Luca Munaron

Zinc-containing bioactive glasses: surface reactivity and behaviour towards endothelial cells

This paper reports a physico-chemical study devoted to reactivity towards hydroxo-carbonate apatite (HCA) formation of bioactive glass 45S5 (H glass; commercially known as Bioglass) and of two preparations of zinc-doped 45S5-derived systems (HZ5, HZ20), immersed in Tris(hydroxymethyl)aminomethane (Tris) and Dulbeccos modified Eagles medium (DMEM) buffer solutions. The activity/toxicity of the glasses was also tested using endothelial cells (EC). Zn caused a drastic reduction in the overall leaching activity of glasses and, at high Zn concentration (HZ20), the formation of HCA on the glass surface was thoroughly inhibited. The presence of Zn also decreased the increment of pH after glass immersion in both Tris and DMEM solution. EC are known to be very sensitive to pH changes and, for this reason, the rapid increase in pH brought about by H glass dissolution is likely to affect cell adhesion and spreading, whereas the high zinc release from HZ20 causes a drastic reduction in cell proliferation after a long contact time (approximately 1 week). This study shows that only HZ5 glass containing 5 wt.% Zn presents at the same time: reduced solubility, bioactivity (monitored by HCA formation) and conditions allowing EC growth over a 6-day period.

Intracellular calcium, endothelial cells and angiogenesis.

The proliferation and motility of vascular endothelial cells (ECs) are critical steps in angiogenesis and are strictly controlled by different extracellular signals. Among mitogens, peptides binding to tyrosine kinase receptors (i.e. VEGFs and FGFs) are well known and are released by several cell types, including ECs and tumor cells. The binding of mitogens to their specific receptors triggers intracellular signaling cascades, involving a number of messengers working in a sort of network. In particular, in this review we describe the increases of calcium levels in the cytosol, a universal, evolutionary conserved and highly versatile signal involved in the regulation of ECs proliferation and motility. Most mitogens, including angiogenic factors, generate cytosolic calcium rises through two mechanisms: entry from extracellular medium, through the opening of calcium permeable channels in the plasma membrane, or release from intracellular organelles (mainly endoplasmic reticulum, ER). Calcium entry, the main topic of this review, can be dependent on previously IP(3)-activated emptying of calcium stores (store-dependent or capacitative calcium entry--CCE), or independent on it (non capacitative calcium entry, NCCE). The intracellular pathways underlying calcium entry are under investigation and recently arachidonic acid (AA) and nitric oxide (NO) metabolism have been suggested to play a key role, at least in some cell types. Even if some calcium entry blockers are under clinical trial with encouraging results, a better knowledge about the molecular nature of calcium channels and their intracellular regulation, together with a more detailed description of spatiotemporal dynamics of intracellular calcium events, could lead to new and more specific strategies in therapeutical approach to cancer progression and angiogenesis.

Intracellular calcium signals and control of cell proliferation: how many mechanisms?

The progression through the cell cycle in non‐transformed cells is under the strict control of extracellular signals called mitogens, that act by eliciting complex cascades of intracellular messengers. Among them, increases in cytosolic free calcium concentration have been long realized to play a crucial role; however, the mechanisms coupling membrane receptor activation to calcium signals are still only partially understood, as are the pathways of calcium entry in the cytosol. This article centers on the role of calcium influx from the extracellular medium in the control of proliferative processes, and reviews the current understanding of the pathways responsible for this influx and of the second messengers involved in their activation.

TRPV4 mediates tumor-derived endothelial cell migration via arachidonic acid-activated actin remodeling

Changes in intracellular calcium [Ca2+]i levels control critical cytosolic and nuclear events that are involved in the initiation and progression of tumor angiogenesis in endothelial cells (ECs). Therefore, the mechanism(s) involved in agonist-induced Ca2+i signaling is a potentially important molecular target for controlling angiogenesis and tumor growth. Several studies have shown that blood vessels in tumors differ from normal vessels in their morphology, blood flow and permeability. We had previously reported a key role for arachidonic acid (AA)-mediated Ca2+ entry in the initial stages of tumor angiogenesis in vitro. In this study we assessed the mechanism involved in AA-induced EC migration. We report that TRPV4, an AA-activated channel, is differentially expressed in EC derived from human breast carcinomas (BTEC) as compared with ‘normal’ EC (HMVEC). BTEC display a significant increase in TRPV4 expression, which was correlated with greater Ca2+ entry, induced by AA or 4αPDD (a selective TRPV4 agonist) in the tumor-derived ECs. Wound-healing assays revealed a key role of TRPV4 in regulating cell migration of BTEC but not HMVEC. Knockdown of TRPV4 expression completely abolished AA-induced BTEC migration, suggesting that TRPV4 mediates the pro-angiogenic effects promoted by AA. Furthermore, pre-incubation of BTEC with AA induced actin remodeling and a subsequent increase in the surface expression of TRPV4. This was consistent with the increased plasma membrane localization of TRPV4 and higher AA-stimulated Ca2+ entry in the migrating cells. Together, the data presented herein demonstrate that: (1) TRPV4 is differentially expressed in tumor-derived versus ‘normal’ EC; (2) TRPV4 has a critical role in the migration of tumor-derived but not ‘normal’ EC migration; and (3) AA induces actin remodeling in BTEC, resulting in a corresponding increase of TRPV4 expression in the plasma membrane. We suggest that the latter is critical for migration of EC and thus in promoting angiogenesis and tumor growth.

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.

OXYTOCIN INHIBITS THE PROLIFERATION OF MDA-MB231 HUMAN BREAST-CANCER CELLS VIA CYCLIC ADENOSINE MONOPHOSPHATE AND PROTEIN KINASE A

Oxytocin (OT) inhibits the proliferation of breast‐cancer cells in vitro via a specific G‐coupled receptor. To elucidate the intracellular mechanism involved in this biological effect, different G‐coupled receptor mediators have been investigated in untreated and OT‐treated MDA‐MB231 breast‐carcinoma cells. In these cells, after OT treatment, a significant cAMP increase was observed using a radioimmunoassay procedure, whereas the Ca2+ (determined with the fluorescent probe fura‐2) and the inositol phosphate (determined after cell labeling with myo(2‐3H)‐inositol) concentrations were not modified, contrary to what has been observed in myometrial and myo‐epithelial cells. The PKA inhibitor PKI (6‐22) amide reverted the effect of OT, indicating that the anti‐proliferative effect of the peptide is strictly related to the cAMP–PKA pathway. OT treatment did not modify tyrosine phosphorylation either. Our results indicate that in breast epithelial cells devoid of contractile activity, cAMP is the intracellular mediator of OT action, whereas the Ca2+‐phosphoinositide system is not involved. Int. J. Cancer 72:340–344, 1997.

Endothelial Calcium Machinery and Angiogenesis: Understanding Physiology to Interfere with Pathology

Endothelial cells (ECs) play a pivotal role in physiological and altered tissue neovascularization. They face multiple morphological, biochemical and functional changes during the different phases of angiogenesis, under the regulation of a great number of proangiogenic and antiangiogenic signals, including soluble and insoluble factors, cell-cell and cell-matrix interactions. ECs mutual contacts (and also interactions with other cell types, such as pericytes and smooth vascular muscle cells), motility, proliferation, apoptosis and differentiation are all calcium-dependent events finely tuned in space and time. Most of the angiogenic-related peptidic factors (VEGF, bFGF and others) promote an increase of cytosolic free calcium concentration in ECs, giving rise to calcium-activated intracellular cascades engaged in the different steps of the angiogenic process. A better knowledge of such signals could allow to set new diagnostic and therapeutical approaches aimed to interfere with altered neovascularization, particularly during cancer progression. This review reports the state of the art about endothelial angiogenic-related calcium signaling and discusses the most attractive perspectives for the future.

ARACHIDONIC ACID MEDIATES CALCIUM INFLUX INDUCED BY BASIC FIBROBLAST GROWTH FACTOR IN BALB-C 3T3 FIBROBLASTS

Basic fibroblast growth factor (bFGF), a peptide acting as a mitogen in different cell types, is able to induce a long lasting non capacitative calcium influx from the extracellular medium in Balb-c 3T3 mouse fibroblasts. This effect is mediated by the tyrosine kinase activity of bFGF receptors and the opening of voltage independent, agonist activated calcium channels. In this paper we investigate the signal transduction steps involved in this process using single cell calcium fluorimetry and electrophysiological techniques. One of the pathways initiated by the binding of growth factors to their tyrosine kinase receptors is the activation of cytosolic phospholipase A2 (cPLA2) and the release of arachidonic acid (AA) from the plasma membrane with the subsequent production of eicosanoids. We show here that, in our preparation, this pathway is involved in the opening of the bFGF-activated calcium permeable channels, through the activation of mitogen activated protein kinase (MAPK) and cPLA2. Evidence for direct involvement of AA is given by the finding that: (i) bFGF induces AA release from Balb-c 3T3 cells; (ii) blockers of AA metabolism are not effective; and (iii) the application of either arachidonic acid or its non metabolizable analogue 5,8,11,14-eicosatetraynoic acid (ETYA) reproduces the responses described for bFGF. Finally, single channel analysis indicates that bFGF, AA and ETYA can activate the same calcium permeable channel.

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. J. Cell. Physiol. 197: 370–378, 2003© 2003 Wiley‐Liss, Inc.

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.