Arachidonic acid-induced Ca2+ entry is involved in early steps of tumor angiogenesis.

Growth factor-induced intracellular calcium signals in endothelial cells regulate cytosolic and nuclear events involved in the angiogenic process. Among the intracellular messengers released after proangiogenic stimulation, arachidonic acid (AA) plays a key role and its effects are strictly related to calcium homeostasis and cell proliferation. Here, we studied AA-induced intracellular calcium signals in endothelial cells derived from human breast carcinomas (B-TEC). AA promotes B-TEC proliferation and organization of vessel-like structures in vitro. The effect is directly mediated by the fatty acid without a significant contribution of its metabolites. AA induces Ca(2+)(i) signals in the entire capillary-like structure during the early phases of tubulogenesis in vitro. No such responses are detectable in B-TECs organized in more structured tubules. In B-TECs growing in monolayer, AA induces two different signals: a Ca(2+)(i) increase due to Ca(2+) entry and an inhibition of store-dependent Ca(2+) entry induced by thapsigargin or ATP. An inhibitor of Ca(2+) entry and angiogenesis, carboxyamidotriazole, significantly and specifically decreases AA-induced B-TEC tubulogenesis, as well as AA-induced Ca(2+) signals in B-TECs. We conclude that (a) AA-activated Ca(2+) entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation and tubulogenesis, and it is down-regulated during the reorganization of tumor-derived endothelial cells in capillary-like structures; and (b) inhibition of AA-induced Ca(2+) entry may contribute to the antiangiogenic action of carboxyamidotriazole.

Arachidonic acid and calcium signals in human breast tumor-derived endothelial cells: a proteomic study.

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 upon proangiogenic stimulation, arachidonic acid (AA) and its metabolites play a key role, and their effects are strictly related to calcium homeostasis. In human breast tumor-derived endothelial cells (B-TECs) AA stimulates proliferation and tubulogenesis in a calcium-dependent way. Here, to characterize the proteins whose expression is regulated by AA-induced calcium entry, we used a proteomic approach (two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, 2-DE and MALDI-MS) and we compared the proteomes of B-TECs stimulated with AA in presence or in absence of calcium entry (with addition to the culture medium of the calcium chelator EGTA, which completely prevents calcium fluxes throughout the plasma membrane). We found that six proteins increased their levels of expression, all higher when AA-induced calcium entry was abolished. These proteins have been identified by mass spectrometry and database search, and their potential roles in AA-stimulated pathway and in angiogenesis are discussed.

The secret marriage between calcium and tumor angiogenesis.

Endothelial cell biochemistry and responsiveness to a wide variety of external stimula is regulated by intracellular calcium concentration. During the last twenty years, electrophysiology and functional imaging based on the use of fluorescent probes provided several informations about the dynamics and role of calcium at the single cell level: highly diverse extracellular agonists, such as proangiogenic growth factors and vasoactive compounds, trigger increases in intracellular calcium and specific informations are transduced for proliferation, differentiation, death, movement in physiological and pathological conditions. Obviously, the investigation at multicellular and tissutal levels is much more complex. In this review we discuss the potential specific roles of calcium signaling in tumor angiogenesis progression trying to address two key questions: (i) how can this ion play specific roles in the angiogenesis regulation; and (ii) could it be used as a target to interfere with or prevent tumor vascularization?

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.

Multiple roles of protein kinase a in arachidonic acid-mediated Ca2+ entry and tumor-derived human endothelial cell migration.

We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca(2+) entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca(2+) signaling in the same cells. B-TEC stimulation with 5 µmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser(1177), and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI(14-22) completely prevented both AA- and NO-induced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N(G)-nitro-l-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs.