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AIMS: Previous work found that during the first wave of the COVID-19 pandemic, 34% of patients with lung cancer treated with curative-intent radiotherapy in the UK had a change to their centre's usual standard of care treatment (Banfill et al. Clin Oncol 2022;34:19-27). We present the impact of these changes on patient outcomes. MATERIALS AND METHODS: The COVID-RT Lung database was a prospective multicentre UK cohort study including patients with stage I-III lung cancer referred for and/or treated with radical radiotherapy between April and October 2020. Data were collected on patient demographics, radiotherapy and systemic treatments, toxicity, relapse and death. Multivariable Cox and logistic regression were used to assess the impact of having a change to radiotherapy on survival, distant relapse and grade ≥3 acute toxicity. The impact of omitting chemotherapy on survival and relapse was assessed using multivariable Cox regression. RESULTS: Patient and follow-up forms were available for 1280 patients. Seven hundred and sixty-five (59.8%) patients were aged over 70 years and 603 (47.1%) were female. The median follow-up was 213 days (119, 376). Patients with stage I-II non-small cell lung cancer (NSCLC) who had a change to their radiotherapy had no significant increase in distant relapse (P = 0.859) or death (P = 0.884); however, they did have increased odds of grade ≥3 acute toxicity (P = 0.0348). Patients with stage III NSCLC who had a change to their radiotherapy had no significant increase in distant relapse (P = 0.216) or death (P = 0.789); however, they did have increased odds of grade ≥3 acute toxicity (P < 0.001). Patients with stage III NSCLC who had their chemotherapy omitted had no significant increase in distant relapse (P = 0.0827) or death (P = 0.0661). CONCLUSION: This study suggests that changes to radiotherapy and chemotherapy made in response to the COVID-19 pandemic did not significantly affect distant relapse or survival. Changes to radiotherapy, namely increased hypofractionation, led to increased odds of grade ≥3 acute toxicity. These results are important, as hypofractionated treatments can help to reduce hospital attendances in the context of potential future emergency situations.
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COVID-19 , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Femenino , Anciano , Anciano de 80 o más Años , Masculino , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Pandemias , Estudios de Cohortes , Estudios Prospectivos , COVID-19/epidemiología , Fraccionamiento de la Dosis de Radiación , Recurrencia Local de Neoplasia/patología , Reino Unido/epidemiología , Estadificación de Neoplasias , Resultado del TratamientoRESUMEN
AIMS: In response to the COVID-19 pandemic, guidelines on reduced fractionation for patients treated with curative-intent radiotherapy were published, aimed at reducing the number of hospital attendances and potential exposure of vulnerable patients to minimise the risk of COVID-19 infection. We describe the changes that took place in the management of patients with stage I-III lung cancer from April to October 2020. MATERIALS AND METHODS: Lung Radiotherapy during the COVID-19 Pandemic (COVID-RT Lung) is a prospective multicentre UK cohort study. The inclusion criteria were: patients with stage I-III lung cancer referred for and/or treated with radical radiotherapy between 2nd April and 2nd October 2020. Patients who had had a change in their management and those who continued with standard management were included. Data on demographics, COVID-19 diagnosis, diagnostic work-up, radiotherapy and systemic treatment were collected and reported as counts and percentages. Patient characteristics associated with a change in treatment were analysed using multivariable binary logistic regression. RESULTS: In total, 1553 patients were included (median age 72 years, 49% female); 93 (12%) had a change to their diagnostic investigation and 528 (34%) had a change to their treatment from their centre's standard of care as a result of the COVID-19 pandemic. Age ≥70 years, male gender and stage III disease were associated with a change in treatment on multivariable analysis. Patients who had their treatment changed had a median of 15 fractions of radiotherapy compared with a median of 20 fractions in those who did not have their treatment changed. Low rates of COVID-19 infection were seen during or after radiotherapy, with only 21 patients (1.4%) developing the disease. CONCLUSIONS: The COVID-19 pandemic resulted in changes to patient treatment in line with national recommendations. The main change was an increase in hypofractionation. Further work is ongoing to analyse the impact of these changes on patient outcomes.
