caused a decrease in the flexural strength of the composites, due to the presence of filler aggregates and voids, though it was still satisfactory for the intended application. The roughness (Ra) and water contact angle had the same trend, ranging from 1.94 microns and 77.2° for unfilled PMMA to 2.45 microns and 105.8° for the composite containing the highest alumina loading, respectively, hence influencing both the protein adsorption and cell adhesion. No cytotoxic effects were found, confirming that all the specimens are biocompatible and capable of sustaining cell growth and proliferation, without remarkable differences at 24 and 48 h. Finally, Al2O3 was able to cause strong cell responses (cell orientation), thus guiding the tissue formation in contact with the composite itself and not enhancing its osteoconductive properties, supporting the PMMA composite’s usage in the envisaged application

Rôle fonctionnel des canaux TRP dans l'angiogenèse et l'invasion du cancer de la prostate

Le cancer de la prostate (CaP) est la deuxième cause de mortalité par cancer chez l'homme et sa lethalité est principalement due aux métastases. Il est donc essentiel de comprendre les mécanismes par lesquels les tumeurs se développent et comment les métastases peuvent se diffuser dans le corps. L'agressivité des tumeurs prostatiques est étroitement liée à la migration des cellules épithéliales et endothéliales (EC) provoquant l'invasion des tissus voisins ainsi que la vascularisation tumorale. Plusieurs canaux de la famille TRP (Transient Receptor Potential) sont dérégulés dans les cellules cancéreuses et ont été suggérés comme marqueurs pronostiques et diagnostiques ainsi que comme des cibles potentielles pour la thérapie du cancer. Dans cette thèse doctorale, j’ai établi le rôle de certains canaux TRP régulant la signature calcique des cellules endothéliales et cancéreuses de la prostate, en se concentrant en particulier sur les canaux qui affectent la migration, une étape clé commune dans la vascularisation et l'invasion tumorales.Nous avons etabli le profil complet d'expression de tous les TRP dans les EC normales et les EC dérivées de tumeurs de la prostate (PTEC), mammaires et rénales. Nous avons identifié trois gènes «associés à la prostate» dont l'expression est corrélée positivement dans les PTEC (TRPV2, TRPC3 et TRPA1). Parmi eux, TRPA1 joue un rôle essentiel dans la régulation de l'angiogenèse du CaP, favorisant la migration des PTEC, la formation du réseau vasculaire et l'angiogenèse par bourgeonnement in vitro et in vivo.En ce qui concerne la motilité des cellules cancéreuses d’origine épithéliale, je me suis concentrée sur TRPM8, canal pour lequel un rôle protecteur dans le CaP métastatique a été proposé via l’altération de la motilité cellulaire. Nous avons tout d’abord validé le rôle anti-métastatique de TRPM8 in vivo, montrant que la surexpression et l'activation de TRPM8 réduisent significativement la croissance tumorale et la dissémination des métastases dans un modèle murin de xénogreffe orthotopique de la prostate. De plus, en étudiant le mécanisme moléculaire sous-jacent à la fonction inhibitrice de TRPM8 sur la migration des cellules du CaP, nous avons constaté que TRPM8 inhibe la migration et l'adhésion des cellules du CaP indépendamment de sa fonction canalaire en piégeant au niveau intracellulaire la petite GTPase Rap1A sous sa forme inactive et en évitant ainsi sa activation sur la membrane plasmique. De plus, nous avons identifié et validé les résidus impliqués dans l'interaction entre TRPM8 et Rap1A: résidus E207 et Y240 dans la séquence de TRPM8 et Y32 dans celle de Rap1A. Nos données révèlent donc le rôle de TRPA1 et TRPM8 dans l'angiogenèse et l'invasion du CaP en affectant la migration cellulaire.Dans la lutte contre les métastases, le développement de systèmes de nano-administration efficaces est aussi crucial que l'identification de nouvelles cibles moléculaires. Dans ce contexte, une deuxième partie de ce projet de doctorat était axé sur l'étude des nanoparticules lipidiques en tant que systèmes d'administration de médicaments appropriés. En particulier, l'utilisation de nanoparticules lipidiques solides (SLN) et de quatsomes (QS) pour l'incorporation de colorants polyméthine (PMD) adaptés à des fins diagnostiques et thérapeutiques a été étudiée. Nous avons démontré que les nanoparticules lipidiques non seulement augmentent la solubilité de la PMD dans des conditions physiologiques, mais améliorent même leurs performances spectroscopiques, faisant des nanoparticules chargés de PMD des candidats potentiels et attrayants pour l'imagerie in vivo et/ou les applications PDT.Dans l'ensemble, cette thèse de doctorat propose les canaux TRP comme nouvelles cibles potentielles dans le traitement du CaP et, en même temps, les nanoparticules lipidiques comme nouveaux outils thérapeutiques pour améliorer l'administration de médicaments dans le traitement du cancer.Prostate cancer (PCa) is the second most lethal tumor among men and its mortality is mainly due to metastasis. Thus, it is critical to understand the mechanisms by which tumors grow and how metastases can diffuse throughout the body. Cell migration of both epithelial and endothelial cells (EC) is required for cancer cell invasion of neighboring tissues as well as for the formation of tumor vasculature. Several Transient Receptor Potential (TRP) channels are deregulated in cancer cells and have been suggested as valuable markers in predicting cancer progression as well as potential targets for pharmaceutical therapy. In the present Ph.D. thesis, I established the role of TRP channels regulating Ca2+ signature in PCa cells and vasculature focusing, in particular, on the channels that affect migration, a common key step in tumor vascularization and invasion.The role of TRP channels in prostatic angiogenesis was studied in prostate tumor-derived EC (PTEC): we fully profiled the expression of all TRPs in normal ECs and TECs derived from PCa, breast, and renal tumors. We identified three ‘prostate-associated’ genes whose expression appears selectively upregulated in PTECs: TRPV2, TRPC3, and TRPA1. Among them, TRPA1 seems to play a critical role in regulating PCa angiogenesis, promoting PTEC migration, vascular network formation, and angiogenic sprouting both in vitro and in vivo.As regards, instead, epithelial PCa cells' motility, emerging evidence indicates that TRPM8 may exert a protective role in metastatic PCa by impairing the motility of these cancer cells. Investigating the molecular mechanism underlying this biological effect, we found that, as previously described for ECs, TRPM8 inhibits PCa cell migration and adhesion independently from its channel function by intracellularly trapping the small GTPase Rap1A in its inactive form and thus avoiding its translocation and activation on the plasma membrane. Moreover, we identified and validated the residues involved in the interaction between TRPM8 and Rap1A: residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A.Our data shed new light on the roles played by TRPA1 and TRPM8 in prostate cancer angiogenesis and invasion by affecting cell migration of endothelial and epithelial cells, respectively.In the fight against metastasis, the development of efficient nanodelivery systems can be as crucial as the identification of new molecular targets in cancer therapy to fill the gap between “drug discovery” and “drug delivery” which is one of the most challenges in clinical perspectives. In this context, the second part of this Ph.D. project focused on the study of lipid nanoparticles as suitable drug delivery systems. In particular, the use of solid lipid nanoparticles (SLN) and quatsomes (QS) for the incorporation of polymethine dyes (PMD) suitable for both diagnostic and therapeutic purposes was investigated. We demonstrated that lipid nanocarriers not only increase the solubility of PMD in physiological conditions but even enhance their spectroscopic performances, making PMD-loaded nanocarriers potential and appealing candidates for in vivo imaging and/or PDT applications.Overall, the present Ph.D. thesis deepens our knowledge of the role of TRP channels in PCa progression, providing new insight into their possible use as new therapeutic targets in PCa treatment and, at the same time, proposes new therapeutic tools to improve drug delivery in cancer therapy

