[Prehospital management of acute coronary syndrome in patients on long-term direct oral anticoagulant treatment]. / Prähospitale Behandlung des akuten Koronarsyndroms unter DOAK-Dauertherapie.

BACKGROUND: Despite an increasing incidence of patients suffering from acute coronary syndrome (ACS) under simultaneous treatment with direct oral anticoagulants (DOAC), neither sufficient scientific data nor uniform guidelines for the anticoagulation treatment of these patients are currently available. OBJECTIVE: The aim of this study was to determine the current practice of preclinical treatment of ACS in patients under DOAC treatment. MATERIAL AND METHODS: An internet and paper-based survey of emergency physicians, specialists of internal medicine, anesthesiologists, emergency and intensive care physicians was performed concerning the prehospital treatment of ACS in patients under long-term DOAC treatment. RESULTS: Overall, 284 questionnaires were answered. Substantial differences in the current treatment of ACS under long-term DOAC therapy were identified. While 39% of the respondents stated that they administer a combination treatment of heparin and acetylsalicylic acid (ASA), 36% renounced the administration of heparin. If a dose reduction was performed, 71% answered that they reduce the heparin dosage. Also, in cases of ST-segment elevation myocardial infarction 48% of the physicians renounced the administration of heparin. CONCLUSION: In Germany there is currently a heterogeneous practice of emergency treatment of ACS patients under DOAC therapy with respect to the administration of heparin and ASA. Therefore, guidelines of the specialist medical societies should address the prehospital emergency anticoagulation management of ACS in patients under therapy with DOAC, which correspond to the needs of patients and emergency physicians.

Early changes in N-terminal pro-B-type natriuretic peptide levels after transcatheter aortic valve replacement and its impact on long-term mortality.

BACKGROUND: N-terminal pro-hormone B-type natriuretic peptide (NT-proBNP) levels correlate with higher peri-procedural mortality after transcatheter aortic valve replacement (TAVR). The long-term prognostic value of NT-proBNP within the first days after TAVR, however, remains unclear. This study examined early changes in NT-proBNP prior to and within 6 days after TAVR, the diagnostic value of this biomarker regarding aortic regurgitation (AR), and its prognostic value regarding one-year mortality. METHODS AND RESULTS: NT-proBNP concentrations were measured in 504 consecutive patients undergoing transapical (TA) or transfemoral (TF) TAVR before and directly after TAVR as well as 4 h and 1, 2, 3, and 6 days after TAVR. The follow-up period was 1 year. NT-proBNP was elevated in all patients at baseline (median 2141 ng/L [IQR 1021-5319 ng/L]). NT-proBNP changes in the first 6 days after TAVR showed significant differences depending on the approach, with a greater and more prolonged rise evident in TA-TAVR patients. NT-proBNP was an independent predictor of mortality in TA patients with AR, with an AUC of 0.794 (95% CI 0.663-0.925; P = 0.003) when measured on day 3 after TAVR. For TF patients with AR and reduced left ventricular systolic function, the AUC for prediction of mortality was 0.897 (95% CI 0.778-1.0; P = 0.004) on day 2. CONCLUSIONS: The prognostic information of early post-procedural NT-proBNP concentrations is superior to pre-procedural values regarding all-cause mortality within 1 year. Post-procedural NT-proBNP must be interpreted in relation to the TAVR approach. NT-proBNP predicts mortality in TF-TAVR patients with AR and reduced left ventricular function.

[Severe caffeine poisoning with rhabdomyolysis]. / Schwere Koffeinintoxikation mit Rhabdomyolyse.

We report the case of a young man who took a large amount of caffeine powder dissolved in water in a suicide attempt. He was found comatose. The initial diagnosis was difficult. In hospital he suffered from rhabdomyolysis with renal failure and sepsis rapidly developed. After renal replacement treatment with hemodialysis, long-term artificial ventilation with tracheotomy and a 3­week stay in the intensive care unit, the patient could be discharged to a rehabilitation center.

Climatic, geomorphologic and hydrologic perturbations as drivers for mid- to late Holocene development of ice-wedge polygons in the western Canadian Arctic.

