RESUMO
Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum.
Assuntos
Catepsina D/fisiologia , Movimento Celular/genética , Proliferação de Células/genética , Células Endoteliais/fisiologia , Omento/citologia , Carcinoma Epitelial do Ovário , Catepsina D/genética , Células Cultivadas , Células Endoteliais/citologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Metástase Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Omento/irrigação sanguínea , Omento/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: New treatment options for metastasised high-grade serous carcinoma (HGSC) are urgently needed. HGSC frequently metastasises to the omentum, inducing angiogenesis in the local omental microvasculature to facilitate tumour growth. We previously showed that HGSC-secreted cathepsin L (CathL) induces pro-angiogenic changes in disease relevant human omental microvascular endothelial cells (HOMECs), suggesting a role in tumour angiogenesis. Here we investigate whether CathL acts by inducing local production of the carbohydrate-binding protein galectin-1 (Gal1), which has been reported to be involved in tumourigenesis in other tumours. METHODS: HOMECs were used for all experiments. Gal1 mRNA and protein levels were measured by RT-PCR and ELISA respectively. Gal1-induced cell proliferation was assessed using WST-1 assay, migration using a transwell assay and in vivo Gal1 expression by immunohistochemistry. RESULTS: CathL transcriptionally regulated HOMEC production and secretion of Gal1 via activation of NFκB (significantly inhibited by sulfasalazine). Gal1 significantly enhanced HOMEC migration (p < 0.001) and proliferation (p < 0.001), suggesting an autocrine action. The latter was significantly reduced by the MEK/ERK1/2 inhibitors U0126 and PD98059 suggesting downstream activation of this pathway. Immunohistochemical analysis of omenta from HGSC patients with or without metastatic disease demonstrated a positive correlation between Gal1 expression and number of microvessels (r = 0.8702, p < 0.001), and area of vessels (r = 0.7283, p < 0.001), supporting a proangiogenic role for Gal1 in omental metastases. CONCLUSION: HOMEC Gal1 transcription and release in response to CathL secreted from metastasising HGSC acts in an autocrine manner on the local microvasculature to induce pro-angiogenic changes, highlighting a potential new therapeutic target.
Assuntos
Catepsina L/metabolismo , Galectina 1/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neovascularização Patológica/genética , Neoplasias Peritoneais/irrigação sanguínea , Neoplasias Peritoneais/patologia , Adulto , Movimento Celular , Proliferação de Células/genética , Células Endoteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Galectina 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Microvasos/patologia , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Gradação de Tumores , Metástase Neoplásica , Omento/irrigação sanguínea , Omento/patologia , Neoplasias Peritoneais/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
Multifocal necrotising leucoencephalopathy is a rare disorder affecting the central nervous system. It is characterised pathologically by microscopic areas of necrosis with pontine predilection but also involvement of extrapontine regions, including the cerebellum, medulla and cerebral hemispheres. It usually occurs on the background of immunosuppression. Here we describe an immunocompetent patient with a recent history of Salmonella infection who presented with subacute neurological deterioration. At postmortem, she had evidence of multifocal necrotising leucoencephalopathy.
Assuntos
Leucoencefalopatias/microbiologia , Infecções por Salmonella/complicações , Idoso , Evolução Fatal , Feminino , Gastroenterite/microbiologia , Humanos , Leucoencefalopatias/patologia , NecroseRESUMO
BACKGROUND: Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction. METHODS: We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression. RESULTS: We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines. CONCLUSIONS: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Doenças Mitocondriais/etiologia , Esclerose Múltipla/complicações , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Apoptose/fisiologia , Encéfalo/citologia , Calnexina/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Feto , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Doenças Mitocondriais/patologia , Esclerose Múltipla/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/ultraestruturaRESUMO
Multiple sclerosis (MS) is normally considered a chronic inflammatory disease of the central nervous system (CNS), where T-cells breaching the blood brain barrier react against proteins of the axonal myelin sheaths, leading to focal plaques and demyelination in the brain and spinal cord. Many current therapies are immunosuppressive in nature and are designed to target the immune system at an early stage of the disease. But there is no cure and MS may evolve into a neurodegenerative disease, where immunomodulatory treatments appear less effective. Neurodegeneration is influenced by oxidative and endoplasmic reticulum (ER) mediated stress which can be induced independently of immune processes. Since 1970, MS patients have been self-managing their long term symptoms using hyperbaric oxygen and reporting improvement in their symptoms, especially bladder control. In contrast, the majority of clinical trial evidence does not support the views of patients. Therefore does oxygen under pressure affect brain tissue by modulating oxidative or ER stress at the cellular level resulting in CNS tissue repair or deterioration? This chapter reviews our understanding and the role of oxidative and ER stress in the context of employing hyperoxia treatments to treat MS and evaluate its effects on neural cells.
