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The stem cell niche in the bone marrow is a hypoxic environment, where the low oxygen tension preserves the pluripotency of stem cells. We have identified mesangiogenic progenitor cells (MPC) exhibiting angiogenic and mesenchymal differentiation capabilities in vitro. The effect of hypoxia on MPC has not been previously explored. In this study, MPCs were isolated from volunteers' bone marrow and cultured under both normoxic and hypoxic conditions (3% O2). MPCs maintained their characteristic morphology and surface marker expression (CD18 + CD31 + CD90-CD73-) under hypoxia. However, hypoxic conditions led to reduced MPC proliferation in primary cultures and hindered their differentiation into mesenchymal stem cells (MSCs) upon exposure to differentiative medium. First passage MSCs derived from MPC appeared unaffected by hypoxia, exhibiting no discernible differences in proliferative potential or cell cycle. However, hypoxia impeded the subsequent osteogenic differentiation of MSCs, as evidenced by decreased hydroxyapatite deposition. Conversely, hypoxia did not impact the angiogenic differentiation potential of MPCs, as demonstrated by spheroid-based assays revealing comparable angiogenic sprouting and tube-like formation capabilities under both hypoxic and normoxic conditions. These findings indicate that hypoxia preserves the stemness phenotype of MPCs, inhibits their differentiation into MSCs, and hampers their osteogenic maturation while leaving their angiogenic potential unaffected. Our study sheds light on the intricate effects of hypoxia on bone marrow-derived MPCs and their differentiation pathways.
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Thymic epithelial cells participate in the maturation and selection of T lymphocytes. This review explores recent insights from single-cell sequencing regarding classifying thymic epithelial cells in both normal and neoplastic thymus. Cortical thymic epithelial cells facilitate thymocyte differentiation and contribute to positive selection. Medullary epithelial cells are distinguished by their expression of AIRE. Cells progress from a pre-AIRE state, containing precursors with cortical and medullary characteristics, termed junctional cells. Mature medullary epithelial cells exhibit promiscuous gene expression and after that downregulate AIRE mRNA. Post-AIRE cells can adopt a Hassall corpuscle-like phenotype or exhibit distinctive differentiation characteristics including tuft cells, ionocytes, neuroendocrine cells, and myoid cells.
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Thymic epithelial tumors, comprising thymic carcinomas and thymomas, are rare neoplasms. They differ in histology, prognosis, and association with autoimmune diseases such as myasthenia gravis. Thymomas, but not thymic carcinomas, often harbor GTF2I mutations. Mutations of CDKN2A, TP53, and CDKN2B are the most common thymic carcinomas. The acquisition of mutations in genes that control chromatin modifications and epigenetic regulation occurs in the advanced stages of thymic carcinomas. Anti-angiogenic drugs and immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown promising results for the treatment of unresectable tumors. Since thymic carcinomas are frankly aggressive tumors, this report presents insights into their oncogenic drivers, categorized under the established hallmarks of cancer.
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AIM: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). MATERIAL AND METHODS: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients' samples and in the Ty82 cell line. RESULTS: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3-5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. CONCLUSIONS: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients' susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP).
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Mesangiogenic progenitor cells (MPCs) have been isolated from human bone marrow (BM) mononuclear cells. They attracted particular attention for the ability to differentiate into exponentially growing mesenchymal stromal cells while retaining endothelial differentiative potential. MPC power to couple mesengenesis and angiogenesis highlights their tissue regenerative potential and clinical value, with particular reference to musculoskeletal tissues regeneration. BM and adipose tissue represent the most promising adult multipotent cell sources for bone and cartilage repair, although discussion is still open on their respective profitability. Culture determinants, as well as tissues of origin, appeared to strongly affect the regenerative potential of cell preparations, making reliable methods for cell isolation and growth a prerequisite to obtain cell-based medicinal products. Our group had established a definite consistent protocol for MPC culture, and here, we present data showing MPCs to be tissue specific.
