Davide Martini

Role of the adrenergic system in a mouse model of oxygen-induced retinopathy: antiangiogenic effects of beta-adrenoreceptor blockade.

PURPOSE Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity (ROP). In OIR mice, this study determined whether blockade of β-adrenergic receptors (β-ARs) with propranolol influences retinal levels of proangiogenic factors, retinal vascularization, and blood-retinal barrier (BRB) breakdown. METHODS Propranolol was administered subcutaneously and picropodophyllin (PPP) intraperitoneally. Intravitreal injections of vascular endothelial growth factor (VEGF) were performed. Messengers of β-ARs, VEGF, its receptors, IGF-1 and IGF-1R were measured with quantitative RT-PCR. VEGF content was determined with ELISA. β-ARs, hypoxia-inducible factor (HIF)-1α, occludin, and albumin were measured with Western blot. Retinal localization of β3-ARs was determined by immunohistochemistry. Retinopathy was assessed by scoring fluorescein-perfused retinas, and plasma extravasation was visualized by Evans blue dye. RESULTS Hypoxia did not influence β-AR expression, except that it increased β3-AR protein with dense β3-AR immunoreactivity localized to engorged retinal tufts. Hypoxia upregulated VEGF, IGF-1, their receptors, and HIF-1α. Propranolol dose-dependently reduced upregulated VEGF and decreased hypoxic levels of IGF-1 mRNA and HIF-1α. Blockade of IGF-1R activity with PPP did not influence propranolols effects on VEGF. Retinal VEGF in normoxic mice or VEGF in brain, lungs, and heart of the OIR mice were unaffected by propranolol. Propranolol ameliorated the retinopathy score, restored occludin and albumin, and reduced hypoxia-induced plasma extravasation without influencing the vascular permeability induced by intravitreal VEGF. CONCLUSIONS This is the first demonstration that β-AR blockade is protective against retinal angiogenesis and ameliorates BRB dysfunction in OIR. Although the relevance of these results to infant ROP is uncertain, the findings may help to establish potential pharmacologic targets based on β3-AR pharmacology.

Antiangiogenic effects of β2 -adrenergic receptor blockade in a mouse model of oxygen-induced retinopathy.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07530.x

Modulation of the neuronal response to ischaemia by somatostatin analogues in wild‐type and knock‐out mouse retinas

Somatostatin acts at five G protein‐coupled receptors, sst1‐sst5. In mouse ischaemic retinas, the over‐expression of sst2 (as in sst1 knock‐out mice) results in the reduction of cell death and glutamate release. In this study, we reported that, in wild‐type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischaemia‐induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over‐expressing ischaemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti‐ischaemic effect of somatostatin agonists in the presence of sst2 over‐expression, we tested sst2 desensitisation because of internalisation or altered receptor function. We observed that (i) sst2 was not internalised, (ii) among G protein‐coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischaemia, (iii) both GRK1 and RGS1 were down‐regulated by octreotide in wild‐type ischaemic retinas, (iv) octreotide down‐regulated GRK1 but not RGS1 in sst2 over‐expressing ischaemic retinas. These results demonstrate that sst2 activation protects against retinal ischaemia. However, in the presence of sst2 over‐expression sst2 is functionally desensitised by agonists, possibly because of sustained RGS1 levels.

Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission

Somatostatin‐14 (SRIF) co‐localizes with γ‐aminobutyric acid (GABA) in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of seizures has been proposed, although its exact contribution requires some clarification. In particular, SRIF knockout (KO) mice do not exhibit spontaneous seizures, indicating that compensatory changes may occur in KO. In the KO hippocampus, we examined whether specific SRIF receptors and/or the cognate peptide cortistatin‐14 (CST) compensate for the absence of SRIF. We found increased levels of both sst2 receptors (sst2) and CST, and we explored the functional consequences of sst2 compensation on bursting activity and synaptic responses in hippocampal slices. Bursting was decreased by SRIF in wild‐type (WT) mice, but it was not affected by either CST or sst2 agonist and antagonist. sst4 agonist increased bursting frequency in either WT or KO. In WT, but not in KO, its effects were blocked by agonizing or antagonizing sst2, suggesting that sst2 and sst4 are functionally coupled in the WT hippocampus. Bursting was reduced in KO as compared with WT and was increased upon application of sst2 antagonist, while SRIF, CST and sst2 agonist had no effect. At the synaptic level, we observed that in WT, SRIF decreased excitatory postsynaptic potentials which were, in contrast, increased by sst2 antagonist in KO. We conclude that sst2 compensates for SRIF absence and that its upregulation is responsible for reduced bursting and decreased excitatory transmission in KO mice. We suggest that a critical density of sst2 is needed to control hippocampal activity.

