Biomedical subjects

Antonello Villa

Publications and source records attributed to Antonello Villa.

14 recordsLinked to original sources

PubMed2006

Spatial and temporal expression of POF1B, a gene expressed in epithelia.

Mammalian epithelia possess specialized cellular components that provide an impermeable barrier between two different environments. In particular, in the skin, mitotically dividing cells undergo a programmed set of morphological and biochemical changes leading to the establishment of the epidermal permeability barrier (EPB) to prevent escape of moisture and entrance of toxic molecules. Many different skin proteins are involved in the process but not all have been identified. We report here the results of the expression studies of a novel gene, highly and specifically expressed in the granular layer of the epidermis and in the epithelia of the oro-pharyngeal and gastro-intestinal tracts. Our data show that during mouse development Pof1b expression is activated in the external layers of the epidermis just prior to formation of the EPB.

Animals↗

PubMed2006

Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes.

Human tumors constitutively release endosome-derived microvesicles, transporting a broad array of biologically active molecules with potential modulatory effects on different immune cells. Here, we report the first evidence that tumor-released microvesicles alter myeloid cell function by impairing monocyte differentiation into dendritic cells and promoting the generation of a myeloid immunosuppressive cell subset. CD14+ monocytes isolated from healthy donors and differentiated with interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in the presence of tumor-derived microvesicles turned into HLA-DR(-/low) cells, retaining CD14 expression and failing to up-regulate costimulatory molecules, such as CD80 and CD86. These phenotypic changes were paralleled by a significant release of different cytokines, including IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and a dose-dependent suppressive activity on activated T-cell-proliferation and cytolytic functions, which could be reversed by anti-TGF-beta-neutralizing antibodies. Microvesicles isolated from plasma of advanced melanoma patients, but not from healthy donors, mediated comparable effects on CD14+ monocytes, skewing their differentiation toward CD14+HLA-DR-/low cells with TGF-beta-mediated suppressive activity on T-cell-functions. Interestingly, a subset of TGF-beta-secreting CD14+HLA-DR- cells mediating suppressive activity on T lymphocytes was found to be significantly expanded in peripheral blood of melanoma patients compared with healthy donors. These data suggest the development in cancer patients of an immunosuppressive circuit by which tumors promote the generation of suppressive myeloid cells through the release of circulating microvesicles and without the need for cell-to-cell contact. Therapeutic interventions on the crucial steps of this pathway may contribute to restore tumor/immune system interactions favoring T-cell-mediated control of tumor growth in cancer patients.

Apoptosis↗

PubMed2006

Glomerular podocytes contain neuron-like functional synaptic vesicles.

Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.

Animals↗

PubMed2006

Differential availability/processing of decorin precursor in arterial and venous smooth muscle cells.

The existence of specific differentiation markers for arterial smooth muscle (SM) cells is still a matter of debate. A clone named MM1 was isolated from a library of monoclonal antibodies to adult porcine aorta, which in vivo binds to arterial but not venous SM cells, except for the pulmonary vein. MM1 immunoreactivity in Western blotting involved bands in the range of M(r) 33-226 kDa, in both arterial and venous SM tissues. However, immunoprecipitation experiments revealed that MM1 bound to a 100-kDa polypeptide that was present only in the arterial SM extract. By mass spectrometry analysis of tryptic digests from MM1-positive 130- and 120-kDa polypeptides of aorta SM extract, the antigen recognized by the antibody was identified as a decorin precursor. Using a crude decorin preparation from this tissue MM1 reacted strongly with the 33-kDa polypeptide and this pattern did not change after chondroitinase ABC treatment. In vitro, decorin immunoreactivity was found in secreted grainy material produced by confluent arterial SM cells, although lesser amounts were also seen in venous SM cells. Western blotting of extracts from these cultures showed the presence of the 33-kDa band but not of the high-molecular-weight components, except for the 100-kDa monomer. The 100/33-kDa combination was more abundant in arterial SM cells than in the venous counterpart. In the early phase of neointima formation, induced by endothelial injury of the carotid artery or vein-to-artery transposition, the decorin precursor was not expressed, but it was up-regulated in the SM cells of the media underlying the neointima in both models. Collectively, these data suggest a different processing/utilization of the 100-kDa monomer of proteoglycan decorin in arterial and venous SM cells, which is abolished after vein injury.

Animals↗

PubMed2005

TEL/ARG induces cytoskeletal abnormalities in 293T cells.

We previously identified TEL/ARG as a novel fusion transcript consisting of the oligomerization domain of TEL and the kinase domain of ARG, in a case of acute myeloid leukemia. We report here the existence of an alternatively spliced TEL/ARG transcript lacking part of a F-actin binding domain of ARG, and the phenotype of TEL/ARG expressing 293T cells. In 293T cells, both TEL/ARG forms co-localized with the cellular beta-actin and were associated with a morphologic change of the cells, consisting in cell rounding and detachment from the tissue culture plastic. We identified the Rho inhibitor p190RhoGAP, a critical regulator of cellular adhesion, as a target of the aberrant kinase.

Alternative Splicing↗

PubMed2005

Junctional adhesion molecule-A-deficient polymorphonuclear cells show reduced diapedesis in peritonitis and heart ischemia-reperfusion injury.

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. Here we report that JAM-A is required for the correct infiltration of polymorphonuclear leukocytes (PMN) into an inflamed peritoneum or in the heart upon ischemia-reperfusion injury. The defect was not observed in mice with an endothelium-restricted deficiency of the protein but was still detectable in mice transplanted with bone marrow from JAM-A(-/-) donors. Microscopic examination of mesenteric and heart microvasculature of JAM-A(-/-) mice showed high numbers of PMN adherent on the endothelium or entrapped between endothelial cells and the basement membrane. In vitro, in the absence of JAM-A, PMN adhered more efficiently to endothelial cells and basement membrane proteins, and their polarized movement was strongly reduced. This paper describes a nonredundant role of JAM-A in controlling PMN diapedesis through the vessel wall.

Animals↗

PubMed2005

VE-cadherin is a critical molecule for trophoblast-endothelial cell interaction in decidual spiral arteries.

Fetal cytotrophoblasts colonize the decidual spiral arteries during pregnancy and partially replace the endothelium by an as yet unknown mechanism. To clarify this issue, we cocultured trophoblast cells (TCs) and decidual endothelial cells (DECs) isolated from first trimester placentae and found by electron microscopic analysis that TCs adhered to DECs and migrated through the interendothelial junctions within 24 h. Since extravillous TCs were shown by FACS analysis to express vascular-endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM)-1, we investigated the role of these junctional molecules in TC adhesion to DECs and transendothelial migration of cytotrophoblasts. Both VE-cadherin and PECAM-1 were present at the contact sites between TCs and DECs in decidual sections. TC adhesion and migration were markedly inhibited by mAbs to VE-cadherin and marginally by mAb to PECAM-1. Increased expression of VE-cadherin was observed at the contact areas between TCs and DECs, whereas PECAM-1 was found to be redistributed from intercellular junctions. The induction of apoptosis of DECs by TCs, as the mechanism responsible for their replacement, was ruled out by the negative staining with TUNEL of DECs cocultured with TCs and the absence of DNA fragmentation. In conclusion, VE-cadherin is involved in transendothelial migration of TCs, and replacement of DECs by TCs is not the result of apoptosis.

Antigens, CD↗

PubMed2005

Low-dose cisplatin protects human neuroblastoma SH-SY5Y cells from paclitaxel-induced apoptosis.

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin alpha, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis.

Antineoplastic Agents↗

PubMed2004

Chromogranin A protects vessels against tumor necrosis factor alpha-induced vascular leakage.

Elevated levels of circulating chromogranin A (CgA), a protein stored in the secretory granules of many neuroendocrine cells and neurons, have been detected in the blood of patients with neuroendocrine tumors or heart failure. The pathophysiological role of increased secretion of CgA is unknown. Using mice bearing subcutaneous tumors genetically engineered to secrete CgA in circulation, we have found that increased blood levels of this protein prevent vascular leakage induced by tumor necrosis factor-alpha (TNF) in the liver venous system. Structure-activity studies, carried out with CgA fragments administered to normal mice, showed that an active site is located within residues 7-57 of CgA. Accordingly, an anti-CgA antibody directed to residues 53-57 inhibited the effect of circulating CgA, either endogenously produced or exogenously administered, on liver vessels. Studies of the mechanism of action showed that CgA inhibits TNF-induced VE-cadherin down-regulation and barrier alteration of cultured endothelial cells, in an indirect manner. Other effectors, such as thrombin and vascular endothelial growth factor were partially inhibited by CgA N-terminal fragments in in vitro permeability assays. These findings suggest that circulating CgA could help regulate the endothelial barrier function and to protect vessels against TNF-induced plasma leakage in pathological conditions characterized by increased production of TNF and CgA, such as cancer or heart failure.

Animals↗

PubMed2003

Expression of laminin receptors in schwann cell differentiation: evidence for distinct roles.

Schwann cells require laminin-2 throughout nerve development, because mutations in the alpha2 chain in dystrophic mice interfere with sorting of axons before birth and formation of myelin internodes after birth. Mature Schwann cells express several laminin receptors, but their expression and roles in development are poorly understood. Therefore, we correlated the onset of myelination in nerve and synchronized myelinating cultures to the appearance of integrins and dystroglycan in Schwann cells. Only alpha6beta1 integrin is expressed before birth, whereas dystroglycan and alpha6beta4 integrin appear perinatally, just before myelination. Although dystroglycan is immediately polarized to the outer surface of Schwann cells, alpha6beta4 appears polarized only after myelination. We showed previously that Schwann cells lacking beta1 integrin do not relate properly to axons before birth. Here we show that the absence of beta1 before birth is not compensated by other laminin receptors, whereas coexpression of both dystroglycan and beta4 integrin is likely required for beta1-null Schwann cells to myelinate after birth. Finally, both beta1-null and dystrophic nerves contain bundles of unsorted axons, but they are predominant in different regions: in spinal roots in dystrophic mice and in nerves in beta1-null mice. We show that differential compensation by laminin-1, but not laminin receptors may partially explain this. These data suggest that the action of laminin is mediated by beta1 integrins during axonal sorting and by dystroglycan, alpha6beta1, and alpha6beta4 integrins during myelination.

Animals↗

PubMed2003

IL-1 beta scavenging by the type II IL-1 decoy receptor in human neutrophils.

IL-1 elicits its cellular effects by binding a heterodimeric receptor consisting of IL-1RI and the accessory protein, IL-1RAcPr. In addition, it binds to IL-1RII, which lacking signaling function has been ascribed a decoy role. The fate of the ligand following interaction with the decoy receptor was examined in human polymorphonuclear cells (PMN), which express predominantly (>90%) IL-1RII. Incubation of PMN with IL-1beta results in a rapid decrease in cell surface-associated ligand accompanied by a concomitant increase in internalized IL-1 with 50-60% of IL-1beta located intracellularly within 1 h at 37 degrees C. The use of blocking Abs revealed that IL-1 internalization is mediated exclusively by the decoy receptor. The results of inhibitor analysis demonstrate that internalization requires ATP synthesis and involves clathrin-mediated endocytosis. Following removal of the ligand, the receptor was rapidly re-expressed on the cell surface. Cyclohexamide, a protein synthesis inhibitor, had no effect upon the process, suggesting that the re-expressed receptor was recycled. In addition, human keratinocytes stably transfected with IL-1RII (HaCAT 811) also internalized the IL-1RII with 43% cell surface receptor internalized after 90 min. Immunofluorescence microscopy revealed colocalization of the internalized receptor with wheat germ agglutinin-labeled internalized glycoproteins and early endosome Ag-1, a protein associated with the early endosome compartments, indicative of cellular uptake of IL-1RII by endocytosis. In contrast, little or no internalization was observed in other cells of immune origin. These results suggest that the decoy receptor IL-1RII can act as a scavenger of IL-1, representing a novel autoregulatory mechanism of the IL-1 system.

Arsenicals↗

PubMed2003

Targeting of alpha-kinase-anchoring protein (alpha KAP) to sarcoplasmic reticulum and nuclei of skeletal muscle.

The sarcoplasmic reticulum (SR) plays a key role in excitation/contraction coupling of skeletal muscle. The SR is composed of two continuous yet heterogeneous membrane compartments, the free or longitudinal SR and cisternal SR. Cisternal SR is made up of free SR membrane, enriched in Ca(2+) pumps, and junctional SR (jSR) membrane, enriched in ryanodine-sensitive Ca(2+)-release channels, and contains calsequestrin within its lumen. Protein phosphorylation mediated by the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) has significant, distinct regulatory roles in both Ca(2+) uptake and Ca(2+) release. Kinase-anchoring proteins (KAPs) constitute a novel mechanism for achieving cell compartmentalization of effectors in phosphorylation pathways. Here, targeting of alpha KAP, a CaM kinase II-anchoring protein encoded within the alpha-CaM kinase II gene, was studied in transgenic skeletal muscle fibres of the adult rat soleus. The transgenes were epitope-tagged versions of alpha KAP and of a deletion mutant, allowing their specific immunodetection against the wild-type background. Our results show that alpha KAP is largely localized at the free SR and thus near the Ca(2+) pump, a protein that can be modulated by CaM kinase II phosphorylation. Only minor co-localization was observed with the jSR ryanodine-sensitive Ca(2+)-release channel, which is a potential CaM kinase II target. In non-muscle cells, recombinant alpha KAP is targeted to endoplasmic reticulum (ER). Both ER and SR targeting requires the N-terminal hydrophobic region of alpha KAP. An unexpected additional specific localization that does not require the N-terminus was found in the nucleus, providing a first clue of how CaM kinase II can fulfil its nuclear functions in skeletal muscle.

Amino Acid Sequence↗

PubMed2002

Multimodal quantal release at individual hippocampal synapses: evidence for no lateral inhibition.

Most CNS synapses investigated thus far contain a large number of vesicles docked at the active zone, possibly forming individual release sites. At the present time, it is unclear whether these vesicles can be discharged independently of one another. To investigate this problem, we recorded miniature excitatory currents by whole-cell and single-synapse recordings from CA3-CA1 hippocampal neurons and analyzed their stochastic properties. In addition, spontaneous release was investigated by ultrastructural analysis of quickly frozen synapses, revealing vesicle intermediates in docking and spontaneous fusion states. In these experiments, no signs of inhibitory interactions between quanta could be detected up to 1 msec from the previous discharge. This suggests that exocytosis at one site does not per se inhibit vesicular fusion at neighboring sites. At longer intervals, the output of quanta diverged from a random memoryless Poisson process because of the presence of a bursting component. The latter, which could not be accounted for by random coincidences, was independent of Ca2+ elevations in the cytosol, whether from Ca2+ flux through the plasma membrane or release from internal stores. Results of these experiments, together with the observation of spontaneous pairs of omega profiles at the active zone, suggest that multimodal release is produced by an enduring activation of an integrated cluster of release sites.

Animals↗

PubMed2002

Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts.

Integrins are membrane receptors that mediate interactions between cells and the extracellular matrix. We recently showed that the osteoclast integrin alpha(v)beta(3) exists in two different conformations, so-called 'basal' and 'activated', with each exhibiting a distinct function. In this study we demonstrate that, in non-resorbing osteoclasts, the 'activated' form of alpha(v)beta(3) accumulates in the motile areas of the plasma membrane. During bone resorption this conformation is prevalent in the ruffled membrane, whereas the 'basal' form of alpha(v)beta(3) is also present in the sealing zone. Moreover, hepatocyte growth factor (HGF) and macrophage colony stimulating factor (M-CSF), two molecules involved in osteoclastogenesis and osteoclast survival, modulate alpha(v)beta(3) conformation in vitro. Preincubation with HGF or M-CSF induces a shift of conformation of alpha(v)beta(3) in primary human osteoclasts (OCs) and in the osteoclast-like cell line (GCT 23). Activated integrin promotes osteoclast migration to the alpha(v)beta(3) ligand osteopontin and enhances bone resorption. Thus, HGF and M-CSF modulate the alpha(v)beta(3) conformational states required for osteoclast polarization and resorption. The capacity of growth factors to alter the affinity of alpha(v)beta(3) toward its ligands offers a potential explanation for the diverse responses of osteoclasts to the same ligand.

Antibodies, Monoclonal↗