RESUMEN

Mammalian Ste-20-like Kinases 1 and 2 (MST1/2) are core serine-threonine kinases of the Hippo pathway regulating several cellular processes, including cell cycle arrest and cell death. Here, we discovered a novel alternative splicing variant of the MST2 encoding gene, STK3, in malignant cells and tumor datasets. This variant, named STK3∆7 or MST2∆7 (for mRNA or protein, respectively), resulted from the skipping of exon 7. MST2∆7 exhibited increased ubiquitylation and interaction with the E3 ubiquitin-protein ligase CHIP compared to the full-length protein (MST2FL). Exon 7 in STK3 encodes a segment within the kinase domain, and its exclusion compromised MST2 interaction with and phosphorylation of MOB, a major MST1/2 substrate. Nevertheless, MST2∆7 was capable of interacting with MST1 and MST2FL. Unlike MST2FL, overexpression of MST2∆7 did not lead to increased cell death and growth arrest. Strikingly, we observed the exclusion of STK3 exon 7 in 3.2-15% of tumor samples from patients of several types of cancer, while STK3∆7 was seldomly found in healthy tissues. Our study identified a novel STK3 splicing variant with loss of function and the potential to disturb tissue homeostasis by impacting on MST2 activities in the regulation of cell death and quiescence.

Asunto(s)

Empalme Alternativo , Proliferación Celular , Proteínas Serina-Treonina Quinasas , Serina-Treonina Quinasa 3 , Humanos , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Exones/genética , Células HEK293 , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasa 3/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/genética

RESUMEN

Inflammation resolution is an active process that involves cellular events such as apoptosis and efferocytosis, which are key steps in the restoration of tissue homeostasis. Hepatocyte growth factor (HGF) is a growth factor mostly produced by mesenchymal-origin cells and has been described to act via MET receptor tyrosine kinase. The HGF/MET axis is essential for determining the progression and severity of inflammatory and immune-mediated disorders. Here, we investigated the effect of blocking the HGF/MET signalling pathway by PF-04217903 on the resolution of established models of neutrophilic inflammation. In a self-resolving model of gout induced by MSU crystals, HGF expression on periarticular tissue peaked at 12 h, the same time point that neutrophils reach their maximal accumulation in the joints. The HGF/MET axis was activated in this model, as demonstrated by increased levels of MET phosphorylation in neutrophils (Ly6G+ cells). In addition, the number of neutrophils was reduced in the knee exudate after PF-04217903 treatment, an effect accompanied by increased neutrophil apoptosis and efferocytosis and enhanced expression of Annexin A1, a key molecule for inflammation resolution. Reduced MPO activity, IL-1ß and CXCL1 levels were also observed in periarticular tissue. Importantly, PF-04217903 reduced the histopathological score and hypernociceptive response. Similar findings were obtained in LPS-induced neutrophilic pleurisy. In human neutrophils, the combined use of LPS and HGF increased MET phosphorylation and provided a prosurvival signal, whereas blocking MET with PF-04217903 induced caspase-dependent neutrophil apoptosis. Taken together, these data demonstrate that blocking HGF/MET signalling may be a potential therapeutic strategy for inducing the resolution of neutrophilic inflammatory responses.

Asunto(s)

Factor de Crecimiento de Hepatocito , Neutrófilos , Humanos , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Factor de Crecimiento de Hepatocito/uso terapéutico , Lipopolisacáridos/farmacología , Inflamación/metabolismo , Apoptosis , Proteínas Proto-Oncogénicas c-met/metabolismo , Homeostasis

RESUMEN

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications.