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus , in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.

Beta-adrenoreceptor agonism influences retinal responses to hypoxia in a model of retinopathy of prematurity.

PURPOSE In a mouse model of oxygen-induced retinopathy (OIR), a well-established model of retinopathy of prematurity (ROP), blocking beta-adrenoreceptors (β-ARs), and, in particular, β2-ARs, counteracts retinal responses to hypoxia. In the present work, we determined the effects of the β-AR agonist isoproterenol on retinal angiogenesis and β-AR signaling to better clarify the role of sympathetic transmission in ROP. METHODS Isoproterenol was administered subcutaneously. Protein kinase A activity was determined by a colorimetric assay to assess drug effectiveness. Blood pressure and heart-to-body weight ratio were measured. Vascular endothelial growth factor (VEGF) and norepinephrine were measured with ELISA. Retinal neovascularization was assessed by CD31 immunohistochemistry. β-AR-coupled adenylyl cyclase (AC) activity was measured with a competition assay. β-ARs, G-protein-coupled receptor kinase (GRK)2, and β-arrestins were determined by Western blot. Association of β-arrestins with β2-ARs was assessed by immunoprecipitation. RESULTS Isoproterenol-induced modulation of protein kinase A activity suggests that the drug was effective at the receptor level. Isoproterenol did not affect cardiovascular parameters, but decreased retinal levels of VEGF and reduced pathogenic neovascularization, likely through an influence on sympathetic transmission. In fact, isoproterenol downregulated β2-AR expression, recovered the hypoxia-induced increase in β-AR-coupled AC activity, and increased GRK2 and β-arrestins, which promote β-AR desensitization through the uncoupling of G-protein-coupled receptors from G proteins. Immunoprecipitation studies demonstrated that β-AR desensitization involved β2-ARs. CONCLUSIONS Our findings suggest that hypoxia-induced retinal neovascularization depends at least in part on increased sympathetic transmission, as reduction of sympathetic drive by agonist-induced β2-AR desensitization inhibits some of the hallmarks of OIR.

Vascular endothelial growth factor up-regulation in the mouse hippocampus and its role in the control of epileptiform activity

The vascular endothelial growth factor (VEGF) signalling pathway may represent an endogenous anti‐convulsant in the rodent hippocampus although its exact contribution requires some clarification. In mouse hippocampal slices, the potassium channel blocker 4‐aminopyridine (4‐AP) in the absence of external Mg2+(0 Mg2+) produces both ictal and interictal activity followed by a prolonged period of repetitive interictal activity. In this model, we demonstrated that exogenous VEGF has clear effects on ictal and interictal activity as it reduces the duration of ictal‐like events, but decreases the frequency and intensity of interictal discharges. VEGF affects epileptiform activity through its receptor VEGFR‐2. We also demonstrated for the first time that the synaptic action of VEGF in the hippocampus is through VEGFR‐2‐mediated effects on NMDA and GABAB receptors and that VEGF does not affect the NMDA excytatory postsynaptic potential paired‐pulse facilitation ratio. Exogenous VEGF does not affect the AMPA‐mediated responses and the dendritic or the somatic GABAA inhibitory postsynaptic potentials. In addition, VEGF drastically reduces 0 Mg2+/4‐AP‐induced glutamate release through VEGFR‐2 activation. In vitro epileptiform activity is sufficient to increase hippocampal expression of VEGF and VEGFR‐2, and this up‐regulation may serve a neuroprotective and/or anti‐convulsant role. VEGFR‐2 up‐regulation has been localized to the CA1 region, which suggests that VEGF signalling may protect CA1 pyramidal cells from hyperexcitability. These results indicate that VEGF controls epileptic activity by influencing both glutamatergic and GABAergic transmission and further advance our understanding of the conditions required for endogenous VEGF up‐regulation, and the mechanisms by which VEGF achieves an anti‐convulsant effect.

Expression, localization, and functional coupling of the somatostatin receptor subtype 2 in a mouse model of oxygen-induced retinopathy.

Purpose. In the mouse model of oxygen-induced retinopathy (OIR), somatostatin-14 (SRIF) acting at the SRIF receptor subtype 2 (sst(2)) inhibits angiogenic responses to hypoxia through a downregulation of vascular endothelial growth factor. Information about where SRIF-sst(2) interactions take place is lacking, and downstream effectors mediating SRIF-sst(2) antiangiogenic actions are unknown. Methods. In the OIR model, retinal expression of SRIF was evaluated with RT-PCR and radioimmunoassay. The bindings of [(125)I]LTT-SRIF-28 and [(125)I]Tyr(3)-octreotide were measured in coronal sections of the eye. With Western blot analysis, the authors evaluated the levels of sst(2A) and the expression and activity of the signal transducer and activator of transcription (STAT)3. The analysis of STAT3 was performed in hypoxic mice treated with the sst(2) agonist octreotide or with the sst(2) antagonist D-Tyr(8) cyanamid 154806 (CYN). Retinal localization of sst(2A) was assessed by single and double immunohistochemistry with an endothelial cell marker. Results. In the hypoxic retina, both SRIF and sst(2) levels as well as [(125)I]Tyr(3)-octreotide binding were downregulated. In addition, sst(2A) immunostaining was decreased in the neuroretina but was increased in capillaries. Hypoxia increased both the expression and the activity of STAT3. This increase was inhibited by octreotide but was strengthened by CYN. Conclusions. These data suggest that sst(2) expressed by capillaries may be responsible for the antiangiogenic effects of SRIF and that downstream effectors in this action include the transcription factor STAT3. These results support the possibility of using sst(2)-selective ligands in the treatment of proliferative retinopathies and indicate STAT3 as an additional target for a novel therapeutic approach.

Functional effects of somatostatin receptor 1 activation on synaptic transmission in the mouse hippocampus

Somatostatin‐14 (SRIF) co‐localizes with GABA in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of hippocampal activity has been proposed, although the exact contribution of each SRIF receptor (sst1–sst5) in mediating SRIF action requires some clarification. We used hippocampal slices of wild‐type and sst1 knockout (KO) mice and selective pharmacological tools to provide conclusive evidence for a role of sst1 in mediating SRIF inhibition of synaptic transmission. With single‐ and double‐label immunohistochemistry, we determined the distribution of sst1 in hippocampal slices and we quantified sst1 colocalization with SRIF. With electrophysiology, we found that sst1 activation with CH‐275 inhibited both the NMDA‐ and the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐mediated responses. Results from sst1 KO slices confirmed the specificity of CH‐275 effects; sst1 activation did not affect the inhibitory transmission which was in contrast increased by sst4 activation with L‐803,087 in both wild‐type and sst1 KO slices. The AMPA‐mediated responses were increased by L‐803,087. Functional interaction between sst1 and sst4 is suggested by the finding that their combined activation prevented the CH‐275‐induced inhibition of AMPA transmission. The involvement of pre‐synaptic mechanisms in mediating inhibitory effects of sst1 on excitatory transmission was demonstrated by the finding that CH‐275 (i) increased the paired‐pulse facilitation ratio, (ii) did not influence the AMPA depolarization in the presence of tetrodotoxin, and (iii) inhibited glutamate release induced by epileptiform treatment. We conclude that SRIF control of excitatory transmission through an action at sst1 may represent an important contribution to the regulation of hippocampal activity.

The Secondary Metabolite Euplotin C Induces Apoptosis-Like Death in the Marine Ciliated Protist Euplotes vannus

ABSTRACT. The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non‐producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6‐diamino‐2‐phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro‐apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.