Assuntos
Encéfalo/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/terapia , Estresse Oxidativo/fisiologia , Oxigênio/uso terapêutico , Encéfalo/patologia , Humanos , Esclerose Múltipla/patologia , Neurônios/metabolismo , Neurônios/patologiaRESUMO
BACKGROUND: Some neurodegenerative diseases have an element of neuroinflammation that is triggered by viral nucleic acids, resulting in the generation of type I interferons. In the cGAS-STING pathway, microbial and host-derived DNA bind and activate the DNA sensor cGAS, and the resulting cyclic dinucleotide, 2'3-cGAMP, binds to a critical adaptor protein, stimulator of interferon genes (STING), which leads to activation of downstream pathway components. However, there is limited work demonstrating the activation of the cGAS-STING pathway in human neurodegenerative diseases. METHODS: Post-mortem CNS tissue from donors with multiple sclerosis (n = 4), Alzheimer's disease (n = 6), Parkinson's disease (n = 3), amyotrophic lateral sclerosis (n = 3) and non-neurodegenerative controls (n = 11) were screened by immunohistochemistry for STING and relevant protein aggregates (e.g., amyloid-ß, α-synuclein, TDP-43). Human brain endothelial cells were cultured and stimulated with the STING agonist palmitic acid (1-400 µM) and assessed for mitochondrial stress (release of mitochondrial DNA into cytosol, increased oxygen consumption), downstream regulator factors, TBK-1/pIRF3 and inflammatory biomarker interferon-ß release and changes in ICAM-1 integrin expression. RESULTS: In neurodegenerative brain diseases, elevated STING protein was observed mainly in brain endothelial cells and neurons, compared to non-neurodegenerative control tissues where STING protein staining was weaker. Interestingly, a higher STING presence was associated with toxic protein aggregates (e.g., in neurons). Similarly high STING protein levels were observed within acute demyelinating lesions in multiple sclerosis subjects. To understand non-microbial/metabolic stress activation of the cGAS-STING pathway, brain endothelial cells were treated with palmitic acid. This evoked mitochondrial respiratory stress up to a ~2.5-fold increase in cellular oxygen consumption. Palmitic acid induced a statistically significant increase in cytosolic DNA leakage from endothelial cell mitochondria (Mander's coefficient; p < 0.05) and a significant increase in TBK-1, phosphorylated transcription factor IFN regulatory factor 3, cGAS and cell surface ICAM. In addition, a dose response in the secretion of interferon-ß was observed, but it failed to reach statistical significance. CONCLUSIONS: The histological evidence shows that the common cGAS-STING pathway appears to be activated in endothelial and neural cells in all four neurodegenerative diseases examined. Together with the in vitro data, this suggests that the STING pathway might be activated via perturbation of mitochondrial stress and DNA leakage, resulting in downstream neuroinflammation; hence, this pathway may be a target for future STING therapeutics.
RESUMO
OBJECTIVES: Despite an increasing research demand for human microvascular endothelial cells, isolation of primary endothelial cells from human tissue remains difficult. The omentum, a highly vascular visceral adipose tissue, could provide an excellent source of these cells. METHODS: A reliable method to isolate HOMECs has been developed. It consists of initial enzymatic digestion (to deplete cell contaminants), followed by further digestion, selective filtration, and immunoselection using Dynabeads coated with CD31 antibody. Cultures were characterized for expression of endothelial cell markers and their ability to undergo VEGF-dependent in vitro tube structure formation. RESULTS: Omental-derived cultures of microvascular endothelial cells were achieved with <5% contamination of other cell types. The endothelial origin of cells was confirmed by the constitutive expression of a range of vascular endothelial markers (CD31, CD105, vWF) and internalization of DiI-AcLDL. Furthermore, cultures were negative for lymphatic endothelial markers, underwent in vitro angiogenesis, and exhibited typical endothelial morphology. CONCLUSIONS: This isolation method produces homogeneous HOMEC cultures that can be maintained in vitro for at least six passages without loss of cellular features characterizing endothelial cells.
