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1.
BMC Biol ; 19(1): 124, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34134693

RESUMO

BACKGROUND: Doxorubicin (Dox) is an anti-cancer anthracycline drug that causes double-stranded DNA breaks. It is highly effective against several types of tumours; however, it also has adverse effects on regenerative populations of normal cells, such as human cardiac mesenchymal progenitor cells (hCmPCs), and its clinical use is limited by cardiotoxicity. Another known effect of Dox is nucleolar disruption, which triggers the ubiquitously expressed nucleolar phosphoprotein Nucleophosmin (NPM) to be released from the nucleolus into the cell, where it participates in the orchestration of cellular stress responses. NPM has also been observed in the extracellular space in response to different stress stimuli; however, the mechanism behind this and its functional implications are as yet largely unexplored. The aim of this study was to establish whether Dox could elicit NPM secretion in the extracellular space and to elucidate the mechanism of secretion and the effect of extracellular NPM on hCmPCs. RESULTS: We found that following the double-strand break formation in hCmPCs caused by Dox, NPM was rapidly secreted in the extracellular space by an active mechanism, in the absence of either apoptosis or necrosis. Extracellular release of NPM was similarly seen in response to ultraviolet radiation (UV). Furthermore, we observed an increase of NPM levels in the plasma of Dox-treated mice; thus, NPM release also occurred in vivo. The treatment of hCmPCs with extracellular recombinant NPM induced a decrease of cell proliferation and a response mediated through the Toll-like receptor (TLR)4. We demonstrated that NPM binds to TLR4, and via TLR4, and nuclear factor kappa B (NFkB) activation/nuclear translocation, exerts proinflammatory functions by inducing IL-6 and COX-2 gene expression. Finally, we found that in hCmPCs, NPM secretion could be driven by an autophagy-dependent unconventional mechanism that requires TLR4, since TLR4 inhibition dramatically reduced Dox-induced secretion. CONCLUSIONS: We hypothesise that the extracellular release of NPM could be a general response to DNA damage since it can be elicited by either a chemical agent such as Dox or a physical genotoxic stressor such as UV radiation. Following genotoxic stress, NPM acts similarly to an alarmin in hCmPCs, being rapidly secreted and promoting cell cycle arrest and a TLR4/NFκB-dependent inflammatory response.

2.
Vasc Biol ; 3(1): R1-R14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537555

RESUMO

Coronavirus disease 2019 (COVID-19) is a new infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is frequently characterized by a marked inflammatory response with severe pneumonia and respiratory failure associated with multiorgan involvement. Some risk factors predispose patients to develop a more severe infection and to an increased mortality; among them, advanced age and male gender have been identified as major and independent risk factors for COVID-19 poor outcome. The renin-angiotensin-aldosterone system (RAAS) is strictly involved in COVID-19 because angiotensin converting enzyme 2 (ACE2) is the host receptor for SARS-CoV-2 and also converts pro-inflammatory angiotensin (Ang) II into anti-inflammatory Ang(1-7). In this review, we have addressed the effect of aging and gender on RAAS with emphasis on ACE2, pro-inflammatory Ang II/Ang II receptor 1 axis and anti-inflammatory Ang(1-7)/Mas receptor axis.

3.
Sci Rep ; 11(1): 671, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436662

RESUMO

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo.


Assuntos
Animais Geneticamente Modificados/crescimento & desenvolvimento , Proliferação de Células , Evolução Molecular , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Fluorescência Verde/genética , Luciferases/genética , Regeneração , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
Vasc Biol ; 2(1): R45-R58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32923974

