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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.

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INTRODUCTION: Microvascular remodelling is a symptom of cardiovascular disease. Despite the mechanical environment being recognized as a major contributor to the remodelling process, it is currently only understood in a rudimentary way. OBJECTIVE: A morphological and mechanical evaluation of the resistance vasculature in health and diabetes mellitus. METHODS: The cells and extracellular matrix of human subcutaneous resistance arteries from abdominal fat biopsies were imaged using two-photon fluorescence and second harmonic generation at varying transmural pressure. The results informed a two-layer mechanical model. RESULTS: Diabetic resistance arteries reduced in wall area as pressure was increased. This was attributed to the presence of thick, straight collagen fibre bundles that braced the outer wall. The abnormal mechanical environment caused the internal elastic lamina and endothelial and vascular smooth muscle cell arrangements to twist. CONCLUSIONS: Our results suggest diabetic microvascular remodelling is likely to be stress-driven, comprising at least 2 stages: (1) Laying down of adventitial bracing fibres that limit outward distension, and (2) Deposition of additional collagen in the media, likely due to the significantly altered mechanical environment. This work represents a step towards elucidating the local stress environment of cells, which is crucial to build accurate models of mechanotransduction in disease.

Asunto(s)

Grasa Abdominal/irrigación sanguínea , Arterias/patología , Diabetes Mellitus Tipo 2/patología , Remodelación Vascular , Anciano , Presión Arterial , Arterias/fisiopatología , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/fisiopatología , Tejido Elástico/patología , Femenino , Colágenos Fibrilares , Humanos , Masculino , Mecanotransducción Celular , Microscopía de Fluorescencia por Excitación Multifotónica , Persona de Mediana Edad , Estrés Mecánico , Resistencia Vascular

RESUMEN

BACKGROUND: Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). METHODS: We treated OC cells with the chemotherapeutic-doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor-thapsigargin (TG)-and/or an ER stress inhibitor-TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. RESULTS: Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. CONCLUSION: Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

Asunto(s)

Calreticulina , Estrés del Retículo Endoplásmico , Neoplasias Ováricas , Apoptosis , Calreticulina/metabolismo , Carcinoma Epitelial de Ovario , Femenino , Fluoresceína-5-Isotiocianato , Humanos , Tapsigargina/farmacología

RESUMEN

Tumour metastasis to the brain is a complex process involving crosstalk between the circulating tumour cells and the blood brain barrier (BBB). Astrocytes, which reside in the abluminal surface of the microvasculature of the BBB, are now known to play an essential role in tumour cell migration and invasion into the brain parenchyma. For instance, pro-inflammatory astrocyte secretions, including TNF-α, IL-6, CXCL10 as well as polyunsaturated fatty acids interact with circulating tumour cells to promote migration and proliferation. Additionally, astrocyte and tumour cell derived MMPs play a vital role in tumour cell invasion through the BBB. Understanding these complex interactions between tumour cells and astrocytes in the tumour microenvironment may contribute to the development of novel therapeutics for brain metastasis. Therefore, in this review, we present key interactions within the neurovascular unit of the BBB in the tumour microenvironment that significantly aids cancer metastasis, focusing particularly on astrocytes.

Asunto(s)

Neoplasias Encefálicas , Células Neoplásicas Circulantes , Astrocitos , Barrera Hematoencefálica , Encéfalo , Humanos , Microambiente Tumoral

RESUMEN

Significantly reduced levels of the anti-inflammatory gaseous transmitter hydrogen sulfide (H2S) are observed in diabetic patients and correlate with microvascular dysfunction. H2S may protect the microvasculature by preventing loss of the endothelial glycocalyx. We tested the hypothesis that H2S could prevent or treat retinal microvascular endothelial dysfunction in diabetes. Bovine retinal endothelial cells (BRECs) were exposed to normal (NG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) ± the slow-release H2S donor NaGYY4137 in vitro. Glycocalyx coverage (stained with WGA-FITC) and calcein-labeled monocyte adherence were measured. In vivo, fundus fluorescein angiography (FFA) was performed in normal and streptozotocin-induced (STZ) diabetic rats. Animals received intraocular injection of NaGYY4137 (1 µM) or the mitochondrial-targeted H2S donor AP39 (100 nM) simultaneously with STZ (prevention) or on day 6 after STZ (treatment), and the ratio of interstitial to vascular fluorescence was used to estimate apparent permeability. NaGYY4137 prevented HG-induced loss of BREC glycocalyx, increased monocyte binding to BRECs (p ≤ 0.001), and increased overall glycocalyx coverage (p ≤ 0.001). In rats, the STZ-induced increase in apparent retinal vascular permeability (p ≤ 0.01) was significantly prevented by pre-treatment with NaGYY4137 and AP39 (p < 0.05) and stabilized by their post-STZ administration. NaGYY4137 also reduced the number of acellular capillaries (collagen IV + /IB4-) in the diabetic retina in both groups (p ≤ 0.05). We conclude that NaGYY4137 and AP39 protected the retinal glycocalyx and endothelial permeability barrier from diabetes-associated loss of integrity and reduced the progression of diabetic retinopathy (DR). Hydrogen sulfide donors that target the glycocalyx may therefore be a therapeutic candidate for DR.

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Cancer remains a major and leading health problem worldwide. Lack of early diagnosis, chemoresistance, and recurrence of cancer means vast research and development are required in this area. The complexity of the tumor microenvironment in the biological milieu poses greater challenges in having safer, selective, and targeted therapies. Existing strategies such as chemotherapy, radiotherapy, and antiangiogenic therapies moderately improve progression-free survival; however, they come with side effects that reduce quality of life. Thus, targeting potential candidates in the microenvironment, such as extracellular cathepsin D (CathD) which has been known to play major pro-tumorigenic roles in breast and ovarian cancers, could be a breakthrough in cancer treatment, specially using novel treatment modalities such as immunotherapy and nanotechnology-based therapy. This chapter discusses CathD as a pro-cancerous, more specifically a proangiogenic factor, that acts bi-functionally in the tumor microenvironment, and possible ways of targeting the protein therapeutically.

Asunto(s)

Neoplasias de la Mama/patología , Catepsina D , Neoplasias Ováricas/patología , Microambiente Tumoral , Animales , Neoplasias de la Mama/tratamiento farmacológico , Catepsina D/antagonistas & inhibidores , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Calidad de Vida , Microambiente Tumoral/efectos de los fármacos

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OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS: Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.

Asunto(s)

Barrera Hematorretinal , Permeabilidad Capilar , Diabetes Mellitus Experimental , Retinopatía Diabética , Animales , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/fisiopatología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/fisiopatología , Masculino , Ratas

RESUMEN

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.

Asunto(s)

Catepsina L/metabolismo , Galectina 1/genética , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neovascularización Patológica/genética , Neoplasias Peritoneales/irrigación sanguínea , Neoplasias Peritoneales/patología , Adulto , Movimiento Celular , Proliferación Celular/genética , Células Endoteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Galectina 1/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Microvasos/patología , Persona de Mediana Edad , FN-kappa B/metabolismo , Clasificación del Tumor , Metástasis de la Neoplasia , Epiplón/irrigación sanguínea , Epiplón/patología , Neoplasias Peritoneales/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/genética

RESUMEN

BACKGROUND: In this study, we investigate the expression of markers of angiogenesis and microvessel density (MVD) in cases of microcystic, elongated and fragmented (MELF) pattern, with its prognostic role in the survival of endometrioid endometrial adenocarcinomas (EA) patients. METHODS: In this study, 100 cases of EA, 49 cases with MELF pattern and 51 without, were immunohistochemically stained for galectin-1, vascular endothelial growth factor (VEGF), and MVD. Morphometry and statistical (univariate and multivariate) analyses were performed to assess overall survival (OS) and disease-free survival. RESULTS: The expression of VEGF (p<.001) and galectin-1 (p<.001), as well as MVD area (p<.001) and number of vessels/mm2 (p<.050), were significantly higher in the +MELF pattern group compared to the -MELF group. A low negative correlation between MELFpattern and the number of days of survival (p<.001, r=-0.47) was also found. A low positive correlation of MELF-pattern with galectin-1 expression (p<.001, r=0.39), area of vessels/mm2 (p<.001, r=0.36), outcome of EA (p<.001, r=0.42) and VEGF expression (p<.001, r=0.39) suggests potential pathological relevance of these factors in the prognosis of EA. A univariate survival analysis indicated a role for all parameters of survival. Multivariate Cox proportional hazard regression analysis revealed that only area of vessels/mm2 (hazard ratio [HR], 1.018; 95% confidence interval [CI], 1.002 to 1.033), galectin-1 (HR, 1.049; 95% CI, 1.025 to 1.074) and VEGF (HR, 1.049; 95% CI, 1.022 to 1.077) play key roles in OS. CONCLUSIONS: This study reports an increase in MVD, VEGF and galectin-1 expression in EA with MELF pattern and suggests that MELF pattern, along with the angiogenic profile, may be a prognostic factor in EA.