Assuntos
Separação Celular/métodos , Células Endoteliais/citologia , Microvasos/citologia , Omento/citologia , Antígenos de Diferenciação/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Microvasos/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Omento/irrigação sanguínea , Omento/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
BACKGROUND: Thousands of people with multiple sclerosis (MS) have used self-administered oxygen therapy in the UK. Clinical trials have been performed, with scant evidence that people with MS have been consulted to explore how they benefit from or how to optimize this treatment. The conventional MS disease disability scores used in trials seldom reflect the effects individuals report when using oxygen therapy to treat their symptoms. METHODS: Three people with MS and the manager of an MS Centre formed a public involvement group and collaborated with clinicians and scientists to inform a lab-based study to investigate the physiological effects of oxygen therapy on microvascular brain endothelial cells. RESULTS: People with MS often use oxygen therapy at a later stage when their symptoms worsen and only after using other treatments. The frequency of oxygen therapy sessions and hyperbaric pressure is individualized and varies for people with MS. Despite direct comparisons of efficacy proving difficult, most individuals are exposed to 100% O2 at 1.5 atmosphere absolute (ATA; 1140 mmHg absolute) for 60 min. In a laboratory-based study human brain endothelial cells were exposed in vitro to 152 mmHg O2 for 60 min with and without pressure, as this equates to 20% O2 achievable via hyperbarics, which was then replicated at atmospheric pressure. A significant reduction in endothelial cells ICAM-1 (CD54) implicated in inflammatory cell margination across the blood brain barrier was observed under oxygen treatment. CONCLUSIONS: By collaborating with people living with MS, we were able to design laboratory-based experimental protocols that replicate their treatment regimens to advance our understanding of the physiological effects of hyperbaric oxygen treatment on brain cells and their role in neuroinflammation.
Assuntos
Oxigenoterapia Hiperbárica , Esclerose Múltipla , Encéfalo , Células Endoteliais , Humanos , Esclerose Múltipla/terapia , OxigênioRESUMO
BACKGROUND: Metastasis still remains the major cause of therapeutic failure, poor prognosis and high mortality in epithelial ovarian cancer (EOC) patients. Previously, we showed that EOC cells secrete a range of factors with potential pro-angiogenic activity, in disease-relevant human omental microvascular endothelial cells (HOMECs), including the lysosomal protease cathepsin L (CathL). Thus, the aim of this study was to examine potential pro-proliferative and pro-migratory effects of CathL in HOMECs and the activated signalling pathways, and whether these proangiogenic responses are dependent on CathL-catalytic activity. METHODS: HOMECs proliferation was investigated using WST-1, BrdU and CyQUANT assays. Cell migration was examined using a Cultrex Cell 96 transwell migration assay. Enzyme activity was assayed at various pHs using the CathL-specific fluorogenic substrate FY-CHO. Activation of cell signalling pathways was tested using a commercially available phosphokinase array and intact cellbased ELISAs. RESULTS: We showed for the first time that CathL has a potent pro-proliferative and pro-migratory effect on HOMECs. For instance, CathL significantly increases HOMEC proliferation (134.8±14.7% vs control 100%) and migration (146.6±17.3% vs control 100%). Our data strongly suggest that these proangiogenic effects of CathL are mediated via a non-proteolytic mechanism. Finally, we show that CathL-induced activation of the ERK1/2 pathway is involved in inducing these cellular effects in HOMECs. CONCLUSION: These data suggest that CathL acts as an extracellular ligand and plays an important pro-angiogenic, and thus pro-metastatic, role during EOC metastasis to the omentum, by activating the omental microvasculature, and thus can potentially be targeted therapeutically in the future.
