RESUMO
Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS), diagnosed at a mean age of 32 years. CNS glia are crucial players in the onset of MS, primarily involving astrocytes and microglia that can cause/allow massive oligodendroglial cells death, without immune cell infiltration. Current therapeutic approaches are aimed at modulating inflammatory reactions during relapsing episodes, but lack the ability to induce very significant repair mechanisms. In this review article, different experimental approaches based mainly on the application of different cell types as therapeutic strategies applied for the induction of myelin repair and/or the amelioration of the disease are discussed. Regarding this issue, different cell sources were applied in various experimental models of MS, with different results, both in significant improvements in remyelination and the reduction of neuroinflammation and glial activation, or in neuroprotection. All cell types tested have advantages and disadvantages, which makes it difficult to choose a better option for therapeutic application in MS. New strategies combining cell-based treatment with other applications would result in further improvements and would be good candidates for MS cell therapy and myelin repair.
Assuntos
Esclerose Múltipla , Remielinização , Humanos , Adulto , Bainha de Mielina/fisiologia , Esclerose Múltipla/metabolismo , Remielinização/fisiologia , Oligodendroglia/metabolismo , NeurogliaRESUMO
The perception of noxious environmental stimuli by nociceptive sensory neurons is an essential mechanism for the prevention of tissue damage. Etv4 is a transcriptional factor expressed in most nociceptors in dorsal root ganglia (DRG) during the embryonic development. However, its physiological role remains unclear. Here, we show that Etv4 ablation results in defects in the development of the peripheral peptidergic projections in vivo, and in deficits in axonal elongation and growth cone morphology in cultured sensory neurons in response to NGF. From a mechanistic point of view, our findings reveal that NGF regulates Etv4-dependent gene expression of molecules involved in extracellular matrix (ECM) remodeling. Etv4-null mice were less sensitive to noxious heat stimuli and chemical pain, and this behavioral phenotype correlates with a significant reduction in the expression of the pain-transducing ion channel TRPV1 in mutant mice. Together, our data demonstrate that Etv4 is required for the correct innervation and function of peptidergic sensory neurons, regulating a transcriptional program that involves molecules associated with axonal growth and pain transduction.
Assuntos
Fator de Crescimento Neural , Nociceptividade , Proteínas Proto-Oncogênicas c-ets/metabolismo , Animais , Gânglios Espinais/metabolismo , Camundongos , Fator de Crescimento Neural/genética , Nociceptividade/fisiologia , Dor/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
BACKGROUND AND AIMS: Liver fibrosis results from cycles of liver damage and scar formation. We herein aimed at analysing neural crest cells and/or bone marrow stromal cells contribution to the liver. METHODS: Two liver fibrosis and one hepatectomy model were applied on double-transgenic loxP-Cre mouse lines. RESULTS: Increased numbers of glia with more complex processes were found in fibrotic livers. During embryonic development, only few cells were traced in the liver and bone marrow, in a minor fraction of mice of different neural crest reporter strains analysed: therefore, a neural crest origin of such cells is doubtful. In the fibrotic liver, a significantly higher incidence of endothelial cells and hepatocyte-like cells expressing the reporter gene Tomato were found in Wnt1-Cre-Tom and GLAST-CreERT2-Tom mice. Consistently, during early fibrogenesis stromal Wnt1-traced cells, with progenitor (CFU-F) properties, get likely mobilized to peripheral blood. Circulating adult Wnt1-traced cells are stromal cells and lack from the expression of other bone marrow and endothelial progenitor cells markers. Furthermore, in a 70% hepatectomy model GLAST+ Wnt1-traced pericytes were found to be mobilized from the bone marrow and the incidence of GLAST-traced hepatocyte-like cells was increased. Finally, GLAST-traced hepatocyte like-cells were found to maintain the expression of stromal markers. CONCLUSIONS: Our data suggest a gliosis process during liver fibrogenesis. While neural crest cells probably do not contribute with other liver cell types than glia, GLAST+ Wnt1-traced bone marrow pericytes are likely a source of endothelial and hepatocyte-like cells after liver injury and do not contribute to scarring.