RESUMEN

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) analogues reduce the risk of macrovascular disease in diabetes; however, little is known about their microvascular effects. This research examined the microvascular actions of the GLP-1 analogues liraglutide and exenatide in individuals with and without type 2 diabetes (study 1). It also explored the involvement of the GLP-1 receptor (study 2) and the nitric oxide pathway in mediating the microvascular effects of the analogues. METHODS: Trial design: Studies 1 and 2 had a randomised, controlled, double-blind study design. Study 1 participants, intervention and methods: three participant groups were recruited: individuals with well-controlled type 2 diabetes, and obese and lean individuals without diabetes (21 participants per group). Liraglutide (0.06 mg), exenatide (0.5 µg) and saline (154 mmol/l NaCl; 0.9%) control were microinjected into separate sites in the dermis (forearm) in a randomised order, blinded to operator and participant. Skin microvascular perfusion was assessed by laser Doppler perfusion imaging. Outcomes were stabilised response (mean skin perfusion between 7.5 and 10 min post microinjection) and total response (AUC, normalised for baseline perfusion). Perfusion response to GLP-1 analogues was compared with saline within each group as well as between groups. Study 2 participants, intervention and methods: in healthy individuals (N = 16), liraglutide (0.06 mg) and saline microinjected sites were pretreated with saline or the GLP-1 receptor blocker, exendin-(9,39), in a randomised order, blinded to participant and operator. Outcomes were as above (stabilised response and total perfusion response). Perfusion response to liraglutide was compared between the saline and the exendin-(9,39) pretreated sites. In vitro study: the effects of liraglutide and exenatide on nitrate levels and endothelial nitric oxide synthase phosphorylation (activation) were examined using human microvascular endothelial cells. RESULTS: Study 1 results: both analogues increased skin perfusion (stabilised response and total response) in all groups (n = 21 per group, p < 0.001), with the microvascular responses similar across groups (p ≥ 0.389). Study 2 results: liraglutide response (stabilised response and total response) was not influenced by pretreatment with exendin-(9,39) (70 nmol/l) (N = 15, one dataset excluded) (p ≥ 0.609). Liraglutide and exenatide increased nitrate production and endothelial nitric oxide synthase (eNOS) phosphorylation (p ≤ 0.020). CONCLUSIONS/INTERPRETATION: Liraglutide and exenatide increased skin microvascular perfusion in individuals with and without well-controlled diabetes, potentially mediated, at least in part, by NO. TRIAL REGISTRATION: ClinicalTrials.gov NCT01677104. FUNDING: This work was supported by Diabetes UK (grant numbers: 09/0003955 and 12/0004600 [RW and JM Collins Legacy, Funded Studentship]).

Asunto(s)

Diabetes Mellitus Tipo 2/tratamiento farmacológico , Exenatida/uso terapéutico , Péptido 1 Similar al Glucagón/análogos & derivados , Liraglutida/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Exenatida/administración & dosificación , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Modelos Lineales , Liraglutida/administración & dosificación , Masculino , Microcirculación/efectos de los fármacos , Persona de Mediana Edad

RESUMEN

Overexpression and secretion of the enzymes cathepsin D (CathD) and cathepsin L (CathL) is associated with metastasis in several human cancers. As a superfamily, extracellularly, these proteins may act within the tumor microenvironment to drive cancer progression, proliferation, invasion and metastasis. Therefore, it is important to discover novel therapeutic treatment strategies to target CathD and CathL and potentially impede metastasis. Graphene oxide (GO) could form the basis of such a strategy by acting as an adsorbent for pro-metastatic enzymes. Here, we have conducted research into the potential of targeted anti-metastatic therapy using GO to adsorb these pro-tumorigenic enzymes. Binding of CathD/L to GO revealed that CathD/L were adsorbed onto the surface of GO through its cationic and hydrophilic residues. This work could provide a roadmap for the rational integration of CathD/L-targeting agents into clinical settings.

RESUMEN

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.

Asunto(s)

Catepsina L/metabolismo , Proliferación Celular , Endotelio Vascular/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Patológica/patología , Epiplón/patología , Apoptosis , Movimiento Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Femenino , Humanos , Neovascularización Patológica/metabolismo , Epiplón/metabolismo

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BACKGROUND: Protein-graphene interactions have the potential to play a pivotal role in the future directions of nanomedicine. These interactions lead to diverse processes such as generation of protein coronas, nano-bio interfaces, particle wrapping, and biocatalytic processes that could determine the ultimate fate of graphene nanocomposites in biologic systems. However, such interactions and their effects on the bioavailability of graphene have not yet been widely appreciated, despite the fact that this is the primary surface in contact with cells. METHODS: This paper reports on the integrative physiochemical interaction between trypsin and graphene quantum dots (GQDs) to determine their potential biologic identity in enzyme engineering. This interaction was measured by a wide range of analytical methods. RESULTS: Definitive binding and modulation of trypsin-GQDs was demonstrated for the first time by use of vibrational spectroscopy and wetting transparency, which revealed that trypsin was absorbed on GQDs' surface through its cationic and hydrophilic residues. Our findings suggested that trypsin's active sites were stabilized and protected by the GQDs, which were likely to be responsible for the high bioavailability of GQDs in enzymes. CONCLUSION: Our work demonstrates the efficacy of GQDs as an enzyme modulator with high specificity, and their great application potential in enzyme engineering as well as enzyme-based therapies.

