Chiara Ristori

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

Tissue response to poly(ether)urethane-polydimethylsiloxane-fibrin composite scaffolds for controlled delivery of pro-angiogenic growth factors

The development of a scaffold able to mimic the mechanical properties of elastic tissues and to induce local angiogenesis by controlled release of angiogenic growth factors could be applied in the treatment of several ischemic diseases. For this purpose a composite scaffold made of a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (semi-IPN) and fibrin loaded growth factors (GFs), such as VEGF and bFGF, was manufactured using spray, phase-inversion technique. To evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, three different scaffold formulations were developed as follows: 1) bare PEtU-PDMS; 2) PEtU-PDMS/Fibrin; and 3) PEtU-PDMS/Fibrin + GFs. Scaffolds were characterized in vitro respect to their morphology, VEGF and bFGF release kinetics and bioactivity. The induction of in vivo angiogenesis after subcutaneous and ischemic hind limb scaffold implantation in adult Wistar rats was evaluated at 7 and 14 days by immunohistological analysis (IHA), while Laser Doppler Perfusion Imaging (LDPI) was performed in the hind limbs at 0, 3, 7, 10 and 14 days. IHA of subcutaneously implanted samples showed that at 7 and 14 days the PEtU-PDMS/Fibrin + GFs scaffold induced a statistically significant increase in number of capillaries compared to bare PEtU-PDMS scaffold. IHA of ischemic hind limb showed that at 14 days the capillary number induced by PEtU-PDMS/Fibrin + GFs scaffolds was higher than that of PEtU-PDMS/Fibrin scaffolds. Moreover, at both time-points PEtU-PDMS/Fibrin scaffolds induced a significant increase in number of capillaries compared to bare PEtU-PDMS scaffolds. LDPI showed that at 10 and 14 days the ischemic/non-ischemic blood perfusion ratio was significantly greater in the PEtU-PDMS/Fibrin + GFs than in the other scaffolds. In conclusion, this study showed that the semi-IPN composite scaffold acting as a pro-angiogenic GFs delivery system has therapeutic potential for the local treatment of ischemic tissue and wound healing.

Inhibition of Na+/K+ ATPase potentiates synaptic transmission in tactile sensory neurons of the leech

Increasing evidence indicates that modulation of Na+/K+ ATPase activity is involved in forms of neuronal and synaptic plasticity. In tactile (T) neurons of the leech Hirudo medicinalis, Na+/K+ ATPase is the main determinant of the afterhyperpolarization (AHP), which characterizes the firing of these mechanosensory neurons. Previously, it has been reported that cAMP (3′,5′‐cyclic adenosine monophosphate), which mediates the effects of serotonin (5HT) in some forms of learning in the leech, negatively modulates Na+/K+ ATPase activity, thereby reducing the AHP amplitude in T neurons. Here, we show that a transient inhibition of Na+/K+ ATPase can affect the synaptic connection between two ipsilateral T neurons. Bath application of 10 nm dihydroouabain (DHO), an ouabain analogue, causes an increase in the amplitude of the synaptic potential (SP) recorded in the postsynaptic element when a test stimulus is applied in the presynaptic neuron. Iontophoretic injection of cAMP into the presynaptic T neuron also produces an increase of SP. Simulations carried out by using a computational model of the T neuron suggest that a reduction of the pump rate and a consequent depression of the AHP might facilitate the conduction of action potentials to the synaptic terminals. Moreover, nearly intact leeches injected with 10 nm DHO respond with a swimming episode more quickly to an electrical stimulation, which selectively activates T neurons exhibiting sensitization of swimming induction. Collectively, our results show that inhibition of Na+/K+ ATPase is critical for short‐term plasticity.

Study protocol: safety and efficacy of propranolol in newborns with Retinopathy of Prematurity (PROP-ROP): ISRCTN18523491

BackgroundDespite new therapeutic approaches have improved the prognosis of newborns with retinopathy of prematurity (ROP), an unfavourable structural and functional outcome still remains high. There is high pressure to develop new drugs to prevent and treat ROP. There is increasing enthusiasm for anti-VEGF drugs, but angiogenic inhibitors selective for abnormal blood vessels would be considered as an optimal treatment.In an animal experimental model of proliferative retinopathy, we have recently demonstrated that the pharmacological blockade of beta-adrenoreceptors improves retinal neovascularization and blood retinal barrier breakdown consequent to hypoxia. The purpose of this study is to evaluate the propranolol administration in preterm newborns suffering from a precocious phase of ROP in terms of safety and efficacy in counteracting the progression of retinopathy.Methods/DesignPreterm newborns (gestational age at birth lower than 32 weeks) with stage 2 ROP (zone II-III without plus) will be randomized, according to their gestational age, to receive propranolol added to standard treatment (treatment adopted by the ETROP Cooperative Group) or standard treatment alone. Propranolol will be administered until retinal vascularization will be completely developed, but not more than 90 days. Forty-four participants will be recruited into the study. To evaluate the safety of propranolol administration, cardiac and respiratory parameters will be continuously monitored. Blood samplings will be performed to check renal, liver and metabolic balance. To evaluate the efficacy of propranolol, the progression of the disease, the number of laser treatments or vitrectomies, the incidence of retinal detachment or blindness, will be evaluated by serial ophthalmologic examinations. Visual function will be evaluated by means of behavioural standardized tests.DiscussionThis pilot study is the first research that explores the possible therapeutic role of beta blockers in ROP. The objective of this research is highly ambitious: to find a treatment simple, inexpensive, well tolerated and with few adverse effects, able to counteract one of the major complications of the prematurity. Any favourable results of this research could open new perspectives and original scenarios about the treatment or the prevention of this and other proliferative retinopathies.Trial RegistrationCurrent Controlled Trials ISRCTN18523491; ClinicalTrials.gov Identifier NCT01079715; EudraCT Number 2010-018737-21

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.

Effects of Somatostatin Analogues on Retinal Angiogenesis in a Mouse Model of Oxygen-Induced Retinopathy: Involvement of the Somatostatin Receptor Subtype 2

PURPOSE To determine whether selective activation or blockade of the somatostatin (SRIF) receptor 2 (sst(2)) with two SRIF analogues, octreotide and D-Tyr(8) cyanamid 154806 (CYN), influences retinal vascularization and levels of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and -2 in a mouse model of oxygen-induced retinopathy (OIR). METHODS Wild-type (WT), sst(1)-knockout (KO), and sst(2)-KO mice were used. The OIR model was used to test the effects of octreotide and CYN administered subcutaneously. Retinopathy was assessed by a retinal scoring system using fluorescein-perfused retinal wholemounts. Retinal levels of VEGF, VEGFR-1, and -2 were evaluated with quantitative RT-PCR, Western blot, and ELISA. RESULTS In both WT and sst(1)-KO mice, OIR-induced neovascularization was reduced by octreotide, whereas it was increased by CYN. No effects of octreotide and CYN on retinal neovascularization were observed in sst(2)-KO retinas. Hypoxia upregulated the expression of VEGF and its receptors. Compared with WT retinas, the increase in VEGF, but not in VEGF receptors, was less pronounced in sst(1)-KO retinas in which sst(2) is known to be overexpressed. The hypoxia-induced increase in VEGF and its receptors was affected by SRIF analogues, with ameliorative effects of octreotide and worsening effects of CYN, which were more pronounced in the presence of sst(2) overexpression. CONCLUSIONS These data suggest that sst(2) regulates angiogenic responses to the hypoxic insult through a modulation of retinal levels of VEGF and its receptors. The present results further support the possibility of the use of sst(2)-selective ligands in the treatment of retinopathy.

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.

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.