RESUMO
Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.
Assuntos
Reprogramação Celular , Proteínas com Homeodomínio LIM/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Miócitos de Músculo Liso/citologia , Fatores de Transcrição/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Autorrenovação Celular , Separação Celular , Células Cultivadas , Células Clonais , Inativação Gênica , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Ratos Endogâmicos F344 , Telomerase/metabolismo , Transativadores/metabolismoRESUMO
Monocyte/macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX(3)CR1 is an essential regulator of monocyte/macrophage derived smooth muscle cell differentiation in the vessel wall after injury. Here we hypothesised the contribution of CX(3)CR1- CX(3)CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX(3)CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX(3)CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX(3)CL1-CX(3)CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX(3)CR1 system in vivo, Matrigel plugs were implanted in competent CX(3)CR1(+/gfp) and functionally deficient CX(3)CR1(gfp/gfp) mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX(3)CR1(gfp/gfp) but not in CX(3)CR1(+/gfp) mice. In experimental plaque neovascularization immature MV phenotype was observed in CX(3)CR1(gfp/gfp) mice, lacking CX(3)CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX(3)CR1(+/gfp) mice. This was associated with increased extravasation of platelets into the intima of CX(3)CR1(gfp/gfp) but not functionally competent CX(3)CR1 mice. Pharmacologic targeting using CX(3)CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX(3)CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype.
Assuntos
Endotélio Vascular/metabolismo , Macrófagos/metabolismo , Microvasos/metabolismo , Monócitos/metabolismo , Receptores de Quimiocinas/genética , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Receptor 1 de Quimiocina CX3C , Permeabilidade Capilar , Movimento Celular/efeitos dos fármacos , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Colágeno/genética , Colágeno/metabolismo , Combinação de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde , Laminina/genética , Laminina/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/efeitos dos fármacos , Microvasos/patologia , Monócitos/efeitos dos fármacos , Monócitos/patologia , Neovascularização Patológica , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteoglicanas , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismoRESUMO
AIMS: We have previously reported the cardioprotective effects of endothelial progenitor cell (EPC)-conditioned media (CM) therapy post-myocardial infarction (MI). In the present study, we have determined the insulin-like growth factor-1 (IGF-1) contribution to EPC CM effects on cardiomyocyte survival, contractility, and angiogenesis in vivo. METHODS AND RESULTS: Conditioned media from porcine EPC were administered intracoronary in the presence and absence of specific neutralizing antibodies to IGF-1 or control IgG in a porcine model of MI. X-vivo (non-conditioned) medium was used as a control. Functional, histological, and biochemical parameters were evaluated at 24 h and 8-week post-therapy. Conditioned media therapy significantly abrogated infarct zone (IZ) apoptosis, hypocontractility, and impaired left ventricular (LV) relaxation observed in control infarcts acutely (24 h post-MI). At 8 weeks following treatment, CM therapy augmented LV contractility and relaxation, IZ angiogenesis and inhibited infarct size expansion, wall expansion, and wall thinning. All of these acute and chronic beneficial effects of CM therapy were vitiated by neutralizing antibodies to IGF-1 but not by control IgG. Moreover, the addition of neutralizing IGF-1 antibody to control medium had no effect on these structural or functional changes in the heart post-treatment. CONCLUSION: Insulin-like growth factor-1 within the EPC CM mediates potent acute myocardial repair and chronic remodelling effects post-MI. These findings may provide a rationale for comparative trials of specific growth factors vs. current progenitor cell strategies.
Assuntos
Cardiotônicos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/fisiologia , Transplante de Células-Tronco/métodos , Animais , Anticorpos Neutralizantes/fisiologia , Apoptose/fisiologia , Biomarcadores/metabolismo , Sobrevivência Celular , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/fisiologia , Células Endoteliais/transplante , Feminino , Ventrículos do Coração/patologia , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/imunologia , Contração Miocárdica/fisiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Neovascularização Fisiológica/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco/fisiologia , Sus scrofa , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/terapiaRESUMO
The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a specific and potent inducer of apoptosis in cancer cells, but the resistance of many tumour cells to TRAIL still represents a major hurdle for the clinical treatment of tumours with TRAIL. As apoptosis is regulated by the balance of activities of several anti-apoptotic factors and pro-apoptotic factors, we analysed the relative contribution of the two sides and found that down-regulation of Bcl-x(L) and in particular XIAP, but not c-Flip, sensitised the TRAIL resistant pancreatic cancer cell line Panc-1. A combination of both XIAP and Bcl-x(L) knock-downs showed no substantial added benefit indicating that both act in the same pathway. Notably, the degree of sensitisation by silencing of anti-apoptotic genes was further elevated by concomitantly increasing the pro-apoptotic potential in Panc-1 cells through over-expression of TRAIL-R1 or IFN-γ-mediated increases in caspase-8 levels. Similar sensitisation effects were obtained for another TRAIL-resistant pancreatic tumour cell line, AsPC-1. Our findings demonstrate that modulation of the balance between anti- and pro-apoptotic pathways from both sides by inhibition of apoptosis-antagonists and stimulation of pro-apoptotic factors provides the best way to enhance the anti-tumourigenic effect of TRAIL.