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COVID-19 , Neoplasias Pulmonares , Anciano , Prueba de COVID-19 , Estudios de Cohortes , Femenino , Humanos , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/radioterapia , Masculino , Pandemias , Estudios Prospectivos , SARS-CoV-2 , Reino Unido/epidemiologíaRESUMEN
AIMS: The neutrophil-lymphocyte ratio (NLR) and the absolute lymphocyte count (ALC) have been proposed as prognostic markers in non-small cell lung cancer (NSCLC). The objective of this study was to examine the association of NLR/ALC before and after curative-intent radiotherapy for NSCLC on disease recurrence and overall survival. MATERIALS AND METHODS: A retrospective study of consecutive patients who underwent curative-intent radiotherapy for NSCLC across nine sites in the UK from 1 October 2014 to 1 October 2016. A multivariate analysis was carried out to assess the ability of pre-treatment NLR/ALC, post-treatment NLR/ALC and change in NLR/ALC, adjusted for confounding factors using the Cox proportional hazards model, to predict disease recurrence and overall survival within 2 years of treatment. RESULTS: In total, 425 patients were identified with complete blood parameter values. None of the NLR/ALC parameters were independent predictors of disease recurrence. Higher pre-NLR, post-NLR and change in NLR plus lower post-ALC were all independent predictors of worse survival. Receiver operator curve analysis found a pre-NLR > 2.5 (odds ratio 1.71, 95% confidence interval 1.06-2.79, P < 0.05), a post-NLR > 5.5 (odds ratio 2.36, 95% confidence interval 1.49-3.76, P < 0.001), a change in NLR >3.6 (odds ratio 2.41, 95% confidence interval 1.5-3.91, P < 0.001) and a post-ALC < 0.8 (odds ratio 2.86, 95% confidence interval 1.76-4.69, P < 0.001) optimally predicted poor overall survival on both univariate and multivariate analysis when adjusted for confounding factors. Median overall survival for the high-versus low-risk groups were: pre-NLR 770 versus 1009 days (P = 0.34), post-NLR 596 versus 1287 days (P ≤ 0.001), change in NLR 553 versus 1214 days (P ≤ 0.001) and post-ALC 594 versus 1287 days (P ≤ 0.001). CONCLUSION: NLR and ALC, surrogate markers for systemic inflammation, have prognostic value in NSCLC patients treated with curative-intent radiotherapy. These simple and readily available parameters may have a future role in risk stratification post-treatment to inform the intensity of surveillance protocols.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Humanos , Neoplasias Pulmonares/radioterapia , Recuento de Linfocitos , Linfocitos , Recurrencia Local de Neoplasia/radioterapia , Neutrófilos , Pronóstico , Estudios RetrospectivosRESUMEN
AIMS: There is a paucity of evidence on which to produce recommendations on neither the clinical nor the imaging follow-up of lung cancer patients after curative-intent radiotherapy. In the 2019 National Institute for Health and Care Excellence lung cancer guidelines, further research into risk-stratification models to inform follow-up protocols was recommended. MATERIALS AND METHODS: A retrospective study of consecutive patients undergoing curative-intent radiotherapy for non-small cell lung cancer from 1 October 2014 to 1 October 2016 across nine UK trusts was carried out. Twenty-two demographic, clinical and treatment-related variables were collected and multivariable logistic regression was used to develop and validate two risk-stratification models to determine the risk of disease recurrence and death. RESULTS: In total, 898 patients were included in the study. The mean age was 72 years, 63% (562/898) had a good performance status (0-1) and 43% (388/898), 15% (134/898) and 42% (376/898) were clinical stage I, II and III, respectively. Thirty-six per cent (322/898) suffered disease recurrence and 41% (369/898) died in the first 2 years after radiotherapy. The ASSENT score (age, performance status, smoking status, staging endobronchial ultrasound, N-stage, T-stage) was developed, which stratifies the risk for disease recurrence within 2 years, with an area under the receiver operating characteristic curve (AUROC) for the total score of 0.712 (0.671-0.753) and 0.72 (0.65-0.789) in the derivation and validation sets, respectively. The STEPS score (sex, performance status, staging endobronchial ultrasound, T-stage, N-stage) was developed, which stratifies the risk of death within 2 years, with an AUROC for the total score of 0.625 (0.581-0.669) and 0.607 (0.53-0.684) in the derivation and validation sets, respectively. CONCLUSIONS: These validated risk-stratification models could be used to inform follow-up protocols after curative-intent radiotherapy for lung cancer. The modest performance highlights the need for more advanced risk prediction tools.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Anciano , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Estudios Retrospectivos , Factores de Riesgo , Reino Unido/epidemiologíaRESUMEN