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Hematopoiesis is hosted, supported and regulated by a special bone marrow (BM) microenvironment known as "niche." BM niches have been classified based on micro-anatomic distance from the bone surface into "endosteal" and "central" niches. Whilst different blood vessels have been found in both BM niches in mice, our knowledge of the human BM architecture is much more limited. Here, we have used a combination of markers including NESTIN, CD146, and αSMA labeling different blood vessels in benign human BM. Applying immunohistochemical/immunofluorescence techniques on BM trephines and performing image analysis on almost 300 microphotographs, we detected high NESTIN expression in BM endothelial cells (BMECs) of small arteries (A) and endosteal arterioles (EA), and also in very small vessels we named NESTIN+ capillary-like tubes (NCLTs), not surrounded by sub-endothelial perivascular cells that occasionally reported low levels of NESTIN expression. Statistically, NCLTs were detected within 40 µm from bone trabecula, frequently found in direct contact to the bone line and spatially correlated with hematopoietic stem/progenitor cells. Our results support the expression of NESTIN in human BMECs of EA and A in accordance with the updated classification of murine BM micro-vessels. NCLTs for their peculiar characteristics and micro-anatomical localization have been here proposed as transitional vessels possibly involved in regulating human hematopoiesis.
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Multiple myeloma (MM) progresses mainly in the bone marrow where the involvement of a specific microenvironment plays a critical role in maintaining plasma cell growth, spread, and survival. In active disease, the switch from a pre-vascular/non-active phase to a vascular phase is coupled with the impairment of bone turnover. Previously, we have isolated Mesangiogenic Progenitor Cells (MPCs), a bone marrow population that showed mesengenic and angiogenic potential, both in vitro and in vivo. MPC differentiation into musculoskeletal tissue and their ability of sprouting angiogenesis are mutually exclusive, suggesting a role in the imbalancing of the microenvironment in multiple myeloma. MPCs from 32 bone marrow samples of multiple myeloma and 23 non-hematological patients were compared in terms of frequency, phenotype, mesengenic/angiogenic potential, and gene expression profile. Defective osteogenesis was recorded for MM-derived MPCs that showed longer angiogenic sprouting distances respect to non-hematological MPCs, retaining this capability after mesengenic induction. This altered MPCs differentiation potential was not detected in asymptomatic myelomatous disease. These in vitro experiments are suggestive of a forced angiogenic fate in MPCs isolated from MM patients, which also showed increased sprouting activity. Taking together our results suggest a possible role of these cells in the "angiogenic switch" in the MM micro-environment.
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Long-term glial cell treatment with the proinflammatory cytokine TNF-alpha has been demonstrated to increase the functional responsiveness of A(2B) adenosine receptors (A(2B) ARs), which in turn synergize with the cytokine inducing chronic astrogliosis. In the present study, we investigated the short-term effects of TNF-alpha on A(2B) AR functional responses in human astroglial cells (ADF), thus simulating the acute phase of cerebral damage which is characterized by both cytokine and adenosine high level release. Short-term TNF-alpha cell treatment caused A(2B) AR phosphorylation inducing, in turn, impairment in A(2B) AR-G protein coupling and cAMP production. These effects occurred in a time-dependent manner with a maximum following 3-h cell exposure. Moreover, we showed PKC intracellular kinase is mainly involved in the TNF-alpha-mediated regulation of A(2B) AR functional responses. The results may indicate the A(2B) AR functional impairment as a cell defense mechanism to counteract the A(2B) receptor-mediated effects during the acute phase of brain damage, underlying A(2B) AR as a target to modulate early inflammatory responses.
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Astrócitos/patologia , Receptor A2B de Adenosina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Isquemia Encefálica/patologia , Células Cultivadas , AMP Cíclico/biossíntese , Humanos , Inflamação , Cinética , Fosforilação , Proteína Quinase C , Receptor A2B de Adenosina/efeitos dos fármacosRESUMO
The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are reported. High-affinity ligands at benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) were obtained when the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of heteroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a 'hydrogen-bond acceptor ring' at position 3 is synergic with an electron donor substituent at position 8, while a 'hydrogen-bond donor ring' is synergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize and qualitatively interpret the GABA(A)/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been performed, with the aim of assessing the preferred geometry of protein-ligand complex.
