RESUMO
The adult heart displays poor reparative capacities after injury. Cell transplantation and tissue engineering approaches have emerged as possible therapeutic options. Several stem cell populations have been largely used to treat the infarcted myocardium. Nevertheless, transplanted cells displayed limited ability to establish functional connections with the host cardiomyocytes. In this study, we provide a new experimental tool, named 3D eX vivo muscle engineered tissue (X-MET), to define the contribution of mechanical stimuli in triggering functional remodeling and to rescue cardiac ischemia. We revealed that mechanical stimuli trigger a functional remodeling of the 3D skeletal muscle system toward a cardiac muscle-like structure. This was supported by molecular and functional analyses, demonstrating that remodeled X-MET expresses relevant markers of functional cardiomyocytes, compared to unstimulated and to 2D- skeletal muscle culture system. Interestingly, transplanted remodeled X-MET preserved heart function in a murine model of chronic myocardial ischemia and increased survival of transplanted injured mice. X-MET implantation resulted in repression of pro-inflammatory cytokines, induction of anti-inflammatory cytokines, and reduction in collagen deposition. Altogether, our findings indicate that biomechanical stimulation induced a cardiac functional remodeling of X-MET, which showed promising seminal results as a therapeutic product for the development of novel strategies for regenerative medicine.
Assuntos
Isquemia Miocárdica , Camundongos , Animais , Isquemia Miocárdica/terapia , Miocárdio , Miócitos Cardíacos , Engenharia Tecidual/métodos , Fenômenos Fisiológicos CardiovascularesRESUMO
Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis. Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR. Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.
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Circular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1. Particularly, the interaction with CKAP5 mRNA, which interestingly overlaps the back-splicing junction, enhances CKAP5 translation, regulating microtubule function in cancer cells and sustaining cell-cycle progression. Finally, we show that circZNF609 downregulation increases the sensitivity of several cancer cell lines to different microtubule-targeting chemotherapeutic drugs and that locked nucleic acid (LNA) protectors against the pairing region on circZNF609 phenocopy such effects. These data set an example of how the small effects tuned by circZNF609/CKAP5 mRNA interaction might have a potent output in tumor growth and drug response.
Assuntos
Carcinogênese , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neoplasias/metabolismo , RNA Circular/metabolismo , RNA Mensageiro/metabolismo , Animais , Antineoplásicos/farmacologia , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células K562 , Masculino , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Microtúbulos/patologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , RNA Circular/genética , RNA Mensageiro/genética , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: We tested the hypothesis that chronic treatment with the direct renin inhibitor aliskiren improves vascular function in resistance and conduit arteries of type two diabetic and hypertensive patients. METHOD: Sixteen patients with mild essential hypertension and with a previous diagnosis of noninsulin-dependent diabetes mellitus were included in the study. Patients were then randomized to aliskiren (150âmg once daily, nâ=â9), or ramipril (5âmg once daily, nâ=â7). Each patient underwent a biopsy of the subcutaneous tissue and small arteries were dissected and mounted on a pressurized micromyograph to evaluate endothelium dependent vasorelaxation in response to acetylcholineâ±âN omega-nitro-L-arginine methyl ester hydrochloride in vessels precontracted with norepinephrine. Endothelial function has been quantified also in large conduit arteries by flow-mediated dilation. RESULTS: A similar office blood pressure-lowering effect was observed with the two drugs, although changes in DBP were not statistically significant in the ramipril group. Aliskiren significantly improved endothelium-dependent relaxation in subcutaneous resistance arteries, as well as increased flow-mediated dilation in conduit arteries, whereas the effects induced by ramipril did not reach statistical significance. Only aliskiren significantly increased the expression of p1177-endothelial nitric oxide synthase in the endothelium. Both aliskiren and ramipril had a negligible effect on markers of oxidative stress. CONCLUSION: Aliskiren restored endothelial function and induced a more prompt peripheral vasodilation in hypertensive and diabetic patients possibly through the increased production of nitric oxide via the enhanced expression and function of the active phosphorylated form of endothelial nitric oxide synthase.
Assuntos
Diabetes Mellitus , Hipertensão , Amidas/farmacologia , Pressão Sanguínea , Endotélio Vascular , Fumaratos/farmacologia , Humanos , Hipertensão/tratamento farmacológico , Óxido Nítrico , Renina , VasodilataçãoRESUMO
In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Imidazóis/farmacologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neuroblastoma/tratamento farmacológico , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos NOD , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Neuroblastoma/imunologia , Neuroblastoma/patologia , Receptores de Células Matadoras Naturais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.
