PAKing up to the endothelium

Angiogenesis recapitulates the growth of blood vessels that progressively expand and remodel into a highly organized and stereotyped vascular network. During adulthood, endothelial cells that formed the vascular wall retain their plasticity and can be engaged in neo-vascularization in response to physiological stimuli, such as hypoxia, wound healing and tissue repair, ovarian cycle and pregnancy. In addition, numerous human diseases and pathological conditions are characterized by an excessive, uncontrolled and aberrant angiogenesis. The signalling pathways involving the small Rho GTPase, Rac and its downstream effector the p21-activated serine/threonine kinase (PAK) had recently emerged as pleiotropic modulators in these processes. Indeed, Rac and PAK were found to modulate endothelial cell biology, such as sprouting, migration, polarity, proliferation, lumen formation, and maturation. Elucidating the Rac/PAK molecular circuitry will provide essential information for the development of new therapeutic agents designed to normalize the blood vasculature in human diseases.Comment: Cell Signal (2009) epub ahead of prin

Endothelial Secreted Factors Suppress Mitogen Deprivation-Induced Autophagy and Apoptosis in Glioblastoma Stem-Like Cells

International audienceRapidly growing and highly vascularized tumors, such as glioblastoma multiforme, contain heterogeneous areas within the tumor mass, some of which are inefficiently supplied with nutrients and oxygen. While the cell death rate is elevated in such zones, tumor cells are still suspected to grow and survive independently of extracellular growth factors. In line with this, glioblastoma stem-like cells (GSCs) are found closely associated with brain vasculature in situ, and as such are most likely in a protected microenvironment. However, the behavior of GSCs under deprived conditions has not been explored in detail. Using a panel of 14 patient-derived GSCs, we report that ex vivo mitogen deprivation impaired self-renewal capability, abolished constitutive activation of the mTor pathway, and impinged on GSC survival via the engagement of autophagic and apoptotic cascades. Moreover, pharmacological inhibition of the mTor pathway recapitulated the mitogen deprivation scenario. In contrast, blocking either apoptosis or autophagy, or culturing GSCs with endothelial-secreted factors partly restored mTor pathway activation and rescued GSC survival. Overall, our data suggest that GSCs are addicted to mTor, as their survival and self-renewal are profoundly dependent on this signaling axis. Thus, as mTor governs the fate of GSCs under both deprivation conditions and in the presence of endothelial factors, it could be a key target for therapeutic purposes

Evaluation of transcriptionally regulated genes identifies NCOR1 in hormone receptor negative breast tumors and lung adenocarcinomas as a potential tumor suppressor gene

Regulation of transcription is a key process in cellular homeostasis. It depends on regulators that either repress or stimulate the transcription of genes, therefore controlling different biological functions. The Nuclear Receptor Corepressor 1 (NCOR1) is one of those co-repressors that regulate the transcription by facilitating the recruitment of HDAC1, 2, 3, 4, 5 and 7. In our article, by using an in silico approach, we evaluate the mutational status of NCOR1 in breast and lung tumors. We identified that NORC1 is mutated in more than 3% of breast tumors and lung adenocarcinomas and linked this fact with detrimental outcome in some subtypes, particularly in those that are hormone receptor negative. In addition to these findings, as mutations in this gene are deleterious, we confirmed that high levels of this gene were linked with good prognosis in the same tumor subtypes. Findings in the same direction were identified in lung adenocarcinomas, with mutations associated with detrimental prognosis and high expression with better outcome. In conclusion, hereby we describe the presence and prognostic role of mutations in the NCOR1 gene in hormone receptor negative breast and lung adenocarcinomas, and we also confirm that NCOR1 is a tumor suppressor gene. Further studies should be performed to explore therapeutic mechanisms to restore its function

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis

Semaphorin 3A elevates endothelial cell permeability through PP2A inactivation

International audienceVE-cadherin-mediated cell-cell junction weakening increases paracellular permeability in response to both angiogenic and inflammatory stimuli. Although Semaphorin 3A has emerged as one of the few known anti-angiogenic factors to exhibit pro-permeability activity, little is known about how it triggers vascular leakage. Here we report that Semaphorin 3A induced VE-cadherin serine phosphorylation and internalisation, cell-cell junction destabilisation, and loss of barrier integrity in brain endothelial cells. In addition, high-grade glioma-isolated tumour-initiating cells were found to secrete Semaphorin 3A, which promoted brain endothelial monolayer permeability. From a mechanistic standpoint, Semaphorin 3A impinged upon the basal activity of the serine phosphatase PP2A and disrupted PP2A interaction with VE-cadherin, leading to cell-cell junction disorganization and increased permeability. Accordingly, both pharmacological inhibition and siRNA-based knockdown of PP2A mimicked Semaphorin 3A effects on VE-cadherin. Hence, local Semaphorin 3A production impacts on the PP2A/VE-cadherin equilibrium and contributes to elevated vascular permeability

Mitogen deprivation triggers both autophagy and apoptosis.

