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1.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
2.
J Cell Physiol ; 236(7): 4857-4873, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33432663

RESUMO

Cardiovascular diseases (CVDs) are the leading cause of death globally and the number of cardiovascular patients, which is estimated to be over 30 million in 2018, represent a challenging issue for the healthcare systems worldwide. Therefore, the identification of novel molecular targets to develop new treatments is an ongoing challenge for the scientific community. In this context, sphingolipids (SLs) have been progressively recognized as potent bioactive compounds that play crucial roles in the modulation of several key biological processes, such as proliferation, differentiation, and apoptosis. Furthermore, SLs involvement in cardiac physiology and pathophysiology attracted much attention, since these molecules could be crucial in the development of CVDs. Among SLs, ceramide and sphingosine-1-phosphate (S1P) represent the most studied bioactive lipid mediators, which are characterized by opposing activities in the regulation of the fate of cardiac cells. In particular, maintaining the balance of the so-called ceramide/S1P rheostat emerged as an important novel therapeutical target to counteract CVDs. Thus, this review aims at critically summarizing the current knowledge about the antithetic roles of ceramide and S1P in cardiomyocytes dysfunctions, highlighting how the modulation of their metabolism through specific molecules, such as myriocin and FTY720, could represent a novel and interesting therapeutic approach to improve the management of CVDs.


Assuntos
Ceramidas/metabolismo , Transtornos Cerebrovasculares/patologia , Lisofosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Idoso , Animais , Transtornos Cerebrovasculares/epidemiologia , Transtornos Cerebrovasculares/mortalidade , Doença das Coronárias/patologia , Humanos , Camundongos , Doença Arterial Periférica/patologia , Embolia Pulmonar/patologia , Cardiopatia Reumática/patologia , Esfingosina/metabolismo , Trombose Venosa/patologia
3.
Front Cell Dev Biol ; 8: 593508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33262987

RESUMO

Herein we unveil that Hypoxia-inducible factor-1α (HIF-1α) directly regulates WNT7A expression during myogenesis. In fact, chromatin immunoprecipitation (ChiP) and site-directed mutagenesis experiments revealed two distinct hypoxia response elements (HREs) that are specific HIF-1α binding sites on the WNT7A promoter. Remarkably, a pharmacological activation of HIF-1α induced WNT7A expression and enhanced muscle differentiation. On the other hand, silencing of WNT7A using CRISPR/Cas9 genome editing blocked the effects of HIF-1α activation on myogenesis. Finally, treatment with prolyl hydroxylases (PHDs) inhibitors improved muscle regeneration in vitro and in vivo in a cardiotoxin (CTX)-induced muscle injury mouse model, paving the way for further studies to test its efficacy on acute and chronic muscular pathologies.

4.
Biochem J ; 477(17): 3401-3415, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32869836

RESUMO

Cardiac fibrosis is a key physiological response to cardiac tissue injury to protect the heart from wall rupture. However, its progression increases heart stiffness, eventually causing a decrease in heart contractility. Unfortunately, to date, no efficient antifibrotic therapies are available to the clinic. This is primarily due to the complexity of the process, which involves several cell types and signaling pathways. For instance, the transforming growth factor beta (TGF-ß) signaling pathway has been recognized to be vital for myofibroblasts activation and fibrosis progression. In this context, complex sphingolipids, such as ganglioside GM3, have been shown to be directly involved in TGF-ß receptor 1 (TGF-R1) activation. In this work, we report that an induced up-regulation of sialidase Neu3, a glycohydrolytic enzyme involved in ganglioside cell homeostasis, can significantly reduce cardiac fibrosis in primary cultures of human cardiac fibroblasts by inhibiting the TGF-ß signaling pathway, ultimately decreasing collagen I deposition. These results support the notion that modulating ganglioside GM3 cell content could represent a novel therapeutic approach for cardiac fibrosis, warranting for further investigations.


