Biomedical subjects

Luca Munaron

Publications and source records attributed to Luca Munaron.

10 recordsLinked to original sources

PubMed2006

Cytosolic calcium microdomains by arachidonic acid and nitric oxide in endothelial cells.

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plasmamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors.

Animals↗

PubMed2006

Interaction between TRPC channel subunits in endothelial cells.

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.

Animals↗

PubMed2006

Oxytocin induces proliferation and migration in immortalized human dermal microvascular endothelial cells and human breast tumor-derived endothelial cells.

Oxytocin either increases or inhibits cell growth in different cell subtypes. We tested here the effect of oxytocin on cell proliferation and migration of human dermal microvascular endothelial cells (HMEC) and tumor-associated endothelial cells purified from human breast carcinomas (B-TEC). Oxytocin receptors were expressed in both cell subtypes at mRNA and protein levels. Through oxytocin receptor, oxytocin (1 nmol/L-1 mumol/L) significantly increased cell proliferation and migration in both HMEC and B-TEC, and addition of a selective oxytocin antagonist fully reverted these effects. To verify whether a different expression of adhesion molecule-related genes could be responsible for the oxytocin-induced cell migration, untreated and treated cells were compared applying a microarray technique. In HMEC, oxytocin induced the overexpression of the matrix metalloproteinase (MMP)-17, cathepsin D, and integrin beta(6) genes. In B-TEC, oxytocin significantly switched on the gene profile of some MMP (MMP-11 and MMP-26) and of integrin beta(6). The up-regulation of the integrin beta(6) gene could be involved in the oxytocin-induced cell growth, because this subunit is known to determine activation of mitogen-activated protein kinase-extracellular signal-regulated kinase 2, which is involved in the oxytocin mitogenic effect. In B-TEC, oxytocin also increased the expression of caveolin-1 at gene and protein levels. Because oxytocin receptor localization within caveolin-1-enriched membrane domains is necessary for activation of the proliferative (instead of the inhibitory) response to oxytocin, its enhanced expression can be involved in the oxytocin-induced B-TEC growth as well. Altogether, these data indicate that oxytocin contributes to cell motility and growth in HMEC and B-TEC.

Breast Neoplasms↗

PubMed2005

Regulation of noncapacitative calcium entry by arachidonic acid and nitric oxide in endothelial cells.

Several peptides, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), activate the release of arachidonic acid (AA) and nitric oxide (NO) in endothelial cells (ECs). Both messengers are involved in EC proliferation and vascular permeability and control calcium homeostasis in different ways. Interestingly, it has been recently suggested that NO acts as a downstream mediator of AA-induced calcium entry in smooth muscle cells and isolated mouse parotid cells. In this paper, we have investigated the complex relationships that link intracellular calcium, AA, and NO in cultured endothelial cells. Using different experimental approaches, mainly simultaneous Ca2+ and NO fluorimetric confocal imaging, we provide evidence for a complex pathway leading to noncapacitative calcium entry (NCCE) in bovine aortic endothelial cells (BAECs). In particular, AA is able to induce NCCE through two different pathways: one dependent on eNOS recruitment and NO release, the other NO-independent. Finally, we show that NO increase is involved in the control of BAEC proliferation.

Analysis of Variance↗

PubMed2005

Calcium signals activated by arachidonic acid in embryonic chick ciliary ganglion neurons.

Arachidonic acid (AA, 20:4) has been reported to modulate a variety of calcium-permeable ionic channels, both in the plasma membrane and in the endoplasmic reticulum. We have studied the effects of AA on calcium signaling in a well-characterized model of developing peripheral neurons, embryonic chick ciliary ganglion neurons in culture. When given at low non-micellar concentrations (5 microM), in the majority of cells AA directly activated a delayed and long-lasting increase in [Ca2+]i, involving both the cytoplasm and the nucleoplasm, that was completely reversed by abolition of extracellular calcium. Other fatty acids (FAs), either saturated like arachidic acid (20:0), or unsaturated like linoleic (18:2) and docosahexaenoic acid (22:6), shared its ability to activate calcium influx. This entry was not suppressed by voltage-dependent calcium channel inhibitors omega-conotoxin and nifedipine, by the voltage-independent calcium channel antagonist LOE-908, by pre-treatment with blockers of AA metabolic pathways or with pertussis toxin. The arachidonate-activated calcium pathway was permeable to Mn2+ and blocked by La3+, Gd3+ and Ni2+. In a neuronal subpopulation, AA at the same concentration was also able to elicit calcium release from thapsigargin-sensitive intracellular stores; we provide evidence that cytochrome P450 epoxygenase is involved in this process.

Animals↗

PubMed2004

Blocking Ca2+entry: a way to control cell proliferation.

Ca(2+) signalling is involved in virtually all cellular processes: among the others, it controls cell survival, proliferation and death regulating a plethora of intracellular enzymes located in the cytoplasm, nucleus and organelles. Changes in the cytosolic free Ca(2+) concentration may be due either to release from the intracellular Ca(2+) stores or to influx from the extracellular medium, through the opening of plasma membrane calcium-permeable channels. In particular, Ca(2+) entry from the extracellular space is a mechanism able to sustain long lasting intracellular Ca(2+) elevations: this signal, activated by many growth factors and mitogens in normal and tumoral tissues, is linked to DNA transcription and duplication, finally leading to cell proliferation. In the last years many informations have been provided about the transduction mechanisms related to Ca(2+) entry induced by mitogenic factors, mostly binding to tyrosine kinase receptors, but also to G-protein coupled ones. Nevertheless, some key points remain to be fully clarified: among them, the molecular structure of the Ca(2+) channels involved, their regulation by intracellular messengers, and the modes through which specificity is achieved. The increasing knowledge on Ca(2+) entry-dependent control of proliferation may provide a more satisfactory understanding of pathological alterations, including cancer progression and angiogenesis. A detailed description of the mechanisms that trigger Ca(2+) entry, and in particular the definition of calcium-permeable channels and their modulators at the molecular levels, will greatly improve our possibility to take advantage of Ca(2+) entry regulation as a therapeutic approach for the control of cell proliferation, designing antibodies or molecules with low side effects and specific channel blocker functions. The review will focus on this topic.

Animals↗

PubMed2003

Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach.

In physiological conditions, endothelial cell proliferation is strictly controlled by several growth factors, among which bFGF and VEGF are the most effective. Both bind to specific tyrosine kinase receptors and trigger intracellular signal cascades. In particular, bFGF stimulates the release of arachidonic acid (AA) and its metabolites in many types of endothelial cells in culture. In bovine aortic endothelial cells, it has been suggested that AA is released by the recruitment of cytosolic phospholipase A2 (cPLA2). AA metabolites are involved in the control of both endothelial cell motility (mostly via the cyclooxygenase pathway) and proliferation (via the lipoxygenase (LOX) cascade). On the other hand, evidence has been provided for a proliferative role of AA-induced calcium influx. By using a pharmacological approach, we have tried to elucidate the contribution to bovine aortic endothelial proliferation of the different pathways leading to production of AA and its metabolites. Two main informations were obtained by our experiments: first, AA release is not entirely due to cPLA2 involvement, but also to DAG lipase recruitment; second, cyclooxygenase derivatives play a role in the control of cell proliferation, and not only of motility. Moreover, by combining proliferation assays and single cell calcium measurements, we show that the blocking effect of carboxyamido-triazole (CAI), an inhibitor of tumor growth and angiogenesis acting on calcium influx-dependent pathways, including AA metabolism, is at least in part due to a direct effect on AA-induced calcium influx.

Animals↗

PubMed2002

Oxytocin is a growth factor for Kaposi's sarcoma cells: evidence of endocrine-immunological cross-talk.

Oxytocin receptors (OTRs) are expressed in numerous tissues, including human normal endothelium. Here we investigated the expression and biological significance of OTRs in Kaposi's sarcoma (KS), an intensely angioproliferative disease of possible vascular origin with a prominent inflammatory component. Immunohistochemistry and in situ hybridization studies showed OTR expression in tumor cells of cutaneous classic and AIDS-related KS lesions. OTR mRNA and protein were also detected on cultured KS-IMM spindle cells by reverse transcription-PCR and immunofluorescence procedures. In these cells, OTR expression was up-regulated by the supernatants of resting CD4+ and CD8+ lymphocytes through a still unidentified factor. Functionality of OTRs was demonstrated because OT treatment of KS-IMM cells led to a significant increase in cell proliferation, coupled to the increase of intracellular calcium, but did not effect cell migration in vitro or angiogenesis in vivo. In addition, we demonstrated that CD4+ and CD8+ cells produce OT themselves, thus constituting an intralesional source of peptide. These results indicate that: (a) functioning OTRs are expressed in KS cells and modulated by the inflammatory counterpart of KS lesions; (b) via OTRs, OT stimulates KS-IMM cell proliferation and could, therefore, be considered a new possible relevant growth factor involved in KS progression; and finally (c) the evidence of OT synthesis by CD4+ and CD8+ lymphocytes strongly suggests the existence of local endocrine-immunological cross-talk in Kaposi's sarcoma.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Expression and functional role of bTRPC1 channels in native endothelial cells.

We have analyzed the expression and localization of bovine transient receptor potential-C1 (bTRPC1) in bovine aortic endothelial cells, and its possible involvement in the store-independent calcium influx induced by basic fibroblast growth factor (bFGF). RT-PCR experiments confirmed the existence of two btrpc1 mRNA isoforms; conversely, the btrpc3 gene was not transcribed. Anti-TRPC1 antibody revealed the presence of the protein in the membrane-rich compartment only. Application of anti-TRPC1 during the response to bFGF caused a partial but significant reduction of calcium entry. This is the first evidence of TRP channel involvement in a non-capacitative calcium influx induced by a biologically relevant agonist such as the angiogenic factor bFGF in native endothelial cells.

Alternative Splicing↗

PubMed2002

Calcium signalling and control of cell proliferation by tyrosine kinase receptors (review).

Most peptidic growth factors (GFs) that act as mitogens for different cell types bind to tyrosine kinase receptors (RTKs) and trigger complex intracellular signal transduction pathways finally leading to cell proliferation. Among the early events induced by GFs, cytosolic calcium increase plays a key role, and in particular calcium entry from extracellular medium appears to be a widespread signal. Even if a great amount of data has been provided during the last decades, several issues remain to be fully clarified: the nature of the calcium-permeable channels involved, their regulation by intracellular messengers, and the mechanisms underlying calcium-dependent cell proliferation. This review focuses on the relationship between calcium signals and angiogenesis, a process in which endothelial cell proliferation, mainly triggered by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), is a critical step.

Animals↗