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1.
Nature ; 539(7630): 560-564, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27851739

RESUMO

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.


Assuntos
Técnicas de Cultura de Células/métodos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Intestinos/citologia , Organoides/citologia , Organoides/crescimento & desenvolvimento , Células-Tronco/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Forma Celular , Fibronectinas/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Camundongos , Proteólise , Nicho de Células-Tronco
2.
Int J Mol Sci ; 23(2)2022 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-35054998

RESUMO

Hit finding, scaffold hopping, and structure-activity relationship studies are important tasks in rational drug discovery. Implementation of these tasks strongly depends on the availability of compounds similar to a known bioactive molecule. SwissSimilarity is a web tool for low-to-high-throughput virtual screening of multiple chemical libraries to find molecules similar to a compound of interest. According to the similarity principle, the output list of molecules generated by SwissSimilarity is expected to be enriched in compounds that are likely to share common protein targets with the query molecule and that can, therefore, be acquired and tested experimentally in priority. Compound libraries available for screening using SwissSimilarity include approved drugs, clinical candidates, known bioactive molecules, commercially available and synthetically accessible compounds. The first version of SwissSimilarity launched in 2015 made use of various 2D and 3D molecular descriptors, including path-based FP2 fingerprints and ElectroShape vectors. However, during the last few years, new fingerprinting methods for molecular description have been developed or have become popular. Here we would like to announce the launch of the new version of the SwissSimilarity web tool, which features additional 2D and 3D methods for estimation of molecular similarity: extended-connectivity, MinHash, 2D pharmacophore, extended reduced graph, and extended 3D fingerprints. Moreover, it is now possible to screen for molecular structures having the same scaffold as the query compound. Additionally, all compound libraries available for screening in SwissSimilarity have been updated, and several new ones have been added to the list. Finally, the interface of the website has been comprehensively rebuilt to provide a better user experience. The new version of SwissSimilarity is freely available starting from December 2021.


Assuntos
Descoberta de Drogas/métodos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas , Software , Navegador , Bases de Dados de Produtos Farmacêuticos , Desenho de Fármacos , Humanos , Ligantes , Relação Quantitativa Estrutura-Atividade , Interface Usuário-Computador
3.
J Sex Med ; 18(5): 875-888, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33863684

RESUMO

BACKGROUND: An increased fibrosis of the corpora cavernosa is a prevalent process that underlies most cases of erectile dysfunction. Apelin, an endogenous circulating peptide, has been documented as an important effector on cardiovascular homeostasis, controlling vascular function and reducing fibrosis in multiple pathological conditions. Recently, initial studies have shown that Apelin, acting through the APJ receptor, also modulates penile erection, however, the role of this system on penile structure and intracorporal collagen remodeling has not been investigated yet. AIMS: Here we sought to investigate the effect of chronic Apelin treatment on the corpus cavernosum structure of hyperchOlesterolemic mice. METHODS: Apolipoprotein gene-deleted (ApoE-/-) mice were fed with a Western diet for 11 weeks and received Apelin-13 (2 mg/kg/day) or vehicle during the last 3 weeks. Penile samples were obtained for histological and biochemical analyses to assess the intracorporal collagen content and key proteins expression. Furthermore, the effect of Apelin-13 was evaluated in cultured NIH3T3 mouse fibroblasts stimulated with TGF-ß. OUTCOME: Local expression of Apelin-13 in mouse corpus cavernosum and its protective effect against fibrosis. RESULTS: Apelin and APJ receptor were expressed (gene and protein) within the corpus cavernosum of ApoE-/- mice, indicating a local modulation of the Apelin system. Interestingly, 3 weeks of Apelin-13 treatment strongly reduced intracavernosal collagen content. In addition, Apelin-13 enhanced total matrix metalloproteinase (MMP) activity in the mice penis, which was associated with an increased protein expression of MMP-1, MMP-3, MMP-8, and MMP-9, while tissue inhibitor of metalloproteinase were unaltered. These beneficial actions were not associated with changes in nNOS or eNOS protein expression, intracavernosal reactive oxygen species content, or atherosclerotic plaque deposition. Additionally, in cultured fibroblast, Apelin-13 inhibited TGF-ß-induced fibroblast to myofibroblast differentiation and collagen production, possibly through the activation of ERK1/2 kinase. CLINICAL TRANSLATION: These results point out Apelin/APJ system as a potential target to treat intracavernosal fibrosis-related disorders. STRENGTH & LIMITATIONS: These results provide the first evidence of the Apelin system's positive role on erectile tissue structure/remodeling. Nevertheless, additional functional study addressing erectile response would bring extended validation regarding the relevance of such effect. CONCLUSION: These results suggest a local modulation of the Apelin system within the corpus cavernosum. Remarkably, Apelin-13 reduced intracavernosal fibrosis in hypercholesterolemic mice by: (i) enhancing MMPs expression and activity; and (ii) inhibiting fibroblast differentiation into myofibroblast. Altogether, these results suggest an essential protective role of Apelin, indicating Apelin/APJ system as a promising candidate for the development of fibrosis-associated erectile dysfunction treatments. Sturny M, Anguenot L Costa-Fraga FP, et al. Apelin-13 Protects Corpus Cavernosum Against Fibrosis Induced by High-Fat Diet in an MMP-Dependent Mechanism. J Sex Med 2021;18:875-888.


Assuntos
Dieta Hiperlipídica , Disfunção Erétil , Animais , Apelina , Dieta Hiperlipídica/efeitos adversos , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/etiologia , Disfunção Erétil/prevenção & controle , Fibrose , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Metaloproteinases da Matriz , Camundongos , Células NIH 3T3 , Ereção Peniana , Pênis
4.
J Sex Med ; 17(11): 2129-2140, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32943375

RESUMO

BACKGROUND: The renin-angiotensin system (RAS) plays an important role in erectile function. The RAS contains 2 major axes: one deleterious, composed of ACE-Ang II-AT1 receptor, and another protective, composed of ACE2-Ang-(1-7)-Mas receptor. While aging is a well-known cause for development of male sexual disorders, little is known about local regulation of the RAS in age-related erectile dysfunction (ED). AIM: The present study aimed to assess regulation of the RAS in aging-associated ED rat model and evaluate possible options for disease management through pharmacological modulation of the RAS. METHODS: Penile tissues were harvested from 3-, 12-, and 24-month-old Wistar rats. Local expression of major RAS components and ED markers was measured by RT-PCR. Protein expression of RAS components was assessed by western blot. Collagen deposition was measured by Sirius Red and immunohistochemical staining. Evaluation of collagen content was also performed in penile sections of Mas-knockout mice by Sirius Red and Masson's trichrome stainings. Finally, the effect of Ang-(1-7) pretreatment on TGF-ß-induced myofibroblast activation was studied in primary cavernosal and immortalized fibroblasts. OUTCOMES: Experimental results highlighted the essential role of the RAS in modulation of cavernosal fibrosis. RESULTS: The present study demonstrates local expression of angiotensinogen mRNA alongside with major RAS components, which suggests local autonomous functioning of the RAS within penile tissue. Gene expression analysis revealed strong positive correlation between ACE-Ang II-AT1 axis with markers for inflammation and fibrosis. While corpus cavernosum from 24-month-old rats was characterized by increased collagen deposition, protein expression of ACE, AT1, and Mas was shown to be upregulated in the penile tissue of this group. At the same time, penile sections from Mas-knockout mice (FVB/N background) were also shown to have increased collagen deposition. Finally, it was demonstrated that Ang-(1-7) treatment of primary cavernosal and immortalized fibroblasts was able to alleviate TGF-ß-induced fibroblast-to-myofibroblast transition. CLINICAL TRANSLATION: The present study suggests Ang-(1-7) treatment as a possible strategy for pharmacological management of fibrosis-associated ED in aging. STRENGTHS & LIMITATIONS: The link between the RAS and penile fibrosis, indicated by a holistic screening of different ED markers, was confirmed by in vivo and in vitro data. However, results, presented in the manuscript, need to be further reinforced by human data. Important to note, the main goal of the study was to characterize RAS regulation in aging condition rather than state any causal relationships. CONCLUSION: Present study characterizes RAS regulation in aging-associated ED and indicates its important role in cavernosal fibrosis. Bragina ME, Costa-Fraga F, Sturny M, et al. Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis. J Sex Med 2020;17:2129-2140.


Assuntos
Induração Peniana , Sistema Renina-Angiotensina , Idoso , Angiotensina I , Angiotensina II/metabolismo , Animais , Fibrose , Humanos , Masculino , Camundongos , Ereção Peniana , Fragmentos de Peptídeos , Peptidil Dipeptidase A/metabolismo , Ratos , Ratos Wistar
5.
Platelets ; 30(5): 563-571, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30183501

RESUMO

Despite significant advances in the treatment of cardiovascular diseases, antiplatelet therapies are still associated with a high risk of hemorrhage. In order to develop new drugs, methods to measure platelet function must be adapted for the high-throughput screening (HTS) format. Currently, all assays capable of assessing platelet function are either expensive, complex, or not validated, which makes them unsuitable for drug discovery. Here, we propose a simple, low-cost, and high-throughput-compatible platelet function assay, validated for the 384-well plate. In the proposed assay, agonist-induced platelet activity was assessed by three different methods: (i) measurement of light absorbance, which decreases with platelet aggregation; (ii) luminescence measurement, based on ATP release from activated platelets and luciferin-luciferase reaction; and (iii) automated bright-field microscopy of the wells and further quantification of platelet image area, described here for the first time. Brightfield imaging results were validated by demonstrating the similarity of dose-response curves obtained with absorbance and luminescence measurements after stimulating platelets, pre-incubated with prostaglandin E1 or tirofiban, and demonstrating the similarity of dose-response curves obtained with agonists. Assay quality was confirmed using the Z'-factor, a statistical parameter used to validate the robustness and suitability of an HTS assay. The results showed that, under high rotations per minute (1200 RPM), an acceptable Z'-factor score is reached for absorbance measurements (Z'-factor - 0.58) and automated brightfield imaging (Z'-factor - 0.52), without the need of replicates, while triplicates must be used to achieve an acceptable Z'-factor score (0.54) for luminescence measurements. Using low platelet concentration (4 × 104/µl - 10 µl), the brightfield imaging test was further validated using washed platelets. Furthermore, drug screening was performed with compounds selected by structure-based virtual screening. Taken together, this study presents an optimized and validated assay for HTS to be used as a tool for antiplatelet drug discovery.


Assuntos
Ensaios de Triagem em Larga Escala , Testes de Função Plaquetária , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Testes de Função Plaquetária/métodos , Testes de Função Plaquetária/normas , Plasma Rico em Plaquetas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
Adv Sci (Weinh) ; : e2405949, 2024 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-39159239

RESUMO

Approaches to analyze and cluster T-cell receptor (TCR) repertoires to reflect antigen specificity are critical for the diagnosis and prognosis of immune-related diseases and the development of personalized therapies. Sequence-based approaches showed success but remain restrictive, especially when the amount of experimental data used for the training is scarce. Structure-based approaches which represent powerful alternatives, notably to optimize TCRs affinity toward specific epitopes, show limitations for large-scale predictions. To handle these challenges, TCRpcDist is presented, a 3D-based approach that calculates similarities between TCRs using a metric related to the physico-chemical properties of the loop residues predicted to interact with the epitope. By exploiting private and public datasets and comparing TCRpcDist with competing approaches, it is demonstrated that TCRpcDist can accurately identify groups of TCRs that are likely to bind the same epitopes. Importantly, the ability of TCRpcDist is experimentally validated to determine antigen specificities (neoantigens and tumor-associated antigens) of orphan tumor-infiltrating lymphocytes (TILs) in cancer patients. TCRpcDist is thus a promising approach to support TCR repertoire analysis and TCR deorphanization for individualized treatments including cancer immunotherapies.

7.
Methods Mol Biol ; 1614: 165-174, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28500603

RESUMO

Binding assay is a common technique used to characterize ability of a ligand to interact with a specific biological target. A number of parameters, such as binding affinity, receptor density, and association/dissociation rate constants, can be measured by means of this technique. In most cases, implementation of the binding assay requires specific infrastructure for labeling and detecting the ligand, which impedes realization of this technique in a standard laboratory. Here we describe a simple fluorescence-based binding assay for angiotensin peptides and receptors, which does not require complex equipment and can be used for initial screening of the novel ligands or mutational studies.


Assuntos
Angiotensinas/metabolismo , Fluorescência , Corantes Fluorescentes/química , Receptores de Angiotensina/análise , Animais , Ligação Competitiva , Células CHO , Cricetulus , Ligantes , Ligação Proteica
8.
Vascul Pharmacol ; 74: 103-113, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26304699

RESUMO

Angiotensin (Ang) II contributes to the development of atherosclerosis, while Ang-(1-7) has atheroprotective actions. Accordingly, angiotensin-converting enzyme 2 (ACE2), which breaks-down Ang II and forms Ang-(1-7), has been suggested as a target against atherosclerosis. Here we investigated the actions of diminazene, a recently developed ACE2 activator compound, in a model of vulnerable atherosclerotic plaque. Atherosclerotic plaque formation was induced in the carotid artery of ApoE-deficient mice by a shear stress (SS) modifier device. The animals were treated with diminazene (15mg/kg/day) or vehicle. ACE2 was strongly expressed in the aortic root and low SS-induced carotid plaques, but poorly expressed in the oscillatory SS-induced carotid plaques. Diminazene treatment did not change the lesion size, but ameliorated the composition of aortic root and low SS-induced carotid plaques by increasing collagen content and decreasing both MMP-9 expression and macrophage infiltration. Interestingly, these beneficial effects were not observed in the oscillatory SS-induced plaque. Additionally, diminazene treatment decreased intraplaque ICAM-1 and VCAM-1 expression, circulating cytokine and chemokine levels and serum triglycerides. In summary, ACE2 was distinctively expressed in atherosclerotic plaques, which depends on the local pattern of shear stress. Moreover, diminazene treatment enhances the stability of atherosclerotic plaques.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/tratamento farmacológico , Diminazena/farmacologia , Placa Aterosclerótica/metabolismo , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aterosclerose/metabolismo , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Molécula 1 de Adesão Intercelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/metabolismo , Placa Aterosclerótica/tratamento farmacológico , Molécula 1 de Adesão de Célula Vascular/metabolismo
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