BioTEA: Containerized Methods of Analysis for Microarray-Based Transcriptomics Data

Tens of thousands of gene expression data sets describing a variety of model organisms in many different pathophysiological conditions are currently stored in publicly available databases such as the Gene Expression Omnibus (GEO) and ArrayExpress (AE). As microarray technology is giving way to RNA-seq, it becomes strategic to develop high-level tools of analysis to preserve access to this huge amount of information through the most sophisticated methods of data preparation and processing developed over the years, while ensuring, at the same time, the reproducibility of the results. To meet this need, here we present bioTEA (biological Transcript Expression Analyzer), a novel software tool that combines ease of use with the versatility and power of an R/Bioconductor-based differential expression analysis, starting from raw data retrieval and preparation to gene annotation. BioTEA is an R-coded pipeline, wrapped in a Python-based command line interface and containerized with Docker technology. The user can choose among multiple options—including gene filtering, batch effect handling, sample pairing, statistical test type—to adapt the algorithm flow to the structure of the particular data set. All these options are saved in a single text file, which can be easily shared between different laboratories to deterministically reproduce the results. In addition, a detailed log file provides accurate information about each step of the analysis. Overall, these features make bioTEA an invaluable tool for both bioinformaticians and wet-lab biologists interested in transcriptomics. BioTEA is free and open-source

Oral Cavity as a Source of Mesenchymal Stem Cells Useful for Regenerative Medicine in Dentistry

The use of mesenchymal stem cells (MSCs) for regenerative purposes has become common in a large variety of diseases. In the dental and maxillofacial field, there are emerging clinical needs that could benefit from MSC-based therapeutic approaches. Even though MSCs can be isolated from different tissues, such as bone marrow, adipose tissue, etc., and are known for their multilineage differentiation, their different anatomical origin can affect the capability to differentiate into a specific tissue. For instance, MSCs isolated from the oral cavity might be more effective than adipose-derived stem cells (ASCs) for the treatment of dental defects. Indeed, in the oral cavity, there are different sources of MSCs that have been individually proposed as promising candidates for tissue engineering protocols. The therapeutic strategy based on MSCs can be direct, by using cells as components of the tissue to be regenerated, or indirect, aimed at delivering local growth factors, cytokines, and chemokines produced by the MSCs. Here, the authors outline the major sources of mesenchymal stem cells attainable from the oral cavity and discuss their possible usage in some of the most compelling therapeutic frontiers, such as periodontal disease and dental pulp regeneration