RESUMO
BACKGROUND: The two ends of arteriovenous graft (AVG) are anastomosed to the upper limb vessels by surgery for hemodialysis therapy. However, the size of upper limb vessels varies to a large extent among different individuals. METHODS: According to the shape and size of neck vessels quantified from the preoperative computed tomography angiographic scan, the ethylene-vinyl acetate (EVA)-based AVG was produced in H-shape by the three-dimensional (3D) printer and then sterilized. This study investigated the function of this novel 3D-printed AVG in vitro and in vivo. RESULTS: This 3D-printed AVG can be implanted in the rabbit's common carotid artery and common jugular vein with ease and functions in vivo. The surgical procedure was quick, and no suture was required. The blood loss was minimal, and no hematoma was noted at least 1 week after the surgery. The blood flow velocity within the implanted AVG was 14.9 ± 3.7 cm/s. Additionally, the in vitro characterization experiments demonstrated that this EVA-based biomaterial is biocompatible and possesses a superior recovery property than ePTFE after hemodialysis needle cannulation. CONCLUSIONS: Through the 3D printing technology, the EVA-based AVG can be tailor-made to fit the specific vessel size. This kind of 3D-printed AVG is functioning in vivo, and our results realize personalized vascular implants. Further large-animal studies are warranted to examine the long-term patency.
RESUMO
Arteriovenous fistula (AVF) is the vascular access of choice for renal replacement therapy. However, AVF is susceptible to calcification with a high prevalence of 40%-65% in chronic hemodialysis patients. Repeated needle puncture for hemodialysis cannulation results in intimal denudation of AVF. We hypothesized that exposure to blood shear stress in the medial layer promotes venous smooth muscle cell (SMC) osteogenesis. While previous studies of shear stress focused on arterial-type SMCs, SMCs isolated from the vein had not been investigated. This study established a venous cell model of AVF using the fluid shear device, combined with a high phosphate medium to mimic the uremic milieu. Osteogenic gene expression of venous SMCs upon mechanical and chemical cues was analyzed in addition to the activated cell signaling pathways. Our findings indicated that upon shear stress and high phosphate environment, mechanical stimulation (shear stress) had an additive effect in up-regulation of an early osteogenic marker, Runx2. We further identified that the integrin ß1-ERK1/2 signaling pathway was responsible for the molecular basis of venous SMC osteogenesis upon shear stress exposure. Mitochondrial biogenesis also took part in the early stage of this venopathy pathogenesis, evident by the up-regulated mitochondrial transcription factor A and mitochondrial DNA polymerase γ in venous SMCs. In conclusion, synergistic effects of fluid shear stress and high phosphate induce venous SMC osteogenesis via the ERK1/2 pathway through activating the mechanosensing integrin ß1 signaling. The present study identified a promising druggable target for reducing AVF calcification, which deserves further in vivo investigations.
Assuntos
Calcinose/patologia , Integrina beta1/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos de Músculo Liso/patologia , Osteogênese , Fosfatos/efeitos adversos , Estresse Mecânico , Calcinose/etiologia , Calcinose/metabolismo , Sinais (Psicologia) , Fístula/etiologia , Fístula/metabolismo , Fístula/patologia , Humanos , Integrina beta1/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Miócitos de Músculo Liso/metabolismo , Diálise Renal/efeitos adversos , Resistência ao Cisalhamento , Transdução de SinaisRESUMO
BACKGROUND: Life-long peritoneal dialysis (PD) as a renal replacement therapy is limited by peritoneal fibrosis. Previous studies showed immunomodulatory and antifibrotic effects of adipose-derived mesenchymal stem cells (ADSCs) on peritoneal fibrosis. However, the role of the peritoneal macrophage in this process remains uninvestigated. METHODS: We examined the therapeutic effects of ADSC and bone marrow-derived mesenchymal stem cells (BM-MSC) in the rat model of dialysis-induced peritoneal fibrosis using methylglyoxal. In addition, treatment of macrophages with the conditioned medium of ADSC and BM-MSC was performed individually to identify the beneficial component of the stem cell secretome. RESULTS: In the in vivo experiments, we found dialysis-induced rat peritoneal fibrosis was attenuated by both ADSC and BM-MSC. Interestingly, ADSC possessed a more prominent therapeutic effect than BM-MSC in ameliorating peritoneal membrane thickening while also upregulating epithelial cell markers in rat peritoneal tissues. The therapeutic effects of ADSC were positively associated with M2 macrophage polarization. In the in vitro experiments, we confirmed that interleukin-6 (IL-6) secreted by MSCs upon transforming