RESUMO
BACKGROUND: Metastasis is one of the most significant prognostic factors in osteosarcoma (OS). The goal of this study was to construct a clinical prediction model for OS patients in a population cohort and to evaluate the factors influencing the occurrence of pulmonary metastasis. METHODS: We collected data from 612 patients with osteosarcoma (OS), and 103 clinical indicators were collected. After the data were filtered, the patients were randomly divided into training and validation cohorts by using random sampling. The training cohort included 191 patients with pulmonary metastasis in OS and 126 patients with non-pulmonary metastasis, and the validation cohort included 50 patients with pulmonary metastasis in OS and 57 patients with non-pulmonary metastasis. Univariate logistics regression analysis, LASSO regression analysis and multivariate logistic regression analysis were performed to identify potential risk factors for pulmonary metastasis in patients with osteosarcoma. A nomogram was developed that included risk influencing variables selected by multivariable analysis, and used the concordance index (C-index) and calibration curve to validate the model. Receiver operating characteristic curve (ROC), decision analysis curve (DCA) and clinical impact curve (CIC) were employed to assess the model. In addition, we used a predictive model on the validation cohort. RESULTS: Logistic regression analysis was used to identify independent predictors [N Stage + Alkaline phosphatase (ALP)+Thyroid stimulating hormone (TSH)+Free triiodothyronine (FT3)]. A nomogram was constructed to predict the risk of pulmonary metastasis in patients with osteosarcoma. The performance was evaluated by the concordance index (C-index) and calibration curve. The ROC curve provides the predictive power of the nomogram (AUC = 0.701 in the training cohort, AUC = 0.786 in the training cohort). Decision curve analysis (DCA) and clinical impact curve (CIC) demonstrated the clinical value of the nomogram and higher overall net benefits. CONCLUSIONS: Our study can help clinicians effectively predict the risk of lung metastases in osteosarcoma with more readily available clinical indicators, provide more personalized diagnosis and treatment guidance, and improve the prognosis of patients. MINI ABSTRACT: A new risk model was constructed to predict the pulmonary metastasis in patients with osteosarcoma based on multiple machine learning.
Assuntos
Neoplasias Ósseas , Neoplasias Pulmonares , Osteossarcoma , Humanos , Prognóstico , Modelos Estatísticos , Aprendizado de MáquinaRESUMO
Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.
Assuntos
Cistos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Pró-Proteína Convertases/genética , Mutação de Sentido Incorreto/genética , Tunicamicina , Actinas/genética , Modelos Animais de Doenças , Fígado/patologia , Cistos/genética , Serina-Treonina Quinases TOR/genética , MamíferosRESUMO
BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ductos Biliares Intra-Hepáticos/patologia , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/patologia , Mutação , Proteínas de Peixe-Zebra/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose , Ductos Biliares Intra-Hepáticos/metabolismo , Estudos de Casos e Controles , Colestase Intra-Hepática/metabolismo , Doença Crônica , Feminino , Edição de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Sequenciamento do Exoma , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Hepatic macrophages are key components of the liver immunity and consist of two main populations. Liver resident macrophages, known as Kupffer cells in mammals, are crucial for maintaining normal liver homeostasis. Upon injury, they become activated to release proinflammatory cytokines and chemokines and recruit a large population of inflammatory monocyte-derived macrophages to the liver. During the progression of liver diseases, macrophages are highly plastic and have opposing functions depending on the signaling cues that they receive from the microenvironment. A comprehensive understanding of liver macrophages is essential for developing therapeutic interventions that target these cells in acute and chronic liver diseases. Mouse studies have provided the bulk of our current knowledge of liver macrophages. The emergence of various liver disease models and availability of transgenic tools to visualize and manipulate macrophages have made the teleost zebrafish (Danio rerio) an attractive new vertebrate model to study liver macrophages. In this review, we summarize the origin and behaviors of macrophages in healthy and injured livers in zebrafish. We highlight the roles of macrophages in zebrafish models of alcoholic and non-alcoholic liver diseases, hepatocellular carcinoma, and liver regeneration, and how they compare with the roles that have been described in mammals. We also discuss the advantages and challenges of using zebrafish to study liver macrophages.
