Massimo Dal Monte

The pathophysiology of retinopathy of prematurity : an update of previous and recent knowledge

Retinopathy of prematurity (ROP) is a disease that can cause blindness in very low birthweight infants. The incidence of ROP is closely correlated with the weight and the gestational age at birth. Despite current therapies, ROP continues to be a highly debilitating disease. Our advancing knowledge of the pathogenesis of ROP has encouraged investigations into new antivasculogenic therapies. The purpose of this article is to review the findings on the pathophysiological mechanisms that contribute to the transition between the first and second phases of ROP and to investigate new potential therapies. Oxygen has been well characterized for the key role that it plays in retinal neoangiogenesis. Low or high levels of pO2 regulate the normal or abnormal production of hypoxia‐inducible factor 1 and vascular endothelial growth factors (VEGF), which are the predominant regulators of retinal angiogenesis. Although low oxygen saturation appears to reduce the risk of severe ROP when carefully controlled within the first few weeks of life, the optimal level of saturation still remains uncertain. IGF‐1 and Epo are fundamentally required during both phases of ROP, as alterations in their protein levels can modulate disease progression. Therefore, rhIGF‐1 and rhEpo were tested for their abilities to prevent the loss of vasculature during the first phase of ROP, whereas anti‐VEGF drugs were tested during the second phase. At present, previous hypotheses concerning ROP should be amended with new pathogenetic theories. Studies on the role of genetic components, nitric oxide, adenosine, apelin and β‐adrenergic receptor have revealed new possibilities for the treatment of ROP. The genetic hypothesis that single‐nucleotide polymorphisms within the β‐ARs play an active role in the pathogenesis of ROP suggests the concept of disease prevention using β‐blockers. In conclusion, all factors that can mediate the progression from the avascular to the proliferative phase might have significant implications for the further understanding and treatment of ROP.

Purification and characterization of two odorant-binding proteins from nasal tissue of rabbit and pig

1. Soluble proteins showing binding activity to 2-isobutyl-3-methoxypyrazine have been purified to homogeneity from rabbit and pig nasal tissue; their characteristics are similar to the bovine odorant-binding protein and are to be considered members of the same family. 2. The rabbit protein is a homodimer with subunits of Mr 19k and an isoelectric point of 4.7, whereas the pig protein appears to consist of a single polypeptide chain of Mr 22k and an isoelectric point of 4.2. 3. Both proteins bind 2-isobutyl-3-methoxypyrazine with dissociation constants in the micromolar range. 4. Antibodies against the bovine OBP react well with the rabbit protein, and slightly with the porcine one.

Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

Somatostatin (SRIF) and SRIF receptors in the mouse retina

In the retina, somatostatin (SRIF) acts as a neuromodulator by interacting with specific SRIF subtype (sst) receptors. The aim of this study was to detect mRNAs for sst(1-5) receptors by semiquantitative RT-PCR and to determine the cellular localization of either SRIF or individual SRIF receptor immunoreactivities. Size, density and absolute number of immunolabeled somata were measured using computer-assisted image analysis. With RT-PCR we found that all five sst receptor mRNAs were expressed, with highest levels of sst(2) and sst(4) receptors. SRIF immunolabeling was localized to sparse-occurring amacrine cells in the inner nuclear layer (INL) and to displaced amacrine cells in the ganglion cell layer (GCL). sst(2A) receptors were localized to protein kinase- (PKC) immunoreactive (IR) rod bipolar cells, calbindin- (CaBP-) IR horizontal cells, tyrosine hydroxylase- (TH-) IR amacrine cells and glycinergic amacrine cells. None of the sst(2A)-IR amacrine cells were found to express parvalbumin (PV) immunoreactivity. sst(4) receptor immunolabeling was localized to CaBP-IR and CaBP-non-IR cells in the GCL that originated long process bundles in the GC axon layer. These cells were not observed after optic nerve transection and they were therefore interpreted as ganglion cells. Quantitative analysis showed that all of the PKC-IR rod bipolar cells, CaBP-IR horizontal cells, and TH-IR amacrine cells and 5% of the glycinergic amacrine cells expressed sst(2A) receptors. In addition, 4-6% of the putative ganglion cells expressed sst(4) receptors. The localization of SRIF to sparse-occurring retinal neurons, together with the widespread expression of sst(2A) and sst(4) receptors suggests that SRIF acts at multiple levels of retinal circuitry. These results provide a database for investigations of the functional retinal networks in mice with genetic alterations of somatostatinergic transmission.

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

Genetic deletion of somatostatin receptor 1 alters somatostatinergic transmission in the mouse retina

In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst(1-5)). Among them, the sst1 receptor has been localised to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localisation and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localised to SRIF-containing amacrine cells, whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF, whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain.

Somatostatin inhibits potassium-evoked glutamate release by activation of the sst2 somatostatin receptor in the mouse retina

In the mammalian retina, somatostatin (SRIF-14) acts through distinct receptor subtypes (sst1–5). Among them, sst2 has been localized to numerous retinal cells, including photoreceptors and rod bipolar cells (RBCs). The specific role of sst2 in the retina is largely undetermined. In this study, we characterized retinas of mice with targeted deletion of sst2 (sst2 KO) and we investigated functions of sst2 in respect to its possible modulation of glutamate (GLU) release, as measured by HPLC. In contrast with wild-type (WT) mice, sst2 mRNA and sst2A immunoreactivity were no longer detectable in the retina of sst2 KO mice. In retinal explants of WT mice, SRIF and its analogue octreotide that displays high selectivity for sst2, similarly reduced the evoked release of GLU without affecting its basal level. In sst2 KO retinas, SRIF or octreotide did not affect GLU release indicating that they act at sst2. Unexpectedly, the compound CYN-154806, although introduced as the first potent sst2 antagonist, reduced the evoked release of GLU with equipotency to SRIF and octreotide. Its inhibitory effect was no longer observed in sst2 KO retinas, indicating that this substance acts at sst2 receptors as an agonist. In conclusion, SRIF controls evoked release of GLU through sst2 receptors and this control may represent part of a mechanism by which SRIF regulates GLU concentration in the retina.

Oral propranolol for retinopathy of prematurity: Risks, safety concerns, and perspectives

OBJECTIVE To evaluate safety and efficacy of oral propranolol administration in preterm newborns affected by an early phase of retinopathy of prematurity (ROP). STUDY DESIGN Fifty-two preterm newborns with Stage 2 ROP were randomized to receive oral propranolol (0.25 or 0.5 mg/kg/6 hours) added to standard treatment or standard treatment alone. To evaluate safety of the treatment, hemodynamic and respiratory variables were continuously monitored, and blood samples were collected weekly to check for renal, liver, and metabolic balance. To evaluate efficacy of the treatment, the progression of the disease (number of laser treatments, number of bevacizumab treatments, and incidence of retinal detachment) was evaluated by serial ophthalmologic examinations, and plasma soluble E-selectin levels were measured weekly. RESULTS Newborns treated with propranolol showed less progression to Stage 3 (risk ratio 0.52; 95% CI 0.47-0.58, relative reduction of risk 48%) or Stage 3 plus (relative risk 0.42 95% CI 0.31-0.58, relative reduction of risk 58%). The infants required fewer laser treatments and less need for rescue treatment with intravitreal bevacizumab (relative risk 0.48; 95% CI 0.29-0.79, relative reduction of risk 52 %), a 100% relative reduction of risk for progression to Stage 4. They also had significantly lower plasma soluble E-selectin levels. However, 5 of the 26 newborns treated with propranolol had serious adverse effects (hypotension, bradycardia), in conjunction with episodes of sepsis, anesthesia induction, or tracheal stimulation. CONCLUSION This pilot study suggests that the administration of oral propranolol is effective in counteracting the progression of ROP but that safety is a concern.

Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices

Somatostatin‐14 [somatotropin release‐inhibiting factor (SRIF)] reduces hippocampal epileptiform activity but the contribution of its specific receptors (sst1−5) is poorly understood. We have focused on the role of sst1 and sst2 in mediating SRIF modulation of epilepsy using hippocampal slices of wild‐type (WT) and sst1 or sst2 knockout (KO) mice. Recordings of epileptiform discharge induced by Mg2+‐free medium with 4‐aminopyridine were performed from the CA3 region before and after the application of SRIF compounds. In WT mice, SRIF and the sst1 agonist CH‐275 reduce epilepsy whereas sst1 blockade with its antagonist SRA‐880 increases the bursting discharge. Activation of sst2 does not affect the bursting frequency unless its agonist octreotide is applied with SRA‐880, indicating that sst1 masks sst2‐mediated modulation of epilepsy. In sst1 KO mice: (i) the bursting frequency is lower than in WT; (ii) SRIF, CH‐275 and SRA‐880 are ineffective on epilepsy and (iii) octreotide is also devoid of effects, whereas blockade of sst2 with the antagonist d‐Tyr8 Cyn 154806 increases the bursting frequency. In sst2 KO mice, the SRIF ligand effects are similar to those in WT. In the whole hippocampus of sst1 KO mice, sst2 mRNA, protein and binding are higher than in WT and reverse transcription‐polymerase chain reaction of the CA3 subarea confirms an increase of the sst2 messenger. We conclude that sst1 mediates inhibitory actions of SRIF and that interactions between sst1 and sst2 may prevent sst2 modulation of epilepsy. We suggest that, in sst1 KO mice, activation of over‐expressed sst2 reduces the bursting frequency, indicating that sst2 density represents the rate‐limiting factor for sst2‐mediated modulation of epilepsy.

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