RESUMO

Regenerative medicine is a new therapeutic modality that aims to mend tissue damage by encouraging the reconstitution of physiological integrity. It represents an advancement over conventional therapies that allow reducing the damage but result in disease chronicization. Age-related decline in spontaneous capacity of repair, especially in organs like the heart that have very limited proliferative capacity, contributes in reducing the benefit of conventional therapy. ncRNAs are emerging as key epigenetic regulators of cardiovascular regeneration. Inhibition or replacement of miRNAs may offer reparative solutions to cardiovascular disease. The first part of this review article is devoted to illustrating novel therapies emerging from research on miRNAs. In the second part, we develop new therapeutic concepts emerging from genetics of longevity. Prolonged survival, as in supercentenarians, denotes an exceptional capacity to repair and cope with risk factors and diseases. These characteristics are shared with offspring, suggesting that the regenerative phenotype is heritable. New evidence indicates that genetic traits responsible for prolongation of health span in humans can be passed to and benefit the outcomes of animal models of cardiovascular disease. Genetic studies have also focused on determinants of accelerated senescence and related druggable targets. Evolutionary genetics assessing the genetic basis of adaptation and comparing successful and unsuccessful genetic changes in response to selection within populations represent a powerful basis to develop novel therapies aiming to prolong cardiovascular and whole organism health.

5.
Curr Probl Cardiol ; 45(12): 100406, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30704792

RESUMO

Aging is a major risk factor for heart failure, one of the leading causes of death in Western society. The mechanisms that underlie the different forms of heart failure have been elucidated only in part and the role of noncoding RNAs is still poorly characterized. Specifically, microRNAs (miRNAs), a class of small noncoding RNAs that can modulate gene expression at the posttranscriptional level in all cells, including myocardial and vascular cells, have been shown to play a role in heart failure with reduced ejection fraction. In contrast, miRNAs role in heart failure with preserved ejection fraction, the predominant form of heart failure in the elderly, is still unknown. In this review, we will focus on age-dependent miRNAs in heart failure and on some other conditions that are prevalent in the elderly and are frequently associated with heart failure with preserved ejection fraction.


Assuntos
Envelhecimento , Insuficiência Cardíaca , MicroRNAs , Idoso , Envelhecimento/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , MicroRNAs/genética
6.
JACC Basic Transl Sci ; 4(2): 248-250, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31061926
7.
Drug Deliv Transl Res ; 9(5): 935-944, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30859393

RESUMO

In rodents with acute myocardial infarction (AMI), high mobility group box 1 (HMGB1) injection has produced controversial results. Given the lack of data in large mammals, we searched the dose that would promote angiogenesis and expression of specific regenerative genes in sheep with AMI (protocol 1) and, subsequently, use this dose to study long-term effects on infarct size and left ventricular (LV) function (protocol 2). Protocol 1: Sheep with AMI received 250 µg (high-dose, n = 7), 25 µg (low-dose, n = 7) HMGB1, or PBS (placebo, n = 7) in 10 intramyocardial injections (0.2 ml each) in the peri-infarct area. Seven days later, only the high-HMGB1-dose group exhibited higher microvascular densities, Ki67-positive cardiomyocytes, and overexpression of VEGF, Ckit, Tbx20, Nkx2.5, and Gata4. Protocol 2: Sheep with AMI received HMGB1 250 µg (n = 6) or PBS (n = 6). At 60 days, HMGB1-treated sheep showed smaller infarcts (8.5 ± 2.11 vs. 12.2 ± 1.97% LV area, P < 0.05, ANOVA-Bonferroni) and higher microvascular density (capillaries, 1798 ± 252 vs. 1266 ± 250/mm2; arterioles, 18.3 ± 3.9 vs. 11.7 ± 2.2/mm2; both P < 0.01). Echocardiographic LV ejection fraction, circumferential shortening, and wall thickening increased from day 3 to 60 with HMGB1 (all P < 0.05). Conclusion: in ovine AMI, high-dose HMGB1 induces angio-arteriogenesis, reduces infarct size, and improves LV function at 2 months post-treatment.


Assuntos
Cardiotônicos/administração & dosagem , Proteína HMGB1/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Animais , Feminino , Masculino , Microvasos/efeitos dos fármacos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Ovinos , Função Ventricular Esquerda/efeitos dos fármacos
8.
Cell Mol Life Sci ; 76(2): 211-229, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30306212

RESUMO

High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein involved in transcription regulation, DNA replication and repair and nucleosome assembly. HMGB1 is passively released by necrotic tissues or actively secreted by stressed cells. Extracellular HMGB1 acts as a damage-associated molecular pattern (DAMPs) molecule and gives rise to several redox forms that by binding to different receptors and interactors promote a variety of cellular responses, including tissue inflammation or regeneration. Inhibition of extracellular HMGB1 in experimental models of myocardial ischemia/reperfusion injury, myocarditis, cardiomyopathies induced by mechanical stress, diabetes, bacterial infection or chemotherapeutic drugs reduces inflammation and is protective. In contrast, administration of HMGB1 after myocardial infarction induced by permanent coronary artery ligation ameliorates cardiac performance by promoting tissue regeneration. HMGB1 decreases contractility and induces hypertrophy and apoptosis in cardiomyocytes, stimulates cardiac fibroblast activities, and promotes cardiac stem cell proliferation and differentiation. Interestingly, maintenance of appropriate nuclear HMGB1 levels protects cardiomyocytes from apoptosis by preventing DNA oxidative stress, and mice with HMGB1cardiomyocyte-specific overexpression are partially protected from cardiac damage. Finally, higher levels of circulating HMGB1 are associated to human heart diseases. Hence, during cardiac injury, HMGB1 elicits both harmful and beneficial responses that may in part depend on the generation and stability of the diverse redox forms, whose specific functions in this context remain mostly unexplored. This review summarizes recent findings on HMGB1 biology and heart dysfunctions and discusses the therapeutic potential of modulating its expression, localization, and oxidative-dependent activities.


Assuntos
Proteína HMGB1/metabolismo , Cardiopatias/patologia , Alarminas/metabolismo , Animais , Biomarcadores/metabolismo , Cardiopatias/metabolismo , Humanos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocardite/metabolismo , Miocardite/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
9.
Clin Sci (Lond) ; 132(22): 2423-2436, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30389857

RESUMO

Early recognition of vulnerable carotid plaques could help in identifying patients at high stroke risk, who may benefit from earlier revascularisation. Nowadays, different biomarkers of plaque instability have been unravelled, among these miRNAs are promising tools for the diagnosis and treatment of atherosclerosis. Inflammation, reactive oxygen species (ROS) and endothelial dysfunction play a key role in unstable plaques genesis. We showed that miR-200c induces endothelial dysfunction, ROS production and a positive mechanism among miR-200c and miR-33a/b, two miRNAs involved in atherosclerosis progression. The goal of the present study was to determine whether miR-200c could be an atherosclerosis biomarker. Carotid plaques of patients that underwent carotid endarterectomy (CEA) were assayed for miR-200c expression. miR-200c was up-regulated in carotid plaques (n=22) and its expression was higher in unstable (n=12) compared with stable (n=10) plaques. miR-200c positively correlated with instability biomarkers (i.e. monocyte chemoattractant protein-1, cicloxigenase-2 (COX2), interleukin 6 (IL6), metalloproteinase (MMP) 1 (MMP1), 9 (MMP9)) and miR-33a/b. Moreover, miR-200c negatively correlated with stability biomarkers (i.e. zinc finger E-box binding homoeobox 1 (ZEB1), endothelial nitric oxide (NO) synthase (eNOS), forkhead boxO1 (FOXO1) and Sirtuin1 (SIRT1)) (stable plaques = 15, unstable plaques = 15). Circulating miR-200c was up-regulated before CEA in 24 patients, correlated with miR-33a/b and decreased 1 day after CEA. Interestingly, 1 month after CEA, circulating miR-200c is low in patients with stable plaques (n=11) and increased to control levels, in patients with unstable plaques (n=13). Further studies are needed to establish whether miR-200c represents a circulating biomarker of plaque instability. Our results show that miR-200c is an atherosclerotic plaque progression biomarker and suggest that it may be clinically useful to identify patients at high embolic risk.


Assuntos
Artérias Carótidas/patologia , Estenose das Carótidas/genética , MicroRNAs/genética , Placa Aterosclerótica , Idoso , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/cirurgia , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/patologia , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas , Feminino , Regulação da Expressão Gênica , Marcadores Genéticos , Humanos , Masculino , MicroRNAs/sangue , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco , Ruptura Espontânea , Fatores de Tempo , Resultado do Tratamento , Ultrassonografia
10.
Arterioscler Thromb Vasc Biol ; 38(9): 2079-2090, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30026277

RESUMO

Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.


Assuntos
Senescência Celular/fisiologia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sirtuína 1/metabolismo , Calcificação Vascular , Adulto , Envelhecimento/patologia , Animais , Aorta/metabolismo , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Fatores de Transcrição SOX9/metabolismo , Regulação para Cima , Adulto Jovem
12.
Cell Death Dis ; 8(8): e3020, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28837147

RESUMO

Doxorubicin (DOXO) treatment is limited by its cardiotoxicity, since it causes cardiac-progenitor-cell depletion. Although the cardioprotective role of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF1/CXCR4) axis is well established, its involvement during DOXO-induced cardiotoxicity has never been investigated. We showed that in a mouse model of DOXO-induced cardiomyopathy, CXCR4+ cells were increased in response to DOXO, mainly in human cardiac mesenchymal progenitor cells (CmPC), a subpopulation with regenerative potential. Our in vitro results showed a CXCR4 induction after 24 h of DOXO exposure in CmPC. SDF1 administration protected from DOXO-induced cell death and promoted CmPC migration. CXCR4 promoter analysis revealed zinc finger E-box binding homeobox 1 (ZEB1) binding sites. Upon DOXO treatment, ZEB1 binding decreased and RNA-polymerase-II increased, suggesting a DOXO-mediated transcriptional increase in CXCR4. Indeed, DOXO induced the upregulation of miR-200c, that directly targets ZEB1. SDF1 administration in DOXO-treated mice partially reverted the adverse remodeling, decreasing left ventricular (LV) end diastolic volume, LV ejection fraction and LV anterior wall thickness in diastole, recovering LV end systolic pressure and reducing±dP/dt. Moreover, in vivo administration of SDF1 partially reverted DOXO-induced miR-200c and p53 protein upregulation in mouse hearts. In addition, downmodulation of ZEB1 mRNA and protein by DOXO was significantly increased by SDF1. In keeping, p21 mRNA, that is induced by p53 and inhibited by ZEB1, is induced by DOXO treatment and is decreased by SDF1 administration. This study showed new players of the DOXO-induced cardiotoxicity, that can be exploited to ameliorate DOXO-associated cardiomyopathy.


Assuntos
Doxorrubicina/farmacologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Receptores CXCR4/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Receptores CXCR4/genética , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
13.
Biochim Biophys Acta Mol Basis Dis ; 1863(11): 2693-2704, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28716707

RESUMO

Myocardial infarction (MI) is a major health burden worldwide. Extracellular High mobility group box 1 (HMGB1) regulates tissue healing after injuries. The reduced form of HMGB1 (fr-HMGB1) exerts chemotactic activity by binding CXCL12 through CXCR4, while the disulfide form, (ds-HMGB1), induces cytokines expression by TLR4. Here, we assessed the role of HMGB1 redox forms and the non-oxidizable mutant (3S) on human cardiac fibroblast (hcFbs) functions and cardiac remodeling after infarction. Among HMGB1 receptors, hcFbs express CXCR4. Fr-HMGB1 and 3S, but not ds-HMGB1, promote hcFbs migration through Src activation, while none of HMGB1 redox forms induces proliferation or inflammatory mediators. 3S is more effective than fr-HMGB1 in stimulating hcFbs migration and Src phosphorylation being active at lower concentrations and in oxidizing conditions. Notably, chemotaxis toward both proteins is CXCR4-dependent but, in contrast to fr-HMGB1, 3S does not require CXCL12 since hcFbs migration persists in the presence of the CXCL12/CXCR4 inhibitor AMD3100 or an anti-CXCL12 antibody. Interestingly, 3S interacts with CXCR4 and induces a different receptor conformation than CXCL12. Mice undergoing MI and receiving 3S exhibit adverse LV remodeling owing to an excessive collagen deposition promoted by a higher number of myofibroblasts. On the contrary, fr-HMGB1 ameliorates cardiac performance enhancing neoangiogenesis and reducing the infarcted area and fibrosis. Altogether, our results demonstrate that non-oxidizable HMGB1 induce a sustained cardiac fibroblasts migration despite the redox state of the environment and by altering CXCL12/CXCR4 axis. This affects proper cardiac remodeling after an infarction.


Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Fibroblastos/metabolismo , Proteína HMGB1/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Receptores CXCR4/metabolismo , Feminino , Fibroblastos/patologia , Humanos , Masculino , Infarto do Miocárdio/patologia , Miocárdio/patologia , Oxirredução
15.
Aesthet Surg J ; 37(5): 591-602, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28052909

RESUMO

Background: Fat grafts enriched with cells of the stromal vascular fraction (SVF), especially adipose-derived stromal cells (ASCs), exhibit significantly improved retention over non enriched, plain fat. Different types of liposuction cannulae may yield lipoaspirates with different subpopulations of cells. Moreover, preparation of adipose tissue for transplantation typically involves centrifugation, which creates a density gradient of fat. Objectives: The authors sought to determine whether liposuction with a barbed or smooth cannula altered the enrichment of the SVF, and specifically ASCs, in low-density (LD) and high-density (HD) fractions of centrifuged adipose tissue. Methods: Fat was harvested from 2 abdominal sites of 5 healthy women with a barbed or smooth multihole blunt-end cannula. After centrifugation, LD and HD fat fractions were digested with collagenase and analyzed by polychromatic flow cytometry to identify and enumerate distinct populations of cells. Results: Overall cell yield and the number of immune cells were consistently higher in HD fractions than in LD fractions, regardless of the cannula employed. More living cells, and specifically more ASCs, populated the HD fractions of lipoaspirates obtained with a barbed cannula than with a smooth cannula. Conclusions: In this study, lipoaspiration with a barbed cannula and isolation of the HD layer of centrifuged adipose tissue yielded maximal amounts of SVF cells, including ASCs.


Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/transplante , Separação Celular/métodos , Lipectomia/instrumentação , Coleta de Tecidos e Órgãos/instrumentação , Transplantes/citologia , Adulto , Cânula , Centrifugação , Feminino , Citometria de Fluxo/métodos , Humanos , Lipectomia/métodos , Pessoa de Meia-Idade , Células Estromais/transplante , Coleta de Tecidos e Órgãos/métodos
16.
Oncotarget ; 8(2): 2628-2646, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27793050

RESUMO

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y-dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Lamina Tipo A/metabolismo , Complexos Multiproteicos/metabolismo , Transcrição Genética , Animais , Fator de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lamina Tipo A/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Elementos de Resposta
17.
Aging Cell ; 16(2): 262-272, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27995756

RESUMO

To understand why livers from aged donors are successfully used for transplants, we looked for markers of liver aging in 71 biopsies from donors aged 12-92 years before transplants and in 11 biopsies after transplants with high donor-recipient age-mismatch. We also assessed liver function in 36 age-mismatched recipients. The major findings were the following: (i) miR-31-5p, miR-141-3p, and miR-200c-3p increased with age, as assessed by microRNAs (miRs) and mRNA transcript profiling in 12 biopsies and results were validated by RT-qPCR in a total of 58 biopsies; (ii) telomere length measured by qPCR in 45 samples showed a significant age-dependent shortage; (iii) a bioinformatic approach combining transcriptome and miRs data identified putative miRs targets, the most informative being GLT1, a glutamate transporter expressed in hepatocytes. GLT1 was demonstrated by luciferase assay to be a target of miR-31-5p and miR-200c-3p, and both its mRNA (RT-qPCR) and protein (immunohistochemistry) significantly decreased with age in liver biopsies and in hepatic centrilobular zone, respectively; (iv) miR-31-5p, miR-141-3p and miR-200c-3p expression was significantly affected by recipient age (older environment) as assessed in eleven cases of donor-recipient extreme age-mismatch; (v) the analysis of recipients plasma by N-glycans profiling, capable of assessing liver functions and biological age, showed that liver function recovered after transplants, independently of age-mismatch, and recipients apparently 'rejuvenated' according to their glycomic age. In conclusion, we identified new markers of aging in human liver, their relevance in donor-recipient age-mismatches in transplantation, and offered positive evidence for the use of organs from old donors.


Assuntos
Envelhecimento/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Transplante de Fígado , Fígado/metabolismo , MicroRNAs/metabolismo , Doadores de Tecidos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Transportador 2 de Aminoácido Excitatório , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Imuno-Histoquímica , Luciferases/metabolismo , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Telômero/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Adulto Jovem
18.
Pharmacol Ther ; 171: 43-55, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27742569

RESUMO

Organ-specific mesenchymal cells naturally reside in the stroma, where they are exposed to some environmental variables affecting their biology and functions. Risk factors such as diabetes or aging influence their adaptive response. In these cases, permanent epigenetic modifications may be introduced in the cells with important consequences on their local homeostatic activity and therapeutic potential. Numerous results suggest that mesenchymal cells, virtually present in every organ, may contribute to tissue regeneration mostly by paracrine mechanisms. Intriguingly, the heart is emerging as a source of different cells, including pericytes, cardiac progenitors, and cardiac fibroblasts. According to phenotypic, functional, and molecular criteria, these should be classified as mesenchymal cells. Not surprisingly, in recent years, the attention on these cells as therapeutic tools has grown exponentially, although only very preliminary data have been obtained in clinical trials to date. In this review, we summarized the state of the art about the phenotypic features, functions, regenerative properties, and clinical applicability of mesenchymal cells, with a particular focus on those of cardiac origin.


Assuntos
Células-Tronco Mesenquimais/citologia , Miocárdio/citologia , Regeneração/fisiologia , Animais , Epigênese Genética , Fibroblastos/citologia , Coração/fisiologia , Humanos , Pericitos/citologia , Fenótipo , Fatores de Risco
20.
Int J Cardiol ; 212: 324-35, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27057951

RESUMO

BACKGROUND: Critical limb ischemia (CLI) is a major health problem with no adequate treatment. Since CLI is characterized by insufficient tissue vascularization, efforts have focused on the discovery of novel angiogenic factors. Cyclophilin A (CyPA) is an immunophilin that has been shown to promote angiogenesis in vitro and to enhance bone marrow (BM) cell mobilization in vivo. However, its potential as an angiogenic factor in CLI is still unknown. Thus, this study aimed to evaluate whether CyPA might induce neo-angiogenesis in ischemic tissues. METHODS AND RESULTS: Wild-type C57Bl/6j mice underwent acute hind-limb ischemia (HLI) and received a single intramuscular administration of recombinant CyPA or saline. Limb perfusion, capillary density and arteriole number in adductor muscles were significantly increased after CyPA treatment. Interestingly, BM-derived CD117(+) cell recruitment was significantly higher in ischemic adductor tissue of mice treated with CyPA versus saline. Therefore, the effect of CyPA on isolated BM-derived CD117(+) cells in vitro was evaluated. Low concentrations of CyPA stimulated CD117(+) cell proliferation while high concentrations promoted cell death. Moreover, CyPA enhanced CD117(+) cell adhesion and migration in a dose-dependent manner. Mechanistic studies revealed that CyPA up-regulated CXCR4 in CD117(+) cells and in adductor muscles after ischemia. Additionally, SDF-1/CXCR4 axis inhibition by the CXCR4 antagonist AMD3100 decreased CyPA-mediated CD117(+) cell recruitment in the ischemic limb. CONCLUSION: CyPA induces neo-angiogenesis by recruiting BM-derived CD117(+) cell into ischemic tissues, at least in part, through SDF-1/CXCR4 axis.


Assuntos
Ciclofilina A/administração & dosagem , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Ciclofilina A/farmacologia , Modelos Animais de Doenças , Membro Posterior/efeitos dos fármacos , Humanos , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos
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