Asunto(s)

Productos Biológicos , Células Madre Mesenquimatosas , Somatomedinas , Animales , Plaquetas/metabolismo , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Medios de Cultivo/química , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Proteómica , Reproducibilidad de los Resultados , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/metabolismo , Somatomedinas/análisis , Somatomedinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Hepatocyte growth factor (HGF) has been proved to protect the liver against α-naphthylisothiocyanate (ANIT)-induced cholestasis by acting as an antioxidant agent and redirecting toxic biliary solutes towards blood for urinary excretion. However, this may represent an additional potential risk for kidney integrity, which is already compromised by the cholestatic process itself (cholemic nephropathy). Therefore, in the present work, we studied the renal damage caused by ANIT-induced cholestasis and whether it is aggravated or, on the contrary, counteracted by HGF; if the latter holds, the involvement of its antioxidant properties will be ascertained. ANIT-induced cholestatic deleterious renal effects were corroborated by the presence of urine bile salts, impairment of renal function, and the alterations of renal damage markers, such as HSP72, creatinine clearance, and albuminuria. HGF fully reverted all these, and the cast formation in the tubules was significantly decreased. These findings were associated with the control of renal oxidative stress. In summary, despite HGF enhancing the overload of potentially harmful biliary constituents that the kidney should remove from the bloodstream as an alternative depuration organ in cholestasis, it simultaneously protects the kidney from this damage by counteracting the prooxidant effects resulting from this harmful exposure.

Asunto(s)

Colestasis/tratamiento farmacológico , Factor de Crecimiento de Hepatocito/farmacología , Enfermedades Renales/fisiopatología , 1-Naftilisotiocianato/efectos adversos , 1-Naftilisotiocianato/farmacología , Animales , Antioxidantes/farmacología , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/fisiopatología , Colestasis/sangre , Colestasis/metabolismo , Modelos Animales de Enfermedad , Factor de Crecimiento de Hepatocito/metabolismo , Riñón/metabolismo , Enfermedades Renales/metabolismo , Hígado/metabolismo , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Telocytes are cells present in the stroma of various tissues including the prostate. The detection of telocytes is still very much dependent on obtaining ultrastructural data that show the presence of telopodes, which are cytoplasmic projections that alternate between dilated regions, the podoms, and thin segments, the podomers. These structures are the distinctive characteristics of the telocytes. Thus, in vitro assays are important for the study of telocytes, which are more easily identified in culture, which also enables the experimental manipulation of these cells. The isolation of telocytes per se does not allow the analysis of the behavior of these cells in relation to other cell types in a given organ. In this sense, in the prostate, explants could be a useful tool for the study of telocytes. The present study obtained prostatic explants and evaluated the influence of recombinant proteins, scattering factor (SCF) and stromal-derived factor 1 (SDF-1), which could impact on the migration of CD34-positive cells. Telocytes migrate out of explants and SDF-1 stimulates the proliferation and formation of telocyte networks in vitro. Telocytes are not smooth muscle cell progenitors in the prostate; on the contrary, they are CD90- and CD44-negative cells and, hence, have limited progenitor capacity. The present study demonstrated that explants are useful tools to elucidate the nature of telocytes and their functions.

Asunto(s)

Quimiocina CXCL12/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Telocitos/metabolismo , Animales , Antígenos CD34/metabolismo , Técnicas de Cultivo de Célula/métodos , Gerbillinae , Masculino , Próstata/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Telocitos/fisiología

RESUMEN

There is a constant need for improving embryo culture conditions in assisted reproduction. One possibility is to use mesenchymal stem/stromal cells derived from menstrual blood (mbMSCs), with an endometrial origin. In this study, we sought to analyze the expansion of mouse embryos in a direct coculture model with mbMSCs. Our results showed that after five passages, mbMSCs presented a spindle-shaped morphology, with surface markers that were comparable with the normal mesenchymal cell phenotype. mbMSCs could differentiate into adipogenic and osteogenic lineages and secrete angiopoetin-2 and hepatocyte growth factor. The coculture experiments employed 103 two-cell-stage embryos that were randomly divided into two groups: control (n = 50), embryos cultured in GV-Blast medium, and cocultured mbMSCs (n = 53), embryos cocultured with GV-Blast and mbMSCs. Typically, two to three embryos were placed in a well with 200 µL of culture medium and observed until developmental day 5. After 5 days, the cocultured group had more embryos in the blastocyst stage (69.8%) when compared with the control group (30%) (p < 0.001). It was also found that nearly 57% of blastocysts in the cocultured group reached the hatching stage, while only 13% achieved this stage in the control group (p < 0.001). Analyses of cultured mbMSCs and growth media, in the presence or absence of an embryo, were also performed. Immunofluorescence detected similar levels of collagen I and III and fibronectin in both mbMSCs and cocultured mbMSCs, and similar amounts of growth factors, VEGF, PDGF-AA, and PDGF-BB, were also observed in the conditioned medium, regardless of embryo presence. The present study describes, for the first time, an easy, noninvasive, and autologous method that could potentially increase blastocyst growth rates during assisted reproductive procedures (i.e., in vitro fertilization). It is proposed that this mbMSC coculture strategy enriches the embryonic microenvironment and promotes embryo development. This technique may complement or replace existing assisted reproduction methods and is directly relevant to the field of personalized medicine. Impact statement The study demonstrates a novel and potentially personalized assisted reproduction approach. The search for alternative and autologous methods provides assisted reproduction patients with a better chance of a successful pregnancy. In this study, mesenchymal cells derived from menstrual blood resembled the outside uterine surface and could potentially be employed for improving embryo outgrowth. Our protocol enriches the embryonic microenvironment and facilitates high-quality single-embryo transfer.

Asunto(s)

Desarrollo Embrionario/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Angiopoyetina 2/metabolismo , Blastocisto/citología , Blastocisto/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Medios de Cultivo Condicionados , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Endometrio/citología , Endometrio/metabolismo , Femenino , Fibronectinas/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos

RESUMEN

Wound scarring remains a major challenge for plastic surgeons. Transforming growth factor (TGF)-ß plays a key role in the process of scar formation. Previous studies have demonstrated that truncated TGF-ß type II receptor (t-TGF-ßRII) is unable to continue signal transduction but is still capable of binding to TGF-ß, thereby blocking the TGF-ß signaling pathway. Hepatocyte growth factor (HGF) is a multifunctional growth factor that promotes tissue regeneration and wound healing. Theoretically, the combination of HGF and t-TGF-ßRII would be expected to exert a synergistic effect on promoting wound healing and reducing collagen formation. In the present study, lentivirus-mediated transfection of the two genes (t-TGF-ßRII/HGF) into fibroblasts in vitro and in a rat model in vivo was used. The results demonstrated that the expression of t-TGF-ßRII and HGF in NIH-3T3 cells was successfully induced. The expression of both molecules significantly reduced collagen I and III expression, and also inhibited fibroblast proliferation. Furthermore, histological examination and scar quantification revealed less scarring in the experimental wound in a rat model. Moreover, on macroscopic inspection, the experimental wound exhibited less visible scarring compared with the control. Therefore, the present study demonstrated that the combination gene therapy of t-TGF-ßRII and HGF promoted wound healing, with less scarring and more epithelial tissue formation, not only by suppressing the overgrowth of collagen due to its antifibrotic effect, but also by promoting tissue regeneration.

Asunto(s)

Cicatriz/metabolismo , Colágeno/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Transfección , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Proliferación Celular , Cicatriz/patología , Ratones , Modelos Animales , Ratas , Ratas Sprague-Dawley

RESUMEN

Wound scarring remains a major challenge for plastic surgeons. Transforming growth factor (TGF)-β plays a key role in the process of scar formation. Previous studies have demonstrated that truncated TGF-β type II receptor (t-TGF-βRII) is unable to continue signal transduction but is still capable of binding to TGF-β, thereby blocking the TGF-β signaling pathway. Hepatocyte growth factor (HGF) is a multifunctional growth factor that promotes tissue regeneration and wound healing. Theoretically, the combination of HGF and t-TGF-βRII would be expected to exert a synergistic effect on promoting wound healing and reducing collagen formation. In the present study, lentivirus-mediated transfection of the two genes (t-TGF-βRII/HGF) into fibroblasts in vitro and in a rat model in vivo was used. The results demonstrated that the expression of t-TGF-βRII and HGF in NIH-3T3 cells was successfully induced. The expression of both molecules significantly reduced collagen I and III expression, and also inhibited fibroblast proliferation. Furthermore, histological examination and scar quantification revealed less scarring in the experimental wound in a rat model. Moreover, on macroscopic inspection, the experimental wound exhibited less visible scarring compared with the control. Therefore, the present study demonstrated that the combination gene therapy of t-TGF-βRII and HGF promoted wound healing, with less scarring and more epithelial tissue formation, not only by suppressing the overgrowth of collagen due to its antifibrotic effect, but also by promoting tissue regeneration.

Asunto(s)

Animales , Conejos , Ratas , Transfección , Colágeno/metabolismo , Cicatriz/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Cicatriz/patología , Ratas Sprague-Dawley , Modelos Animales , Proliferación Celular

RESUMEN

INTRODUCTION AND OBJECTIVES: Chronic liver inflammation may lead to hepatic cirrhosis, limiting its regenerative capacity. The clinical standard of care is transplantation, although stem cell therapy may be an alternative option. The study aim was to induce endogenous hematopoietic stem cells (HSCs) with granulocyte colony stimulating factor (G-CSF) and/or intravenous administration of adipose tissue-derived mesenchymal stem cells (MSCs) to decrease hepatic fibrosis in an experimental model. MATERIAL AND METHODS: A liver fibrosis model was developed with female Wistar rats via multiple intraperitoneal doses of carbon tetrachloride. Three rats were selected to confirm cirrhosis, and the rest were set into experimental groups to evaluate single and combined therapies of G-CSF-stimulated HSC mobilization and intravenous MSC administration. RESULTS: Treatment with MSCs and G-CSF significantly improved alanine amino transferase levels, while treatment with G-CSF, MSCs, and G-CSF+MSCs decreased aspartate amino transferase levels. Hepatocyte growth factor (HGF) and interleukin 10 levels increased with MSC treatment. Transforming growth factor ß levels were lower with MSC treatment. Interleukin 1ß and tumor necrosis factor alpha levels decreased in all treated groups. Histopathology showed that MSCs and G-CSF reduced liver fibrosis from F4 to F2. CONCLUSIONS: MSC treatment improves liver function, decreases hepatic fibrosis, and plays an anti-inflammatory role; it promotes HGF levels and increased proliferating cell nuclear antigen when followed by MSC treatment mobilization using G-CSF. When these therapies were combined, however, fibrosis improvement was less evident.

Asunto(s)

Tejido Adiposo/citología , Movilización de Célula Madre Hematopoyética/métodos , Cirrosis Hepática/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Alanina Transaminasa/metabolismo , Animales , Antígenos CD34 , Tetracloruro de Carbono/toxicidad , Terapia Combinada , Modelos Animales de Enfermedad , Femenino , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Células Madre Hematopoyéticas , Factor de Crecimiento de Hepatocito/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Células Madre Mesenquimatosas , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The extracellular matrix (ECM) is a crucial component of the tumor microenvironment involved in numerous cellular processes that contribute to cancer progression. It is acknowledged that tumor⁻stromal cell communication is driven by a complex and dynamic network of cytokines, growth factors and proteases. Thus, the ECM works as a reservoir for bioactive molecules that modulate tumor cell behavior. The hepatocyte growth factor (HGF) produced by tumor and stromal cells acts as a multifunctional cytokine and activates the c-MET receptor, which is expressed in different tumor cell types. The HGF/c-MET signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion and metastasis. Moreover, c-MET activation can be promoted by several ECM components, including proteoglycans and glycoproteins that act as bridging molecules and/or signal co-receptors. In contrast, c-MET activation can be inhibited by proteoglycans, matricellular proteins and/or proteases that bind and sequester HGF away from the cell surface. Therefore, understanding the effects of ECM components on HGF and c-MET may provide opportunities for novel therapeutic strategies. Here, we give a short overview of how certain ECM components regulate the distribution and activation of HGF and c-MET.

Asunto(s)

Matriz Extracelular/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Humanos , Neoplasias/patología , Transducción de Señal

RESUMEN

Acute kidney injury is mostly reversible, and hepatocyte growth factor (HGF) has a relevant role in the tissue repair. MicroRNA (miR)-26a is an endogenous modulator of HGF. The role of miR-26a in the kidney repair process was evaluated in Wistar rats submitted to an acute kidney injury model of rhabdomyolysis induced by glycerol (6 mL/kg). Animals were evaluated 3, 12, 48, 96, and 120 hours after glycerol injection. Serum creatinine (SCr) and gene expression of HGF, c-met, signal transducer and activator of transcription 3 (STAT3), and miR-26a were estimated. Also, tubular NK52E cells were transfected with anti-miR26a and stimulated with Fe3+ for 24 hours to mimic the effects of myoglobin in vitro. SCr was highest after 48 hours. After 96 hours, SCr started to decrease, characterizing the recovery phase, with normalization after 120 hours. HGF expression increased during the onset phase (3 hours), with a low relationship with miR-26a. In contrast, in the recovery phase, the increase in miR-26a was coincident with HGF messenger RNA suppression, suggesting that in the recovery phase, miR-26a may have a role in HGF modulation. Fe3+ induced cellular death after 3 hours and proliferation after 24 hours. There was no correlation between miR-26a and STAT3 during the death phase; however, during the proliferation phase, an increase in STAT3 was paralleled with a decrease in miR-26a. miR-26a silencing induced increases in cell viability and the phosphorylated form of STAT3 protein expression in cells receiving Fe3+ . In conclusion, miR-26a may have a key role in modulating HGF levels after its proliferative effects have been triggered.

Asunto(s)

Lesión Renal Aguda/genética , Glicerol/efectos adversos , Factor de Crecimiento de Hepatocito/genética , MicroARNs/genética , Factor de Transcripción STAT3/genética , Lesión Renal Aguda/sangre , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Animales , Línea Celular , Creatinina/sangre , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Masculino , Fosforilación , Proteínas Proto-Oncogénicas c-met/metabolismo , Ratas , Ratas Wistar , Rabdomiólisis/inducido químicamente , Factor de Transcripción STAT3/metabolismo , Transducción de Señal

RESUMEN

INTRODUCTION AND AIM: Hepatocyte growth factor (HGF) has been shown to ameliorate liver inflammation and fibrosis; however, the mechanism underlying its effects in non-alcoholic steatohepatitis (NASH) is unclear. This study aimed to analyse the relationship between the JAK2-STAT3 signalling pathway and the ameliorating effect of HGF on NASH. MATERIAL AND METHODS: Mice were fed a high-fat diet (HFD) for 16 weeks, and then plasma and hepatic tissues were collected. Histological and clinical chemistry assays were performed to assess liver disease. The mRNA and protein levels of JAK2, STAT3, and c-Met were assessed by real-time PCR and western blotting, respectively. RESULTS: Serum ALT, AST, and TG levels were increased in NASH mice. Histological analysis showed different degrees of steatosis, inflammatory infiltrates, and fibrosis in HFD animals. Exogenous administration of recombinant human (rh) HGF via the tail vein for 14 days markedly decreased ALT and AST to levels lower than those in the control group. Compared with the levels in HFD mice, c-Met, p-c-Met, JAK2, p-JAK2, and p-STAT3 levels were increased in mice that were administered HGF (P < 0.05). Furthermore, silencing of HGF or blocking of its receptor c-Met affected JAK2 and STAT3 protein phosphorylation. CONCLUSIONS: Excess HGF highly probable improved NASH liver function. Combined with its ligand, c-Met, HGF may promote the phosphorylation of JAK2-STAT3 and inhibit inflammation in NASH. Therefore, it may be potentially useful treatment for NASH.

Asunto(s)

Antiinflamatorios/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Janus Quinasa 2/metabolismo , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Citocinas/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Mediadores de Inflamación/sangre , Janus Quinasa 2/genética , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos BALB C , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Fosforilación , Proteínas Tirosina Quinasas Receptoras/genética , Factor de Transcripción STAT3/genética , Triglicéridos/sangre

RESUMEN

Bone marrow cells (BMC) migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF) and reduction of pro-inflammatory cytokines (IL-17A and IL-6). Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration.

Asunto(s)

Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Comunicación Celular , Colestasis/terapia , Citocinas/metabolismo , Hepatocitos/metabolismo , Cirrosis Hepática/terapia , Animales , Movimiento Celular , Proliferación Celular , Quimiocina CXCL12/metabolismo , Colestasis/complicaciones , Colestasis/genética , Colestasis/patología , Colágeno/metabolismo , Citocinas/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

We investigated the paracrine effects of bone marrow mesenchymal stem cells (BMSCs) on the proliferation, apoptosis, and alpha-actin-2 (ACTA2) expression of hepatic stellate cells (HSCs), and explored the possible mechanisms of hepatocyte growth factor (HGF). We established a co-culture system by culturing BMSCs on the upper layer and HSCs on the lower layer of a 6-well Transwell plate. Normal HSCs were cultured alone as a control. Cell apoptosis was determined by flow cytometry. We detected the expression of ACTA2 mRNA and ACTA2 protein in HSC using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. ACTA2 in HSCs was detected by fluorescent staining, and HGF in the co-culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic rate of HSCs in the experiment group was 2.6 times that in the control group (P < 0.05). The expression levels of ACTA2 mRNA and ACTA2 protein were significantly inhibited in HSCs compared with the control group (P < 0.05). HGF concentration in the co-culture supernatant was 0.43 ± 0.47 mM in the experimental group, which was significantly higher than in the control group (0.16 ± 0.43 mM) (P < 0.05). The paracrine effect of BMSCs, which was caused by the suppression of ACTA2 and HGF expression, induced HSC apoptosis.

Asunto(s)

Actinas/genética , Actinas/metabolismo , Células Estrelladas Hepáticas/citología , Células Madre Mesenquimatosas/citología , Comunicación Paracrina , Apoptosis , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo , Células Estrelladas Hepáticas/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos

RESUMEN

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.

Asunto(s)

Factor I del Crecimiento Similar a la Insulina/metabolismo , Cirrosis Hepática/terapia , Macrófagos/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Tioacetamida/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

BACKGROUND: Bone marrow multipotent mesenchymal stromal cells (MSCs) are a diverse subset of precursors that contribute to the homeostasis of the hematopoietic niche. MSCs can be isolated and expanded in vitro and have unique immunomodulatory and regenerative properties that make them attractive for the treatment of autoimmune diseases, including type 1 diabetes (T1D). Whether autologous or allogeneic MSCs are more suitable for therapeutic purposes has not yet been established. While autologous MSCs may present abnormal function, allogeneic cells may be recognized and rejected by the host immune system. Thus, studies that investigate biological characteristics of MSCs isolated from T1D patients are essential to guide future clinical applications. METHODS: Bone marrow-derived MSCs from recently diagnosed type 1 diabetes patients (T1D-MSCs) were compared with those from healthy individuals (C-MSCs) for morphological and immunophenotypic characteristics and for differentiation potential. Bioinformatics approaches allowed us to match absolute and differential gene expression of several adhesion molecules, immune mediators, growth factors, and their receptors involved with hematopoietic support and immunomodulatory properties of MSCs. Finally, the differentially expressed genes were collated for functional pathway enrichment analysis. RESULTS: T1D-MSCs and C-MSCs were similar for morphology, immunophenotype, and differentiation potential. Our absolute gene expression results supported previous literature reports, while also detecting new potential molecules related to bone marrow-derived MSC functions. T1D-MSCs showed intrinsic abnormalities in mRNA expression, including the immunomodulatory molecules VCAM-1, CXCL12, HGF, and CCL2. Pathway analyses revealed activation of sympathetic nervous system and JAK STAT signaling in T1D-MSCs. CONCLUSIONS: Collectively, our results indicate that MSCs isolated from T1D patients present intrinsic transcriptional alterations that may affect their therapeutic potential. However, the implications of these abnormalities in T1D development as well as in the therapeutic efficacy of autologous MSCs require further investigation.