Assuntos
Fibroblastos/metabolismo , Gangliosídeo G(M3)/metabolismo , Regulação Enzimológica da Expressão Gênica , Miocárdio/metabolismo , Neuraminidase/biossíntese , Regulação para Cima , Fibroblastos/patologia , Fibrose , Humanos , Miocárdio/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo
5.
Bioorg Med Chem ; 28(14): 115563, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32616179

RESUMO

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hemaglutininas/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Vírus da Doença de Newcastle/efeitos dos fármacos , Ácidos Siálicos/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hemaglutininas/metabolismo , Humanos , Estrutura Molecular , Neuraminidase/metabolismo , Vírus da Doença de Newcastle/enzimologia , Ácidos Siálicos/síntese química , Ácidos Siálicos/química , Relação Estrutura-Atividade
6.
Cells ; 9(3)2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204578

RESUMO

Bone is an active tissue where bone mineralization and resorption occur simultaneously. In the case of fracture, there are numerous factors required to facilitate bone healing including precursor cells and blood vessels. To evaluate the interaction between bone marrow-derived mesenchymal stem cells (BMSC)-the precursor cells able to differentiate into bone-forming cells and human umbilical vein endothelial cells (HUVEC)-a cell source widely used for the study of blood vessels. We performed direct coculture of BMSC and HUVEC in normoxia and chemically induced hypoxia using Cobalt(II) chloride and Dimethyloxaloylglycine and in the condition where oxygen level was maintained at 1% as well. Cell proliferation was analyzed by crystal violet staining. Osteogenesis was examined by Alizarin Red and Collagen type I staining. Expression of angiogenic factor-vascular endothelial growth factor (VEGF) and endothelial marker-von Willebrand factor (VWF) were demonstrated by immunohistochemistry and enzyme-linked immunosorbent assay. The quantitative polymerase chain reaction was also used to evaluate gene expression. The results showed that coculture in normoxia could retain both osteogenic differentiation and endothelial markers while hypoxic condition limits cell proliferation and osteogenesis but favors the angiogenic function even after 1 of day treatment.


Assuntos
Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Osteogênese , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Hipóxia Celular , Proliferação de Células , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fator de von Willebrand/metabolismo
7.
Magn Reson Imaging ; 68: 127-135, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32004712

RESUMO

Preclinical cardiac MR is challenging and time-consuming. A fast and comprehensive acquisition protocol and standardized image post-processing may improve preclinical research, reducing acquisition time, costs and variability of results. In the present study, we evaluated the feasibility of a contrast-enhanced 3D IntraGate steady-state cine sequence (ce-3D-IG-cine) with short acquisition time (11 min) for a single-shot combined characterization of left ventricle (LV) remodeling and infarct size (IS) in a mouse model of acute ischemia-reperfusion injury. Sixteen male C57BL/6N mice underwent 7T cardiac MR (Bruker, BioSpec 70/30) including optimized ce-3D-IG-cine (total scan time 11 min) at day 1, 5 and 28 after surgery. LV end-diastolic volume (EDVMR) and ejection fraction (EFMR) extracted from MR were compared to ones from short-axis (SA-EDVecho, SA-EFecho) and parasternal long-axis (LA-EDVecho, LA-EFecho) echocardiography. IS was manually and semiautomatically segmented from ce-3D-IG-cine using different standard deviation (SD +2, +3, +4, +5, +6 in respect to a reference tissue). Mice were sacrificed at day 28, immediately after imaging. IS at day 28 was compared to injury burden at histology. MR and echocardiographic morpho-functional parameters were compared, as IS from MR and histology. Bland-Altman plots were used to assess the agreement in ischemic burden segmentation. Volumetric and functional parameters measured on ce-3D-IG-cine correlated to the correspondent echocardiographic parameter (EDVMR vs SA-EDVecho: ρ = 0.813; EDVMR vs LA-EDVecho: ρ = 0.845; EFMR vs SA-EFecho ρ = 0.612; EFMR vs LA-EFecho ρ = 0.791; p < 0.001 in all cases). Manually segmented IS strongly correlated with the scar at histology (ρ = 0.904, p < 0.001). A threshold of +3SD showed the highest performance for semiautomatic assessment of IS compared to manual segmentation (ρ = 0.965, p < 0.001), with an overall reproducibility of 73%, and a peak reproducibility of 80% at day 1. The ce-3D-IG-cine sequence, manually or semiautomatically segmented using 3SD threshold, allows fast and comprehensive LV morpho-functional and structural characterization in myocardial ischemia-reperfusion injury model.


