Preventing parastomal hernias with systematic intraperitoneal specifically designed mesh.

BACKGROUND: Parastomal hernia is a very common complication after stoma formation. Current surgical techniques for repairing parastomal hernia have unsatisfactory results. We aim to assess our preliminary experience with prophylactic mesh placement at the time of stoma formation. METHODS: Data were prospectively recorded. A specifically designed mesh made of polyvinyl fluoride with central conduit (Dynamesh IPST®) was fixed using an intra-peritoneal onlay technique. Safety was evaluated by means of surgical data and frequency of mesh-related complications, efficacy by the rate of parastomal hernias. RESULTS: Thirty-four patients were included in the study. Three of them died before a year of follow up (not related to the stoma), so they were excluded. The other 31 patients (11 women and 20 men) were prospectively followed up after different pathologies resulting in a permanent colostomy. Twelve months after surgery CT-Scan imaging revealed two (6.4%) parastomal hernias, one of them already clinically suspected. During the follow up, 29% of the patients (n = 9) developed another type of hernia (incisional, inguinal or both). In five patients (16.1%) a light stomal retraction of the otherwise slightly prominent ostomy was observed. Median clinical follow-up was 17.5 months (range 12-34). CONCLUSION: Prophylactic parastomal mesh placement might be a safe and effective procedure with a potential to reduce the risk of parastomal hernia. Routine use of this technique should be further analysed.

The ER Stress Sensor PERK Coordinates ER-Plasma Membrane Contact Site Formation through Interaction with Filamin-A and F-Actin Remodeling.

Loss of ER Ca2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca2+. Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton.

The Oxygen Sensor PHD2 Controls Dendritic Spines and Synapses via Modification of Filamin A.

Neuronal function is highly sensitive to changes in oxygen levels, but how hypoxia affects dendritic spine formation and synaptogenesis is unknown. Here we report that hypoxia, chemical inhibition of the oxygen-sensing prolyl hydroxylase domain proteins (PHDs), and silencing of Phd2 induce immature filopodium-like dendritic protrusions, promote spine regression, reduce synaptic density, and decrease the frequency of spontaneous action potentials independently of HIF signaling. We identified the actin cross-linker filamin A (FLNA) as a target of PHD2 mediating these effects. In normoxia, PHD2 hydroxylates the proline residues P2309 and P2316 in FLNA, leading to von Hippel-Lindau (VHL)-mediated ubiquitination and proteasomal degradation. In hypoxia, PHD2 inactivation rapidly upregulates FLNA protein levels because of blockage of its proteasomal degradation. FLNA upregulation induces more immature spines, whereas Flna silencing rescues the immature spine phenotype induced by PHD2 inhibition.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

VEGF modulates NMDA receptors activity in cerebellar granule cells through Src-family kinases before synapse formation.

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).

VEGF mediates commissural axon chemoattraction through its receptor Flk1.

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.

HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of PlGF.

Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris.

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.

Grb4 and GIT1 transduce ephrinB reverse signals modulating spine morphogenesis and synapse formation.

Dendritic spines are small protrusions emerging from dendrites that receive excitatory input. The process of spine morphogenesis occurs both in the developing brain and during synaptic plasticity. Molecules regulating the cytoskeleton are involved in spine formation and maintenance. Here we show that reverse signaling by the transmembrane ligands for Eph receptors, ephrinBs, is required for correct spine morphogenesis. The molecular mechanism underlying this function of ephrinBs involves the SH2 and SH3 domain-containing adaptor protein Grb4 and the G protein-coupled receptor kinase-interacting protein (GIT) 1. Grb4 binds by its SH2 domain to Tyr392 in the synaptic localization domain of GIT1. Phosphorylation of Tyr392 and the recruitment of GIT1 to synapses are regulated by ephrinB activation. Disruption of this pathway in cultured rat hippocampal neurons impairs spine morphogenesis and synapse formation. We thus show an important role for ephrinB reverse signaling in spine formation and have mapped the downstream pathway involved in this process.

Gold glyconanoparticles as new tools in antiadhesive therapy.

Gold glyconanoparticles (GNPs) have been prepared as new multivalent tools that mimic glycosphingolipids on the cell surface. GNPs are highly soluble under physiological conditions, stable against enzymatic degradation and nontoxic. Thereby GNPs open up a novel promising multivalent platform for biological applications. It has recently been demonstrated that specific tumor-associated carbohydrate antigens (glycosphingolipids and glycoproteins) are involved in the initial step of tumor spreading. A mouse melanoma model was selected to test glyconanoparticles as possible inhibitors of experimental lung metastasis. A carbohydrate-carbohydrate interaction is proposed as the first recognition step for this process. Glyconanoparticles presenting lactose (lacto-GNPs) have been used successfully to significantly reduce the progression of experimental metastasis. This result shows for the first time a clear biological effect of lacto-GNPs, demonstrating the potential application of this glyconanotechnology in biological processes.

Chemical transformations on botryane skeleton. Effect on the cytotoxic activity.

Eighteen compounds with a botryane skeleton have been obtained through chemical transformations of various toxins from the fungus Botrytis cinerea. During the course of these transformations, the C-10 carbon of the botryane skeleton was found to exhibit an interesting high regioselectivity to oxidizing and reducing agents. In addition, the cytotoxicity of 27 botryane derivatives was determined in vitro against Hs578T, MDA-MB-231, HT-1080, U87-MG, IMR-90, and HUVEC cell lines. The results of this study confirm that the cytotoxicity of botrydial (1) and its derivatives is related to the presence of a 1,5-dialdehyde functionality.

Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis.

Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism and mitochondrial cytochrome c release. Vascular-like structure formation was further blocked by caspase inhibitors such as z-VAD or Ac-DEVD-CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti-apoptotic human Bcl-2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel-like structure formation. Caspase inhibitors blocked VEGF- or bFGF-induced HUVEC angiogenesis on 2- or 3-D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF-dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.

Decreased B16F10 melanoma growth and impaired vascularization in telomerase-deficient mice with critically short telomeres.

Endothelial cell function and angiogenesis are modulated by aging. However, the underlying molecular mechanisms are largely unknown. Here we show that in telomerase-deficient mice Terc(-/-), short telomeres result in a sharp decrease in angiogenesis in both Matrigel implants and murine melanoma grafts. In the latter model, decreased microvessel counts in late generation Terc(-/-) mice led to diminished tumor cell proliferation and increased tumor cell apoptosis, resulting in a lower tumor growth rate. Our results indicate that telomere length is a key molecular determinant of angiogenic potential in vivo and that telomere length modifiers and telomerase inhibitors could be useful antiangiogenic agents.