Assuntos
Interferon gama/farmacologia , Neoplasias Pancreáticas/terapia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/deficiência , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Apoptose/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Células HCT116 , Humanos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Recombinantes/farmacologia , Transdução Genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína bcl-X/deficiência , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
BACKGROUND: Insulin-like growth factor-1 (IGF-1) is recognized as an important regulator of cardiac structure and cardiomyocyte homeostasis. The prosurvival and antiapoptotic effects of IGF-1 have been investigated in vitro and in rodent models of myocardial infarction (MI). However, the clinical application of IGF-1 has been hampered by dose-dependent side effects both acutely and during chronic administration. We hypothesized that single, low-dose IGF-1 (LD-IGF-1) administered locally and early in the reperfusion phase after acute MI in a large animal model would avoid significant side effects but would have prosurvival effects that would manifest in long-term structural and functional improvement after MI treatment. METHODS AND RESULTS: Forty-four female Landrace pigs underwent intracoronary administration of LD-IGF-1 or saline 2 hours into the reperfusion phase of acute left anterior descending artery occlusion MI. In the area of infarction, IGF-1 receptor and signaling responses were activated at 30 minutes and cardiomyocyte cell death attenuated at 24 hours after LD-IGF-1 but not saline treatment. Hemodynamic and structural studies using pressure-volume loop, CT, and triphenyltetrazolium chloride analysis 2 months post-MI confirmed a marked reduction in infarct size, attenuation of wall thinning, and augmentation of wall motion in the LD-IGF-1-treated but not in the saline-treated animals. These regional structural benefits were associated with global reductions in left ventricular volumes and significant improvement in left ventricular systolic and diastolic function. CONCLUSIONS: One-time LD-IGF-1 effects potent acute myocardial salvage in a preclinical model of left anterior descending artery occlusive MI, extending to long-term benefits in MI size, wall structure, and function and underscoring its potential as an adjunctive therapeutic agent.
Assuntos
Fator de Crescimento Insulin-Like I/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Fatores de Tempo , Animais , Apoptose/efeitos dos fármacos , Protocolos Clínicos , Feminino , Hemodinâmica , Humanos , Fator de Crescimento Insulin-Like I/efeitos adversos , Modelos Animais , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Receptor IGF Tipo 1/metabolismo , Volume Sistólico , Suínos , Sais de Tetrazólio/metabolismoRESUMO
The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in many cancer cells. However, a significant proportion of tumours are TRAIL-resistant erecting a major hurdle for a successful TRAIL-based treatment regimen in the future. In this context, it would be a major advantage to be able to identify the tumours that respond to TRAIL. The existence of two apoptosis-inducing receptors (TRAIL-R1 and TRAIL-R2) and two receptors that cannot transmit an apoptotic signal and have an inhibitory function (TRAIL-R3 and TRAIL-R4) make TRAIL signalling complicated. We analysed the surface expression of all four membrane-bound TRAIL receptors in cancer cell lines of various origin and primary cancer and normal cells and found a good correlation between TRAIL-sensitivity and the expression of TRAIL-R1 alone, but an even better correlation when a ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4 was analysed. Experimental overexpression of TRAIL-R1 alone or in combination with TRAIL-R4 in PANC-1 cells confirmed our correlation results. Similar to the surface expression-apoptosis correlation analysis we found a high correlation between TRAIL-sensitivity and the mRNA level ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4. A value of <0.85 for the ratio predicted TRAIL resistance in both protein and RNA analysis. Hence, TRAIL receptor RNA expression analysis by real-time PCR might be a feasible approach to predict possible TRAIL-responses in individual tumour samples.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Fator de Necrose Tumoral/genética , Membro 10c de Receptores do Fator de Necrose Tumoral/biossíntese , Membro 10c de Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/farmacologia , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated, but its exact role is controversial. We studied different cell lines displaying varying responses to TRAIL and found that TRAIL can activate NF-kappaB in all our cancer cell lines regardless of their TRAIL sensitivity. Inhibition of NF-kappaB via adenoviral expression of the IkappaB-alpha super-repressor only sensitized the TRAIL-resistant pancreatic cancer cell line Panc-1. Panc-1 cells harbor constitutively activated NF-kappaB, pointing to a possible role of preactivated NF-kappaB in protection from TRAIL. Furthermore, we could reduce X-linked inhibitor of apoptosis protein (XIAP) levels in Panc-1 cells by inhibition of constitutively activated NF-kappaB and sensitize Panc-1 cells to TRAIL by RNA interference against XIAP. These results implicate elevated XIAP levels caused by high basal NF-kappaB activity in TRAIL resistance and suggest that therapeutic strategies involving TRAIL can be abetted by inhibition of NF-kappaB and/or XIAP only in tumor cells with constitutively activated NF-kappaB.
Assuntos
Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Neoplasias/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Regulação para Cima , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose , Regulação para Baixo , Células HeLa , Humanos , Proteínas I-kappa B/fisiologia , Neoplasias/genética , Interferência de RNA , Células Tumorais CultivadasRESUMO
Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.