Blockade of VEGF-C signaling inhibits lymphatic malformations driven by oncogenic PIK3CA mutation

Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3CA(H1047R) mutation, resulting in constitutive activation of the p110 alpha PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CA(H1047R)-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110 alpha activation determining the LM subtype. In the postnatal vasculature, PIK3CA(H1047R) promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways. Lymphatic malformation (LM) is a debilitating often incurable vascular disease. Using a mouse model of LM driven by a disease-causative PIK3CA mutation, the authors show that vascular growth is dependent on the upstream lymphangiogenic VEGF-C signalling, permitting effective therapeutic intervention.Peer reviewe

Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

<p>Abstract</p> <p>Background</p> <p>Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD₅₀of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.</p> <p>Results</p> <p>Secretion level of the fusion protein produced <it>in vitro </it>in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced <it>in vitro </it>was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). <it>In vitro </it>nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.</p> <p>Conclusion</p> <p>Both the pharmacokinetic study and the <it>in vitro </it>nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced <it>in vitro </it>and <it>in vivo</it>. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.</p

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Electrodriven separations : possibilities for analysing phenols and polyphenols

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Non-hotspot PIK3CA mutations are more frequent in CLOVES than in common or combined lymphatic malformations.

Theragnostic management, treatment according to precise pathological molecular targets, requests to unravel patients' genotypes. We used targeted next-generation sequencing (NGS) or digital droplet polymerase chain reaction (ddPCR) to screen for somatic PIK3CA mutations on DNA extracted from resected lesional tissue or lymphatic endothelial cells (LECs) isolated from lesions. Our cohort (n = 143) was composed of unrelated patients suffering from a common lymphatic malformation (LM), a combined lymphatic malformation [lymphatico-venous malformation (LVM), capillaro-lymphatic malformation (CLM), capillaro-lymphatico-venous malformation (CLVM)], or a syndrome [CLVM with hypertrophy (Klippel-Trenaunay-Weber syndrome, KTS), congenital lipomatous overgrowth-vascular malformations-epidermal nevi -syndrome (CLOVES), unclassified PIK3CA-related overgrowth syndrome (PROS) or unclassified vascular (lymphatic) anomaly syndrome (UVA)]. We identified a somatic PIK3CA mutation in resected lesions of 108 out of 143 patients (75.5%). The frequency of the variant allele ranged from 0.54 to 25.33% in tissues, and up to 47% in isolated endothelial cells. We detected a statistically significant difference in the distribution of mutations between patients with common and combined LM compared to the syndromes, but not with KTS. Moreover, the variant allele frequency was higher in the syndromes. Most patients with an common or combined lymphatic malformation with or without overgrowth harbour a somatic PIK3CA mutation. However, in about a quarter of patients, no such mutation was detected, suggesting the existence of (an)other cause(s). We detected a hotspot mutation more frequently in common and combined LMs compared to syndromic cases (CLOVES and PROS). Diagnostic genotyping should thus not be limited to PIK3CA hotspot mutations. Moreover, the higher mutant allele frequency in syndromes suggests a wider distribution in patients' tissues, facilitating detection. Clinical trials have demonstrated efficacy of Sirolimus and Alpelisib in treating patients with an LM or PROS. Genotyping might lead to an increase in efficacy, as treatments could be more targeted, and responses could vary depending on presence and type of PIK3CA-mutation

Effect of TNF-α and DRM destabilization on AA release.

<p>Calu-3 cells were incubated overnight with either ³H-labelled AA, treated with 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, with or without preincubation with 15 µM pyrrolidine for 45 min, or with a combination of the different treatments. For combined treatments, TNF-α was added for the last 10 min of incubation. After incubation, supernatants were collected and radioactivity measured by a scintillation counter. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent increment with respect to control. Asterisks denote p<0.05 with respect to control unless indicated otherwise, n = 3.</p

Effect of TNF-α and DRM destabilization on eicosanoid production.

<p>Calu-3 cells were treated with either 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, with or without preincubation with 15 µM pyrrolidine for 45 min, or with a combination of the different treatments. For combined treatments, TNF-α was added for the last 10 min of incubation. After incubation the supernatant was collected, either immediately or after 3 h of incubation in fresh medium, and subjected to ELISA for LTB4 and PGE2 determination. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent of control values. Asterisks denote p<0.05 with respect to control, n≥3.</p

The direct interaction between NBD1 and p11 may connect CFTR to the cPLA2α/ANXA1 complex.

<p>A: SPR association curves of NBD1 and p11 (analyte) at serial concentrations of the latter. B: SPR association of NBD1 with p11 and cPLA2α/ANXA1 complex. p11 (400 µg/ml) and a preincubated cPLA2α/ANXA1 (100 µg/ml each) complex were sequentially co-injected as analytes (cPLA2α/ANXA1 was injected before dissociation of p11). C: Negative SPR association of NBD1 with a preincubated cPLA2α/ANXA1 complex (injected as analyte at 100 µg/ml each). D: Negative SPR association of NBD1 with ANXA1 (injected as analyte at 100 µg/ml).</p

Hypothetical model linking CFTR/DRM interaction with cytokine and eicosanoid release.

<p>TNF-α exerts two effects that seem to be dissociated: eicosanoid release and IL-8 synthesis with the participation of DRM, in which CFTR, cPLA2α, ANXA1, TNFR1 and c-Src are transiently recruited. Both DRM destabilization (mβCD) and CFTR inhibition (Inh172) lead to increased eicosanoid release. However, they counteract in some conditions the other effect of TNF-α -in the case of Inh172, only at long term (12 h).</p