Assuntos
Neoplasias Cerebelares/tratamento farmacológico , Cromonas/administração & dosagem , Portadores de Fármacos/química , Proteínas Hedgehog/antagonistas & inibidores , Meduloblastoma/tratamento farmacológico , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Colanos/química , Cromonas/farmacocinética , Modelos Animais de Doenças , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Transgênicos , Micelas , Polietilenoglicóis/química , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Distribuição TecidualRESUMO
Angiotensin (1-7) production increases during AT1R (angiotensin type-1 receptor) blockade. The contribution of Ang (1-7) (angiotensin [1-7]) and its receptor (MasR) to the favorable effect of angiotensin receptor blockers on remodeling and function of resistance arteries remains unclear. We sought to determine whether MasR contributes to the improvement of vascular structure and function during chronic AT1R blockade. Spontaneously hypertensive rats were treated with Ang (1-7) or olmesartan ± MasR antagonist A-779, or vehicle, for 14 days. Blood pressure was measured by tail cuff methodology. Mesenteric arteries were dissected and mounted on a pressurized micromyograph to evaluate media-to-lumen ratio (M/L) and endothelial function. Expression of MasR and eNOS (endothelial nitric oxide synthase) was evaluated by immunoblotting, plasma nitrate by colorimetric assay, and reactive oxygen species production by dihydroethidium staining. Independently of blood pressure, olmesartan significantly reduced M/L and improved NO bioavailability, A-779 prevented these effects. Likewise, Ang (1-7) significantly reduced M/L and NO bioavailability. MasR expression was significantly increased by Ang (1-7) as well as by olmesartan, and it was blunted in the presence of A-779. Both Ang (1-7) and olmesartan increased eNOS expression and plasma nitrite which were reduced by A-779. Superoxide generation was attenuated by olmesartan and Ang (1-7) and was blunted in the presence of A-779. These MasR-mediated actions were independent of AT2R activation since olmesartan and Ang (1-7) increased MasR expression and reduced M/L in Ang II (angiotensin II)-infused AT2R knockout mice, independently of blood pressure control. A-779 prevented these effects. Hence, MasR activation may contribute to the favorable effects of AT1R antagonism on NO bioavailability and microvascular remodeling, independently of AT2R activation and blood pressure control.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Hipertensão/fisiopatologia , Imidazóis/farmacologia , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Tetrazóis/farmacologia , Remodelação Vascular/efeitos dos fármacos , Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Pressão Sanguínea/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/metabolismo , Fragmentos de Peptídeos/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Ratos Endogâmicos SHR , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Remodelação Vascular/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy. Here we demonstrate that Notch3 is able to sustain UPR in T-ALL cells, as Notch3 silencing favored a Bip-dependent IRE1α inactivation under ER stress conditions, leading to increased apoptosis via upregulation of the ER stress cell death mediator CHOP. By using Juglone, a naturally occurring naphthoquinone acting as an anticancer agent, to decrease Notch3 expression and induce ER stress, we observed an increased ER stress-associated apoptosis. Altogether our results suggest that Notch3 inhibition may prevent leukemia cells from engaging a functional UPR needed to compensate the Juglone-mediated ER proteotoxic stress. Notably, in vivo administration of Juglone to human T-ALL xenotransplant models significantly reduced tumor growth, finally fostering the exploitation of Juglone-dependent Notch3 inhibition to perturb the ER stress/UPR signaling in Notch3-dependent T-ALL subsets.
RESUMO
The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Correpressoras/efeitos dos fármacos , Proteínas Hedgehog/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Neoplasias/tratamento farmacológico , Fenformin/uso terapêutico , Animais , Antineoplásicos/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Camundongos , Fenformin/farmacologiaRESUMO
Colorectal cancer is characterized by well-known genetic defects and approximately 50% of cases harbor oncogenic Ras mutations. Increased expression of Notch ligand Jagged1 occurs in several human malignancies, including colorectal cancer, and correlates with cancer progression, poor prognosis, and recurrence. Herein, we demonstrated that Jagged1 was constitutively processed in colorectal cancer tumors with mutant Kras, which ultimately triggered intrinsic reverse signaling via its nuclear-targeted intracellular domain Jag1-ICD. This process occurred when Kras/Erk/ADAM17 signaling was switched on, demonstrating that Jagged1 is a novel target of the Kras signaling pathway. Notably, Jag1-ICD promoted tumor growth and epithelial-mesenchymal transition, enhancing colorectal cancer progression and chemoresistance both in vitro and in vivo. These data highlight a novel role for Jagged1 in colorectal cancer tumor biology that may go beyond its effect on canonical Notch activation and suggest that Jag1-ICD may behave as an oncogenic driver that is able to sustain tumor pathogenesis and to confer chemoresistance through a noncanonical mechanism. SIGNIFICANCE: These findings present a novel role of the transcriptionally active Jag1-ICD fragment to confer and mediate some of the activity of oncogenic KRAS.