Ice-wedge polygons are widespread periglacial features and influence landscape hydrology and carbon storage. The influence of climate and topography on polygon development is not entirely clear, however, giving high uncertainties to projections of permafrost development. We studied the mid- to late Holocene development of modern ice-wedge polygon sites to explore drivers of change and reasons for long-term stability. We analyzed organic carbon, total nitrogen, stable carbon isotopes, grain size composition and plant macrofossils in six cores from three polygons. We found that all sites developed from aquatic to wetland conditions. In the mid-Holocene, shallow lakes and partly submerged ice-wedge polygons existed at the studied sites. An erosional hiatus of ca 5000 years followed, and ice-wedge polygons re-initiated within the last millennium. Ice-wedge melt and surface drying during the last century were linked to climatic warming. The influence of climate on ice-wedge polygon development was outweighed by geomorphology during most of the late Holocene. Recent warming, however, caused ice-wedge degradation at all sites. Our study showed that where waterlogged ground was maintained, low-centered polygons persisted for millennia. Ice-wedge melt and increased drainage through geomorphic disturbance, however, triggered conversion into high-centered polygons and may lead to self-enhancing degradation under continued warming.

Keratinocyte Sonic Hedgehog Upregulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion.

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

Pulmonary hypertensive vasculopathy in parenchymal lung diseases and/or hypoxia: Number 1 in the Series "Pathology for the clinician" Edited by Peter Dorfmüller and Alberto Cavazza.

Pulmonary hypertension (PH) with complicating chronic lung diseases and/or hypoxia falls into group 3 of the updated classification of PH. Patients with chronic obstructive lung disease (COPD), diffuse lung disease (such as idiopathic pulmonary fibrosis (IPF)) and with sleep disordered breathing are particularly exposed to the risk of developing PH. Although PH in such a context is usually mild, a minority of patients exhibit severe haemodynamic impairment, defined by a mean pulmonary arterial pressure (mPAP) of ≥35 mmHg or mPAP values ranging between 25 mmHg and 35 mmHg with a low cardiac index (<2 L·min-1·m-2). The overlap between lung parenchymal disease and PH heavily affects life expectancy in such a patient population and complicates their therapeutic management. In this review we illustrate the pathological features and the underlying pathophysiological mechanisms of pulmonary circulation in chronic lung diseases, with an emphasis on COPD, IPF and obstructive sleep apnoea syndrome.

Breast Cancer Survival of BRCA1/BRCA2 Mutation Carriers in a Hospital-Based Cohort of Young Women.

Background: The primary aim of the study was to investigate prognosis and long-term survival in young breast cancer patients with a BRCA1 or BRCA2 germline mutation compared with noncarriers. The secondary aim was to investigate whether differences in survival originate from associations with tumor characteristics, second cancers, and/or treatment response. Methods: We established a cohort of invasive breast cancer patients diagnosed younger than age 50 years in 10 Dutch hospitals between 1970 and 2003. BRCA1/2 testing of most prevalent mutations was mainly done using DNA isolate from formalin-fixed paraffin-embedded nontumor tissue. Survival estimates were derived using Cox regression and competing risk models. Results: In 6478 breast cancer patients, we identified 3.2% BRCA1 and 1.2% BRCA2 mutation carriers. BRCA1 mutation carriers had a worse overall survival independent of clinico-pathological/treatment characteristics, compared with noncarriers (adjusted hazard ratio [HR] = 1.20, 95% confidence interval [CI] = 0.97 to 1.47), though only statistically significant in the first five years of follow-up (adjusted HR = 1.40, 95% CI = 1.07 to 1.84). A large part of the worse survival was explained by incidence of ovarian cancers. Breast cancer-specific, disease-free, and metastasis-free survival results were less pronounced and mostly statistically nonsignificant but in the same direction with those of overall survival. Overall survival was worse, although not statistically significantly, within the ER-negative or ER-positive, grade 3, and small tumor subgroups. The worse survival was most pronounced in non-chemotherapy-treated patients (adjusted HR = 1.54, 95% CI = 1.08 to 2.19). Power for BRCA2 mutation carriers was limited; only after five years' follow-up overall survival was worse (adjusted HR = 1.47, 95% CI = 1.00 to 2.17). Conclusions: BRCA1/2 mutation carriers diagnosed with breast cancer before age 50 years are prone to a worse survival, which is partly explained by differences in tumor characteristics, treatment response, and second ovarian cancers.

Release kinetics of high-sensitivity cardiac troponins I and T and troponin T upstream open reading frame peptide (TnTuORF) in clinically induced acute myocardial infarction.

CONTEXT: Troponin T upstream open reading frame peptide (TnTuORF) may be useful as a novel biomarker in acute cardiac syndromes. OBJECTIVE: The study examined the early release kinetics of TnTuORF. MATERIALS AND METHODS: We analyzed the time course of the release of cardiac troponins I and T and TnTuORF in patients (n = 31) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH). RESULTS: Fifteen minutes after TASH, the levels of both troponins increased significantly (cTnT median: 18 ng/L versus 27 ng/L; cTnI median: 15 ng/L versus 25 ng/L). TnTuORF showed no variation. DISCUSSION: We observed a significantly greater increase in cTnI compared with cTnT. CONCLUSION: Our results demonstrate that troponin assays allow early detection of myocardial injury, whereas TnTuORF levels remain unchanged in this setting.

Thrombomodulin-dependent protein C activation is required for mitochondrial function and myelination in the central nervous system.

Essentials The role of protein C (PC) activation in experimental autoimmune encephalitis (EAE) is unknown. PC activation is required for mitochondrial function in the central nervous system. Impaired PC activation aggravates EAE, which can be compensated for by soluble thrombomodulin. Protection of myelin by activated PC or solulin is partially independent of immune-modulation. SUMMARY: Background Studies with human samples and in rodents established a function of coagulation proteases in neuro-inflammatory demyelinating diseases (e.g. in multiple sclerosis [MS] and experimental autoimmune encephalitis [EAE]). Surprisingly, approaches to increase activated protein C (aPC) plasma levels as well as antibody-mediated inhibition of PC/aPC ameliorated EAE in mice. Hence, the role of aPC generation in demyelinating diseases and potential mechanisms involved remain controversial. Furthermore, it is not known whether loss of aPC has pathological consequences at baseline (e.g. in the absence of disease). Objective To explore the role of thrombomodulin (TM)-dependent aPC generation at baseline and in immunological and non-immunological demyelinating disease models. Methods Myelination and reactive oxygen species (ROS) generation were evaluated in mice with genetically reduced TM-mediated protein C activation (TMPro/Pro ) and in wild-type (WT) mice under control conditions or following induction of EAE. Non-immunological demyelination was analyzed in the cuprizone-diet model. Results Impaired TM-dependent aPC generation already disturbs myelination and mitochondrial function at baseline. This basal phenotype is linked with increased mitochondrial ROS and aggravates EAE. Reducing mitochondrial ROS (p66Shc deficiency), restoring aPC plasma levels or injecting soluble TM (solulin) ameliorates EAE in TMPro/Pro mice. Soluble TM additionally conveyed protection in WT-EAE mice. Furthermore, soluble TM dampened demyelination in the cuprizone-diet model, demonstrating that its myelin-protective effect is partially independent of an immune-driven process. Conclusion These results uncover a novel physiological function of TM-dependent aPC generation within the CNS. Loss of TM-dependent aPC generation causes a neurological defect in healthy mice and aggravates EAE, which can be therapeutically corrected.

Increasing time trends of thin melanomas in The Netherlands: What are the explanations of recent accelerations?

BACKGROUND: A disproportional increase in in situ or thin melanomas may point at underlying causes such as increased melanoma awareness, as well as 'overdiagnosis' of melanoma in diagnostically equivocal small lesions. OBJECTIVES: The purposes of this study were to estimate trends in melanoma incidence by sex, Breslow thickness (thin melanomas subdivided into four subgroups: <0.25 mm, 0.25-0.49 mm, 0.50-0.74 mm, and 0.75-1.0 mm), age and location, and to compare these with trends in subgroups of thicker melanomas. METHODS: Data on all histologically confirmed in situ and invasive melanomas diagnosed between 1994 and 2010 were retrieved from the Netherlands Cancer Registry. Trends in European standardised rates (ESRs) were assessed using joinpoint analysis, and expressed as estimated annual percentage change (EAPC). RESULTS: Between 1994 and 2010, 34,156 persons were diagnosed with an in situ or thin melanoma. The ESR of in situ melanomas doubled for males and females with a recent steeper rise in incidence (EAPC 12% (95% confidence interval [CI]: 8.1-16) and 13% (95% CI: 5.9-20), respectively). ESR for thin melanomas amongst males approximately doubled with a steep, but non-significant acceleration compared to other thickness categories since 2006 for <0.25 mm melanomas (EAPC 26% (95% CI: 2.1-35)). For female patients with thin melanomas the ESRs increased almost two-fold, except for <0.25 mm melanomas. CONCLUSIONS: The incidence rates of in situ, thin and thick melanomas increased similarly between 1994 and 2010. Recently steep increases were found for in situ melanomas and thin melanomas in men. Explanations are 'overdiagnosis' in conjunction with increased ultraviolet exposure (natural and artificial) and therefore a 'true' increase, increased awareness, early detection, diagnostic drift and changed market forces in the Dutch health care system.

Gross morphology and histology of the alimentary tract of the convict cichlid Amatitlania nigrofasciata.

The primary objectives of this study were to document the macroscopic and histological structure of the alimentary tract (AT) of the convict cichlid Amatitlania nigrofasciata, because there are no data available for this omnivorous freshwater fish of the family Cichlidae. The morphology of the AT of A. nigrofasciata resembles that of related species. While having morphological criteria of the AT typical of most omnivorous fishes, such as a blind sac stomach and medium length intestine, A. nigrofasciata also has some structural peculiarities: the oesophagus is lined by a uniform stratified squamous epithelial layer with interspersed goblet cells along its entire length. Additionally, it has well-developed layers of the tunica muscularis including muscle fibre bundles that ascend into its mucosal folds. Occasionally, taste buds are present. In the transitional area between oesophagus and stomach, a prominent torus-like closure device is present. The mucosa of the stomach cannot be divided into different regions according to mucosal and morphological properties. The simple pattern of intestinal loops of A. nigrofasciata has few variations, irrespective of sex, mass and length of the individual fish. The first segment of the intestine is characterized by the largest mucososerosal ratio and the most complex mucosal surface architecture. A distinction of midgut and hindgut was not possible in A. nigrofasciata due to lack of defining structural components as described for other fish species.

"Lentiginous melanoma": full-fledged melanoma or melanoma precursor?

On the basis of a critical review of published literature, it is concluded that there is as yet insufficient evidence to conclude that the melanocytic lesion, which is currently known as "lentiginous melanoma," is a full-fledged melanoma, with the capacity to metastasize to distant sites and to cause the demise of the patient. It is proposed that this lesion is better designated as "lentiginous SAMPUS," that is, a superficial atypical melanocytic proliferation of uncertain significance, with a lentiginous, or predominantly lentiginous, arrangement of the junctional component. As there is uncertainty regarding its actual metastatic potential or the likelihood of progression to melanoma NOS, the lesion should be removed completely, with free surgical margins.

Nitric oxide donors augment interleukin-1ß-induced vascular endothelial growth factor in airway smooth muscle cells.

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1ß (IL-1ß) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48 h) PASMC were stimulated with IL-1ß alone or with NO donor, L-arginine and/or NO synthase inhibitor L-NAME for 4 and 24 h. IL-1ß did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24 h. VEGF release was inhibited by L-NAME (531.8 ± 52 pg/ml), but restored and further elevated by L-arginine (1,529 ± 287 pg/ml). IL-1ß up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by L-NAME (1.1-fold) and augmented by L-arginine (3.8-fold) at 4 h. Restoration of a NO pathway by L-arginine in L-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [(3)H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1ß, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.

Angiogenesis and vascular remodeling in chronic airway diseases.

Asthma and chronic obstructive pulmonary disease remain a global health problem, with increasing morbidity and mortality. Despite differences in the causal agents, both diseases exhibit various degrees of inflammatory changes, structural alterations of the airways leading to airflow limitation. The existence of transient disease phenotypes which overlap both diseases and which progressively decline the lung function has complicated the search for an effective therapy. Important characteristics of chronic airway diseases include airway and vascular remodeling, of which the molecular mechanisms are complex and poorly understood. Recently, we and others have shown that airway smooth muscle (ASM) cells are not only structural and contractile components of airways, rather they bear capabilities of producing large number of pro-inflammatory and mitogenic factors. Increase in size and number of blood vessels both inside and outside the smooth muscle layer as well as hyperemia of bronchial vasculature are contributing factors in airway wall remodeling in patients with chronic airway diseases, proposing for the ongoing mechanisms like angiogenesis and vascular dilatation. We believe that vascular changes directly add to the airway narrowing and hyper-responsiveness by exudation and transudation of proinflammatory mediators, cytokines and growth factors; facilitating trafficking of inflammatory cells; causing oedema of the airway wall and promoting ASM accumulation. One of the key regulators of angiogenesis, vascular endothelial growth factor in concerted action with other endothelial mitogens play pivotal role in regulating bronchial angiogenesis. In this review article we address recent advances in pulmonary angiogenesis and remodelling that contribute in the pathogenesis of chronic airway diseases.

DNA microarray and quantitative analysis reveal enhanced myocardial VEGF expression with stunted angiogenesis in human tetralogy of Fallot.

Tetralogy of Fallot (ToF) is a cyanotic congenital heart disease with prominent right ventricular hypertrophy (RVH) associated with impaired myocardial oxygen and nutrient supply. Consequently, the right ventricle may manifest in altered molecular phenotype with a number of adaptive and inherited gene profiles which are largely unknown. The aim of the present study was to investigate the myocardial differential gene expression profile and to assess myocardial vascularisation in patients with ToF. DNA microarray analysis on right ventricular biopsies from ToF-patients operated for primary corrective surgery (referred as ToF-1; n = 12, mean age 0.5 year) and age matched controls (n = 6) was validated by Northern hybridisation and RT-PCR. Employing immunohistochemistry and video image analysis expression of vascular endothelial growth factor (VEGF), vascular density (by α-SMA and CD31 staining) and myocyte cross sectional area (Gomori's reticuline staining) were assessed in ToF-1 and adult patients (referred as ToF-2, n = 12, mean age 30 years) who underwent surgery for pulmonary regurgitation and compared the data with respective age matched controls (n = 6/12). DNA microarray analysis revealed altered expression pattern for 236 genes including enhanced (1.5-2.2-fold) expression of angiogenic factors and their receptors including; VEGF, flt-1, flk-1 angiopoietin-2, FGF-2, FGF-R1, PDGF-A, whereas, flt-4, Tie, TGF-ß, TGF-ß3R showed decreased (1.6-3.4-fold) expression in ToF-patients. Northern blot analysis verified the expression patterns of VEGF and flk-1 in both ToF-1 and ToF-2 patients. VEGF staining in cardiomyocytes was increased in ToF-1 (1.5-fold, p < 0.05) as compared to ToF-2. Video image analysis revealed enhanced vascular density (p < 0.01) with enlarged myocyte cross sectional area (p < 0.01), but vascular wall thickness remained unchanged in ToF-1 patients as compared to age matched controls. Our data suggest that RVH is associated with profound changes in gene profile for a number of genes, where VEGF/VEGF-R system contributes to enhance, but stunted myocardial angiogenesis in patients with ToF.

Surveillance of second-degree relatives from melanoma families with a CDKN2A germline mutation.

BACKGROUND: Lifetime melanoma risk of mutation carriers from families with a germline mutation in the CDKN2A gene is estimated to be 67%. The necessity to include family members in a melanoma surveillance program is widely endorsed, but there is no consensus on which family members should be invited. METHODS: In a retrospective follow-up study, we investigated the yield of surveillance of first- and second-degree relatives of melanoma and pancreatic cancer patients from 21 families with the "p16-Leiden" CDKN2A mutation. Melanoma incidence rates were compared with the general population. RESULTS: Three-hundred and fifty-four first-degree relatives and 391 second-degree relatives were included. Forty-five first-degree relatives and 11 second-degree relatives were diagnosed with melanoma. Most (72%) of second-degree relatives diagnosed with melanoma had become a first-degree relative before diagnosis, due to the occurrence of a melanoma in a parent or sibling. Overall, melanoma incidence rate was 2.1 per 1,000 person years [95% confidence interval (CI), 1.2-3.8] in family members still being second-degree relatives at diagnosis, compared with 9.9 per 1,000 person years (95% CI, 7.4-13.3) in first-degree relatives. The standardized morbidity ratio for melanoma of second-degree relatives compared with the general population was 12.9 (95% CI, 7.2-23.4). CONCLUSION: Second-degree relatives from families with the p16-Leiden mutation in CDKN2A have a considerably increased melanoma risk compared with the general population. IMPACT: This study provides justification for the surveillance of second-degree relatives from families with a CDKN2A germline mutation.

[Revision of the national guideline 'Melanoma']. / Revisie van de landelijke richtlijn 'Melanoom'.

Melanoma is in the top ten of the most common types of cancer in the Netherlands. Incidence is increasing steadily by about 4% every year. The relative 5-year survival rate for patients with a melanoma with Breslow thickness < 1mm is about 98%. The national guideline 'Melanoma version 2.0' is the result of an evidence based revision focussed on the most important bottlenecks encountered in clinical practice. The most important changes concern indications for sentinel node procedures (in patients with tumours stage 1b and higher) and multidisciplinary consultation (patients with stage 3 and 4 tumours). The guideline is intended for all professionals involved in diagnosis, treatment and support of patients with melanoma of the skin.- Guideline summary cards and the 'Melanoma Pathway' ('Zorgpad Melanoom') are available at www.iknl.nl.- An English translation of the quideline will be available in April 2013 at www.oncoline.nl.

Azithromycin attenuates fibroblast growth factors induced vascular endothelial growth factor via p38(MAPK) signaling in human airway smooth muscle cells.

The airways in asthma and COPD are characterized by an increase in airway smooth muscle (ASM) mass and bronchial vascular changes associated with increased expression of pro-angiogenic growth factors, such as fibroblast growth factors (FGF-1 and FGF-2) and vascular endothelial growth factor (VEGF). We investigated the contribution of FGF-1/-2 in VEGF production in ASM cells and assessed the influence of azithromycin and dexamethasone and their underlying signaling mechanisms. Growth-synchronized human ASM cells were pre-treated with MAPK inhibitors, U0126 for ERK1/2(MAPK) and SB239063 for p38(MAPK) as well as with dexamethasone or azithromycin, 30 min before incubation with FGF-1 or FGF-2. Expression of VEGF (VEGF-A, VEGF121, and VEGF165) was assessed by quantitative PCR, VEGF release by ELISA and MAPK phosphorylation by Western blotting. Both FGF-1 and FGF-2 significantly induced mRNA levels of VEGF-A, VEGF121, and VEGF165. The VEGF protein release was increased 1.8-fold (FGF-1) and 5.5-fold (FGF-2) as compared to controls. Rapid transient increase in ERK1/2(MAPK) and p38(MAPK) phosphorylation and subsequent release of VEGF from FGF-1 or FGF-2-treated ASM cells were inhibited by respective blockers. Furthermore, azithromycin and dexamethasone significantly reduced both the VEGF release and the activation of p38(MAPK) pathway in response to FGF-1 or FGF-2 treatment. Our Results demonstrate that FGF-1 and FGF-2 up-regulate VEGF production via ERK1/2(MAPK) and p38(MAPK) pathways. Both azithromycin and dexamethasone elicited their anti-angiogenic effects via p38(MAPK) pathway in vitro, thereby suggesting a possible therapeutic approach to tackle VEGF-mediated vascular remodeling.

Targeting BRAFV600E in an inducible murine model of melanoma.

The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.

Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis.

Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.