Assuntos
Ecocardiografia , Ventrículos do Coração/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética , Traumatismo por Reperfusão/diagnóstico por imagem , Animais , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes
8.
Curr Med Chem ; 27(21): 3448-3462, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30605049

RESUMO

The development of new therapeutic applications for adult and embryonic stem cells has dominated regenerative medicine and tissue engineering for several decades. However, since 2006, induced Pluripotent Stem Cells (iPSCs) have taken center stage in the field, as they promised to overcome several limitations of the other stem cell types. Nonetheless, other promising approaches for adult cell reprogramming have been attempted over the years, even before the generation of iPSCs. In particular, two years before the discovery of iPSCs, the possibility of synthesizing libraries of large organic compounds, as well as the development of high-throughput screenings to quickly test their biological activity, enabled the identification of a 2,6-disubstituted purine, named reversine, which was shown to be able to reprogram adult cells to a progenitor-like state. Since its discovery, the effect of reversine has been confirmed on different cell types, and several studies on its mechanism of action have revealed its central role in inhibitory activity on several kinases implicated in cell cycle regulation and cytokinesis. These key features, together with its chemical nature, suggested a possible use of the molecule as an anti-cancer drug. Remarkably, reversine exhibited potent cytotoxic activity against several tumor cell lines in vitro and a significant effect in decreasing tumor progression and metastatization in vivo. Thus, 15 years since its discovery, this review aims at critically summarizing the current knowledge to clarify the dual role of reversine as a dedifferentiating agent and anti-cancer drug.


Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Morfolinas , Purinas
9.
J Org Chem ; 84(9): 5460-5470, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30892893

RESUMO

Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.


Assuntos
Lactonas/química , Ácido N-Acetilneuramínico/química , Estereoisomerismo
10.
Int J Mol Sci ; 19(10)2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-30332812

RESUMO

Despite considerable improvements in the treatment of cardiovascular diseases, heart failure (HF) still represents one of the leading causes of death worldwide. Poor prognosis is mostly due to the limited regenerative capacity of the adult human heart, which ultimately leads to left ventricular dysfunction. As a consequence, heart transplantation is virtually the only alternative for many patients. Therefore, novel regenerative approaches are extremely needed, and several attempts have been performed to improve HF patients' clinical conditions by promoting the replacement of the lost cardiomyocytes and by activating cardiac repair. In particular, cell-based therapies have been shown to possess a great potential for cardiac regeneration. Different cell types have been extensively tested in clinical trials, demonstrating consistent safety results. However, heterogeneous efficacy data have been reported, probably because precise end-points still need to be clearly defined. Moreover, the principal mechanism responsible for these beneficial effects seems to be the paracrine release of antiapoptotic and immunomodulatory molecules from the injected cells. This review covers past and state-of-the-art strategies in cell-based heart regeneration, highlighting the advantages, challenges, and limitations of each approach.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Coração/fisiologia , Regeneração/fisiologia , Animais , Ensaios Clínicos como Assunto , Humanos , Modelos Biológicos
11.
Stem Cells Int ; 2018: 4706943, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30210549

RESUMO

Gangliosides, the sialic acid-conjugated glycosphingolipids present in the lipid rafts, have been recognized as important regulators of cell proliferation, migration, and apoptosis. Due to their peculiar localization in the cell membrane, they modulate the activity of several key cell receptors, and increasing evidence supports their involvement also in stem cell differentiation. In this context, herein we report the role played by the ganglioside GM1 in the osteogenic differentiation of human tendon stem cells (hTSCs). In particular, we found an increase of GM1 levels during osteogenesis that is instrumental for driving the process. In fact, supplementation of the ganglioside in the medium significantly increased the osteogenic differentiation capability of hTSCs. Mechanistically, we found that GM1 supplementation caused a reduction in the phosphorylation of the platelet-derived growth factor receptor-ß (PDGFR-ß), which is a known inhibitor of osteogenic commitment. These results were further corroborated by the observation that GM1 supplementation was able to revert the inhibitory effects on osteogenesis when the process was inhibited with exogenous PDGF.