<p>a-e) GSCs were deprived from mitogens for 1 to 3 days. GSCs growing in mitogen-supplemented medium were use as a control (C). a) Electron microscopy analysis of mitogen-starved GSC#1. <i>Left panels</i>. Cells presented morphological signs of apoptosis, including cytoplasmic blebbing, nuclear fragmentation and chromatin condensation. Scale bars: 2 μm. <i>Right panels</i>. Different stages of autophagy can be seen, such as pre-autophagosome and early autophagosome (upper panels) and late autophagosomes (autolysosomes/amphisomes, lower panels). Scale bars: 50 nm. b) Western blot analysis was conducted using the indicated antibodies in GSC#1. ** non-processed form; * processed form. c-e) LC3B puncta, Atg12 aggregates and mitochondrial cytochrome c release were analyzed by confocal microscopy. Scale bars: 5 μm (c,d) and 2.5 μm (e). f) GSC#1 were pre-treated with vehicle (DMSO) or QVD (10 μM) for 45 min and mitogen-deprived for 3 days in the absence or presence of the drug. Flow cytometry analysis (10.000 events) of Annexin V-FITC/PI staining was used to measure cell death in GSC#1. Number of cells either viable (Annexin V/PI –/–) or in early (Annexin V/PI +/–), late apoptosis (Annexin V/PI +/+) and necrotic (Annexin V/PI –/+) phases was expressed as percentage of total population. g) GSC#1 were transfected with siRNA against Beclin-1 or a control siRNA (siC) for 48 hours. Cells were mitogen-deprived as indicated and collected 5 days post-transfection. Beclin-1 protein levels were analyzed by western-blot and cell death was examined as in f). h) GSC#1 were treated with vehicle (DMSO) or Chloroquine (CQ) (25 μM, XX min) in mitogen-supplemented medium (C) and in mitogen-deprived conditions for 3 days (3d). Flow cytometry analysis (10.000 events) of Annexin V-FITC/PI staining was used to measure cell death in GSC#1 as in f). Graph represents mean+s.d. of three independent experiments. Each panel is representative of three independent experiments.</p

Mitogen deprivation reduced glioblastoma stem-like cell survival.

<p>a-h) GSCs were deprived from mitogens for 1 to 3 days. GSCs growing in mitogen-supplemented medium were used as a control (C). a) The number of secondary neurospheres per field of view (NS/FOV) was counted after 3 days of deprivation in GSCs #1-4. b) Sox2 levels (green) were analyzed by confocal in GSCs #1-4. Nuclei were counterstained with DAPI (blue). Scale bar: 15 μm. c-d) Expression of Sox2 and Nestin as well as GADPH as a control, was evaluated by RT-PCR, while GFAP expression was analyzed by confocal analysis. Differentiation of GSC#4 was induced as described in methods. e) PI incorporation was measured by flow cytometry (10.000 events) in GSCs #1–14 and the percentage of cell death was estimated. Graph represents mean+s.d. of three independent experiments. Student’s t-test: ^***<i>P</i><0.001, ^**<i>P</i><0.01. f) Flow cytometry analysis (10.000 events) of Annexin V-FITC/Propidium Iodide (PI) staining was used to measure cell death in GSC#1. Number of cells either viable (Annexin V/PI –/–) or in early (Annexin V/PI +/–), late apoptosis (Annexin V/PI +/+) and necrotic (Annexin V/PI –/+) phases was expressed as percentage of total population. g) DNA profile was analyzed by flow cytometry with PI staining in GSC#1. Percentage of apoptotic cells was calculated based on sub-G₁ peak. Student’s t-test: ^***<i>P</i><0.001. h) Western blot analysis was conducted using the indicated antibodies in GSC#1. Each panel is representative of three independent experiments.</p

mTOR inhibition provokes autophagy and apoptosis in GSC.

<p>a-c) GSC#1 were treated with DMSO (–), LY294002 (LY, 10 μM), Rapamycin (RP, 50nM), PP242 (PP, 1 μM) and PI103 (PI, 10 μM). a) Protein lysates were analyzed after 1 day by western blot for the indicated antibodies. b) LC3B puncta were examined under confocal microscope after 3 days. Scale bars: 5 μm. c) Flow cytometry analysis of Annexin V-FITC/PI staining was performed as described in 2f). d-g) GSC#1 were pre-treated with vehicle (DMSO) or QVD (10 μM) for 45 min and mitogen-deprived for 3 days in the absence or presence of the drug (d-e). Alternatively, GSC#1 were transfected with siRNA against Beclin-1 or a control siRNA (siC) for 48 hours. Cells were mitogen-deprived as indicated and collected 5 days post-transfection (f-g). Protein extracts were analyzed by western blot for the indicated antibodies (d, f). ** non-processed form; * processed form. LC3B staining was examined by confocal microscopy (e, g). Scale bars: 5 μm. Graph represents mean+s.d. of three independent experiments. Each panel is representative of three independent experiments.</p