Assuntos
Crista Neural , Pericitos , Animais , Medula Óssea , Células Endoteliais , Fígado , Regeneração Hepática , Camundongos , Camundongos TransgênicosRESUMO
Schwann cell precursors (SCPs) are frequently regarded as neural crest-derived cells (NCDCs) found in contact with axons during nerve formation. Nevertheless, cells with SCPs properties can be found up to the adulthood. They are well characterized with regard to both gene expression profile and cellular behavior -for instance, proliferation, migratory capabilities and survival requirements-. They differ in origin regarding their anatomic location: even though most of them are derived from migratory NCCs, there is also contribution of the boundary cap neural crest cells (bNCCs) to the skin and other tissues. Many functions are known for SCPs in normal development, including nerve fasciculation and target innervation, arterial branching patterning and differentiation, and other morphogenetic processes. In addition, SCPs are now known to be a source of many neural (glia, endoneural fibroblasts, melanocytes, visceral neurons, and chromaffin cells) and non-neural-like (mesenchymal stromal cells, able e.g., to generate dentine-producing odontoblasts) cell types. Until now no reports of endoderm-like derivatives were reported so far. Interestingly, in the Schwann cell lineage only early SCPs are likely able to differentiate into melanocytes and bone marrow mesenchymal stromal cells. We have also herein discussed the literature regarding their role in repair as well as in disease mechanisms, such as in diverse cancers. Moreover, many caveats in our knowledge of SCPs biology are highlighted all through this article. Future research should expand more into the relevance of SCPs in pathologies and in other regenerative mechanisms which might bring new unexpected clinically-relevant knowledge.
Assuntos
Células-Tronco Neurais/fisiologia , Células de Schwann/fisiologia , Animais , Humanos , Doenças do Sistema Nervoso/fisiopatologiaRESUMO
Some late embryonic and adult postmigratory neural crest-derived cells (NCDCs) from diverse tissues were shown to grow as multipotent neurospheres. Neural crest stem cells (NCSCs) contained in these spheres were found to give rise not only to neuroectodermal derivatives but also to some of the progeny of the other embryonic germ layers. In this review, evidences regarding the in vivo properties of NCDCs contributing to NCSCs are discussed. Even though in many cases the final proof for the phenotype identity of in vivo cells generating NCSCs is lacking, some evidences suggest that such postmigratory NCDCs would differ from neural crest cells. The streamline of this review follows a historical perspective that helps understanding the advancements in knowledge of this field of research and highlighting its importance, in an appropriate context. Finally, the potential for regenerative medicine purpose of NCDCs and more specifically of tissues that can be a source of peripheral glia progenitors in the adult is underlined.
Assuntos
Células-Tronco Adultas/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Adulto , Humanos , Células-Tronco Multipotentes/citologia , Neuroglia/citologia , Medicina RegenerativaRESUMO
BACKGROUND: Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hMø) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hMø in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hMø behavior in the context of liver fibrosis resolution. METHODS: Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8 weeks). In vivo gene expression analyses, in vitro experiments using hMø isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of Mø were performed. RESULTS: One day after treatment, hMø from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hMø from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hMø, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hMø depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hMø abrogated such effects on the expression of the most highly regulated genes. CONCLUSIONS: Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Cirrose Hepática/terapia , Macrófagos/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Tioacetamida/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Mesenchymal stem/stromal cells (MSCs) are progenitors which share plastic-adherence capacity and cell surface markers but have different properties according to their cell and tissue sources and to culture conditions applied. Many recent publications suggest that MSCs can differentiate into hepatic-like cells, which can be a consequence of either a positive selection of rare in vivo pluripotent cells or of the original plasticity of some cells contributing to MSC cultures. A possible role of MSCs in hereditary transmission of obesity and/or diabetes as well as properties of MSCs regarding immunomodulation, cell fusion and exosome release capacities are discussed according to recent literature. Limitations in methods used to track MSCs in vivo especially in the context of liver cirrhosis are addressed as well as strategies explored to enhance their migratory, survival and proliferation properties, which are known to be relevant for their future clinical use. Current knowledge regarding mechanisms involved in liver cirrhosis amelioration mediated by naïve and genetically modified MSCs as well as the effects of applying preconditioning and combined strategies to improve their therapeutic effects are evaluated. Finally, first reports of GMP guidelines and biosafety issues in MSCs applications are discussed.
Assuntos
Diferenciação Celular/fisiologia , Cirrose Hepática/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Animais , Proliferação de Células/fisiologia , Humanos , Cirrose Hepática/fisiopatologia , Regeneração Hepática/fisiologia , Modelos Biológicos , Medicina Regenerativa/métodos , Medicina Regenerativa/tendênciasRESUMO
Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.