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Modelos Moleculares , Óxidos/química , Pirazóis/química , Receptores de GABA-A/metabolismo , Triazinas/síntese química , Triazinas/farmacologia , Animais , Ansiedade/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , Etanol/farmacologia , Ligantes , Masculino , Memória/efeitos dos fármacos , Estrutura Molecular , Ratos , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Sono/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Tempo , Triazinas/química , Triazinas/uso terapêuticoRESUMO
The Polycomb gene BMI1 expression exerts a negative predictive impact on several hematological malignancies, such as acute and chronic myeloid leukemia (CML), myelofibrosis, and follicular lymphoma. As already demonstrated in CML, BMI1 is responsible for the resistance to the tyrosine kinase inhibitors (TKIs) in a BCR-ABL1-independent way. Even if, it is unknown where BMI1 in CML is expressed (in progenitors or more mature cells). We decided, therefore, to evaluate if and where the BMI1 protein is located, focusing mainly on the CD34+/CD38-/CD26+ CML progenitors. To begin we measured, by flow cytometry, the proportion of CD34+/CD26+ cells in 31 bone marrow samples from 20 CML patients, at diagnosis and during treatment with imatinib. After that the bone marrow blood smears were stained with antibodies anti-CD26, BCR-ABL1, and BMI1. These smears were observed by a confocal laser microscope and a 3D reconstruction was then performed. At diagnosis, CD34+/CD26+ cells median value/µL was 0.48; this number increased from diagnosis to the third month of therapy and then reduced during treatment with imatinib. The number and behavior of the CD26+ progenitors were independent from the BCR-ABL1 expression, but they summed up what previously observed about the BMI1 expression modulation. In this work we demonstrate for the first time that in CML the BMI1 protein is co-expressed with BCR-ABL1 only in the cytoplasm of the CD26+ precursors; on the contrary, in other hematological malignancies where BMI1 is commonly expressed (follicular lymphoma, essential thrombocytemia, acute myeloid leukemia), it was not co-localized with CD26 or, obviously, with BCR-ABL1. Once translated into the clinical context, if BMI1 is a marker of stemness, our results would suggest the combination of the BMI1 inhibitors with TKIs as an interesting object of research, and, probably, as a promising way to overcome resistance in CML patients.
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Novel N-substituted indol-3-ylglyoxylamides (10-37) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR). In an effort to achieve affinity-based selectivity among BzR subtypes, these compounds were designed to probe the LDi and L2 lipophilic regions. Taking the alpha1-selective benzylindolylglyoxylamides Ia and Ib as leads, we varied the substituent on the benzylamide phenyl ring (compounds 10-23) or replaced the benzyl moiety with alkyl groups (compounds 24-37). The above structural changes gave no shift of selectivity from the alpha1 toward the alpha2 or alpha5 subtypes, thus confirming that a ligand which occupies the LDi region probably exhibits alpha1 selectivity, despite its interactions with other lipophilic areas in the receptor binding cleft. Compound 11 (N-(p-methylbenzyl)-5-nitroindol-3-ylglyoxylamide), which selectively binds with a full agonist efficacy at the alpha1 receptor subtype and displays sedative action, can be regarded as an interesting potential zolpidem-like sedative-hypnotic agent.
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Amidas/síntese química , Agonistas de Receptores de GABA-A , Glioxilatos/síntese química , Hipnóticos e Sedativos/síntese química , Indóis/síntese química , Amidas/química , Amidas/farmacologia , Animais , Bovinos , Linhagem Celular , Córtex Cerebral/metabolismo , Glioxilatos/química , Glioxilatos/farmacologia , Hipnóticos e Sedativos/química , Hipnóticos e Sedativos/farmacologia , Técnicas In Vitro , Indóis/química , Indóis/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Atividade Motora/efeitos dos fármacos , Ensaio Radioligante , Ratos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Mesangiogenic progenitor cells (MPCs) have shown the ability to differentiate in-vitro toward mesenchymal stromal cells (MSCs) as well as angiogenic potential. MPCs have so far been described in detail as progenitors of the mesodermal lineage and appear to be of great significance in tissue regeneration and in hemopoietic niche regulation. On the contrary, information regarding the MPC angiogenic process is still incomplete and requires further clarification. In particular, genuine MPC angiogenic potential should be confirmed in-vivo. METHODS: In the present article, markers and functions associated with angiogenic cells have been dissected. MPCs freshly isolated from human bone marrow have been induced to differentiate into exponentially growing MSCs (P2-MSCs). Cells have been characterized and angiogenesis-related gene expression was evaluated before and after mesengenic differentiation. Moreover, angiogenic potential has been tested by in-vitro and in-vivo functional assays. RESULTS: MPCs showed a distinctive gene expression profile, acetylated-low density lipoprotein uptake, and transendothelial migration capacity. However, mature endothelial markers and functions of endothelial cells, including the ability to form new capillaries, were absent, thus suggesting MPCs to be very immature endothelial progenitors. MPCs showed marked 3D spheroid sprouting activating the related molecular machinery, a clear in-vitro indication of early angiogenesis. Indeed, MPCs applied to chicken chorioallantoic membrane induced and participated in neovessel formation. All of these features were lost in mesengenic terminally differentiated P2-MSCs, showing definite separation of the two differentiation lineages. CONCLUSION: Our results confirm the bona-fide angiogenic potential of MPCs and suggest that the high variability reported for MSC cultures, responsible for the controversies regarding MSC angiogenic potential, could be correlated to variable percentages of co-isolated MPCs in the different culture conditions so far used.