growth factor-ß1 stimulation promotes M2 macrophage polarization. CONCLUSIONS: In dialysis-induced peritoneal fibrosis, MSCs are situated in an inflammatory environment of TGF-ß1 and secrete IL-6 to polarize macrophages into the M2 phenotype. Our findings reveal a previously unidentified role of tissue macrophage in this antifibrotic process. ADSC has the advantage of abundance and accessibility, making the application values extremely promising. In dialysis-induced peritoneal fibrosis, peritoneal mesothelial cells secrete transforming growth factor-ß1 (TGF-ß1) when exposed to methylglyoxal (MGO)-containing peritoneal dialysate. When situated in TGF-ß1, the inflammatory environment induces mesenchymal stem cells to secrete interleukin-6 (IL-6), IL-6 polarizes macrophages into the M2 phenotype. The dominant peritoneal tissue M2 macrophages, marked by upregulated Arg-1 expression, account for the attenuation of MGO-induced dedifferentiation of peritoneal mesothelial cells to maintain epithelial integrity.
Assuntos
Células-Tronco Mesenquimais , Fibrose Peritoneal , Animais , Interleucina-6 , Macrófagos , Células-Tronco Mesenquimais/patologia , Fibrose Peritoneal/etiologia , Fibrose Peritoneal/patologia , Fibrose Peritoneal/terapia , Ratos , Diálise RenalRESUMO
BACKGROUND: In vitro and clinical studies suggest that the oncogene LMP1 (latent membrane protein 1) encoded by Epstein-Barr virus (EBV) plays a role in the development of nasopharyngeal carcinoma (NPC) and the formation of metastases in immunocompetent individuals. However, whether LMP1 itself is sufficient to drive these events in immunocompetent hosts remains elusive due to the lack of appropriate experimental models. The aim of this study was to study LMP1-dependent tumorigenesis and metastasis in BALB/c mice inoculated with BALB/c-3T3 cells expressing N-LMP1 (a Taiwanese NPC variant). METHODS: Following cancer cell inoculation, metastasis formation was monitored over time using PCR analysis of LMP1 as tumor marker. We also used a luciferase (Luc)-containing N-LMP1 and bioluminescent imaging (BLI) to monitor metastasis formation in a non-invasive manner. RESULTS: N-LMP1 appeared early in draining lymph nodes and in various distant organs before the rapid growth of the primary tumor. Lung metastasis was observed by BLI and further confirmed by histological examination. Furthermore, we detected luciferase signals in the lungs, even before the animals were sacrificed. CONCLUSIONS: Our results demonstrate the high metastatic character of N-LMP1 in immunocompetent hosts. Systemic tumor dissemination occurs even before aggressive tumor growth at the primary site, suggesting that early treatment of primary LMP1-associated tumors and distant micro-metastases is critical to achieve positive results.
Assuntos
Neoplasias Nasofaríngeas/patologia , Metástase Neoplásica/patologia , Proteínas da Matriz Viral/fisiologia , Animais , Herpesvirus Humano 4 , Imunocompetência , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologiaRESUMO
In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Cisplatino/uso terapêutico , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteína p300 Associada a E1A/metabolismo , Endonucleases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/antagonistas & inibidores , Transativadores/genética , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Taxol is a mitotoxin widely used to treat human cancers, including of the breast and ovary. However, taxol resistance (txr) limits treatment efficacy in human patients. To study chemoresistance in ovarian cancer, we established txr ovarian carcinoma cells derived from the SKOV3 cell lineage. The cells obtained were cross-resistant to other mitotoxins such as vincristine while they showed no resistance to the genotoxin cisplatin. Transcriptomic analysis identified 112 highly up-regulated genes in txr cells. Surprisingly, FK506-binding protein 5 (FKBP5) was transiently up-regulated 100-fold in txr cells but showed decreased expression in prolonged culture. Silencing of FKBP5 sensitized txr cells to taxol, whereas ectopic expression of FKBP5 increased resistance to the drug. Modulation of FKBP5 expression produced similar effects in response to vincristine but not to cisplatin. We observed that a panel of newly identified txr genes was trancriptionally regulated by FKBP5 and silencing of these genes sensitized cells to taxol. Notably, immunoprecipitation experiments revealed that FKBP5 forms a protein complex with the androgen receptor (AR), and this complex regulates the transcriptional activity of both proteins. Furthermore, we found that the Akt kinase pathway is regulated by FKBP5. These results indicate that the FKBP5/AR complex may affect cancer cell sensitivity to taxol by regulating expression of txr genes. Our findings suggest that mitotoxin-based treatment against ovarian cancer should be avoided when the Akt/FKBP5/AR axis is activated.