Assuntos
Terapia Genética , Fator de Crescimento Insulin-Like I/genética , Cirrose Hepática/terapia , Fígado/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Adenoviridae/genética , Animais , Proliferação de Células , Fibrose/terapia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-ß, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Caspase/uso terapêutico , Hepatócitos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Ácidos Pentanoicos/uso terapêutico , Animais , Inibidores de Caspase/farmacologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Fibrose , Hepatite/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Ácidos Pentanoicos/farmacologiaRESUMO
We have shown that ex vivo pre-conditioning of bone marrow-derived dendritic cells (DC) with low molecular weight hyaluronan (LMW HA) induces antitumor immunity against colorectal carcinoma (CRC) in mice. In the present study we investigated the effects of LMW HA priming on human-tumor-pulsed monocytes-derived dendritic cells (DC/TL) obtained from healthy donors and patients with CRC. LMW HA treatment resulted in an improved maturation state of DC/TL and an enhanced mixed leucocyte reaction activity in vivo. Importantly, pre-conditioning of DC/TL with LMW HA increased their ability to migrate and reduced their attraction to human tumor derived supernatants. These effects were associated with increased CCR7 expression levels in DC. Indeed, a significant increase in migratory response toward CCL21 was observed in LMW HA primed tumor-pulsed monocyte-derived dendritic cells (DC/TL/LMW HA) when compared to LWM HA untreated cells (DC/TL). Moreover, LMW HA priming modulated other mechanisms implicated in DC migration toward lymph nodes such as the metalloproteinase activity. Furthermore, it also resulted in a significant reduction in DC migratory capacity toward tumor supernatant and IL8 in vitro. Consistently, LMW HA dramatically enhanced in vivo DC recruitment to tumor-regional lymph nodes and reduced DC migration toward tumor tissue. This study shows that LMW HA--a poorly immunogenic molecule--represents a promising candidate to improve human DC maturation protocols in the context of DC-based vaccines development, due to its ability to enhance their immunogenic properties as well as their migratory capacity toward lymph nodes instead of tumors.
Assuntos
Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/imunologia , Células Dendríticas/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Meios de Cultura , Humanos , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Unfortunately, the incidence and mortality associated with HCC are increasing. Therefore, new therapeutic strategies are urgently needed and the use of mesenchymal stromal cells (MSCs) as carrier of therapeutic genes is emerging as a promising option. Different sources of MSCs are being studied for cell therapy and bone marrow-derived cells are the most extensively explored; however, birth associated-tissues represent a very promising source. The aim of this work was to compare the in vitro and in vivo migration capacity between bone marrow MSCs (BM-MSCs) and human umbilical cord perivascular cells (HUCPVCs) towards HCC. We observed that HUCPVCs presented higher in vitro and in vivo migration towards factors released by HCC. The expression of autocrine motility factor (AMF) receptor, genes related with the availability of the receptor on the cell surface (caveolin-1 and -2) and metalloproteinase 3, induced by the receptor activation and important for cell migration, was increased in HUCPVCs. The chemotactic response towards recombinant AMF was increased in HUCPVCs compared to BM-MSCs, and its inhibition in the conditioned medium from HCC induced higher decrease in HUCPVC migration than in BM-MSC. Our results indicate that HUCPVCs could be a useful cellular source to deliver therapeutic genes to HCC.
Assuntos
Medula Óssea/patologia , Carcinoma Hepatocelular/patologia , Movimento Celular/fisiologia , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/patologia , Receptores do Fator Autócrino de Motilidade/metabolismo , Cordão Umbilical/patologia , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Carcinoma Hepatocelular/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Cordão Umbilical/metabolismoRESUMO
BACKGROUND AND AIMS: Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. METHODS: Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. RESULTS: AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. CONCLUSION: AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glucose-6-Fosfato Isomerase/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/metabolismo , Animais , Carcinoma Hepatocelular/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glucose-6-Fosfato Isomerase/farmacologia , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Camundongos , Carga TumoralRESUMO
Mesenchymal stromal cells (MSCs) are more often obtained from adult and extraembryonic tissues, with the latter sources being likely better from a therapeutic perspective. MSCs show tropism towards inflamed or tumourigenic sites. Mechanisms involved in MSC recruitment into tumours are comprehensively analysed, including chemoattractant signalling axes, endothelial adhesion and transmigration. In addition, signals derived from hepatocellular carcinoma (HCC) tumour microenvironment and their influence in MSC tropism and tumour recruitment are dissected, as well as the present controversy regarding their influence on tumour growth and/or metastasis. Finally, evidences available on the use of MSCs and other selected progenitor/stem cells as vehicles of antitumourigenic genes are discussed. A better knowledge of the mechanisms involved in progenitor/stem cell recruitment to HCC tumours is proposed in order to enhance their tumour targeting which may result in improvements in cell-based gene therapy strategies.
Assuntos
Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Terapia Genética/métodos , Humanos , Camundongos , Microambiente TumoralRESUMO
INTRODUCTION: Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. METHODS: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+)) and knock-out (SPARC(-/-)) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/-) and SPARC(+/+) mice using Affymetrix Mouse Gene ST 1.0 array. RESULTS: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/-) mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC(+/+) mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-ß1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. CONCLUSIONS: Overall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.
Assuntos
Glicoproteínas/fisiologia , Cirrose Hepática/fisiopatologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Sequência de Bases , Colágeno/metabolismo , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Osteonectina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The combination of a single low dose of cyclophosphamide (Cy) with the adenovirus-mediated gene transfer of interleukin-12 (AdIL-12) might represent a successful therapy for experimental gastrointestinal tumors. This approach has been proven to revert immunosuppressive mechanisms elicited by cancer cells and to synergistically promote antitumor immunity. In addition, this therapeutic regimen has been shown to be more efficient in achieving complete tumor regressions in mice than the application of a metronomic schedule of Cy plus AdIL-12.
RESUMO
The use of conventional cytotoxic agents at metronomic schedules, alone or in combination with targeted agents or immunotherapy, is being explored as a promising anticancer strategy. We previously reported a potent antitumor effect of a single low-dose cyclophosphamide and interleukin-12 (IL-12) gene therapy against advanced gastrointestinal carcinoma, in mice. Here, we assessed whether the delivery of IL-12 by gene therapy together with metronomic cyclophosphamide exerts antitumor effects in a murine model of colorectal carcinoma. This combination therapy was able, at least in part, to reverse immunosuppression, by decreasing the number of regulatory T cells (Tregs) as well as of splenic myeloid-derived suppressor cells (MDSCs). However, metronomic cyclophosphamide plus IL-12 gene therapy failed to increase the number of tumor-infiltrating T lymphocytes and, more importantly, to induce a specific antitumor immune response. With respect to this, cyclophosphamide at a single low dose displayed a superior anticancer profile than the same drug given at a metronomic schedule. Our results may have important implications in the design of new therapeutic strategies against colorectal carcinoma using cyclophosphamide in combination with immunotherapy.
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BACKGROUND AND OBJECTIVES: Modern imaging techniques have increased the incidental detection of renal atherosclerotic disease (RAD). Because immune activation may hasten RAD progression, identifying cellular immune markers might provide clues to clinical activity. In this study, cellular immune markers were assessed in early RAD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Immune cell markers in peripheral blood of two groups of hypertensive patients with normal carotid and coronary arteries were evaluated: 28 patients had incidental RAD and 22 patients had normal renal arteries; 21 renal arteries obtained at necropsy from individuals with history of hypertension and tissue evidence of RAD were examined and matched with 21 individuals with normal renal arteries. Cell subpopulations were measured by flow cytometry in peripheral blood and direct cell count, respectively, using T and dendritic cells monoclonal antibodies. RESULTS: Peripheral blood of RAD patients showed increased numbers of cells expressing CD3, CD4, CD83, and CD86. CD4 to CD8 ratio was 8.3 ± 1.4 (RAD) to 3.4 ± 0.9 (normal; P<0.001). No differences were found in CD25, CD8, and S100 among groups. Postmortem samples from RAD showed increased CD3+, CD4+, CD86+, and S100+ cells, whereas CD25+ and CD8+ were unmodified between groups. CD4+ to CD8+ ratio was higher in the RAD(PM) group. CONCLUSIONS: These results are consistent with an increased expression of immune cell markers in early RAD. Additional studies will explore if they may potentially turn into treatment targets to prevent disease progression.
