Tullio Genova

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 Ca2+ 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 Ca2+ signaling in the same cells. B-TEC stimulation with 5 μmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser1177, 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 PKI14-22 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. Mol Cancer Res; 8(11); 1466–76. ©2010 AACR.

Cytokine, chemokine, and growth factor profile of platelet-rich plasma

Abstract During wound healing, biologically active molecules are released from platelets. The rationale of using platelet-rich plasma (PRP) relies on the concentration of bioactive molecules and subsequent delivery to healing sites. These bioactive molecules have been seldom simultaneously quantified within the same PRP preparation. In the present study, the flexible Bio-Plex system was employed to assess the concentration of a large range of cytokines, chemokines, and growth factors in 16 healthy volunteers so as to determine whether significant baseline differences may be found. Besides IL-1b, IL-1ra, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17, INF-γ, TNF-α, MCP-1, MIP-1a, RANTES, bFGF, PDGF, and VEGF that were already quantified elsewhere, the authors reported also on the presence of IL-2, IL-5, IL-7, IL-9, IL-10, IL-15 G-CSF, GM-CSF, Eotaxin, CXCL10 chemokine (IP-10), and MIP 1b. Among the most interesting results, it is convenient to mention the high concentrations of the HIV-suppressive and inflammatory cytokine RANTES and a statistically significant difference between males and females in the content of PDGF-BB. These data are consistent with previous reports pointing out that gender, diet, and test system affect the results of platelet function in healthy subjects, but seem contradictory when compared to other quantification assays in serum and plasma. The inconsistencies affecting the experimental results found in literature, along with the variability found in the content of bioactive molecules, urge further research, hopefully in form of randomized controlled clinical trials, in order to find definitive evidence of the efficacy of PRP treatment in various pathologic and regenerative conditions.

Plasma of Argon Affects the Earliest Biological Response of Different Implant Surfaces An In Vitro Comparative Study

The aim of this in vitro study was to evaluate the early cell response and protein adsorption elicited by the argon plasma treatment of different commercially available titanium surfaces via a chair-side device. Sterile disks made of grade 4 titanium (n = 450, 4-mm diameter) with 3 surface topographies (machined, plasma sprayed, and zirconia blasted and acid etched) were allocated to receive 4 testing treatments (2% and 10% protein adsorption and cell adhesion with MC3T3-E1 and MG-63). Furthermore, the specimens were divided to undergo 1) argon plasma treatment (10 W, 1 bar for 12 min) in a plasma reactor, 2) ultraviolet (UV) light treatment for 2 h (positive control group), or 3) no treatment (control group). Pretreatment surface analyses based on a scanning electron microscope and profilometer images were also performed. Profilometric analysis demonstrated that the evaluated specimens perfectly suit the standard parameters. The use of argon plasma was capable of affecting the quantity of proteins adsorbed on the different surfaces, notwithstanding their roughness or topographic features at a low fetal bovine serum concentration (2%). UV light treatment for 2 h attained similar results. Moreover, both the plasma of argon and the UV light demonstrated a significant increase in the number of osteoblasts adherent at 10 min in all tested surfaces. Within its limitations, this in vitro study highlights the potential biological benefits of treating implant surfaces with plasma of argon or UV, irrespective of the roughness of the titanium surface. However, in vivo experiments are needed to confirm these preliminary data and settle the rationale of a treatment that might be clinically relevant in case of bone-reparative deficiencies.

TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein–protein interaction, thus preventing its cytoplasm–plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration.

Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling

Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium.

Overcoming physical constraints in bone engineering: ‘the importance of being vascularized’

Bone plays several physiological functions and is the second most commonly transplanted tissue after blood. Since the treatment of large bone defects is still unsatisfactory, researchers have endeavoured to obtain scaffolds able to release growth and differentiation factors for mesenchymal stem cells, osteoblasts and endothelial cells in order to obtain faster mineralization and prompt a reliable vascularization. Nowadays, the application of osteoblastic cultures spans from cell physiology and pharmacology to cytocompatibility measurement and osteogenic potential evaluation of novel biomaterials. To overcome the simple traditional monocultures in vitro, co-cultures of osteogenic and vasculogenic precursors were introduced with very interesting results. Increasingly complex culture systems have been developed, where cells are seeded on proper scaffolds and stimulated so as to mimic the physiological conditions more accurately. These bioreactors aim at enabling bone regeneration by incorporating different cells types into bio-inspired materials within a surveilled habitat. This review is focused on the most recent developments in the organomimetic cultures of osteoblasts and vascular endothelial cells for bone tissue engineering.

Targeting calcium channels to block tumor vascularization.

Blood vessels and endothelial cells (ECs) are highly versatile in order to accomplish local tissutal needs in the physiological and pathological conditions. Tumor vasculature, in particular, exhibits special morphological and functional features, partly due to the peculiarity of tumor-derived ECs (TECs). This is of great importance for the discovery of selective molecular targets potentially suitable to interfere with tumor growth and spread. In normal ECs, proangiogenic calcium signaling is mediated by different calcium channels, mainly TRPs and Orai, that could play a pivotal role in physiological angiogenesis. They are regulated through multiple mechanisms, involving their interaction with bioactive lipids (arachidonic acid and its metabolites), nitrosylation, sulfhydration, phosphorylation, cytoskeleton-mediated membrane trafficking, and calcium stores depletion. On the other hand, proangiogenic calcium events in TECs have been investigated only recently and their characterization is still preliminary. ECs obtained from human breast and renal carcinomas (B-TECs and R-TECs respectively) display altered calcium signals, which are associated with modified expression and function of TRP channels. Here, we review the state of the art in the field of calcium signaling and tumor vascularization, the related recent literature and patents. Finally, we provide some suggestions for future developments.

Role of surface finishing on the in vitro biological properties of a silicon nitride–titanium nitride (Si3N4–TiN) composite

Silicon nitride (Si3N4) has been introduced clinically as an orthopedic biomaterial for interbody fusion devices and in joint replacements. However, the production of complex shapes through conventional mechanical machining is difficult and expensive and limits interesting applications. Thus, several electrically conductive reinforcements to the Si3N4 matrix, like TiN, have been proposed, generating composites suitable to be wrought by electrical discharge machining (EDM). In this study, Si3N4–TiN with high strength, low density, and good electric conductivity wrought by EDM was studied. The role of surface finishing was investigated comparing the interface generated during the EDM process to that resulting from further polishing. The different topographical features were assessed by electron microscopy, energy dispersive X-ray spectrometry, and profilometry. Surface wettability was also determined based on the measurement of the OCA of water and diiodomethane. The biological responses induced in MC3T3 cells, a widely diffused osteoblast model, were correlated with the surface pattern. The unpolished samples could promote better cell viability, with a more relevant effect on the cytoskeleton arrangement as highlighted by numerous cytoplasmic extensions and filopodia-like structures and the high number of focal adhesions, while MC3T3 cells grown on polished Si3N4–TiN specimens displayed a flat morphology. In addition, the unpolished Si3N4–TiN increased osteocalcin production and calcium deposition. Taken together, these data support the biocompatibility and in vitro osteogenic properties of the electroconductive Si3N4–TiN investigated. Further in vivo studies are required to explore the possible use of bone implants directly obtained by EDM.

Differential sensitivity of prostate tumor derived endothelial cells to sorafenib and sunitinib

BackgroundProstate cancer is the second leading cause of male cancer death in developed countries. Although the role of angiogenesis in its progression is well established, the efficacy of anti-angiogenic therapy is not clearly proved. Whether this could depend on differential responses between tumor and normal endothelial cells has not been tested.MethodsWe isolated and characterized three lines of endothelial cells from prostate cancer and we tested the effect of Sunitinib and Sorafenib, and the combined treatment with the anti-androgen Casodex, on their angiogenic functions.ResultsEndothelial cells isolated from prostate tumors showed angiogenic properties and expression of androgen and vascular endothelial cell growth factor receptors. Sunitinib affected their proliferation, survival and motility while Sorafenib only showed a minor effect. At variance, Sunitinib and Sorafenib showed similar cytotoxic and anti-angiogenic effects on normal endothelial cells. Sorafenib and Sunitinib inhibited vascular endothelial cell growth factor receptor2 phosphorylation of prostate cancer endothelial cells, while they differentially modulated Akt phosphorylation as no inhibitory effect of Sorafenib was observed on Akt activation. The combined treatment of Casodex reverted the observed resistance to Sorafenib both on cell viability and on Akt activation, whereas it did not modify the response to Sunitinib.ConclusionsOur study demonstrates a resistant behavior of endothelial cells isolated from prostate cancer to Sorafenib, but not Sunitinib. Moreover, it shows the benefit of a multi-target therapy combining anti-angiogenic and anti-hormonal drugs to overcome resistance.

Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis

Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial-specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis and provide evidence that endoplasmic reticulum (ER) stress precedes heme-induced paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. Interfering with endothelial heme export represents a valuable model for a deeper understanding of the molecular mechanisms underlying heme-triggered paraptosis and, in the future, might provide a novel tool for the modulation of angiogenesis in pathophysiologic conditions.