Assuntos
Catepsina L/metabolismo , Proliferação de Células , Endotélio Vascular/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Patológica/patologia , Omento/patologia , Apoptose , Movimento Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Humanos , Neovascularização Patológica/metabolismo , Omento/metabolismoRESUMO
The accumulation of senescent cells in tissues is causally linked to the development of several age-related diseases; the removal of senescent glial cells in animal models prevents Tau accumulation and cognitive decline. Senescent cells can arise through several distinct mechanisms; one such mechanism is dysregulation of alternative splicing. In this study, we characterised the senescent cell phenotype in primary human astrocytes in terms of SA-ß-Gal staining and SASP secretion, and then assessed splicing factor expression and candidate gene splicing patterns. Finally, we assessed associations between expression of dysregulated isoforms and premature cognitive decline in 197 samples from the InCHIANTI study of ageing, where expression was present in both blood and brain. We demonstrate here that senescent astrocytes secrete a modified SASP characterised by increased IL8, MMP3, MMP10, and TIMP2 but decreased IL10 levels. We identified significant changes in splicing factor expression for 10/20 splicing factors tested in senescent astrocytes compared with early passage cells, as well as dysregulation of isoform levels for 8/13 brain or senescence genes tested. Finally, associations were identified between peripheral blood GFAPα, TAU3, and CDKN2A (P14ARF) isoform levels and mild or severe cognitive decline over a 3-7-year period. Our data are suggestive that some of the features of cognitive decline may arise from dysregulated splicing of important genes in senescent brain support cells, and that defects in alternative splicing or splicing regulator expression deserve exploration as points of therapeutic intervention in the future.
Assuntos
Astrócitos/patologia , Senescência Celular , Disfunção Cognitiva/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteínas tau/metabolismo , Idoso , Processamento Alternativo , Astrócitos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Humanos , Metaloproteinases da Matriz/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p14ARF/genética , Proteínas tau/genéticaRESUMO
BACKGROUND: Congenital fibrosis of the extraocular muscles types 1 and 3 (CFEOM1/CFEOM3) are autosomal dominant strabismus disorders that appear to result from maldevelopment of ocular nuclei and nerves. We previously reported that most individuals with CFEOM1 and rare individuals with CFEOM3 harbor heterozygous mutations in KIF21A. KIF21A encodes a kinesin motor involved in anterograde axonal transport, and the familial and de novo mutations reported to date predictably alter one of only a few KIF21A amino acids--three within the third coiled-coil region of the stalk and one in the distal motor domain, suggesting they result in altered KIF21A function. To further define the spectrum of KIF21A mutations in CFEOM we have now identified all CFEOM probands newly enrolled in our study and determined if they harbor mutations in KIF21A. RESULTS: Sixteen CFEOM1 and 29 CFEOM3 probands were studied. Three previously unreported de novo KIF21A mutations were identified in three CFEOM1 probands, all located in the same coiled-coil region of the stalk that contains all but one of the previously reported mutations. Eight additional CFEOM1 probands harbored three of the mutations previously reported in KIF21A; seven had one of the two most common mutations, while one harbored the mutation in the distal motor domain. No mutation was detected in 5 CFEOM1 or any CFEOM3 probands. CONCLUSION: Analysis of sixteen CFEOM1 probands revealed three novel KIF21A mutations and confirmed three reported mutations, bringing the total number of reported KIF21A mutations in CFEOM1 to 11 mutations among 70 mutation positive probands. All three new mutations alter amino acids in heptad repeats within the third coiled-coil region of the KIF21A stalk, further highlighting the importance of alterations in this domain in the etiology of CFEOM1.
Assuntos
Cinesinas/química , Cinesinas/genética , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrabismo/genética , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Feminino , Genes Dominantes , Humanos , Padrões de Herança/genética , Masculino , Dados de Sequência Molecular , Linhagem , Estrutura Terciária de ProteínaAssuntos
Seio Cavernoso , Transtornos Cerebrovasculares/complicações , Transtornos Cerebrovasculares/diagnóstico , Meningite/complicações , Meningite/diagnóstico , Idoso , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Seio Cavernoso/diagnóstico por imagem , Seio Cavernoso/patologia , Fluordesoxiglucose F18 , Humanos , Imageamento por Ressonância Magnética , Masculino , Cintilografia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Arrest of metastasising lung cancer cells to the brain microvasculature maybe mediated by interactions between ligands on circulating tumour cells and endothelial E-selectin adhesion molecules; a process likely to be regulated by the endothelial glycocalyx. Using human cerebral microvascular endothelial cells and non-small cell lung cancer (NSCLC) cell lines, we describe how factors secreted by NSCLC cells i.e. cystatin C, cathepsin L, insulin-like growth factor-binding protein 7 (IGFBP7), vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α), damage the glycocalyx and enhance initial contacts between lung tumour and cerebral endothelial cells. METHODS: Endothelial cells were treated with tumour secreted-proteins or lung tumour conditioned medium (CM). Surface levels of E-selectin were quantified by ELISA. Adhesion of A549 and SK-MES-1 cells was examined under flow conditions (1 dyne/cm(2)). Alterations in the endothelial glycocalyx were quantified by binding of fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC). RESULTS: A549 and SK-MES-1 CM and secreted-proteins significantly enhanced endothelial surface E-selectin levels after 30 min and 4 h and tumour cell adhesion after 30 min, 4 and 24 h. Both coincided with significant glycocalyx degradation; A549 and SK-MES-1 CM removing 55 ± 12 % and 58 ± 18.7 % of WGA-FITC binding, respectively. Inhibition of E-selectin binding by monoclonal anti-E-selectin antibody completely attenuated tumour cell adhesion. CONCLUSION: These data suggest that metastasising lung cancer cells facilitate their own adhesion to the brain endothelium by secreting factors that damage the endothelial glycocalyx, resulting in exposure of the previously shielded adhesion molecules and engagement of the E-selectin-mediated adhesion axis.
Assuntos
Neoplasias Encefálicas/secundário , Encéfalo/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/fisiologia , Selectina E/metabolismo , Glicocálix/patologia , Neoplasias Pulmonares/patologia , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Catepsina L/metabolismo , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Cistatina C/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/patologia , Citometria de Fluxo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Pulmão/patologia , Espectrometria de Massas , Microvasos/patologia , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Integrated catalysts as redox sensitisers towards cancer.
Assuntos
Células PC12/efeitos dos fármacos , Quinonas/metabolismo , Quinonas/farmacologia , Animais , Catálise , Sobrevivência Celular/efeitos dos fármacos , Eletroquímica , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Células PC12/metabolismo , Quinonas/química , RatosRESUMO
Epithelial ovarian cancer (EOC) metastasis to the omentum requires implantation and angiogenesis. We propose that prometastatic changes in the omental endothelium (for angiogenesis) and mesothelium (for implantation) are critical. We investigated the expression of angiogenic proteases [cathepsin D (CD), cathepsin L (CL), and matrix metalloproteinase 2 (MMP2) and MMP9] and vascular endothelial growth factor A (VEGFA) in the mesothelium and endothelium of omentum from patients with EOC with omental metastases and control patients with benign ovarian tumors. Endothelial expression of CL, VEGFA, and MMP9 and mesothelial expression of VEGFA, MMP9, and CD were significantly increased in patients with metastasized EOC. High expression of MMP9 and VEGFA in endothelium and mesothelium and CD in mesothelium was positively associated with poor disease-specific survival (DSS). High MMP9 expression in either endothelium or mesothelium and presence of ascites prospectively showed the greatest risk of shorter DSS [hazard ratio (HR)= 6.16, 95% confidence interval (CI) = 1.76-21.6, P = .0045; HR = 11.42, 95% CI = 2.59-50.35, P = .0013; and HR = 6.35, 95% CI = 2.01-20.1, P = .002, respectively]. High endothelial MMP9 expression and ascites were independent predictors of reduced DSS and overall survival, together resulting in worst patient prognosis. Our data show that omental metastasis of EOC is associated with increased proangiogenic protein expression in the omental endothelium and mesothelium.
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BACKGROUND: A subset of T-cells expresses the B-cell marker CD20 and in rheumatoid arthritis secretes Interleukin (IL)-17. IL-17 secreting T-cells (Th17) have also been implicated in the inflammatory response in the central nervous system in multiple sclerosis (MS) and may be a potential target for elimination by biologic therapeutics. ScFvRit:sFasL comprises of a rituximab-derived antibody fragment scFvRit genetically fused to human soluble FasL that specifically eliminated T-cells. OBJECTIVE: To determine the presence and phenotype of CD20+T-cells in blood and brain of MS patients. Second, to determine whether scFvRit:sFasL can selectively eliminate CD20+T-cells. After CD20-selective binding, scFvRit:sFasL is designed to trigger FasL-mediated activation-induced cell death of T-cells, but not B-cells. METHODS: Flow cytometry and immunohistochemistry were used to screen for CD20+inflammatory T-cells in MS blood and brain tissue. ScFvRit:sFasL pro-apoptotic activity was evaluated by Annexin-V/PI staining followed by flow cytometry assessment. RESULTS: Peripheral blood (n=11) and chronic but not active lesions of MS patient brains (n=5) contained CD20+inflammatory T-cells. Activated CD20+T-cells were predominantly CD4+and secreted both IL-17 and INF-γ. ScFvRit:sFasL triggered CD20-restricted FasL-mediated activation-induced cell death in peripheral blood CD20+T-cells, but not CD20+B-cells. CONCLUSION: CD20+inflammatory T-cells are present in blood and chronic brain lesions of MS patients. ScFvRit:sFasL selectively eliminated CD20+T-cells and may eliminate pathogenic T-cells without B-cell depletion.
Assuntos
Antígenos CD20/imunologia , Apoptose/efeitos dos fármacos , Encéfalo/imunologia , Esclerose Múltipla/imunologia , Rituximab/uso terapêutico , Linfócitos T/imunologia , Adulto , Apoptose/imunologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Resultado do Tratamento , Adulto JovemRESUMO
Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA165-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways.
RESUMO
Multiple sclerosis (MS) is primarily considered an inflammatory demyelinating disease, however the role of vasculature in MS pathogenesis is now receiving much interest. MS lesions often develop along blood vessels and alterations in blood brain barrier structure and function, with associated changes in the basement membrane, are pathological features. Nevertheless, the possibility of angiogenesis occurring in MS has received little attention. In this study we used triple label enzyme immunohistochemistry to investigate blood vessel density and endothelial cell proliferation in MS samples (n=39) compared with control tissue to explore evidence of angiogenesis in MS. The results showed that in all MS samples examined blood vessel density increased compared with controls. The greatest increase was found in subacute lesions where numbers of positively stained vessels increased from 43.9+/-8.5% in controls to 84.2+/-13.3% (P=0.001). Furthermore, using an antibody against endoglin (CD105), a specific marker of proliferating endothelial cells, which are characteristic of angiogenesis, we have shown that vessels containing proliferating endothelial cells were more pronounced in all MS tissue examined (normal-appearing white matter, acute, subacute and chronic lesions, P>or=0.027) compared with control and this was greatest in the MS normal-appearing white matter (68.8+/-19.8% versus 10.58+/-6.4%, P=0.003). These findings suggest that angiogenesis may play a role in lesion progression, failure of repair and scar formation.
Assuntos
Vasos Sanguíneos/patologia , Encéfalo/patologia , Células Endoteliais/patologia , Esclerose Múltipla/patologia , Neovascularização Patológica/patologia , Fibras Nervosas Mielinizadas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/fisiopatologia , Capilares/patologia , Capilares/fisiopatologia , Proliferação de Células , Endoglina , Células Endoteliais/fisiologia , Humanos , Pessoa de Meia-Idade , Esclerose Múltipla/fisiopatologia , Neovascularização Patológica/fisiopatologia , Fibras Nervosas Mielinizadas/fisiologia , Receptores de Superfície Celular/metabolismoRESUMO
Astrocytic scar formation occurs subsequent to brain and spinal cord injury and impedes repair. The exact mechanisms of scar formation have yet to be elucidated but it is known that astrocytes within the scar have a different antigenic phenotype from normal or reactive astrocytes. Astrocyte cell culture offers a suitable system to identify factors that induce the scar phenotype as well as factors that reverse this process and that may help identify therapeutic strategies to treat astrogliosis. However, when placed in standard culture conditions, astrocytes become activated/reactive and express molecules characteristic of scar tissue in vivo. In the present study, we made use of this phenomenon to identify culture conditions that change the activated phenotype of cultured astrocytes into one characteristic of normal quiescent astrocytes. In particular, we examined the effect of extracellular matrix (ECM) proteins found in the human brain, on the phenotype of human adult astrocytes. Significantly fewer astrocytes expressed scar properties when grown on tenascin-C (TN-C) than those cultured on other ECM proteins or poly-L-lysine-coated dishes. TN-C also significantly reduced the proliferation rate of the astrocytes in vitro. In addition, further manipulation of culture conditions induced partial astrocyte reactivation. Our findings suggest that astrocytes grown on TN-C revert to a quiescent, nonactivated state that is partially reversible. This raises the possibility that therapeutic strategies aimed at manipulating TN-C levels during CNS injury may help reduce astrocytic scarring.