Assuntos
Biomarcadores Tumorais/análise , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/metabolismo , Receptores Androgênicos/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Immunoblotting , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Paclitaxel/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oncogenic latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is involved in the pathogenesis of human nasopharyngeal carcinoma (NPC) and lymphoma. We and other authors have shown earlier that LMP1 induces apoptosis and inhibits xenograft tumor growth in mice, but the mechanism underlying these processes has not been investigated so far. In the present study, we show that knockdown of LMP1 renders the EBV-positive NPC cell line CG-1 resistant to various genotoxic drugs (cisplatin, etoposide, and adriamycin). LMP1 inhibits the expression of Cabin1, a Ca(2+) regulated protein shown earlier to inhibit calcineurin. Knockdown of calcineurin binding protein (Cabin1) with small hairpin RNA sensitizes CG-1 cells to genotoxic drugs. In contrast, LMP1 overexpression reduces Cabin1 level and renders both CG-1 cells and EBV-negative NPC cell lines sensitive to cisplatin. The c-Jun-N-terminal kinase (JNK) and ERK pathways are required for LMP1-induced suppression of Cabin1 at the transcriptional level. Chromatin immunoprecipitation assays further confirm that the JNK-activated transcription factor AP-1 mediates the LMP1-induced down-regulation of Cabin1 gene expression. LMP1 knockdown also increases the resistance of xenograph tumors to cisplatin in mice, therefore confirming the relevance of our findings in vivo. This study reveals the molecular mechanism underlying the pro-apoptotic activity of LMP1 during cisplatin-based NPC chemotherapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Nasofaríngeas/tratamento farmacológico , Proteínas da Matriz Viral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Carcinoma , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Regiões Promotoras Genéticas , Interferência de RNA , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Transfecção , Carga Tumoral , Proteínas da Matriz Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Growth arrest-specific gene 7 (Gas7) has previously been shown to be involved in neurite outgrowth in vitro; however, its actual role has yet to be determined. To investigate the physiological function of Gas7 in vivo, here we generated a Gas7-deficient mouse strain with a labile Gas7 mutant protein whose functions are similar to wild-type Gas7. METHODOLOGY/PRINCIPAL FINDINGS: Our data show that aged Gas7-deficient mice have motor activity defects due to decreases in the number of spinal motor neurons and in muscle strength, of which the latter may be caused by changes in muscle fiber composition as shown in the soleus. In cross sections of the soleus of Gas7-deficient mice, gross morphological features and levels of myosin heavy chain I (MHC I) and MHC II markers revealed significantly fewer fast fibers. In addition, we found that nerve terminal sprouting, which may be associated with slow and fast muscle fiber composition, was considerably reduced at neuromuscular junctions (NMJ) during aging. CONCLUSIONS/SIGNIFICANCE: These findings indicate that Gas7 is involved in motor neuron function associated with muscle strength maintenance.
Assuntos
Envelhecimento/fisiologia , Atividade Motora/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Sequência de Aminoácidos , Animais , Células do Corno Anterior/metabolismo , Sequência de Bases , Linhagem Celular , Expressão Gênica , Marcação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares Esqueléticas/patologia , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/deficiência , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transporte ProteicoRESUMO
We determined earlier that the hepatoma upregulated protein (HURP) is overexpressed in hepatocellular carcinoma (HCC), but the role of this protein during cancer development and progression remains unknown. Here, we observed that the overexpression of HURP in HEK293 cells promoted the ubiquitination of p53 and its degradation by the proteasome. In contrast, HURP knockdown using short-hairpin RNA reversed these effects. Knockdown of HURP promoted the accumulation of p53 in SK-Hep-1 cells (p53+/-), and these cells showed reduced proliferation, while the p53-mutant Mahlavu cells were not affected. HURP knockdown did not affect the proliferation of H1299 lung carcinoma cells and Hep3B HCC cells which lack p53. Knockdown of HURP also sensitized SK-Hep-1 cells to cisplatin. On the other hand, the expression of exogenous p53 in H1299 and Hep3B cells was decreased following overexpression of HURP, and these cells showed decreased sensitivity to cisplatin-induced apoptosis. Importantly, overexpression of HURP promoted the proliferation of HEK293 cells in an anchorage-independent manner, and inoculation of SK-Hep-1 cancer cells that expressed short-hairpin RNA to knockdown HURP resulted in smaller tumors in nude mice. Gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, was found to be upregulated following HURP expression, and gankyrin knockdown decreased the HURP-mediated downregulation of p53. Notably, we detected a positive correlation between elevated HURP and gankyrin protein levels in HCC patients (r(2) = 0.778; N = 9). Taken together, these results indicate that HURP represents an oncogene that may play a role in HCC progression and chemoresistance.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Neoplasias Hepáticas/genética , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/genética , Ubiquitinação , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Bone marrow (BM)-derived cells were demonstrated within intestines after radiation damage and were reported to be responsible for intestine repair. However, there was a discrepancy between intestine epithelial clonogenic regeneration, and mouse survival after BM transplantation (BMT) and radiation. The contribution of BM to acute intestine repair after radiation needed further investigation. METHODS: Mouse survival, intestine microcolony assay, immunohistochemical studies of both intestine and BM were evaluated in mice after whole body irradiation (WBI) and BMT. Immunoblotting, flowcytometry, and double immunostaining were used to evaluate the amount and the character of stroma cells within intestines of recipient mice after receiving gender-mismatched BMT or BMT from green fluorescence donors. RESULTS: Stromal cell proliferation within the lamina propria correlated with the beneficial effect of BMT to intestine recovery and day-8 survival of mice. Few donor-derived cells were found before the completion of intestine repair. The number of host but not donor-derived myelomonocytic and stromal cells increased dramatically within one week after radiation and BMT. Depletion of myelomonocytic cells of recipient mice abolished the mitigating effect of BMT. CONCLUSIONS: Besides rescuing injured BM from aplasia, BMT triggers trafficking of host CD11b(+) myelomonocytic cells from the host marrow to the radiation-injured intestinal mucosa, enhancing the proliferation of intestinal stroma cells, leading secondarily to epithelial regeneration.
Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Mucosa Intestinal/efeitos da radiação , Irradiação Corporal Total , Animais , Antígeno CD11b/análise , Movimento Celular , Proliferação de Células , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Central dopaminergic system serves two major physiological functions, i.e., motivation activation and motor coordination. The evidence that serotonergic system could modulate these two pathways suggests that serotonin (5-HT) and related agents may possess potential therapeutic values against certain mental or motor disorders caused by dopamine malfunction. This study presents novel modulatory role for serotonergic agents in rat behaviors which have been speculated to be associated with forebrain dopamine system. Three serotonergic agents, including DOI (5-HT2 agonist), ritanserin (5-HT2 antagonist) and amperozide (5-HT2/D2 antagonist) were evaluated, focused particularly on the atypical antipsychotic amperozide. It was found that both amperozide and ritanserin could inhibit amphetamine-induced hyperlocomotion, and only amperozide inhibited nomifensine-induced hyperlocomotion. Amperozide could also reduce significantly the rearing but not sniffing behaviors. Furthermore, DOI and amperozide, but not ritanserin, reduced the haloperidol-induced catalepsy. [corrected] When animals were unilaterally radiofrequency lesioned in either caudate putamen (CP) or nucleus accumbens (NA), amperozide reduced both the ipsi- and contralateral turns in CP-lesioned, but reduced only ipsilateral turns in NA-lesioned rats. Via in vivo microdialysis, we demonstrated that amperozide could increase the extracellular dopamine release in both CP and NA in either intact or para-chlorophenylalanine (p-CPA) serotonin-depleted rats. Overall, we conclude that the modulatory role of amperozide on forebrain dopamine system requires not only 5-HT2/D2 antagonistic but also the blockade of dopamine transporter.