12.
ChemMedChem ; 13(3): 236-240, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29231283

RESUMO

Neuraminidase activity is essential for the infection and propagation of paramyxoviruses, including human parainfluenza viruses (hPIVs) and the Newcastle disease virus (NDV). Thus, many inhibitors have been developed based on the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid inhibitor (DANA) backbone. Along this line, herein we report a series of neuraminidase inhibitors, having C4 (p-toluenesulfonamido and azido substituents) and C5 (N-perfluorinated chains) modifications to the DANA backbone, resulting in compounds with 5- to 15-fold greater potency than the currently most active compound, the N-trifluoroacetyl derivative of DANA (FANA), toward the NDV hemagglutinin-neuraminidase (NDV-HN). Remarkably, these inhibitors were found to be essentially inactive against the human sialidase NEU3, which is present on the outer layer of the cell membrane and is highly affected by the current NDV inhibitor FANA.


Assuntos
Antivirais/síntese química , Azidas/síntese química , Proteína HN/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Vírus da Doença de Newcastle/metabolismo , Sulfonamidas/síntese química , Antivirais/química , Azidas/química , Células HEK293 , Humanos , Ácido N-Acetilneuramínico/química , Neuraminidase/antagonistas & inibidores , Ligação Proteica , Relação Estrutura-Atividade , Sulfonamidas/química
13.
FASEB J ; 31(5): 2146-2156, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28188178

RESUMO

Regeneration of skeletal muscle is a complex process that requires the activation of quiescent adult stem cells, called satellite cells, which are resident in hypoxic niches in the tissue. Hypoxia has been recognized as a key factor to maintain stem cells in an undifferentiated state. Herein we report that hypoxia plays a fundamental role also in activating myogenesis. In particular, we found that the activation of the hypoxia-inducible factor (HIF)-1α under hypoxia, in murine skeletal myoblasts, leads to activation of MyoD through the noncanonical Wnt/ß-catenin pathway. Moreover, chemical inhibition of HIF-1α activity significantly reduces differentiation, thus confirming its crucial role in the process. Furthermore, hypoxia-preconditioned myoblasts, once induced to differentiate under normoxic conditions, tend to form hypertrophic myotubes. These results support the notion that hypoxia plays a pivotal role in activating the regeneration process by directly inducing myogenesis through HIF-1α. Although preliminary, these findings may suggest new perspective for novel therapeutic targets in the treatment of several muscle diseases.-Cirillo, F., Resmini, G., Ghiroldi, A., Piccoli, M., Bergante, S., Tettamanti, G., Anastasia, L. Activation of the hypoxia-inducible factor 1α promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Hipertrofia/metabolismo , Camundongos , Desenvolvimento Muscular/fisiologia , Mioblastos Esqueléticos/metabolismo , RNA Mensageiro/metabolismo , beta Catenina/metabolismo
14.
Int J Cardiol ; 230: 6-13, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28038803

RESUMO

BACKGROUND: Hypoxia is a common feature of many congenital heart defects (CHDs) and significantly contributes to their pathophysiology. Thus, understanding the mechanism underlying cell response to hypoxia is vital for the development of novel therapeutic strategies. Certainly, the hypoxia inducible factor (HIF) has been extensively investigated and it is now recognized as the master regulator of cell defense machinery counteracting hypoxic stress. Along this line, we recently discovered and reported a novel mechanism of HIF activation, which is mediated by sialidase NEU3. Thus, aim of this study was to test whether NEU3 played any role in the cardiac cell response to chronic hypoxia in congenital cyanotic patients. METHODS: Right atrial appendage biopsies were obtained from pediatric patients with cyanotic/non-cyanotic CHDs and processed to obtain mRNA and proteins. Real-Time PCR and Western Blot were performed to analyze HIF-1α and its downstream targets expression, NEU3 expression, and the NEU3 mediated effects on the EGFR signaling cascade. RESULTS: Cyanotic patients showed increased levels of HIF-1α, NEU3, EGFR and their downstream targets, as compared to acyanotic controls. The same patients were also characterized by increased phosphorylation of the EGFR signaling cascade proteins. Moreover, we found that HIF-1α expression levels positively correlated with those recorded for NEU3 in both cyanotic and control patients. CONCLUSIONS: Sialidase NEU3 plays a central role in activating cell response to chronic hypoxia inducing the up-regulation of HIF-1α, and this represent a possible novel tool to treat several CHD pathologies.