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Células-Tronco Adultas/citologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Adipócitos/citologia , Adipócitos/metabolismo , Células-Tronco Adultas/metabolismo , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-IdadeRESUMO
N-(heteroarylmethyl)indol-3-ylglyoxylamides (1-26) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR) to probe the hydrogen bonding properties of the so-called S(1) site of the BzR by means of suitable heterocyclic side chains. SARs were developed in light of our hypothesis of binding modes A and B. Pyrrole and furan derivatives adopting mode A (2, 8, 10, 20, 22) turned out to be more potent (K(i) values < 35 nM) than their analogues lacking hydrogen bonding heterocyclic side chains. These data suggest that the most potent indoles interact with a hydrogen bond acceptor/donor (HBA/D) group located within the S(1) site of the BzR. Compounds 1, 2, 8, 19, 20, and 22, tested at recombinant rat alpha(1)beta(2)gamma(2), alpha(2)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzRs, elicited selectivity for the alpha(1)beta(2)gamma(2) isoform. On the basis of published mutagenesis studies and the present SARs, we speculate that the S(1) HBA/D group might be identified as the hydroxyl of alpha(1)-Tyr209 or of other neighboring amino acids.
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Amidas/farmacologia , Antagonistas de Receptores de GABA-A , Indóis/farmacologia , Amidas/síntese química , Amidas/química , Animais , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Linhagem Celular , Humanos , Ligação de Hidrogênio , Indóis/síntese química , Indóis/química , Ligantes , Estrutura Molecular , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".
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Medula Óssea , Técnicas de Cultura de Células , Células-Tronco/citologia , Células da Medula Óssea/citologia , Diferenciação Celular , HumanosRESUMO
Mesangiogenic Progenitor Cells (MPCs) are human bone marrow-derived multipotent cells, isolated in vitro under selective culture conditions and shown to retain both mesengenic and angiogenic potential. MPCs also co-isolated with multipotent stromal cells (MSCs) when bone marrow primary cultures were set up for clinical applications, using human serum (HS) in place of fetal bovine serum (FBS). MPC culture purity (over 95%) is strictly dependent on HS supplementation with significant batch-to-batch variability. In the present paper we screened different sources of commercially available pooled human AB type serum (PhABS) for their ability to promote MPC production under selective culture conditions. As the majority of "contaminating" cells in MPC cultures were represented by MSC-like cells, we hypothesized a role by differentiating agents present in the sera. Therefore, we tested a number of growth factors (hGF) and found that higher concentrations of FGF-2, EGF, PDGF-AB, and VEGF-A as well as lower concentration of IGF-1 give sub-optimal MPC recovery. Gene expression analysis of hGF receptors was also carried out both in MSCs and MPCs, suggesting that FGF-2, EGF, and PDGF-AB could act promoting MSC proliferation, while VEGF-A contribute to MSC-like cell contamination, triggering MPC differentiation. Here we demonstrated that managing hGF contents, together with applying specific receptors inhibitors (Erlotinib-HCl and Nintedanib), could significantly mitigate the batch-to-batch variability related to serum supplementation. These data represent a fundamental milestone in view of manufacturing MPC-based medicinal products.
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Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we first named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage, while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. In this study, we describe the ex vivo isolation of one novel bone marrow subpopulation (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either fluorescence-activated cell sorting or magnetic-activated cell sorting were applied to characterize Pop#8 as CD64(bright)CD31(bright)CD14(neg). We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in hematopoietic stem cell niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture-expanded MSCs could be the consequence of different culture conditions that select or promote particular subpopulations of precursors.
