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Although erythropoietin-producing human hepatocellular receptor A2 (EphA2) signaling serves an important role in the tumor microenvironment, its contribution to vasculogenic mimicry (VM) formation in gastric cancer cells remains unclear. The aim of the present study was to investigate the role of EphA2 in VM formation induced by cancer-associated fibroblasts (CAFs). The conditioned medium of CAFs (CAF-CM) was prepared from 12 patients with gastric adenocarcinoma. VM was evaluated by the number of tubules and intersections in gastric cancer cells following CAF-CM treatment. The role of EphA2-phosphoinositide 3-kinase (PI3K) in VM was investigated using EphA2-targeted small interfering (si)RNAs (siEphA2), EphA2 inhibitors and PI3K-inhibitors. CAF-CM-induced VM formation was significantly associated with high protein expression levels of EphA2. EphA2 inhibitor and siEphA2 manipulation significantly decreased VM formation by CAF-CM. In siEphA2 cells, decreased expression levels of VM-associated proteins were observed. CAF-CM-induced VM formation was blocked by the PI3K-inhibitor. In conclusion, CAFs facilitate VM formation via EphA2-PI3K signaling in gastric cancer cells. Thus, EphA2-PI3K signaling may be required for CAF-promoted VM formation during gastric tumorigenesis.
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Genetically deregulated tumor cells generate vascular channels by vasculogenic mimicry (VM) that is independent of endothelial blood vessels. The morphological characteristics of VM and the role of EphA2 in the formation of VM were evaluated in 144 clinical samples of gastric adenocarcinoma and AGS gastric cancer cell line. It has long been believed that VM consists of PAS-positive basement membrane and CD31/CD34-negative cells. Interestingly, we found that the luminal surface of gastric tumor cells that form VM channels showed PAS-positive reaction, and that the involvement of CD31/CD34-positive tumor cells in the formation of VM channels. Highly aggressive tumor cells that formed VM were found to express CD31 or CD34, implicating the angiogenic and vasculogenic potential of the genetically deregulated tumor cells. VM occurrence was positively correlated with high expression of EphA2 in our patient cohort, and the indispensable role of EphA2 in VM formation was identified by gene silencing in AGS cells. We also report that Epstein-Barr virus (EBV)-positive tumor cells were involved in the formation of VM channels in EBV-associated gastric cancer samples. Overall, our results suggest that EphA2 signaling promotes tumor metastasis by inducing VM formation during gastric tumorigenesis.
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Adenocarcinoma/metabolismo , Receptor EphA2/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Neovascularización Patológica , Receptor EphA2/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Neoplasias Gástricas/genéticaRESUMEN
PURPOSE: Under physiologic conditions, the binding of erythropoietin-producing hepatocellular (Eph) A2 receptor and its ligand ephrinA1 results in decreased EphA2 level and tumor suppression. However, EphA2 and ephrinA1 are highly expressed in human cancers including gastric adenocarcinoma. In this study, we tested our hypothesis that cancer-associated fibroblasts (CAFs) promote gastric tumorigenesis through EphA2 signaling in a ligand-independent manner. METHODS: Expression of EphA2 protein in primary tumor tissues of 91 patients who underwent curative surgery for gastric adenocarcinoma was evaluated by immunohistochemistry and western blotting. Conditioned medium of cancer-associated fibroblasts (CAF-CM) was used to evaluate the tumorigenic effect of CAFs on gastric cancer cell lines. Epithelial-mesenchymal transition (EMT), cell proliferation, migration, and invasion were assessed. EphrinA1-Fc ligand was used to determine the suppressor role of EphA2 receptor-ligand binding. RESULTS: CAF-CM-induced EMT and promoted cancer cell motility even without cell-cell interaction. Treatment with a selective EphA2 inhibitor (ALW-II-41-27) or EphA2-targeted siRNA markedly reduced CAF-CM-induced gastric tumorigenesis. EphrinA1-Fc ligand treatment showing ligand-dependent tumor suppression diminished the EphA2 expression and EMT progression. In contrast, ephrinA1-targeted siRNA did not significantly affect CAF-CM-mediated increases in EphA2 expression and EMT progression. Treatment with VEGF showed effects like CAF-CM in terms of EphA2 activation and EMT progression. CONCLUSION: CAFs may contribute to gastric tumorigenesis by activating EphA2 signaling pathway in a ligand-independent manner. Our results suggest that ligand-independent activation of EphA2 was triggered by VEGF released from CAF-CM. Our result may partially explain why ligand-dependent tumor suppressor roles of EphA2 are not evident in gastric cancer despite the prominent level of ephrinA1.
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Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Efrina-A2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/fisiología , Femenino , Mucosa Gástrica/metabolismo , Humanos , Ligandos , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño/metabolismo , Receptor EphA2 , Transducción de Señal/fisiología , Estómago/patologíaRESUMEN
BACKGROUND: The objective of this study was to investigate the underlying molecular mechanisms and the therapeutic time window for preventing astrogliosis with erythropoietin (EPO) treatment after in vitro modeled spinal cord injury (SCI). METHODS: Cultured rat spinal cord astrocytes were treated with kainate and scratching to generate an in vitro model of SCI. EPO (100U/mL or 300U/mL) was added immediately or 2, 4, or 8 hours after injury. Some cultures were also treated with AG490, an inhibitor of the EPO-EPO receptor (EpoR) pathway mediator Janus kinase 2 (JAK2). To evaluate neurite extension, rat embryonic spinal cord neurons were seeded onto astrocyte cultures and treated with EPO immediately after injury in the presence or absence of anti-EpoR antibody. RESULTS: EPO treatment at up to 8 hours after injury reduced the expression of axonal growth inhibiting molecules (glial fibrillary acidic protein, vimentin, and chondroitin sulfate proteoglycan), cytoskeletal regulatory proteins (Rho-associated protein kinase and ephephrin A4), and proinflammatory cytokines (tumor necrosis factor-alpha, transforming growth factor-beta, and phosphorylated-Smad3) in a dosedependent manner (Pâ<â.001). Most effects peaked with EPO treatment 2-4hours after injury. Additionally, EPO treatment up to 4 hours after injury promoted expression of the EpoR (>2-fold) and JAK2 (>3-fold) in a dose-dependent manner (Pâ<â.001), whereas co-treatment with AG490 precluded these effects (Pâ<â.001). EPO treatment up to 4hours after injury also enhanced axonal b-III tubulin-immunoreactivity (>12-fold), and this effect was precluded by co-treatment with an anti-EpoR antibody (Pâ<â.001). CONCLUSIONS: EPO treatment within 8 hours after injury reduced astrogliosis, and EPO treatment within 4 hours promoted neurite outgrowth. EPO therapy immediately after spinal cord injury may regulate glia to generate an environment permissive of axonal regeneration.
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Eritropoyetina/administración & dosificación , Gliosis/tratamiento farmacológico , Proyección Neuronal/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Tiempo de Tratamiento , Animales , Astrocitos , Axones/efectos de los fármacos , Células Cultivadas , Esquema de Medicación , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To develop an in vitro model analogous to the environment of traumatic spinal cord injury (SCI), the authors evaluated change of astrogliosis following treatments with kainate and/or scratch, and degree of neurite outgrowth after treatment with a kainate inhibitor. METHODS: Astrocytes were obtained from the rat spinal cord. Then, 99% of the cells were confirmed to be GFAP-positive astrocytes. For chemical injury, the cells were treated with kainate at different concentrations (10, 50 or 100 µM). For mechanical injury, two kinds of uniform scratches were made using a plastic pipette tip by removing strips of cells. For combined injury (S/K), scratch and kainate were provided. Cord neurons from rat embryos were plated onto culture plates immediately after the three kinds of injuries and some cultures were treated with a kainate inhibitor. RESULTS: Astro-gliosis (glial fibrillary acidic protein [GFAP], vimentin, chondroitin sulfate proteoglycan [CSPG], rho-associated protein kinase [ROCK], and ephrin type-A receptor 4 [EphA4]) was most prominent after treatment with 50 µM kainate and extensive scratch injury in terms of single arm (p<0.001) and in the S/K-induced injury model in view of single or combination (p<0.001). Neurite outgrowth in the seeded spinal cord (ß-III tubulin) was the least in the S/K-induced injury model (p<0.001) and this inhibition was reversed by the kainate inhibitor (p<0.001). CONCLUSION: The current in vitro model combining scratch and kainate induced glial scarring and inhibitory molecules and restricted neurite outgrowth very strongly than either the mechanically or chemically-induced injury model; hence, it may be a useful tool for research on SCI.
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BACKGROUNDS/AIMS: Vitamin K may plays a role in controlling hepatocellular carcinoma (HCC) cell growth. In this study, we intended to present 5-year experience of 72 patients receiving oral vitamin K with or without sorafenib. Its end-point was to evaluate the safety of combination therapy using sorafenib and vitamin K. METHODS: An interim analysis was performed as a single-arm cross-sectional study, including 72 HCC patients who underwent liver resection or transplantation and administered oral vitamin K2 alone (n=47) or with sorafenib (n=25). RESULTS: In all patients, administration of vitamin K2 analog 45 mg/day did not show any noticeable adverse side-effect during vitamin K therapy of 23.3±10.6 months, except for one patient who experienced skin rash at the third day of vitamin K therapy. In 25 patients receiving sorafenib and vitamin K for 6 months or longer, any noticeable adverse side-effect suspected of vitamin K origin was not identified yet. A small proportion of patients showed unexpectedly favorable anti-tumor effects after use of vitamin K with or without sorafenib. CONCLUSIONS: Because add-on of oral vitamin K did not increase the adverse side-effects of sorafenib, a combination therapy with these two agents appears to be worthy of further clinical trial with an expectation of synergistic therapeutic effects.
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Sorafenib increases survival of patients with advanced hepatocellular carcinoma (HCC) by inhibiting RAF kinase and receptor tyrosine kinase activity, but involvement of sorafenib in fibrosis and epithelial-mesenchymal transition (EMT) remains unclear. To elucidate effects of sorafenib on EMT progression and matrix metalloproteinase (MMP) activity, levels of E-cadherin, N-cadherin, and MMPs were evaluated in HepG2 human HCC cells induced by hepatocyte growth factor (HGF). Scratching cell migration assay, matrigel cell invasion assay, and immuno histochemistry were performed to examine effects of sorafenib on tumor metastasis and MMP expression. Sorafenib inhibited HGF-induced EMT and suppressed cell migration and invasion. Treatment with sorafenib significantly reduced HGF-enhanced expression of MMPs, suggesting that inhibition of MMP activity contributes to suppression of cellular motility and invasiveness of HepG2 cells. Neutralization of MMP activity by antibodies to MMP2/9, broad-spectrum MMP inhibitor or selective gelatinase inhibitor resulted in significant suppression of HGF-induced EMT and cell migration/invasion. Sorafenib treatment and MMP inactivation inhibited HGF-induced c-MET and MEK/ERK pathways. Sorafenib reduced MMP activity in this HGF-induced tumorigenic model of HCC. These findings provide in vitro evidence that sorafenib suppresses HGF-induced EMT and cell migration/invasion, as well as HGF-induced c-MET and MEK/ERK pathways.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Invasividad Neoplásica/genética , Niacinamida/administración & dosificación , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Proteínas Proto-Oncogénicas c-met/biosíntesis , SorafenibRESUMEN
Vitamin K plays a role in controlling cell growth. Anti-angiogenic effects of sorafenib lead to impairment of vitamin K uptake and induction of des-γ-carboxyprothrombin release by hepatocellular carcinoma (HCC) cells. We examined sorafenib and vitamin K individually and in combination regarding their ability to suppress migration and metastatic potential of HCC cells. HepG2 cells (HCC cell line) were treated with hepatocyte growth factor (HGF). E-Cadherin expression, phospho-MET (p-MET), and phospho-extracellular signal-regulated kinase (p-ERK) levels and cell migration were evaluated. HGF-stimulated HepG2 cells, which were treated with a combination of sorafenib and vitamin K, showed significantly increased expression of E-cadherin and impairment of migration ability compared to when treated with either agent alone. This combination therapy also induced marked inhibition of epithelial-mesenchymal transition phenotype; inhibition of HGF-stimulated cell proliferation, invasion and migration; and inhibition of HGF/c-MET signaling pathway. Levels of p-MET and p-ERK were also significantly reduced by this combination. Our experimental study demonstrated that sorafenib and vitamin K can function synergistically to inhibit the migration and proliferation of HCC cells. Combination therapy with sorafenib and vitamin K appears to be worthy of clinical trial with expectation of synergistic therapeutic effects.
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Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Vitamina K/administración & dosificación , Carcinoma Hepatocelular/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Células Hep G2 , Factor de Crecimiento de Hepatocito/administración & dosificación , Humanos , Neoplasias Hepáticas/patología , Niacinamida/administración & dosificación , SorafenibRESUMEN
OBJECTIVE: The aim of this study was to evaluate transglutaminase type 2 (TG2) expression in human abdominal aortic aneurysm (AAA) tissue and to elucidate a potential role of TG2 in AAA formation. TG2, which is a Ca(2+)-dependent cross-linking enzyme, has been proven important for stabilizing the extracellular matrix. However, there is no evidence of the effect of TG2 on AAA formation in a human model. METHODS: Aortic wall tissues were obtained during surgery in AAA patients (n = 38) and in patients with aortoiliac occlusive disease (Control; n = 4) in the Asan Medical Center from March 2011 to February 2012. In each AAA patient, the aortic neck (Neck) and maximally dilated portion (Max) of the aneurysm were sampled for analysis. TG2 expression was evaluated using immunohistochemistry and Western blotting. In addition, ex vivo experiments of isolated AAA tissue culture with the TG2 inhibitor cystamine and recombinant human TG2 were performed. RESULTS: Among 38 AAA patients, 11 had ruptured (contained or free) AAAs. The mean maximal diameter of AAAs was 6.09 ± 1.46 cm. TG2 expressions of Max were significantly increased compared with those of Control (1.7-fold increase of Control; P = .00). Compared with Control, the intensities of tissue necrosis factor-α, matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitors of metalloproteinase-2 were significantly upregulated in Max (1.7-fold, 1.5-fold, 1.3-fold, and 1.6-fold increases of Control; P = .00, P = .004, P = .046, and P = .007, respectively). Furthermore, double immunofluorescent staining showed that colocalization of TG2/transforming growth factor-ß or TG2/fibronectin was prominent in Max compared with those of Neck or Control. In addition, MMP-2 intensity was upregulated in ruptured AAAs compared with unruptured AAAs, with marginal significance (P = .078). Ex vivo experiments showed that protein expressions of tissue necrosis factor-α, MMP-2, and MMP-9 in cultured AAA tissue were decreased by recombinant human TG2 but were increased by exogenous cystamine. CONCLUSIONS: The TG2 expression in the maximally dilated portion of AAAs was enhanced compared with that of nondilated aorta. It is suggested that TG2 has a potential effect in stabilization of extracellular matrix by inhibition of proinflammatory cytokines and MMPs or by interaction with fibronectin and transforming growth factor-ß.
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Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/enzimología , Rotura de la Aorta/enzimología , Matriz Extracelular/metabolismo , Transglutaminasas/metabolismo , Anciano , Anciano de 80 o más Años , Aorta Abdominal/diagnóstico por imagen , Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Rotura de la Aorta/diagnóstico por imagen , Aortografía/métodos , Western Blotting , Estudios de Casos y Controles , Cistamina/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Fibronectinas/metabolismo , Proteínas de Unión al GTP , Humanos , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteínas Recombinantes/metabolismo , República de Corea , Técnicas de Cultivo de Tejidos , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Tomografía Computarizada por Rayos X , Factor de Crecimiento Transformador beta1/metabolismo , Transglutaminasas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUNDS/AIMS: Mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, The purpose of this study is to investigate the MSCs' differentiation process and therapeutic potentials by comparing isolated MSCs with HGF-treated MSCs in rat's model with thiacetamide (TAA)-induced cirrhosis. METHODS: Male Sprague-Dawley (SD) rats, weighing 100-150 g were used in this study. To induce liver fibrosis, recipient rats were taken with 0.04% thioacetamide (TAA) in the drinking water (400 mg TAA/L) for 8 weeks. The rats underlying liver cirrhosis were divided into 3 groups according to the transplanted materials, compared to normal saline as control (I) and isolated MSCs (II) HGF-treated MSCs. RESULTS: Severe hepatic fibrosis and hepatocyte destruction were detected in the control group. Less hepatic cirrhosis and collagen formation, more hepatocyte regeneration and glycogen storage were detected in isolated MSCs compared to HGF-treated MSCs group, Distribution of red autofluorescence is mainly localized near the sinusoids in isolated MSCs, scattered away the sinusoids in HGF-treated MSCs group. MSCs transdifferentiated into CK-19 postive Oval cells and then to albulmin-producing hepatocytes, HGF treated MSCs differentiated into hepatocyte without the intermediate oval cells phase. HGF treated MSCs became the CK18-positive, MSCs became CD 90-positive. CONCLUSIONS: Significant hepatocyte differentiation occurred in not HGF-treated MSCs but isolated MSCs group unexpectedly. These results suggest that the beneficial effect of MSCs on in rat's model with TAA-induced cirrhosis may occur during early differentiation course of MSCs. Mature hepatocyte itself has a little effect on the accelerated differentiation and functional capacity of hepatic lineage cell-line.
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BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) often recurs after complete surgical resection. Detection of markers of residual circulating cancer cells may predict postoperative HCC recurrence. Human telomerase reverse transcriptase (hTERT) mRNA may be a candidate tumor marker. METHODOLOGY: We prospectively assessed the expression patterns and prognostic value of preoperative peripheral blood hTERT mRNA in patients with HCC undergoing hepatic resection (n=17) or liver transplantation (n=6). As controls, we assessed hTERT mRNA in patients with liver cirrhosis without HCC (n=6) and in living liver donors (n=4). Concentrations of hTERT mRNA were measured by real-time quantitative reverse transcription polymerase chain reaction (RTPCR). RESULTS: No significant difference was observed in the levels of hTERT mRNA between the HCC and control groups. Only alpha-fetoprotein ≥400ng/mL was associated with greater expression levels of hTERT mRNA. At a median follow-up of 30 months, HCC recurred in 10 of 17 resected patients, but in none of the 6 liver transplant recipients. hTERT mRNA concentration was not associated with HCC recurrence after either resection or liver transplantation. CONCLUSIONS: Peripheral blood hTERT mRNA concentration is not a likely marker for the diagnosis or prognosis of HCC, especially in patients undergoing resection. Owing to the small number of transplanted patients assessed, the clinical significance of hTERT mRNA concentration was not objectively verified, suggesting the need for a study in larger numbers of HCC patients undergoing liver transplantation.
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Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Neoplasias Hepáticas/sangre , Recurrencia Local de Neoplasia/sangre , ARN Mensajero/sangre , Telomerasa/sangre , Anciano , Carcinoma Hepatocelular/cirugía , Femenino , Hepatectomía , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/cirugía , Neoplasias Hepáticas/cirugía , Trasplante de Hígado , Masculino , Persona de Mediana Edad , Neoplasia Residual , Células Neoplásicas Circulantes , Valor Predictivo de las Pruebas , Periodo Preoperatorio , Estadísticas no ParamétricasRESUMEN
Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.
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Diferenciación Celular , Hepatocitos/citología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Células Madre Mesenquimatosas/citología , Tioacetamida/farmacología , Animales , Hepatocitos/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Here, we sought to delineate the effect of EPO on the remyelination processes using an in vitro model of demyelination. We report that lysolecithin-induced demyelination elevated EPO receptor (EpoR) expression in oligodendrocyte progenitor cells (OPCs), facilitating the beneficial effect of EPO on the formation of oligodendrocytes (oligodendrogenesis). In the absence of EPO, the resultant remyelination was insufficient, possibly due to a limiting number of oligodendrocytes rather than their progenitors, which proliferate in response to lysolecithin-induced injury. By EPO treatment, lysolecithin-induced proliferation of OPCs was accelerated and the number of myelinating oligodendrocytes and myelin recovery was increased. EPO also enhanced the differentiation of neural progenitor cells expressing EpoR at high level toward the oligodendrocyte-lineage cells through activation of cyclin E and Janus kinase 2 pathways. Induction of myelin-forming oligodendrocytes by high dose of EPO implies that EPO might be the key factor influencing the final differentiation of OPCs. Taken together, our data suggest that EPO treatment could be an effective way to enhance remyelination by promoting oligodendrogenesis in association with elevated EpoR expression in spinal cord slice culture after lysolecithin-induced demyelination.
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Eritropoyetina/farmacología , Vaina de Mielina/fisiología , Neurogénesis/efectos de los fármacos , Oligodendroglía/citología , Regeneración de la Medula Espinal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Animales , Enfermedades Desmielinizantes/inducido químicamente , Lisofosfatidilcolinas/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Receptores de Eritropoyetina/agonistas , Receptores de Eritropoyetina/metabolismo , Médula Espinal/fisiologíaRESUMEN
We investigated the combination of human adipose tissue derived stem cells (ADSC) and in vivo gel-forming methoxy poly (ethyleneglycol)-poly (epsilon-caprolactone) (MPEG-PCL) as a muscle regeneration matrix, with and without inclusion of vascular endothelial cell growth factor (VEGF). VEGF(165)-treated stem cell grafts showed significant proliferation and differentiation into muscle tissue in vivo. Importantly, the inclusion of VEGF enhanced vascularization. This scaffold supported preconditioned ADSC, and allowed them to differentiate into mature muscle tissues in vivo, indicating that ADSC of human origin and MPEG-PCL scaffolds provided an appropriate environment for cellular growth and expansion. Our results thus provide a potential solution to the major obstacle encountered in the engineering of thick complex tissues, which require an adequate blood supply to maintain cell viability during tissue growth and to induce appropriate structural organization. Therefore, the combination of ADSC and in vivo gel-forming MPEG-PCL with VEGF(165) might serve as a suitable non-invasive biomaterial for clinical muscle regeneration applications.
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Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Hidrogeles/química , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Células Madre/efectos de los fármacos , Células Madre/fisiología , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Materiales Biocompatibles/química , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Cristalización/métodos , Ensayo de Materiales , Temperatura , Ingeniería de Tejidos/métodosRESUMEN
OBJECTIVE: To assess the effect of ipsilateral hepatic vein embolization (HVE) performed after portal vein embolization (PVE) on liver regeneration. SUMMARY BACKGROUND DATA: PVE induces shrinkage of the embolized lobe and compensatory enlargement of the non-embolized lobe, but it does not always induce sufficient liver regeneration. There was no effective method to accelerate liver regeneration in addition to PVE yet. METHODS: During a 1-year study period, preoperative HVE were performed on 12 patients who had shown limited liver regeneration after PVE awaiting right hepatectomy. The right hepatic vein was embolized with multiple coils after insertion of vena cava filters or vascular plugs. RESULTS: No HVE procedure-related complications occurred, but embolization of the wrong hepatic vein trunk occurred in 1 patient. The increase in blood liver enzymes after HVE was comparable with that after PVE alone. In 9 patients who underwent hepatectomy, the proportions of future liver remnant volume to total liver volume were 34.8% +/- 1.5% before PVE, 39.7% +/- 0.6% 1 to 2 weeks after PVE, 44.2% +/- 1.1% 2 weeks after HVE, and 64.5% +/- 6.2% 1 week after right hepatectomy. Cirrhotic livers showed lower regeneration rates following HVE after PVE and 1 patient underwent hepatectomy 17 months after HVE. Immunohistochemistry showed that apoptosis occurred more in the liver area affected by both PVE and HVE than in that affected by PVE alone. CONCLUSIONS: Preoperative sequential application of PVE and HVE seems to be safe and effective in facilitating contralateral liver regeneration by inducing more severe liver damage than PVE alone.
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Embolización Terapéutica/métodos , Venas Hepáticas , Neoplasias Hepáticas/terapia , Regeneración Hepática/fisiología , Vena Porta , Anciano , Neoplasias de los Conductos Biliares/mortalidad , Neoplasias de los Conductos Biliares/patología , Neoplasias de los Conductos Biliares/cirugía , Neoplasias de los Conductos Biliares/terapia , Terapia Combinada , Femenino , Estudios de Seguimiento , Hepatectomía/métodos , Humanos , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Cuidados Preoperatorios/métodos , Probabilidad , Estudios Prospectivos , Medición de Riesgo , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Although the neuroprotective effects of erythropoietin (EPO) preconditioning are well known, the potential of postapplied EPO to protect neurons against excitotoxic injury has not been clearly established. Here we show that kainate (KA)-induced excitotoxicity, which plays a key role in secondary spinal cord injury, decreased neuron survival, inhibited neurite extension, and significantly reduced the expression of erythropoietin receptors (EpoR) in cultured spinal neurons. Posttreatment with EPO for 48 hr protected neurons against KA-induced injury, opposing KA-induced apoptosis and promoting regrowth of motoneuron neurites. These neuroprotective effects were paralleled by a restoration of EpoR expression. The importance of the EpoR signaling pathway was demonstrated using an EpoR blocking antibody, which neutralized the neuroprotective action of EPO posttreatment and prevented EPO-induced increases in EpoR expression. We also found that up-regulated EpoR stimulated the Janus kinase 2 (JAK2) pathway, which is known to facilitate neuronal growth and neurite regeneration. Although EPO posttreatment modestly attenuated KA-induced reactive gliosis in mixed neuron-glial cultures, blocking EpoR activity did not alter glial fibrillary acidic protein expression or astrocyte proliferation. In conclusion, 48 hr treatment with EPO following KA exposure induced EpoR-dependent protection against excitotoxic injury, demonstrating that preconditioning is not a prerequisite for neuroprotection by EPO. The neuroprotective effects of EPO posttreatment were mediated by an EpoR-dependent signaling pathway that possibly involves JAK2. The neuroprotective effect of EPO posttreatment against KA excitotoxicity appears to reflect direct effects on neurons and not indirect effects mediated by astrocytes.