Asunto(s)

Células de la Médula Ósea/metabolismo , Diabetes Mellitus Tipo 1/genética , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/genética , Transcriptoma , Adolescente , Adulto , Células de la Médula Ósea/patología , Estudios de Casos y Controles , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Femenino , Perfilación de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Masculino , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , ARN Mensajero/metabolismo , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

UNLABELLED:   BACKGROUND: Uncontrolled hapatic inflammatory response is regarded as the primary pathological mechanism of acute liver failure and impairs the regeneration of hepatocytes and stem cell grafts. Interleukin-1 plays a key role for activating immune and inflammatory response. Recently, siRNA has made quite a few progresses in treating inflammatory response. AIM: To assess the effect of IL-1? siRNA adenovirus on MSC and the therapeutic effect of MSC combined with IL-1? siRNA adenovirus in ALF. MATERIAL AND METHODS: We implanted MSC or/and IL-1? siRNA adenovirus via the tail vein, using CCl4-induced ALF in a mice model. Mice were sacrificed at different time points. Blood samples and liver tissues were collected. Hepatic injury, liver regeneration, cytokines (CXCL1, IL-1?, IL-10, IL-6, VEGF and HGF), animal survival and vital MSC were assessed after cell transplantation. RESULTS: MSC combined with IL-1? siRNA reduced the inflammatory levels and prevented liver failure. These animals administrated with MSC and IL-1? siRNA also exhibited improved liver regeneration and increased survival rates. Immunohistochemistry and fluorescence microscopy revealed the number of vital MSC in ALF + MSC + IL-1? siRNA group were significantly more than that in ALF + MSC group. CONCLUSION: IL-1? siRNA adenovirus could enhance MSC ability of tissue regeneration through increasing its survival rate. Accordingly, combination of IL-1? siRNA adenovirus and MSC had a synergistic effect on acute liver failure.

Asunto(s)

Interleucina-1beta/efectos de los fármacos , Fallo Hepático Agudo , Regeneración Hepática/efectos de los fármacos , Hígado/efectos de los fármacos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Adenoviridae , Animales , Quimiocina CXCL1/efectos de los fármacos , Quimiocina CXCL1/inmunología , Factor de Crecimiento de Hepatocito/metabolismo , Inflamación , Interleucina-10/inmunología , Interleucina-6/inmunología , Hígado/inmunología , Hígado/metabolismo , Regeneración Hepática/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Tratamiento con ARN de Interferencia , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The mechanism by which stem cell-based therapy improves heart function is still unknown, but paracrine mechanisms seem to be involved. Adipose-derived stem cells (ADSCs) secrete several factors, including insulin-like growth factor-1 (IGF-1), which may contribute to myocardial regeneration. Our aim was to investigate whether the overexpression of IGF-1 in ADSCs (IGF-1-ADSCs) improves treatment of chronically infarcted rat hearts. ADSCs were transduced with a lentiviral vector to induce IGF-1 overexpression. IGF-1-ADSCs transcribe100- to 200-fold more IGF-1 mRNA levels compared to nontransduced ADSCs. IGF-1 transduction did not alter ADSC immunophenotypic characteristics even under hypoxic conditions. However, IGF-1-ADSCs proliferate at higher rates and release greater amounts of growth factors such as IGF-1, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) under normoxic and hypoxic conditions. Importantly, IGF-1 secreted by IGF-1-ADSCs is functional given that Akt-1 phosphorylation was remarkably induced in neonatal cardiomyocytes cocultured with IGF-1-ADSCs, and this increase was prevented with phosphatidylinositol 3-kinase (PI3K) inhibitor treatment. Next, we tested IGF-1-ADSCs in a rat myocardial infarction (MI) model. MI was performed by coronary ligation, and 4 weeks after MI, animals received intramyocardial injections of either ADSCs (n = 7), IGF-1-ADSCs (n = 7), or vehicle (n = 7) into the infarcted border zone. Left ventricular function was evaluated by echocardiography before and after 6 weeks of treatment, and left ventricular hemodynamics were assessed 7 weeks after cell injection. Notably, IGF-1-ADSCs improved left ventricular ejection fraction and cardiac contractility index, but did not reduce scar size when compared to the ADSC-treated group. In summary, transplantation of ADSCs transduced with IGF-1 is a superior therapeutic approach to treat MI compared to nontransduced ADSCs, suggesting that gene and cell therapy may bring additional benefits to the treatment of MI.