Assuntos
Cianose/metabolismo , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Neuraminidase/metabolismo , Estudos de Casos e Controles , Pré-Escolar , Doença Crônica , Cianose/complicações , Feminino , Humanos , Hipóxia/etiologia , Lactente , Masculino
15.
Clin Chim Acta ; 463: 122-128, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27780717

RESUMO

BACKGROUND: Myotonic dystrophy (DM) is a genetic disorder caused by nucleotide repeats expansion. Sudden death represents the main cause of mortality in DM patients. Here, we investigated the relationship between serum cardiac biomarkers with clinical parameters in DM patients. METHODS: Case-control study included 59 DM patients and 22 healthy controls. An additional group of 62 controls with similar cardiac defects to DM were enrolled. RESULTS: NT-proBNP, hs-cTnT and CK levels were significantly increased in DM patients compared to healthy subjects (p=0.0008, p<0.0001, p<0.0001). Also, hs-cTnT levels were significantly higher in DM compared to control group with cardiac defects (p=0.0003). Positive correlation was found between hs-cTnT and hs-cTnI in both DM patients and controls (p=0.019, p=0.002). Independently from the age, the risk of DM disease was positively related to an increase in hs-cTnT (p=0.03). On the contrary, the risk of DM was not related to hs-cTnI, but was evidenced a role of PR interval (p=0.03) and CK (p=0.08). CONCLUSIONS: The levels of hs-cTnT were significantly higher in DM patients. Analysis, with anti-cTnT, shows that this increase might be linked to heart problems. This last finding suggests that hs-cTnT might represent a helpful serum biomarker to "predict" cardiac risk in DM disease.


Assuntos
Cardiopatias/sangue , Cardiopatias/diagnóstico , Distrofia Miotônica/sangue , Distrofia Miotônica/complicações , Troponina T/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Cardiopatias/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco
16.
J Biol Chem ; 291(20): 10615-24, 2016 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-26987901

RESUMO

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.


Assuntos
Neuraminidase/metabolismo , Membrana Celular/enzimologia , Endossomos/enzimologia , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Neuraminidase/química , Neuraminidase/genética , Dobramento de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Regulação para Cima
17.
Eur Heart J Suppl ; 18(Suppl E): E1-E7, 2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-28533708

RESUMO

The possibility of generating induced pluripotent stem cells from mouse embryonic fibroblasts and human adult fibroblasts has introduced new perspectives for possible therapeutic strategies to repair damaged hearts. However, obtaining large numbers of adult stem cells is still an ongoing challenge, and the safety of genetic reprogramming with lenti- or retro-viruses has several drawbacks not easy to be addressed. Furthermore, the majority of adult stem cell-based clinical trials for heart regeneration have had generally poor and controversial results. Nonetheless, it is now clear that the injected cells activate the growth and differentiation of progenitor cells that are already present in the heart. This is achieved by the release of signalling factors and/or exosomes carrying them. Along this line, chemistry may play a major role in developing new strategies for activating resident stem cells to regenerate the heart. In particular, this review focuses on small molecule approaches for cell reprogramming, cell differentiation, and activation of cell protection.

18.
Chemistry ; 21(41): 14614-29, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26397189

RESUMO

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.


Assuntos
Ácido N-Acetilneuramínico Citidina Monofosfato/química , Citidina Monofosfato/análogos & derivados , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Receptores ErbB/química , Rim/enzimologia , Ácidos Siálicos/química , Ácidos Siálicos/síntese química , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/química , Citidina Monofosfato/síntese química , Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Humanos , Rim/química , Sialiltransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
J Lipid Res ; 55(3): 549-60, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24449473

RESUMO

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Gangliosídeos/metabolismo , Células-Tronco/metabolismo , Fosfatase Alcalina/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Derme/citologia , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Gangliosídeos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismo , Células-Tronco/citologia
20.
Am J Sports Med ; 41(7): 1653-64, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23393078

RESUMO

BACKGROUND: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. HYPOTHESIS: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. STUDY DESIGN: Controlled laboratory study. METHODS: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. RESULTS: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types--namely, osteoblasts, adipocytes, and skeletal muscle cells. CONCLUSION: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. CLINICAL RELEVANCE: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.


Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular , Células-Tronco Multipotentes/citologia , Manguito Rotador/citologia , Células-Tronco Adultas/fisiologia , Idoso , Diferenciação Celular/fisiologia , Matriz Extracelular/fisiologia , Feminino , Homeostase/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Multipotentes/fisiologia , Esfingolipídeos/fisiologia
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