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Apoptosis/efectos de los fármacos , Eritropoyetina/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Médula Espinal/efectos de los fármacos , Análisis de Varianza , Animales , Astrocitos/fisiología , Caspasa 3/metabolismo , Recuento de Células , Células Cultivadas , Técnicas de Cocultivo/métodos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Humanos , Etiquetado Corte-Fin in Situ , Janus Quinasa 2/metabolismo , Ácido Kaínico/farmacología , Neuronas Motoras/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Técnicas de Cultivo de TejidosRESUMEN
We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Inhibidores Enzimáticos/farmacología , Ácido Gálico/farmacología , Quercetina/farmacología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , NAD/farmacología , Uridina Difosfato Glucosa/farmacologíaRESUMEN
Lead is a neurotoxin that affects the developing central nervous system and may potentially induce apoptotic cell death. We investigated the effect of ascorbic acid against lead-induced neurotoxicity in the developing rat hippocampus. Female Sprague-Dawley rats were divided into three groups: control group, lead-treated group and lead plus ascorbic acid-treated group. Lead (0.2% lead acetate) was administered to female rats during pregnancy and lactation, in their drinking water. During this period, rats in the lead plus ascorbic acid-treated group received 100 mg/kg/day ascorbic acid, orally. At the end of the treatment, neuronal damage, apoptosis and blood lead levels were determined and the levels of Bax and Bcl-2 were immunodetected in the hippocampus of 21-day-old male pups. Histopathological evaluation demonstrated that ascorbic acid significantly attenuates apoptosis in the developing hippocampus and also spares hippocampal CA1, CA3 and dentate gyrus (DG) neurons. Simultaneous administration of ascorbic acid and lead lowered the level of Bax protein and increased Bcl-2 in pup hippocampus and reduced lead level in blood of dams compared with lead-treated only. Based on these results, it seems that ascorbic acid may potentially be beneficial in treating lead-induced brain injury in the developing rat brain.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácido Ascórbico/uso terapéutico , Hipocampo/patología , Degeneración Nerviosa/patología , Degeneración Nerviosa/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/ultraestructura , Etiquetado Corte-Fin in Situ/métodos , Plomo/toxicidad , Intoxicación por Plomo/complicaciones , Masculino , Degeneración Nerviosa/etiología , Embarazo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Proteína X Asociada a bcl-2/metabolismoRESUMEN
There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.
Asunto(s)
Mutación , Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Serina/química , Serina/genética , Serina/metabolismo , Relación Estructura-Actividad , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
The aim of this study was to investigate whether erythropoietin (EPO) protect the spinal GABAergic neurons against kainic acid (KA)-excitotoxic damage in rat spinal cord cell cultures. We performed immunohistochemical staining and Western blotting of glutamate decarboxylase 67 (GAD67), one isoform of GABA-producing enzyme, which was considered to have involved in nonsynaptic functions, such as energy metabolism or trophic support. T exposure to KA significantly reduced the intensity of GAD67 expression in the GABAergic neurons and whole cell lysate, indicating that the excitotoxic damage on the GABAergic neurons may lead to reduction of the GAD67 production following KA-exposure. We found that post-treatment of EPO for 48 h after KA-injury remarkably enhanced the expression level of GAD67 and erythropoietin receptors (EpoR), which were deteriorated by KA. Our confocal images clearly demonstrated a remarkably enhanced expression of EpoR on the surface of the GABAergic neurons by post-treated EPO after KA-damage. This result suggests that the neuroprotective effect of post-treated EPO on the GABAergic neurons can be related to the EPO-mediated EpoR upregulation following KA-excitotoxicity. We observed that the post-applied EPO clearly increased expression of tyrosine Janus kinase 2 (JAK2), which is known to be the first step of EpoR-stimulation. In conclusion, the post-treated EPO is a potent protector of the spinal GABAergic neurons against KA-excitotoxicity and regulates production of GAD67 for the multiple trophic roles after KA-induced disturbance. We suggested that the protective effect of post-treated EPO on the GABAergic neurons is mediated by signal transduction involving EpoR-dependent JAK2 pathway.