Assuntos
Proteína ADAM17/genética , Neoplasias Colorretais/genética , Proteína Jagged-1/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Células HCT116 , Células HT29 , Humanos , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologiaRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease in which dystrophin gene is mutated, resulting in dysfunctional or absent dystrophin protein. The pathology of dystrophic muscle includes degeneration, necrosis with inflammatory cell invasion, regeneration, and fibrous and fatty changes. Nevertheless, the mechanisms by which the absence of dystrophin leads to muscle degeneration remain to be fully elucidated. An imbalance between oxidant and antioxidant systems has been proposed as a secondary effect of DMD. However, the significance and precise extent of the perturbation in redox signaling cascades is poorly understood. We report that mdx dystrophic mice are able to activate a compensatory antioxidant response at the presymptomatic stage of the disease. In contrast, increased circulating levels of IL-6 perturb the redox signaling cascade, even prior to the necrotic stage, leading to severe features and progressive nature of muscular dystrophy.
Assuntos
Interleucina-6/sangue , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/sangue , Transdução de Sinais , Animais , Diafragma/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Necrose , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Skeletal muscle myopathy is universal in cirrhotic patients, however, little is known about the main mechanisms involved. The study aims to investigate skeletal muscle morphological, histological, and functional modifications in experimental models of cirrhosis and the principal molecular pathways responsible for skeletal muscle myopathy. Cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4) administration in mice. Control animals (CTR) underwent bile duct exposure or vehicle administration only. At sacrifice, peripheral muscles were dissected and weighed. Contractile properties of extensor digitorum longus (EDL) were studied in vitro. Muscle samples were used for histological and molecular analysis. Quadriceps muscle histology revealed a significant reduction in cross-sectional area of muscle and muscle fibers in cirrhotic mice with respect to CTR. Kinetic properties of EDL in both BDL and CCl4 were reduced with respect to CTR; BDL mice also showed a reduction in muscle force and a decrease in the resistance to fatigue. Increase in myostatin expression associated with a decrease in AKT-mTOR expressions was observed in BDL mice, together with an increase in LC3 protein levels. Upregulation of the proinflammatory citochines TNF-a and IL6 and an increased expression of NF-kB and MuRF-1 were observed in CCl4 mice. In conclusion, skeletal muscle myopenia was present in experimental models of BDL and CCl4-induced cirrhosis. Moreover, reduction in protein synthesis and activation of protein degradation were the main mechanisms responsible for myopenia in BDL mice, while activation of ubiquitin-pathway through inflammatory cytokines seems to be the main potential mechanism involved in CCl4 mice.
Assuntos
Cirrose Hepática Biliar/complicações , Cirrose Hepática Experimental/complicações , Doenças Musculares/etiologia , Animais , Tetracloreto de Carbono , Modelos Animais de Doenças , Interleucina-6/metabolismo , Ligadura , Cirrose Hepática Biliar/metabolismo , Cirrose Hepática Biliar/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Camundongos , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , NF-kappa B/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca(2+). In the present report the possible involvement of NAADP-controlled Ca(2+) signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this regard, Ca(2+) imaging experiments showed that the response of B16 cells to VEGF stimulation is NAADP-dependent. The whole of these observations indicate that NAADP-controlled Ca(2+) signaling can be relevant not only for neoangiogenesis but also for direct control of tumor cells.
Assuntos
Sinalização do Cálcio , Melanoma/metabolismo , Melanoma/patologia , NADP/análogos & derivados , Neovascularização Patológica/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacocinética , Carbolinas/farmacologia , Carbolinas/toxicidade , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Masculino , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma Experimental , Camundongos , NADP/metabolismo , Metástase Neoplásica , Neovascularização Patológica/tratamento farmacológico , Piperazinas/farmacocinética , Piperazinas/farmacologia , Piperazinas/toxicidade , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.
Assuntos
Coração/fisiologia , Miocárdio/citologia , Esferoides Celulares/citologia , Esferoides Celulares/transplante , Transplante de Células-Tronco , Células-Tronco/citologia , Engenharia Tecidual , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miocárdio/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/metabolismo , Células-Tronco/metabolismo , Alicerces TeciduaisRESUMO
Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD.
Assuntos
Interleucina-6/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Animais , Regulação para Baixo , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos mdx , Desenvolvimento Muscular , Músculo Esquelético/patologia , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Células Satélites de Músculo Esquelético/patologia , Células-Tronco/patologia , Quinase Induzida por NF-kappaBRESUMO
The anti-inflammatory agents glucocorticoids (GC) are the only available treatment for Duchenne muscular dystrophy (DMD). However, long-term GC treatment causes muscle atrophy and wasting. Thus, targeting specific mediator of inflammatory response may be more specific, more efficacious, and with fewer side effects. The pro-inflammatory cytokine interleukin (IL) 6 is overproduced in patients with DMD and in the muscle of mdx, the animal model for human DMD. We tested the ability of inhibition of IL6 activity, using an interleukin-6 receptor (Il6r) neutralizing antibody, to ameliorate the dystrophic phenotype. Blockade of endogenous Il6r conferred on dystrophic muscle resistance to degeneration and alleviated both morphological and functional consequences of the primary genetic defect. Pharmacological inhibition of IL6 activity leaded to changes in the dystrophic muscle environment, favoring anti-inflammatory responses and improvement in muscle repair. This resulted in a functional homeostatic maintenance of dystrophic muscle. These data provide an alternative pharmacological strategy for treatment of DMD and circumvent the major problems associated with conventional therapy.
Assuntos
Músculos/patologia , Músculos/fisiopatologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Receptores de Interleucina-6/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Homeostase , Inflamação/complicações , Inflamação/patologia , Interleucina-6/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/sangue , Distrofia Muscular de Duchenne/sangue , Necrose , Fenótipo , Receptores de Interleucina-6/metabolismoRESUMO
Although in the last decades the molecular underpinnings of the cell cycle have been unraveled, the acquired knowledge has been rarely translated into practical applications. Here, we investigate the feasibility and safety of triggering proliferation in vivo by temporary suppression of the cyclin-dependent kinase inhibitor, p21. Adeno-associated virus (AAV)-mediated, acute knockdown of p21 in intact skeletal muscles elicited proliferation of multiple, otherwise quiescent cell types, notably including satellite cells. Compared with controls, p21-suppressed muscles exhibited a striking two- to threefold expansion in cellularity and increased fiber numbers by 10 days post-transduction, with no detectable inflammation. These changes partially persisted for at least 60 days, indicating that the muscles had undergone lasting modifications. Furthermore, morphological hyperplasia was accompanied by 20% increases in maximum strength and resistance to fatigue. To assess the safety of transiently suppressing p21, cells subjected to p21 knockdown in vitro were analyzed for γ-H2AX accumulation, DNA fragmentation, cytogenetic abnormalities, ploidy, and mutations. Moreover, the differentiation competence of p21-suppressed myoblasts was investigated. These assays confirmed that transient suppression of p21 causes no genetic damage and does not impair differentiation. Our results establish the basis for further exploring the manipulation of the cell cycle as a strategy in regenerative medicine.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células , Aberrações Cromossômicas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dependovirus/classificação , Dependovirus/genética , Fibroblastos , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Camundongos , Contração Muscular/genética , Mutação , Interferência de RNA , RNA Interferente Pequeno/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Sorogrupo , Transdução GenéticaRESUMO
Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-), where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/-) mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.
Assuntos
Isoenzimas/deficiência , Distrofia Muscular Animal/terapia , Proteína Quinase C/deficiência , Animais , Fibrose , Imunoterapia/métodos , Inflamação/prevenção & controle , Camundongos , Distrofia Muscular Animal/patologia , Proteína Quinase C-theta , Resultado do Tratamento , Síndrome de EmaciaçãoRESUMO
Human heart harbors a population of resident progenitor cells that can be isolated by stem cell antigen-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, less than 1%-5% survive and differentiate. Among the major causes of this failure are the distressing protocols used to culture in vitro and implant progenitor cells into damaged hearts. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and were embedded in self-produced extracellular matrix preserving their phenotype and multipotency in the absence of significant apoptosis. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the murine myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such an approach to deliver stem cells to the myocardium. Interestingly, the successful delivery of cells in murine healthy hearts suggests that myocardium displays a continued cell cupidity that is strictly regulated by the limited release of progenitor cells by the adopted source. When an unregulated cell source is added to the system, cells are delivered to the myocardium. The exploitation of this novel concept may pave the way to the setup of new protocols in cardiac cell therapy.
Assuntos
Ventrículos do Coração/transplante , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Idoso , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular , Movimento Celular , Técnicas de Cocultura , Feminino , Perfilação da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Fenótipo , Transplante de Tecidos/métodosRESUMO
Many mutations and deletions in the dystrophin gene, responsible for Duchenne muscular dystrophy (DMD), can be corrected at the posttranscriptional level by skipping specific exons. Here we show that long-term benefit can be obtained in the dystrophic mouse model through the use of adeno-associated viral vectors expressing antisense sequences: persistent exon skipping, dystrophin rescue, and functional benefit were observed 74 weeks after a single systemic administration. The therapeutic benefit was sufficient to preserve the muscle integrity of mice up to old age. These results indicate a possible long-term gene therapy treatment of the DMD pathology.