Asunto(s)

Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Tripsina/metabolismo , Dominio Catalítico , Grafito/química , Interacciones Hidrofóbicas e Hidrofílicas , Luminiscencia , Nanocompuestos/química , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Tripsina/química

RESUMEN

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.

Asunto(s)

Catepsina D/fisiología , Movimiento Celular/genética , Proliferación Celular/genética , Células Endoteliales/fisiología , Epiplón/citología , Carcinoma Epitelial de Ovario , Catepsina D/genética , Células Cultivadas , Células Endoteliales/citología , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Metástasis de la Neoplasia , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Epiplón/irrigación sanguínea , Epiplón/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/metabolismo , Neoplasias Peritoneales/secundario , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología

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The intriguing properties of reduced graphene oxide (rGO) have paved the way for a number of potential biomedical applications such as drug delivery, tissue engineering, gene delivery and bio-sensing. Over the last decade, there have been escalating concerns regarding the possible toxic effects, behaviour and fate of rGO in living systems and environments. This paper reports on integrative chemical-biological interactions of rGO with lung cancer cells, i.e. A549 and SKMES-1, to determine its potential toxicological impacts on them, as a function of its concentration. Cell viability, early and late apoptosis and necrosis were measured to determine oxidative stress potential, and induction of apoptosis for the first time by comparing two lung cancer cells. We also showed the general trend between cell death rates and concentrations for different cell types using a Gaussian process regression model. At low concentrations, rGO was shown to significantly produce late apoptosis and necrosis rather than early apoptotic events, suggesting that it was able to disintegrate the cellular membranes in a dose dependent manner. For the toxicity exposures undertaken, late apoptosis and necrosis occurred, which was most likely resultant from limited bioavailability of unmodified rGO in lung cancer cells.

Asunto(s)

Apoptosis/efectos de los fármacos , Grafito/toxicidad , Necrosis/inducido químicamente , Óxidos/toxicidad , Especies Reactivas de Oxígeno/agonistas , Células A549 , Supervivencia Celular/efectos de los fármacos , Humanos , Necrosis/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Sulfuro de Hidrógeno/metabolismo , Hiperglucemia/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Compuestos Organofosforados/farmacología , Sustancias Protectoras/farmacología , Tionas/farmacología , Animales , Línea Celular , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Transporte de Electrón/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Glucosa/metabolismo , Hiperglucemia/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

Asunto(s)

Catepsina D/metabolismo , Catepsina L/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Animales , Carcinoma Epitelial de Ovario , Catepsina D/genética , Catepsina L/genética , Femenino , Humanos , Metástasis de la Neoplasia , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología

RESUMEN

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.

Asunto(s)

Neoplasias Encefálicas/secundario , Encéfalo/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/patología , Adhesión Celular/fisiología , Selectina E/metabolismo , Glicocálix/patología , Neoplasias Pulmonares/patología , Encéfalo/patología , Neoplasias Encefálicas/patología , Catepsina L/metabolismo , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Medios de Cultivo Condicionados/farmacología , Cistatina C/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/patología , Citometría de Flujo , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Pulmón/patología , Espectrometría de Masas , Microvasos/patología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Together with carbon monoxide (CO), nitric oxide (NO) and hydrogen sulfide (H2S) form a group of physiologically important gaseous transmitters, sometimes referred to as the "gaseous triumvirate". The three molecules share a wide range of physical and physiological properties: they are small gaseous molecules, able to freely penetrate cellular membranes; they are all produced endogenously in the body and they seem to exert similar biological functions. In the cardiovascular system, for example, they are all vasodilators, promote angiogenesis and protect tissues against damage (e.g. ischemia-reperfusion injury). In addition, they have complex roles in inflammation, with both pro- and anti-inflammatory effects reported. Researchers have focused their efforts in understanding and describing the roles of each of these molecules in different physiological systems, and in the past years attention has also been given to the gases interaction or "cross-talk". This review will focus on the role of NO and H2S in inflammation and will give an overview of the evidence collected so far suggesting the importance of their cross-talk in inflammatory processes.

Asunto(s)

Sulfuro de Hidrógeno , Inflamación/metabolismo , Óxido Nítrico , Animales , Humanos , Ratones , Ratas

RESUMEN

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.