Assuntos
Aterosclerose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Artéria Renal/imunologia , Adulto , Antígenos CD/sangue , Doenças Assintomáticas , Aterosclerose/sangue , Aterosclerose/patologia , Autopsia , Antígeno B7-2/sangue , Biomarcadores/sangue , Complexo CD3/sangue , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Imunoglobulinas/sangue , Subunidade alfa de Receptor de Interleucina-2/sangue , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Projetos Piloto , Prognóstico , Estudos Prospectivos , Artéria Renal/patologia , Proteínas S100/sangue , Antígeno CD83RESUMO
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death. Fibrogenesis is an active process characterized by the production of several proinflammatory cytokines, chemokines and growth factors. It involves the activation of hepatic stellate cells (HSCs) which accumulate at the site of injury and are the main source of the extracellular matrix deposits. There are no curative treatments for advanced HCC, thus, new therapies are urgently needed. Mesenchymal stromal cells (MSCs) have the ability to migrate to sites of injury or to remodeling tissues after in vivo administration; however, in several cancer models they demonstrated limited efficacy to eradicate experimental tumors partially due to poor engraftment. Thus, the aim of this work was to analyze the capacity of human MSCs (hMSCs) to migrate and anchor to HCC tumors. We observed that HCC and HSCs, but not nontumoral stroma, produce factors that induce hMSC migration in vitro. Conditioned media (CM) generated from established HCC cell lines were found to induce higher levels of hMSC migration than CM derived from fresh patient tumor samples. In addition, after exposure to CM from HCC cells or HSCs, hMSCs demonstrated adhesion and invasion capability to endothelial cells, type IV collagen and fibrinogen. Consistently, these cells were found to increase metalloproteinase-2 activity. In vivo studies with subcutaneous and orthotopic HCC models indicated that intravenously infused hMSCs migrated to lungs, spleen and liver. Seven days post-hMSC infusion cells were located also in the tumor in both models, but the signal intensity was significantly higher in orthotopic than in subcutaneous model. Interestingly, when orthotopic HCC tumors where established in noncirrhotic or cirrhotic livers, the amount of hMSCs localized in the liver was higher in comparison with healthy animals. A very low signal was found in lungs and spleens, indicating that liver tumors are able to recruit them at high efficiency. Taken together our results indicate that HCC and HSC cells produce factors that efficiently induce hMSC migration toward tumor microenvironment in vitro and in vivo and make MSCs candidates for cell-based therapeutic strategies to hepatocellular carcinoma associated with fibrosis.
Assuntos
Células da Medula Óssea/metabolismo , Carcinoma Hepatocelular/patologia , Movimento Celular , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/patologia , Microambiente Tumoral , Animais , Células da Medula Óssea/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/fisiopatologia , Carcinoma Hepatocelular/terapia , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Endotélio Vascular/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/fisiopatologia , Neoplasias Hepáticas/terapia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties.
Assuntos
Vacinas Anticâncer/imunologia , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/imunologia , Células Dendríticas/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Neoplasias/imunologia , Separação Celular , Neoplasias Colorretais/terapia , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/transplante , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Ácido Hialurônico/imunologia , Imunoterapia , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Immunotherapy-based strategies for gastrointestinal carcinomas (GIC) have been exploited so far, but these approaches have to face strong mechanisms of immune escape induced by tumours. We previously demonstrated that sub-therapeutic doses of an adenovirus expressing IL-12 genes (AdIL-12) mediated a potent antitumour effect against subcutaneous (s.c.) colorectal carcinomas (CRC) in mice pre-treated with low doses of cyclophosphamide (Cy). In our study we used this combination to assess its impact on the immunosuppressive microenvironment. In s.c. CRC model we demonstrated that non-responder mice failed to decrease Tregs in tumour, spleen and peripheral blood. Reconstitution of Tregs into tumour-bearing mice treated with combined therapy abolished the antitumoural effect. In addition, Cy + AdIL-12 modified Tregs functionality by inhibiting the in vitro secretion of IL-10 and TGF-ß and their ability to inhibit dendritic cells activation. Combined treatment decreased the number of myeloid-derived suppressor cells (MDSCs) in comparison to non-treated mice and, interestingly, administration of Tregs restored splenic MDSCs population. Furthermore, combined therapy potently generated specific cytotoxic IFN-γ-secreting CD4+ T cells able to eradicate established CRC tumours after adoptive transfer. Finally, we evaluated the combination on disseminated CRC and pancreatic carcinoma (PC). Cy + AdIL-12 were able to eradicate liver metastatic CRC (47%) and PC tumour nodules (40%) and to prolong animal survival. The results of this study support the hypothesis that Cy + AdIL-12 might be a valid immunotherapeutic strategy for advanced GIC.