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Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Separação Celular/métodos , Mesoderma/citologia , Neovascularização Fisiológica , Células-Tronco/citologia , Adulto , Linhagem da Célula , Forma Celular , Células Cultivadas , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-IdadeRESUMO
Among the very large number of polymeric materials that have been proposed in the field of orthopedics, polyethylene terephthalate (PET) is one of the most attractive thanks to its flexibility, thermal resistance, mechanical strength and durability. Several studies have been proposed that interface nano- or micro-structured surfaces with mesenchymal stromal cells (MSCs), demonstrating the potential of this technology for promoting osteogenesis. All these studies were carried out on biomaterials other than PET, which remains almost uninvestigated in terms of cell shaping, alignment and differentiation. Here, we study the effect of PET 350-depth nanogratings (NGs) with a ridge and lateral groove size of 500 nm (T1) or 1 µm (T2), on bone marrow-derived human MSC (hMSC) differentiation in relation to the osteogenic fate. We demonstrate that these substrates, especially T2, can promote the osteogenic phenotype more efficiently than standard flat surfaces and that this effect is more marked if cells are cultured in osteogenic medium than in basal medium. Finally, we show that the shape and disposition of calcium hydroxyapatite granules on the different substrates was influenced by the substrate symmetry, being more elongated and spatially organized on NGs than on flat surfaces.
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Células da Medula Óssea/citologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Nanoestruturas/química , Osteoblastos/citologia , Osteogênese/fisiologia , Polietilenotereftalatos/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Durapatita , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Polietilenotereftalatos/química , Alicerces TeciduaisRESUMO
Mesangiogenic progenitor cells (MPCs) are a very peculiar population of cells present in the human adult bone marrow, only recently discovered and characterized. Owing to their differentiation potential, MPCs can be considered progenitors for mesenchymal stromal cells (MSCs), and for this reason they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Here, we evaluate the effects of surface nanotopography on MPCs, considering the possibility that this specific physical stimulus alone can trigger MPC differentiation toward the mesenchymal lineage. In particular, we exploit nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we study the MPC-MSC transition by i. analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and ii. quantifying the expression of cell-phenotype characterizing markers by flow cytometry. We demonstrate that the MPC mesengenic differentiation can be induced by the solely interaction with the NGs, in absence of any other external, chemical stimulus. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal.
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A series of 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones (PTBTs), VII, were prepared and tested at the central benzodiazepine receptor (BzR). The skeleton of these compounds was designed by formally combining the N-C=O moieties of the known BzR ligands, triazoloquinoxalines (IV) and triazinobenzimidazoles (ATBIs) (VI). Most of the PTBTs displayed submicromolar/nanomolar potency at the BzR. The 9-chloro derivatives (45-49) were generally found to be more potent than their 9-unsubstituted counterparts (37-44). Compound 45 turned out to be the most potent of the PTBTs (K(i) 2.8 nM). A subset of compounds (37, 42, 45, 49), when tested for their affinity on recombinant rat alpha1beta2gamma2, alpha2beta2gamma2, and alpha5beta3gamma2 GABA(A)/Bz receptor subtypes, showed enhanced affinities for the alpha1beta2gamma2 isoform, with compounds 45 and 49 exhibiting the highest selectivity. Moreover, compounds 45 and 49 were found to display a full agonist efficacy profile at alpha1 and alpha2 receptor subtypes, and an antagonist efficacy at alpha5-containing receptors.
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Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Triazinas/síntese química , Triazóis/síntese química , Animais , Ligação Competitiva , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Cloretos/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inibidores , Ensaio Radioligante , Ratos , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-Atividade , Triazinas/química , Triazinas/farmacologia , Triazóis/química , Triazóis/farmacologiaRESUMO
The present work reports the synthesis of trans-2-amino-5(6)-fluoro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes (4a-f, 5a-f) as a continuation of our studies to better understand the significance of the halo substituent in the trans-1-phenyl-2-aminoindane series and to extend knowledge of the monophenolic ligands of DA receptors. The affinity of the new compounds and related methoxylated precursors (10-15 and 18-23) was estimated in vitro by displacement of [(3)H]SCH23390 (for D(1)-like receptors) or [(3)H]YM-09-151-2 (for D(2)-like receptors) from homogenates of porcine striatal membranes. The results indicate that unsubstituted amines 4a, 5a, 10, and 11 are poorly effective at DA receptors. The introduction of two n-propyl groups on the nitrogen atom (compounds 14, 15, 4c, and 5c) and N-allyl-N-methyl- or N-methyl-N-propyl- substitution (compounds 20-23, 4e, 4f, 5e, 5f) increased the D(2)-like affinities and selectivity. The D(2)-like agonistic activity of selected compounds 15, 20, 21, 4e, 5c, and 5e was proved by evaluating their effects on the cyclic guanosine monophosphate (cGMP) content in rat neostriatal membranes. All tested compounds displayed a potential dopamine D(2)-like agonist profile decreasing basal levels of cGMP. The selective D(2)-like agonism of compounds 20 and 5e was proved by their effects on basal striatal adenylyl cyclase activity.