Assuntos
Modelos Animais de Doenças , Hepatopatias/imunologia , Macrófagos/fisiologia , Animais , Hematopoese , Hepatopatias Alcoólicas/imunologia , Neoplasias Hepáticas/imunologia , Regeneração Hepática , Peixe-ZebraRESUMO
Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. CONCLUSIONS: Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).
Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Bile/metabolismo , Colestase Intra-Hepática/genética , Hepatócitos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Autofagia/genética , Colestase Intra-Hepática/patologia , Feminino , Humanos , Imunossupressores/farmacologia , Lactente , Fígado/patologia , Masculino , Mutação , Sirolimo/farmacologia , Peixe-Zebra/metabolismoRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) occurs more frequently and aggressively in men than in women. Although sex hormones are believed to play a critical role in this disparity, the possible contribution of other factors largely is unknown. We aimed to investigate the role of serotonin on its contribution of sex discrepancy during HCC. METHODS: By using an inducible zebrafish HCC model through hepatocyte-specific transgenic krasV12 expression, differential rates of HCC in male and female fish were characterized by both pharmaceutical and genetic interventions. The findings were validated further in human liver disease samples. RESULTS: Accelerated HCC progression was observed in krasV12-expressing male zebrafish and male fish liver tumors were found to have higher hepatic stellate cell (HSC) density and activation. Serotonin, which is essential for HSC survival and activation, similarly were found to be synthesized and accumulated more robustly in males than in females. Serotonin-activated HSCs could promote HCC carcinogenesis and concurrently increase serotonin synthesis via transforming growth factor (Tgf)b1 expression, hence contributing to sex disparity in HCC. Analysis of liver disease patient samples showed similar male predominant serotonin accumulation and Tgfb1 expression. CONCLUSIONS: In both zebrafish HCC models and human liver disease samples, a predominant serotonin synthesis and accumulation in males resulted in higher HSC density and activation as well as Tgfb1 expression, thus accelerating HCC carcinogenesis in males.
RESUMO
Alcoholic liver disease (ALD) results from alcohol overconsumption and is among the leading causes of liver-related morbidity and mortality worldwide. Elevated expression of vascular endothelial growth factor (VEGF) and its receptors has been observed in ALD, but how it contributes to ALD pathophysiology is unclear. Here, we investigated the impact of VEGF signaling inhibition on an established zebrafish model of acute alcoholic liver injury. Kdrl activity was blocked by chemical inhibitor treatment or by genetic mutation. Exposing 4-day-old zebrafish larvae to 2% ethanol for 24â h induced hepatic steatosis, angiogenesis and fibrogenesis. The liver started self-repair once ethanol was removed. Although inhibiting Kdrl did not block the initial activation of hepatic stellate cells during ethanol treatment, it suppressed their proliferation, extracellular matrix protein deposition and fibrogenic gene expression after ethanol exposure, thus enhancing the liver repair. It also ameliorated hepatic steatosis and attenuated hepatic angiogenesis that accelerated after the ethanol treatment. qPCR showed that hepatic stellate cells are the first liver cell type to increase the expression of VEGF ligand and receptor genes in response to ethanol exposure. Both hepatic stellate cells and endothelial cells, but not hepatic parenchymal cells, expressed kdrl upon ethanol exposure and were likely the direct targets of Kdrl inhibition. Ethanol-induced steatosis and fibrogenesis still occurred in cloche mutants that have hepatic stellate cells but lack hepatic endothelial cells, and Kdrl inhibition suppressed both phenotypes in the mutants. These results suggest that VEGF signaling mediates interactions between activated hepatic stellate cells and hepatocytes that lead to steatosis. Our study demonstrates the involvement of VEGF signaling in regulating sustained liver injuries after acute alcohol exposure. It also provides a proof of principle of using the zebrafish model to identify molecular targets for developing ALD therapies.
Assuntos
Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Regeneração Hepática , Fígado/patologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Etanol , Proteínas da Matriz Extracelular/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Larva/metabolismo , Ligantes , Hepatopatias Alcoólicas/genética , Regeneração Hepática/efeitos dos fármacos , Mutação/genética , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Quinazolinas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genéticaRESUMO
Hepatic stellate cells are liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells. It is becoming increasingly clear that hepatic stellate cells have a profound impact on the differentiation, proliferation, and morphogenesis of other hepatic cell types during liver development and regeneration. In this Review, we summarize and evaluate the recent advances in our understanding of the formation and characteristics of hepatic stellate cells, as well as their function in liver development, regeneration, and cancer. We also discuss how improved knowledge of these processes offers new perspectives for the treatment of patients with liver diseases.
