cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior

Electroformation in a flow chamber with solution exchange as a means of preparation of flaccid giant vesicles

A recently described technique (Estes and Mayer, Biochim. Biophys. Acta 1712 (2005) 152--160) for the preparation of giant unilamellar vesicles (GUVs) in solutions with high ionic strength is examined. By observing a series of osmotic swellings followed by vesicle bursts upon a micropipette transfer of a single POPC GUV from a sucrose solution into an isoosmolar glycerol solution, a value for the permeability of POPC membrane for glycerol, P = (2.09+/-0.82) x 10^{-8} m/s, has been obtained. Based on this result, an alternative mechanism is proposed for the observed exchange of vesicle interior. With modifications, the method of Estes and Mayer is then applied to preparation of flaccid GUVs.Comment: 13 pages, 10 figures, accepted for publication in Colloids and Surfaces B: Biointerface

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke

AIMS: Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. METHODS AND RESULTS: Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. CONCLUSION: These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke

Acid geopolymer materials based on different aluminosilicate sources

Geopolymers synthetized from the reaction of metakaolin (or alumino-silicate) in an alkali medium are studied since decades [1]. However, in order to develop composites, alkali medium is undesirable due to the detrimental effect of alkali ions on fibers. As an alternative, the use of an acid medium seems promising as some authors demonstrated an increase of mechanical properties with acid-based geopolymers [2, 3]. The aim of this work is to investigate the various formulations and to understand the reactional mechanisms. The work is based on the activation of an alumino-silicate source with phosphoric acid to obtain geopolymer The various formulations consist to determine the Al/P ratio leading to consolidation at various temperatures (from 20 to 70°C). The samples were characterized by Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopies and X-Ray Diffraction measurements in order to study their structure. The thermal and mechanical properties were correlated with the microstructure [4]. Consolidated geopolymers were synthetized with different Al/P ratios. The consolidation time decreases with increasing Al/P ratio. For instance, the material realized with Si/Al=1,17 consolidates at 20°C in 15 and 8 days with Al/P=4 and 1, respectively. Tow behaviors can be distinguished samples presenting good fire resistance (Si/Al=1,17 and Al/P=1) or presenting poorly water resistant (Si/Al=1,17 and Al/P=4). Consequently, the impact of the reactivity of the alumino-silicate source on the geopolymerisation kinetics leads to different types of structures (secondary and metastable phases) in relation with the phosphor content. References: [1] J.Davidovits, J. Therm. Anal. 37 (1991) 1633-1656. [2] D. S. Perrera, J Mater Sci, vol. 3, pp. 6562-6566, 2008. [3] H. K. Tchakouté and C. Rüscher, Applied Clay Science, vol. 140, pp. 81-87,2017. [4] H.Celerier and al., under submission

Synchronization of endothelial Dll4-Notch dynamics switch blood vessels from branching to expansion

Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning

Opposite macrophage polarization in different subsets of ovarian cancer: observation from a pilot study

The role of the innate immune system in ovarian cancer is gaining importance. The relevance of tumor-associated macrophages (TAM) is insufficiently understood. In this pilot project, comprising the immunofluorescent staining of 30 biopsies taken from 24 patients with ovarian cancer, we evaluated the presence of total TAM (cluster of differentiation (CD) 68 expression), M1 (major histocompatibility complex (MHC) II expression), and M2 (anti-mannose receptor C type 1 (MRC1) expression), and the blood vessel diameter. We observed a high M1/M2 ratio in low-grade ovarian cancer compared to high-grade tumors, more total TAM and M2 in metastatic biopsies, and a further increase in total TAM and M2 at interval debulking, without beneficial effects of bevacizumab. The blood vessel diameter was indicative for M2 tumor infiltration (Spearman correlation coefficient of 0.65). These data mainly reveal an immune beneficial environment in low-grade ovarian cancer in contrast to high-grade serous ovarian cancer, where immune suppression is not altered by neoadjuvant therapy

Long-lived tumor-associated macrophages in glioma

BACKGROUND: The tumor microenvironment (TME) plays a major tumor-supportive role in glioma. In particular, tumor-associated macrophages (TAMs), which can make up to one third of the tumor mass, actively support tumor growth, invasion and angiogenesis. Predominantly alternatively activated (M2-polarized) TAMs are found in late stage glioma in both human and mouse tumors, as well as in relapse samples from patients. However, whether tumor-educated M2 TAMs can actively contribute to the emergence and growth of relapse is currently debated. METHODS: To investigate whether tumor-educated stromal cells remaining in the brain after surgical removal of the primary tumor can be long-lived and retain their tumor-supporting function, we developed a transplantation mouse model and performed lineage-tracing. RESULTS: We discovered that macrophages can survive transplantation and stay present in the tumor much longer than previously suggested, while sustaining an M2 polarized pro-tumorigenic phenotype. Transplanted tumors showed a more aggressive growth and faster polarization of the TAMs toward an M2 phenotype compared to primary tumors, a process dependent on the presence of few co-transplanted macrophages. CONCLUSIONS: Overall, we propose a new way for tumor-educated TAMs to contribute to glioma aggressiveness by long survival and stable pro-tumorigenic features. These properties could have a relapse-supporting effect