Endothelial dysfunction in small arteries is a ubiquitous, early feature of cardiovascular disease, including hypertension. Dysfunction reflects reduced bioavailability of endothelium-derived nitric oxide (NO) and depressed endothelium-dependent hyperpolarization that enhances vasoreactivity. We measured smooth muscle membrane potential and tension, smooth muscle calcium, and used real-time quantitative polymerase chain reaction in small arteries and isolated tubes of endothelium to investigate how dysfunction enhances vasoreactivity. Rat nonmyogenic mesenteric resistance arteries developed vasomotion to micromolar phenylephrine (α1-adrenoceptor agonist); symmetrical vasoconstrictor oscillations mediated by L-type voltage-gated Ca2+ channels (VGCCs). Inhibiting NO synthesis abolished vasomotion so nanomolar phenylephrine now stimulated rapid, transient depolarizing spikes in the smooth muscle associated with chaotic vasomotion/vasospasm. Endothelium-dependent hyperpolarization block also enabled phenylephrine-vasospasm but without spikes or chaotic vasomotion. Depolarizing spikes were Ca2+-based and abolished by either T-type or L-type VGCCs blockers with depressed vasoconstriction. Removing NO also enabled transient spikes/vasoconstriction to Bay K-8644 (L-type VGCC activator). However, these were abolished by the L-type VGCC blocker nifedipine but not T-type VGCC block. Phenylephrine also initiated T-type VGCC-transient spikes and enhanced vasoconstriction after NO loss in nonmyogenic arteries from spontaneously hypertensive rats. In contrast to mesenteric arteries, myogenic coronary arteries displayed transient spikes and further vasoconstriction spontaneously on loss of NO. T-type VGCC block abolished these spikes and additional vasoconstriction but not myogenic tone. Therefore, in myogenic and nonmyogenic small arteries, reduced NO bioavailability engages T-type VGCCs, triggering transient depolarizing spikes in normally quiescent vascular smooth muscle to cause vasospasm. T-type block may offer a means to suppress vasospasm without inhibiting myogenic tone mediated by L-type VGCCs.
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Canales de Calcio Tipo L/metabolismo , Endotelio Vascular , Hipertensión , Nifedipino/farmacología , Óxido Nítrico/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Fenilefrina/farmacología , Ratas , Resistencia Vascular , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Patients treated with curative-intent lung radiotherapy are in the group at highest risk of severe complications and death from COVID-19. There is therefore an urgent need to reduce the risks associated with multiple hospital visits and their anti-cancer treatment. One recommendation is to consider alternative dose-fractionation schedules or radiotherapy techniques. This would also increase radiotherapy service capacity for operable patients with stage I-III lung cancer, who might be unable to have surgery during the pandemic. Here we identify reduced-fractionation for curative-intent radiotherapy regimes in lung cancer, from a literature search carried out between 20/03/2020 and 30/03/2020 as well as published and unpublished audits of hypofractionated regimes from UK centres. Evidence, practical considerations and limitations are discussed for early-stage NSCLC, stage III NSCLC, early-stage and locally advanced SCLC. We recommend discussion of this guidance document with other specialist lung MDT members to disseminate the potential changes to radiotherapy practices that could be made to reduce pressure on other departments such as thoracic surgery. It is also a crucial part of the consent process to ensure that the risks and benefits of undergoing cancer treatment during the COVID-19 pandemic and the uncertainties surrounding toxicity from reduced fractionation have been adequately discussed with patients. Furthermore, centres should document all deviations from standard protocols, and we urge all colleagues, where possible, to join national/international data collection initiatives (such as COVID-RT Lung) aimed at recording the impact of the COVID-19 pandemic on lung cancer treatment and outcomes.
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Betacoronavirus , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Infecciones por Coronavirus/complicaciones , Fraccionamiento de la Dosis de Radiación , Neoplasias Pulmonares/radioterapia , Neumonía Viral/complicaciones , Guías de Práctica Clínica como Asunto/normas , Carcinoma Pulmonar de Células Pequeñas/radioterapia , COVID-19 , Carcinoma de Pulmón de Células no Pequeñas/virología , Ensayos Clínicos como Asunto , Infecciones por Coronavirus/virología , Humanos , Neoplasias Pulmonares/virología , Metaanálisis como Asunto , Pandemias , Neumonía Viral/virología , Gestión de Riesgos , SARS-CoV-2 , Carcinoma Pulmonar de Células Pequeñas/virología , Revisiones Sistemáticas como AsuntoRESUMEN
BACKGROUND: Intratumoural heterogeneity (ITH) is well recognised in prostate cancer (PC), but its role in high-risk disease is uncertain. A prospective, single-arm, translational study using targeted multiregion prostate biopsies was carried out to study genomic and T-cell ITH in clinically high-risk PC aiming to identify drivers and potential therapeutic strategies. PATIENTS AND METHODS: Forty-nine men with elevated prostate-specific antigen and multiparametric-magnetic resonance imaging detected PC underwent image-guided multiregion transperineal biopsy. Seventy-nine tumour regions from 25 patients with PC underwent sequencing, analysis of mutations, copy number and neoepitopes combined with tumour infiltrating T-cell subset quantification. RESULTS: We demonstrated extensive somatic nucleotide variation and somatic copy number alteration heterogeneity in high-risk PC. Overall, the mutational burden was low (0.93/Megabase), but two patients had hypermutation, with loss of mismatch repair (MMR) proteins, MSH2 and MSH6. Somatic copy number alteration burden was higher in patients with metastatic hormone-naive PC (mHNPC) than in those with high-risk localised PC (hrlPC), independent of Gleason grade. Mutations were rarely ubiquitous and mutational frequencies were similar for mHNPC and hrlPC patients. Enrichment of focal 3q26.2 and 3q21.3, regions containing putative metastasis drivers, was seen in mHNPC patients. We found evidence of parallel evolution with three separate clones containing activating mutations of ß-catenin in a single patient. We demonstrated extensive intratumoural and intertumoural T-cell heterogeneity and high inflammatory infiltrate in the MMR-deficient (MMRD) patients and the patient with parallel evolution of ß-catenin. Analysis of all patients with activating Wnt/ß-catenin mutations demonstrated a low CD8+/FOXP3+ ratio, a potential surrogate marker of immune evasion. CONCLUSIONS: The PROGENY (PROstate cancer GENomic heterogeneitY) study provides a diagnostic platform suitable for studying tumour ITH. Genetic aberrations in clinically high-risk PC are associated with altered patterns of immune infiltrate in tumours. Activating mutations of Wnt/ß-catenin signalling pathway or MMRD could be considered as potential biomarkers for immunomodulation therapies. CLINICAL TRIALS.GOV IDENTIFIER: NCT02022371.
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Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Biopsia/métodos , Epítopos de Linfocito B/inmunología , Dosificación de Gen , Heterogeneidad Genética , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Masculino , Mutación , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Factores de Riesgo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T/inmunología , Linfocitos T/patología , Vía de Señalización WntRESUMEN
Alkali metal intercalation into polyaromatic hydrocarbons (PAHs) has been studied intensely after reports of superconductivity in a number of potassium- and rubidium-intercalated materials. There are, however, no reported crystal structures to inform our understanding of the chemistry and physics because of the complex reactivity of PAHs with strong reducing agents at high temperature. Here we present the synthesis of crystalline K2Pentacene and K2Picene by a solid-solid insertion protocol that uses potassium hydride as a redox-controlled reducing agent to access the PAH dianions, and so enables the determination of their crystal structures. In both cases, the inserted cations expand the parent herringbone packings by reorienting the molecular anions to create multiple potassium sites within initially dense molecular layers, and thus interact with the PAH anion π systems. The synthetic and crystal chemistry of alkali metal intercalation into PAHs differs from that into fullerenes and graphite, in which the cation sites are pre-defined by the host structure.
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The lipid molecule, lysophosphatidylinositol (LPI), is hypothesised to form part of a novel lipid signalling system that involves the G protein-coupled receptor GPR55 and distinct intracellular signalling cascades in endothelial cells. This work aimed to study the possible mechanisms involved in LPI-evoked cytosolic Ca(2+) mobilization in human brain microvascular endothelial cells. Changes in intracellular Ca(2+) concentrations were measured using cell population Ca(2+) assay. LPI evoked biphasic elevation of intracellular calcium concentration, a rapid phase and a sustained phase. The rapid phase was attenuated by the inhibitor of PLC (U 73122), inhibitor of IP(3) receptors, 2-APB and the depletor of endoplasmic reticulum Ca(2+) store, thapsigargin. The sustained phase, on the other hand, was enhanced by U 73122 and abolished by the RhoA kinase inhibitor, Y-27632. In conclusion, the Ca(2+) signal evoked by LPI is characterised by a rapid phase of Ca(2+) release from the endoplasmic reticulum, and requires activation of the PLC-IP(3) signalling pathway. The sustained phase mainly depends on RhoA kinase activation. LPI acts as novel lipid signalling molecule in endothelial cells, and elevation of cytosolic Ca(2+) triggered by it may present an important intracellular message required in gene expression and controlling of vascular tone.
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Señalización del Calcio/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Lisofosfolípidos/farmacología , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Señalización del Calcio/fisiología , Línea Celular Transformada , Citosol/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Atypical cannabinoid O-1602 (5-Methyl-4-[(1R,6R)-3-methyl-6-(1-cyclohexen-1-yl]-1,3-benzenediol) induces vasorelaxation and activates the orphan G protein-coupled receptor GPR55 in human endothelial cells. This study investigates the underlying mechanisms of vasorelaxation induced by this compound. The vasodilator activity was assessed in the rat third order branch of the superior mesenteric artery using a wire myograph. The vasorelaxation was partially endothelium-dependent (pEC50%=5.8±0.3). The endothelial component was antagonized by the putative endothelial receptor antagonists rimonabant (3 µM; pEC50%=5.1±0.2) and O-1918 (10 µM; pEC50%=5.3±0.2) but not by the CB1 and CB2 receptors antagonists AM 251 (10 µM) and AM 630 (10 µM), respectively. The vasorelaxation was not pertussis toxin-sensitive and not mediated through TRPVI receptors or by the release of NO, but was reduced by inhibition of Ca2+ sensitive K+ channels (KCa). In endothelium-denuded vessels, O-1602 abolished CaCl2-induced contraction and the inhibition was apparently reversed by O-1918. O-1602 mediates its vasorelaxant effects partly by an endothelium-dependent pathway involving rimonabant- and O-1918-sensitive targets that are distinct from the classical CB1 and CB2 cannabinoid receptors and might involve activation of KCa. The endothelium-independent relaxation might involve interfering with Ca2+ entry.
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Cannabidiol/farmacología , Ciclohexanos/farmacología , Endotelio Vascular/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Resorcinoles/farmacología , Vasodilatación/efectos de los fármacos , Animales , Cannabidiol/metabolismo , Ciclohexanos/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Masculino , Arterias Mesentéricas/metabolismo , Ratas , Ratas Wistar , Resorcinoles/metabolismo , Vasodilatación/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Lysophosphatidylinositol (LPI), a lipid signalling molecule, activates GPR55 and elevates intracellular Ca(2+). Here, we examine the actions of LPI in the rat resistance mesenteric artery and Ca(2+) responses in endothelial cells isolated from the artery. EXPERIMENTAL APPROACH: Vascular responses were studied using wire myographs. Single-cell fluorescence imaging was performed using a MetaFluor system. Hypotensive effects of LPI were assessed using a Biopac system. KEY RESULTS: In isolated arteries, LPI-induced vasorelaxation was concentration- and endothelium-dependent and inhibited by CID 16020046, a GPR55 antagonist. The CB1 receptor antagonist AM 251 had no effect, whereas rimonabant and O-1918 significantly potentiated LPI responses. Vasorelaxation was reduced by charybdotoxin and iberiotoxin, alone or combined. LPI decreased systemic arterial pressure. GPR55 is expressed in rat mesenteric artery. LPI caused biphasic elevations of endothelial cell intracellular Ca(2+). Pretreatment with thapsigargin or 2-aminoethoxydiphenyl borate abolished both phases. The PLC inhibitor U73122 attenuated the initial phase and enhanced the second phase, whereas the Rho-associated kinase inhibitor Y-27632 abolished the late phase but not the early phase. CONCLUSIONS AND IMPLICATIONS: LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery and a hypotensive agent. The vascular response involves activation of Ca(2+)-sensitive K(+) channels and is not mediated by CB1 receptors, but unexpectedly enhanced by antagonists of the 'endothelial anandamide' receptor. In endothelial cells, LPI utilizes PLC-IP3 and perhaps ROCK-RhoA pathways to elevate intracellular Ca(2+). Overall, these findings support an endothelial site of action for LPI and suggest a possible role for GPR55 in vasculature.
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Células Endoteliales/efectos de los fármacos , Lisofosfolípidos/farmacología , Arterias Mesentéricas/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Animales , Calcio/metabolismo , Células Endoteliales/metabolismo , Estrenos/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Arterias Mesentéricas/metabolismo , Pirrolidinonas/farmacología , Ratas , Ratas Wistar , Receptores de Cannabinoides , Fosfolipasas de Tipo C/metabolismo , Vasodilatadores/farmacologíaRESUMEN
The actions of the anandamide-like mono-unsaturated fatty acid oleoylethanolamide (OEA) were first linked to satiety and control of food intake and recently reported to relax resistance vessels. This study characterizes its vasorelaxant mechanisms. Vasorelaxation to OEA were assessed in third order branches of rat superior mesenteric artery using a wire myograph. The roles of the endothelium, KCa channels, perivascular sensory nerves, NO, cannabinoid receptors, and the phospholipase C (PLC)/inositol trisphosphate (InsP3) and RhoA/ROCK signalling pathways, were assessed. OEA caused concentration- and endothelium-dependent vasorelaxation (pEC50=6.7±0.1, Rmax=93.1±2.5%). L-NAME greatly reduced the response (residual relaxation of only 24.6±12.8%). Capsaicin and pertussis toxin significantly reduced the vasorelaxation. Precontraction with KCl abolished the response. TRAM-34 had no effect, but both iberiotoxin and apamin+charybdotoxin markedly shifted the OEA concentration-response curve to the right (â¼5-fold). O-1918 but not rimonabant attenuated the vasorelaxation. Both the CB1 receptor antagonist, AM251 and the CB2 receptor antagonist, AM630, given alone or in combination, reduced the response to OEA. Inhibition of PLC by U73122, ROCK by Y-27632 and antagonism of inositol trisphosphate (InsP3) receptors by 2-APB abolished OEA vasorelaxation. OEA vasorelaxation involves an endothelial site of action but not the known cannabinoid receptors. It involves Ca(2+) released from InsP3-sensitive endothelial stores by mechanisms involving RhoA kinase and phospholipase C. It is likely that the released Ca(2+) causes NO generation and opening of mainly large-conductance KCa channels. This study demonstrates a possible novel endothelial target that might be important in the control of regional blood flow induced by this lipid molecule.
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Endotelio Vascular/fisiología , Arterias Mesentéricas/fisiología , Ácidos Oléicos/farmacología , Vasodilatación/fisiología , Animales , Endocannabinoides , Técnicas In Vitro , Masculino , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Canales de Potasio Calcio-Activados/fisiología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/fisiología , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/fisiología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/fisiología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/fisiología , Vasodilatación/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiologíaRESUMEN
Our view of the endothelium was transformed around 30 years ago, from one of an inert barrier to that of a key endocrine organ central to cardiovascular function. This dramatic change followed the discoveries that endothelial cells (ECs) elaborate the vasodilators prostacyclin and nitric oxide. The key to these discoveries was the use of the quintessentially pharmacological technique of bioassay. Bioassay also revealed endothelium-derived hyperpolarizing factor (EDHF), particularly important in small arteries and influencing blood pressure and flow distribution. The basic idea of EDHF as a diffusible factor causing smooth muscle hyperpolarization (and thus vasodilatation) has evolved into one of a complex pathway activated by endothelial Ca(2+) opening two Ca(2+) -sensitive K(+) -channels, K(Ca)2.3 and K(Ca)3.1. Combined application of apamin and charybdotoxin blocked EDHF responses, revealing the critical role of these channels as iberiotoxin was unable to substitute for charybdotoxin. We showed these channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, and close to interendothelial gap junctions. Activation of K(Ca) channels hyperpolarizes ECs, and K(+) efflux through them can act as a diffusible 'EDHF' stimulating Na(+) /K(+) -ATPase and inwardly rectifying K-channels. In parallel, hyperpolarizing current can spread from the endothelium to the smooth muscle through myoendothelial gap junctions upon endothelial projections. The resulting radial hyperpolarization mobilized by EDHF is complemented by spread of hyperpolarization along arteries and arterioles, effecting distant dilatation dependent on the endothelium. So the complexity of the endothelium still continues to amaze and, as knowledge evolves, provides considerable potential for novel approaches to modulate blood pressure.
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Factores Biológicos/metabolismo , Endotelio Vascular/fisiología , Animales , Presión Sanguínea/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/fisiopatología , Canales de Potasio Calcio-Activados/metabolismoRESUMEN
Hypoxia contributes to the aggressive and treatment-resistant phenotype of pancreatic ductal adenocarcinoma. Oncolytic vaccinia virus has potential as an anti-tumour agent, but the ability to lyse hypoxic tumour cells is vital for clinical efficacy. We hypothesized that unique aspects of the poxvirus life cycle would protect it from attenuation in hypoxic conditions. We characterized and compared the viral protein production, viral replication, cytotoxicity and transgene expression of Lister strain vaccinia virus in a panel of pancreatic cancer cell lines after exposure to normoxic or hypoxic conditions. Viral protein production was not affected by hypoxia, and high viral titres were produced in both normoxic and hypoxic conditions. Interestingly, there was a 3.5-fold (P<0.001) and 20-fold (P<0.0001) increase in viral cytotoxicity for CFPac1 and MiaPaca2 cell lines, respectively, in hypoxic conditions. Cytotoxicity was equivalent in the remaining cell lines. Levels of transgene expression (luciferase reporter gene) from the vaccinia viral vector were comparable, regardless of the ambient oxygen concentration. The present study suggests that the vaccinia virus is a promising vector for targeting pancreatic cancer and potentially other hypoxic tumour types.
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Hipoxia de la Célula , Técnicas de Transferencia de Gen , Vectores Genéticos , Viroterapia Oncolítica/métodos , Virus Vaccinia/genética , Línea Celular Tumoral , Humanos , Virus Oncolíticos/genética , Transgenes , Proteínas Virales/genética , Replicación ViralRESUMEN
Anandamide activates CB(1) cannabinoid receptors but also has effects, particularly in the vasculature, that cannot be explained by actions at either this or the other cloned cannabinoid receptor, the CB(2) receptor. These effects are probably mediated by a novel G protein-coupled receptor, but genome searching has not revealed a strong candidate. Several approaches have suggested that an orphan receptor, GPR55, is a target for anandamide, but the pharmacology of this receptor is such that it cannot be categorically identified as a cannabinoid receptor. GPR55 appears primarily to be a receptor for lysophosphatidylinositol which may exhibit biased agonism, leading to it also responding to anandamide. GPR55 activates G(alpha12) and G(alpha13) and thence RhoA, leading to an oscillatory intracellular Ca(2+) signal. Further complexity arises from possible interactions between the anandamide-sensitive CB(1) receptor and GPR55. Overall, it appears that GPR55 has several signaling modalities and that, while anandamide can activate systems containing this receptor, GPR55 cannot yet be primarily designated a receptor for this endocannabinoid.
Asunto(s)
Ácidos Araquidónicos/fisiología , Moduladores de Receptores de Cannabinoides/fisiología , Receptores de Cannabinoides/fisiología , Receptores Acoplados a Proteínas G/fisiología , Sitio Alostérico , Animales , Dimerización , Endocannabinoides , Humanos , Ligandos , Patentes como Asunto , Alcamidas Poliinsaturadas , Receptores de Cannabinoides/genética , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes , Transducción de SeñalRESUMEN
Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.
Asunto(s)
Mitocondrias/metabolismo , Daño por Reperfusión/prevención & control , S-Nitrosotioles/farmacología , Compuestos de Sulfhidrilo/metabolismo , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Línea Celular , Complejo I de Transporte de Electrón/metabolismo , Células HeLa , Corazón/efectos de los fármacos , Corazón/fisiopatología , Humanos , Técnicas In Vitro , Masculino , Espectrometría de Masas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/fisiología , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/fisiología , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Óxido Nítrico/metabolismo , Nitrosación/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , S-Nitrosotioles/síntesis química , S-Nitrosotioles/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
Endocannabinoids, such as anandamide and 2-arachidonoylglycerol, are synthesized from membrane phospholipids in the heart and other cardiovascular tissues. They activate cannabinoid CB1 and CB2 receptors, transient receptor potential V1 (TRPV1), peroxisome proliferator-activated receptors, and perhaps a novel vascular G-protein-coupled receptor. Inactivation is by cellular uptake and fatty acid amide hydrolase. Endocannabinoids relax coronary and other arteries and decrease cardiac work but seem not to be involved in tonic regulation of cardiovascular function. They act as a stress response system, which is activated, for example, in myocardial infarction and circulatory shock. Endocannabinoids are largely protective; they decrease tissue damage and arrhythmia in myocardial infarction and may reduce progression of atherosclerosis (CB2 receptor stimulation inhibits lesion progression), and fatty acid amide hydrolase knockout mice (which have enhanced endocannabinoid levels) show decreased cardiac dysfunction with age compared with wild types. However, endocannabinoids may mediate doxorubicin-induced cardiac dysfunction. Their signaling pathways are not fully elucidated but they can lead to changed expression of a variety of genes, including those involved in inflammatory responses. There is potential for therapeutic targeting of endocannabinoids and their receptors, but their apparent involvement in both protective and deleterious actions on the heart means that careful risk assessment is needed before any treatment can be introduced.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Moduladores de Receptores de Cannabinoides/fisiología , Cannabinoides/metabolismo , Endocannabinoides , Miocardio/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides/química , Cannabinoides/farmacología , Cardiotónicos/metabolismo , Humanos , Estructura Molecular , Alcamidas Poliinsaturadas/metabolismo , Receptores de Cannabinoides/metabolismoRESUMEN
BACKGROUND AND PURPOSE: A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal-cannabidiol and is blocked by O-1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel water-soluble agonist, as a vasorelaxant potentially acting at non-CB1, non-CB2 cannabinoid receptors in the vasculature. EXPERIMENTAL APPROACH: VSN16 and some analogues were synthesized and assayed for vasodilator activity in the rat third generation mesenteric artery using wire myography. Also carried out with VSN16 were haemodynamic studies in conscious rats and binding studies to CB1 receptors of rat cerebellum. KEY RESULTS: VSN16 relaxed mesenteric arteries in an endothelium-dependent manner. The vasorelaxation was antagonized by high concentrations of the classical cannabinoid antagonists, rimonabant and AM 251, as well as by O-1918, an antagonist at the abnormal-cannabidiol receptor but not at CB1 or CB2 receptors. It did not affect [3H]CP55,940 binding to CB1 receptors in rat cerebellum. The vasorelaxation was not pertussis toxin-sensitive but was reduced by inhibition of nitric oxide synthesis, Ca(2+)-sensitive K+ channels (KCa) and TRPV1 receptors. In conscious rats VSN16 transiently increased blood pressure and caused a longer-lasting increase in mesenteric vascular conductance. Structure-activity studies on vasorelaxation showed a stringent interaction with the target receptor. CONCLUSIONS AND IMPLICATIONS: VSN16 is an agonist at a novel cannabinoid receptor of the vasculature. It acts on the endothelium to release nitric oxide and activate KCa and TRPV1. As it is water-soluble it might be useful in bringing about peripheral cannabinoid-like effects without accompanying central or severe cardiovascular responses.
Asunto(s)
Benzamidas/farmacología , Endotelio Vascular/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Animales , Apamina/farmacología , Benzamidas/síntesis química , Benzamidas/química , Agonistas de Receptores de Cannabinoides , Antagonistas de Receptores de Cannabinoides , Cannabinoides/farmacología , Cerebelo/metabolismo , Caribdotoxina/farmacología , Ciclohexanoles/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Técnicas In Vitro , Indometacina/farmacología , Masculino , Arterias Mesentéricas/fisiología , Estructura Molecular , NG-Nitroarginina Metil Éster/farmacología , Toxina del Pertussis/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Cannabinoides/metabolismo , Resorcinoles/farmacología , Rimonabant , Canales Catiónicos TRPV/metabolismo , Tritio , Resistencia Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
CB1 and CB2 receptors mediate most responses to cannabinoids but not some of the cardiovascular actions of endocannabinoids such as anandamide and virodhamine, or those of some synthetic agents, like abnormal cannabidiol (abn-cbd). These agents induce vasorelaxation which is antagonised by rimonabant but only at high concentrations relative to those required to block CB1 receptors. Vasorelaxation to anandamide is sensitive to Pertussis toxin (though that to abn-cbd is not), and so is thought to be mediated by a G protein-coupled receptor through Gi/o. An orphan receptor, GPR55, apparently a cannabinoid receptor, is activated by abn-cbd, but is not the receptor mediating vasorelaxation to this agent, as the response persists in vessels from GPR55 knockout mice. However, the activity of anandamide in GPR55 knockout mice is not yet reported and so the role of GPR55 as a cannabinoid receptor mediating vascular responses has yet to be finalised.
Asunto(s)
Cannabinoides/farmacología , Músculo Liso Vascular/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Animales , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
Oleamide (cis-9,10-octadecenoamide), a fatty acid primary amide discovered in the cerebrospinal fluid of sleep-deprived cats, has a variety of actions that give it potential as a signaling molecule, although these actions have not been extensively investigated in the cardiovascular system. The synthetic pathway probably involves synthesis of oleoylglycine and then conversion to oleamide by peptidylglycine alpha-amidating monooxygenase (PAM); breakdown of oleamide is by fatty acid amide hydrolase (FAAH). Oleamide interacts with voltage-gated Na(+) channels and allosterically with GABA(A) and 5-HT(7) receptors as well as having cannabinoid-like actions. The latter have been suggested to be due to potentiation of the effects of endocannabinoids such as anandamide by inhibiting FAAH-mediated hydrolysis. This might underlie an "entourage effect" whereby co-released endogenous nonagonist congeners of endocannabinoids protect the active molecule from hydrolysis by FAAH. However, oleamide has direct agonist actions at CB(1) cannabinoid receptors and also activates the TRPV1 vanilloid receptor. Other actions include inhibition of gap-junctional communication, and this might give oleamide a role in myocardial development. Many of these actions are absent from the trans isomer of 9,10-octadecenoamide. One of the most potent actions of oleamide is vasodilation. In rat small mesenteric artery the response does not involve CB(1) cannabinoid receptors but another pertussis toxin-sensitive, G protein-coupled receptor, as yet unidentified. This receptor is sensitive to rimonabant and O-1918, an antagonist at the putative "abnormal-cannabidiol" or endothelial "anandamide" receptors. Vasodilation is mediated by endothelium-derived nitric oxide, endothelium-dependent hyperpolarization, and also through activation of TRPV1 receptors. A physiological role for oleamide in the heart and circulation has yet to be demonstrated, as has production by cells of the cardiovascular system, but this molecule has a range of actions that could give it considerable modulatory power.