Assuntos
Células Estreladas do Fígado/citologia , Neoplasias Hepáticas/metabolismo , Regeneração Hepática , Fígado/fisiologia , Animais , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Fibrose/metabolismo , Humanos , Fígado/embriologia , Fígado/patologia , Fígado/fisiopatologia , Hepatopatias/metabolismo , Falência Hepática Aguda/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Microscopia de Contraste de Fase/métodosRESUMO
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.
Assuntos
Ductos Biliares Intra-Hepáticos/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Pâncreas/crescimento & desenvolvimento , Fatores de Transcrição SOX9/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Ductos Biliares Intra-Hepáticos/embriologia , Ductos Biliares Intra-Hepáticos/metabolismo , Códon sem Sentido , Regulação da Expressão Gênica no Desenvolvimento , Fígado/embriologia , Fígado/metabolismo , Morfogênese/genética , Pâncreas/embriologia , Pâncreas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
UNLABELLED: Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity, and interactions between these two cell types are potentially critical for their development and function. Here, we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene-expression profile, and increased proliferation and matrix production, in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time-course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells, including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development. CONCLUSION: Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior.
Assuntos
Comunicação Celular , Movimento Celular , Células Endoteliais/citologia , Células Estreladas do Fígado/citologia , Fígado/citologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzoatos/farmacologia , Contagem de Células , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Etanol/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/crescimento & desenvolvimento , Miocárdio/metabolismo , Crista Neural/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico , Transdução de Sinais , Tetra-Hidronaftalenos/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
OBJECT: The lack of primary lymphoid tissue within the central nervous system (CNS) confounds our understanding of the pathogenesis of primary CNS lymphomas (PCNSLs). Comparing the protein expression of PCNSLs and sporadic systemic lymphomas (SSLs) provides a useful strategy for identifying a molecular signature that characterizes disease-associated features and provides information regarding tumor initiation and progression. METHODS: Seven diffuse large B-cell PCNSLs were selected to undergo 2D gel electrophoresis, and profiled proteomes from these PCNSLs were compared with those from 7 diffuse large B-cell SSLs. Distinguishing proteins were sequenced using mass spectrometry. RESULTS: Two-dimensional gel electrophoresis identified an average of 706 proteins from each specimen. Computerized gel analysis and manual reconfirmation revealed a 96% similarity in the proteomes of PCNSLs and SSLs. Comparative analysis identified 9 proteins significantly overexpressed (p < 0.05) and 16 proteins downregulated in PCNSLs. The proteomic findings were further validated using Western blot and immunohistochemical staining. CONCLUSIONS: The similarities in proteomic patterns between PCNSLs and SSLs suggest that these tumor types share structural similarities, acquired during differentiation. The ultimate fate of lymphomatous cells (CNS vs systemic) may be related to differentially expressed proteins, which function in homing and host processing. Elucidating the roles of these differentially expressed proteins will prove valuable in understanding the pathogenesis of PCNSL.