Asunto(s)

Adipocitos/citología , Adipocitos/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/terapia , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

It is hard to bridge the gap between mathematical formulations and biological implementations of Turing patterns, yet this is necessary for both understanding and engineering these networks with synthetic biology approaches. Here, we model a reaction-diffusion system with two morphogens in a monostable regime, inspired by components that we recently described in a synthetic biology study in mammalian cells.1 The model employs a single promoter to express both the activator and inhibitor genes and produces Turing patterns over large regions of parameter space, using biologically interpretable Hill function reactions. We applied a stability analysis and identified rules for choosing biologically tunable parameter relationships to increase the likelihood of successful patterning. We show how to control Turing pattern sizes and time evolution by manipulating the values for production and degradation relationships. More importantly, our analysis predicts that steep dose-response functions arising from cooperativity are mandatory for Turing patterns. Greater steepness increases parameter space and even reduces the requirement for differential diffusion between activator and inhibitor. These results demonstrate some of the limitations of linear scenarios for reaction-diffusion systems and will help to guide projects to engineer synthetic Turing patterns.

Asunto(s)

Modelos Teóricos , Biología Sintética , Animales , Perros , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/metabolismo , Células de Riñón Canino Madin Darby , Unión Proteica , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Taquicininas/antagonistas & inhibidores , Receptores de Taquicininas/metabolismo

RESUMEN

OBJECTIVE: To assess the safety and feasibility of allogeneic human umbilical cord blood (hUCB)-derived mesenchymal stem cell (MSC) transplantation in preterm infants. STUDY DESIGN: In a phase I dose-escalation trial, we assessed the safety and feasibility of a single, intratracheal transplantation of hUCB-derived MSCs in preterm infants at high risk for bronchopulmonary dysplasia (BPD). The first 3 patients were given a low dose (1 × 10(7) cells/kg) of cells, and the next 6 patients were given a high dose (2 × 10(7) cells/kg). We compared their adverse outcomes, including BPD severity, with those of historical case-matched comparison group. RESULTS: Intratracheal MSC transplantation was performed in 9 preterm infants, with a mean gestational age of 25.3 ± 0.9 weeks and a mean birth weight of 793 ± 127 g, at a mean of 10.4 ± 2.6 days after birth. The treatments were well tolerated, without serious adverse effects or dose-limiting toxicity attributable to the transplantation. Levels of interleukin-6, interleukin-8, matrix metalloproteinase-9, tumor necrosis factor α, and transforming growth factor ß1 in tracheal aspirates at day 7 were significantly reduced compared with those at baseline or at day 3 posttransplantation. BPD severity was lower in the transplant recipients, and rates of other adverse outcomes did not differ between the comparison group and transplant recipients. CONCLUSION: Intratracheal transplantation of allogeneic hUCB-derived MSCs in preterm infants is safe and feasible, and warrants a larger and controlled phase II study.

Asunto(s)

Displasia Broncopulmonar/prevención & control , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Biomarcadores/metabolismo , Displasia Broncopulmonar/diagnóstico , Displasia Broncopulmonar/metabolismo , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Citocinas/metabolismo , Estudios de Factibilidad , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Recién Nacido , Recien Nacido Prematuro , Modelos Logísticos , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Índice de Severidad de la Enfermedad , Tráquea/metabolismo , Tráquea/cirugía , Trasplante Homólogo/efectos adversos , Trasplante Homólogo/métodos , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismo