Giuseppina Cantarella

Nerve growth factor–endothelial cell interaction leads to angiogenesis in vitro and in vivo

Nerve growth factor (NGF) has important functions during embryonic development and on various tissues and organs under normal and pathological conditions during the extrauterine life. RT‐PCR analysis and immunological methods demonstrate that human umbilical vein endothelial cells (HUVECs) express the NGF receptors trkANGFR and p75NTR. NGF treatment caused a rapid phosphorylation of trkANGFR in HUVECs, determining a parallel increase of phosphorylated ERK1/2. Accordingly, NGF induced a significant increase in HUVEC proliferation that was abolished by the trkANGFR inhibitor K252a. Also, HUVECs express significant levels of NGF under standard culture conditions that were up‐regulated during serum starvation. Endogenous NGF was responsible for the basal levels of trkANGFR and ERK1/2 phosphorylation observed in untreated HUVEC cultures. Finally, NGF exerted a potent, direct, angiogenic activity in vivo when delivered onto the chorioallantoic membrane of the chicken embryo. The data indicate that NGF may play an important role in blood vessel formation in the nervous system and in several pathological processes, including tumors and inflammatory diseases. Unraveling mechanisms of NGF‐dependent angiogenesis could provide valuable tools for novel therapeutic approaches in antiangiogenic therapy.

The role of antioxidant supplement in immune system, neoplastic, and neurodegenerative disorders: a point of view for an assessment of the risk/benefit profile

This review will discuss some issues related to the risk/benefit profile of the use of dietary antioxidants. Thus, recent progress regarding the potential benefit of dietary antioxidants in the treatment of chronic diseases with a special focus on immune system and neurodegenerative disorders will be discussed here. It is well established that reactive oxygen species (ROS) play an important role in the etiology of numerous diseases, such as atherosclerosis, diabetes and cancer. Among the physiological defense system of the cell, the relevance of antioxidant molecules, such as glutathione and vitamins is quite well established. Recently, the interest of researchers has, for example, been conveyed on antioxidant enzyme systems, such as the heme oxygenase/biliverdin reductase system, which appears modulated by dietary antioxidant molecules, including polyphenols and beta-carotene. These systems possibly counteract oxidative damage very efficiently and finally modulate the activity of oxidative phenomena occurring, for instance, during pathophysiological processes. Although evidence shows that antioxidant treatment results in cytoprotection, the potential clinical benefit deriving from both nutritional and supplemental antioxidants is still under wide debate. In this line, the inappropriate assumption of some lipophylic vitamins has been associated with increased incidence of cancer rather than with beneficial effects.

Endocannabinoids inhibit release of nerve growth factor by inflammation-activated mast cells.

Nerve growth factor (NGF) is a pleiotropic member of the neurotrophin family. Beside its neuronal effects, NGF plays a role in various processes, including angiogenesis. Mast cells release NGF and are among elements contributing to angiogenesis, a process regulated by arrays of factors, including the inhibitory cannabinoids. The possible inhibitory role of cannabinoids on mast cell-related NGF mitogenic effect on endothelial cells was then investigated. Human mastocytic cells HMC-1, challenged with PMA to yield release of NGF, were preincubated with the endocannabinoid PEA. Then, conditioned media were added to HUVEC cultures. PMA-activated HMC-1 cells released substantial amounts of NGF, whereas PEA inhibited PMA-induced NGF release. HUVEC proliferation increased after treatment with media from activated HMC-1 cells, while was reduced with media from HMC-1 cells treated with PEA. To characterize receptors mediating such effects of PEA, RT-PCR and western blot analysis were performed on HMC-1 cells. None of the two cannabinoid CB1 and CB2 receptors was expressed by HMC-1 cells, which on the other hand expressed the orphan receptor GPR55. PEA was ineffective in inhibiting NGF release from HMC-1 cells treated with PMA and transfected with positive GPR55 RNAi, whereas it induced significant reduction of NGF in cells transfected with the corresponding negative control RNAi. Results indicate that NGF released from inflammatory mast cells induces angiogenesis. Cannabinoids attenuate such pro-angiogenic effects of NGF. Finally, cannabinoids could be considered for antiangiogenic treatment in disorders characterized by prominent inflammation.

TRAIL is expressed in the brain cells of Alzheimer's disease patients.

Tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) is a novel cytokine characterized by selective killing activity against tumour cells. We recently found that &bgr;-amyloid-induced apoptosis in a human neuronal cell line was mediated via induction of TRAIL. Here, we show that TRAIL is specifically expressed in the brain of Alzheimers disease (AD) patients and completely absent in the brain of non-demented patients. TRAIL-like immunoreactivity was localized in AD affected regions, such as cerebral cortex, often in the proximity of Congo-red-positive amyloid plaques. These findings suggest that neurons represent an independent and potential source of TRAIL, suggesting that the latter acts redundantly with other noxious stimuli in neurodegenerative diseases characterized by amyloidosis and neuroinflammation.

Divergent effects of corticotropin releasing hormone on endothelial cell nitric oxide synthase are associated with different expression of CRH type 1 and 2 receptors

Endothelium is a target for an array of factors involved in inflammation. Endothelial cells express receptors for CRH, a neuropeptide produced during inflammation. We report both the concentration‐dependent inhibitory effect of CRH upon cytokine‐stimulated nitrite release by H5V murine endothelioma cells, and its stimulatory one in HUVEC cells. Western blot analysis showed that CRH inhibits cytokine‐stimulated iNOS protein in H5V cells, and, instead, potentiated it in HUVEC cells. H5V cells expressed both CRH receptors (CRH‐R1 and R2) mRNAs, whereas HUVEC cells expressed the CRH‐R2 mRNA solely. CRH increased medium nitrites and iNOS protein expression in H5V cells pretreated with the selective CRH‐R1 antagonist CP 154,526. However, the selective CRH‐R2 antagonist anti‐Svg‐30 failed to produce similar effects. In fact, anti‐Svg‐30 inhibited CRH‐induced increase of nitrite release and iNOS expression in HUVEC cells. Our results confirm the activating role of CRH on endothelial cells, although it suggests its possible inhibitory role in the late phase of the inflammatory response. NO‐mediated effects of CRH on endothelial cells could be exploited in therapeutic strategies related to inflammatory and/or degenerative diseases.

Recombinant human TNF-binding protein-1 (rhTBP-1) treatment delays both symptoms progression and motor neuron loss in the wobbler mouse

TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.

Expression of Ca2+-independent and Ca2+-dependent phospholipases A2 and cyclooxygenases in human melanocytes and malignant melanoma cell lines

We provide novel evidence that human melanoma cell lines (M10, M14, SK-MEL28, SK-MEL93, 243MEL, 1074MEL, OCM-1, and COLO38) expressed, at mRNA and protein levels, either Ca(2+)-independent phospholipase A(2) (iPLA(2)) or cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form. Normal human melanocytes contained the lowest levels of both PLA(2)s. Cyclooxygenase-1 and -2 (COX-1 and COX-2) were also expressed in cultured tumor cells as measured by Western blots. The most pronounced overexpression of iPLA(2) and COX-1 was found in two melanoma-derived cells, M14 and COLO38. Normal human melanocytes and the M10 melanoma cell line displayed no COX-2 expression. Using subcellular fractionation, Western blot and confocal microcopy analyses, in paradigmatic SK-MEL28 and SK-MEL93 cells we showed that iPLA(2), COX-1 and even cPLA(2) were equally located in the cytosol, membrane structures and perinuclear region while COX-2 was preferentially associated with the cytosol. Specific inhibitors of these three enzymes significantly reduced the basal proliferation rate either in melanocytes or in melanoma cell lines. These results, coupled with the inhibition of the cell proliferation by electroporation of melanoma cells with cPLA(2) or COX-2 antibodies, demonstrate that a possible correlation between PLA(2)-COX expression and tumor cell proliferation in the melanocytic system does exist. In addition, the high expression level of both PLA(2)s and COXs suggests that eicosanoids modulate cell proliferation and tumor invasiveness.

Protective effects of the sigma agonist pre-084 in the rat retina

Aim: With the rationale that amyloid beta (AB) is toxic to the retina, we here assessed the role of TRAIL, a mediator of AB toxicity and related signal transduction, in a rat model. We also attempted to demonstrate possible protective effects of &sgr;1 receptor agonists in these processes. Methods: AB and the &sgr;1 receptor agonist Pre-084 were injected intravitreally in the anaesthetised rat. In additional experiments, the &sgr;1 receptor antagonist BD1047 was administered to assess specificity of the effects of Pre-084. Western blot analysis was performed on retinas to evaluate the expression of TRAIL and TRAIL receptors in the retina, as well as of Bax and phosphorylated JNK following the different treatments. Lactic dehydrogenase (LDH) levels were measured as a cytotoxicity marker. Results: All TRAIL receptors were expressed in rat retinas. Intravitreal injection of AB in rat eyes induced overexpression of TRAIL and the proapoptotic protein Bax, as well as phosphorylation of JNK. All these effects of AB were abrogated by pretreatment with the &sgr;1 receptor agonist Pre-084. Conclusions: It is likely that TRAIL is a mediator of AB effects on the retina. In light of their specific inhibitory effects upon TRAIL expression, it is plausible to hypothesise that &sgr;1 receptor agonists could represent potential pharmacological tools for restraining AB related retinal damage.

Growth hormone protects human lymphocytes from irradiation-induced cell death.

Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r‐hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro. r‐hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl‐2. r‐hGH restored mitogen‐stimulated release of IL‐2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r‐hGH‐dependent IL‐2 release. These results demonstrate that r‐hGH protects irradiated PBL from death in a specific, receptor‐mediated manner. Such effect of r‐hGH on PBL involves activation of the antiapoptotic gene bcl‐2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer.

Neutralization of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Reduces Spinal Cord Injury Damage in Mice

Spinal cord injury (SCI) is a major cause of disability, its clinical outcome depending mostly on the extent of damage in which proapoptotic cytokines have a crucial function. In particular, the inducers of apoptosis belonging to TNF receptor superfamily and their respective ligands are upregulated after SCI. In this study, the function of the proapoptotic cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in SCI-induced damage was investigated in the mouse. SCI resulted in severe trauma, characterized by prominent inflammation-related damage and apoptosis. Immunostaining for TRAIL and its receptor DR5 was found in the white and gray matter of the perilesional area, as also confirmed by western blotting experiments. Immunoneutralization of TRAIL resulted in improved functional recovery, reduced apoptotic cell number, modulation of molecules involved in the inflammatory response (FasL, TNF-α, IL-1β, and MPO), and the corresponding signaling (caspase-8 and -3 activation, JNK phosphorylation, Bax, and Bcl-2 expression). As glucocorticoid-induced TNF receptor superfamily-related protein (GITR) activated by its ligand (GITRL) contributes to SCI-related inflammation, interactions between TRAIL and GITRL were investigated. SCI was associated with upregulated GITR and GITRL expression, a phenomenon prevented by anti-TRAIL treatment. Moreover, the expression of both TRAIL and DR5 was reduced in tissues from mice lacking the GITR gene (GITR−/−) in comparison with wild-type mice suggesting that TRAIL- and GITRL-activated pathways synergise in the development of SCI-related inflammatory damage. Characterization of new targets within such molecular systems may constitute a platform for innovative treatment of SCI.