Assuntos
Neoplasias do Sistema Nervoso Central/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Estudos de Casos e Controles , Neoplasias do Sistema Nervoso Central/etiologia , Neoplasias do Sistema Nervoso Central/patologia , Eletroforese em Gel Bidimensional , Feminino , Humanos , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , ProteômicaRESUMO
Dynamic changes in the expression of multiple genes appear to be common features that distinguish transformed cells from their normal counterparts. We compared the proteomic profiles of four glioblastoma multiforme (GBM) tissue samples and four normal brain cortex samples to examine the molecular basis of gliomagenesis. Trypsin-digested protein samples were separated by capillary isoelectric focusing with nano-reversed-phase liquid chromatography and were profiled by mass spectrometric sequencing. Wolf-Hirschhorn syndrome candidate 1 (WHSC1), along with 103 other proteins, was found only in the GBM proteomes. Western blot and immunohistochemistry verified our proteomic findings and demonstrated that 30-kDa WHSC1 expression increases with ascending tumor proliferation activity. RNA interference could suppress glioma cell growth by blocking WHSC1 expression. Our novel findings encourage the application of proteomic techniques in cancer research.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Histona-Lisina N-Metiltransferase/biossíntese , Proteínas Repressoras/biossíntese , Western Blotting , Proliferação de Células , Cromatografia Líquida , Humanos , Imuno-Histoquímica , Proteômica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , TransfecçãoRESUMO
PURPOSE: The surgical excision of retinal vascular lesions including hemangioblastomas is rarely practiced. This study investigates the pathological characteristics of 4 patients (3 with von Hippel-Lindau [VHL] disease and 1 with a vasoproliferative retinal tumor) who underwent ocular tumor resection. von Hippel-Lindau is an autosomal dominant disease caused by a defect of the VHL tumor suppressor gene. The VHL protein is required for oxygen-dependent degradation of hypoxia-inducible factor 1alpha. This factor regulates vascular endothelial growth factor (VEGF) and the chemokine receptor CXCR4. Retinal hemangioblastoma is the most common tumor observed in VHL disease. We investigated the expression of CXCR4; its ligand, CXCL12/SDF-1alpha; VEGF; and the VHL gene in VHL disease-associated retinal hemangioblastomas. DESIGN: Interventional case series with immunohistological and molecular pathological analyses. PARTICIPANTS: Immunohistochemistry and molecular pathology of the surgically excised retinal lesions were performed. INTERVENTION: Large retinal hemangioblastomas (1-3 disc diameters) and vasoproliferative tumors were resected surgically after laser photocoagulation in 4 patients. The excised tissues were snap frozen and evaluated by histology. Molecular pathology was performed for the VHL gene, and immunohistochemistry and molecular detection (reverse transcription polymerase chain reaction) were carried out for VEGF, CXCR4, and CXCL12. MAIN OUTCOME MEASURES: Evaluation of clinical presentations and molecular pathology of the excised retinal lesions. RESULTS: Large retinal hemangioblastomas were resected successfully from the 3 VHL cases. Postoperatively, all patients were stable. Molecular analyses disclosed the loss of heterozygosity at the VHL allele locus in the VHL retinal hemangioblastomas but not in the vasoproliferative tumor. High levels of transcript and protein were found for VEGF and CXCR4, whereas low levels of CXCL12 mRNA were expressed in the retinal hemangioblastomas associated with VHL disease. In contrast, very low levels of VEGF and CXCR4 mRNA were detected in the vasoproliferative tumor. Furthermore, increased expression of VEGF and CXCR4 was detected in more active hemangioblastomas. CONCLUSIONS: Surgical resection of large retinal hemangioblastomas may be useful for therapy in selected VHL patients. Activated VHL lesions produce more VEGF. This is the first demonstration of CXCR4 expression in VHL disease-associated retinal hemangioblastomas. We suggest targeting CXCR4 as an additional therapeutic strategy for VHL disease-associated retinal hemangioblastomas.
Assuntos
Regulação Neoplásica da Expressão Gênica , Hemangioblastoma/genética , Receptores CXCR4/genética , Neoplasias da Retina/genética , Doença de von Hippel-Lindau/genética , Adolescente , Adulto , Quimiocina CXCL12 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Feminino , Hemangioblastoma/patologia , Hemangioblastoma/cirurgia , Humanos , Técnicas Imunoenzimáticas , Masculino , Biologia Molecular , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Neoplasias da Retina/patologia , Neoplasias da Retina/cirurgia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/patologia , Doença de von Hippel-Lindau/cirurgiaRESUMO
The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.
Assuntos
Adenosina Trifosfatases/genética , Cobre/metabolismo , Notocorda/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , ATPases Transportadoras de Cobre , Embrião não Mamífero , Dados de Sequência Molecular , Fenótipo , Peixe-Zebra/genéticaRESUMO
Sporadic inclusion body myositis (IBM) is the most frequently acquired inflammatory myopathy of late adult life, yet its diagnostic criteria and pathogenesis remain poorly defined. Because effective treatment is lacking, research efforts have intensified to identify specific markers for this debilitating disorder. In this study, proteomic analysis of 4 cases of sporadic IBM was compared with 5 cases of inflammatory myopathy without clinicopathologic features of IBM to distinguish the IBM-specific proteome. Proteins were separated by 2-dimensional polyacrylamide gel electrophoresis and profiled by mass spectrometric sequencing. Expression of most proteins remained unchanged; however, 16 proteins were upregulated and 6 proteins were downregulated in IBM compared with cases of non-IBM inflammatory myopathy. These IBM-specific proteins included apolipoprotein A-I, amyloid beta precursor protein, and transthyretin, which have been associated with amyloidosis; superoxide dismutase, enolase, and various molecular chaperones indicate perturbations in detoxification, energy metabolism, and protein folding, respectively. The IBM-downregulated proteins mainly serve as carriers for muscle contraction and other normal muscle functions. We further applied Western blot and immunohistochemistry to verify the proteomic findings. This study validates proteomics as a powerful tool in the study of muscle disease and indicates a unique pattern of protein expression in IBM.
Assuntos
Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miosite de Corpos de Inclusão/diagnóstico , Miosite de Corpos de Inclusão/metabolismo , Proteômica/métodos , Idoso , Western Blotting , Regulação para Baixo/fisiologia , Eletroforese em Gel Bidimensional , Metabolismo Energético/fisiologia , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Miosite/diagnóstico , Miosite/metabolismo , Miosite/fisiopatologia , Miosite de Corpos de Inclusão/fisiopatologia , Regulação para Cima/fisiologiaRESUMO
The vertebrate posterior body is formed by a combination of the gastrulation movements that shape the head and anterior trunk and posterior specific cell behaviors. Here, we investigated whether genes that regulate cell movements during gastrulation [no tail (ntl)/brachyury, knypek (kny) and pipetail (ppt)/wnt5] interact to regulate posterior body morphogenesis. Both kny;ntl and ppt;ntl double mutant embryos exhibit synergistic trunk and tail shortening by early segmentation. Gene expression analysis in the compound mutants indicates that anteroposterior germ-layer patterning is largely normal and that the tail elongation defects are not due to failure to specify or maintain posterior tissues. Moreover, ntl interacts with ppt and kny to synergistically regulate the posterior expression of the gene encoding bone morphogenetic protein 4 (bmp4) but not of other known T-box genes, fibroblast growth factor genes or caudal genes. Examination of mitotic and apoptotic cells indicates that impaired tail elongation is not simply due to decreased cell proliferation or increased cell death. Cell tracing in ppt;ntl and kny;ntl mutants demonstrates that the ventral derived posterior tailbud progenitors move into the tailbud. However, gastrulation-like convergence and extension movements and cell movements within the posterior tailbud are impaired. Furthermore, subduction movements of cells into the mesendoderm are reduced in kny;ntl and ppt;ntl mutants. We propose that Ntl and the non-canonical Wnt pathway components Ppt and Kny function in parallel, partially redundant pathways to regulate posterior body development. Our work initiates the genetic dissection of posterior body morphogenesis and links genes to specific tail-forming movements. Moreover, we provide genetic evidence for the notion that tail development entails a continuation of mechanisms regulating gastrulation together with mechanisms unique to the posterior body.
Assuntos
Embrião não Mamífero/fisiologia , Gástrula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Cauda/embriologia , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Padronização Corporal/fisiologia , Genótipo , Movimento/fisiologia , Mutação , Fenótipo , Transdução de Sinais , Cauda/anormalidades , Proteínas Wnt , Peixe-Zebra/genéticaRESUMO
PURPOSE: To investigate the effect on porcine retinal pigment epithelial (RPE) cells of modified intraocular irrigating solutions compared with BSS and BSS plus. METHODS: Confluent cultures of RPE cells were incubated in experimental intraocular irrigating solutions. The cells were then examined for breakdown of DNA by the TUNEL procedure. Fragmentation of the DNA from cells was also confirmed by flow cytometry. RESULTS: Most of the experimentally, treated cells exhibited a shrunken appearance for up to 72 h. There was a steady increase in the number of cells labeled by the TUNEL method in three solutions with time. The data demonstrated that the influence of solutions on the percentage of RPE cell nuclei that gave a clear positive TUNEL stain was, in ascending order: modified solution>BSS>BSS plus. Of the three solutions tested, BSS plus showed least apoptosis. CONCLUSIONS: It is suggested that BSS plus is less harmful to RPE cells than the other solutions tested. Introduction of other adjunctive solutions of antibiotics, mydriatics, miotics, and steroid, resulting in an altered electrolyte balance, pH, or osmolality in the solution, may compromise the safety and efficacy of a properly formulated and packaged solution.