The ß3 adrenoceptor in proliferative retinopathies: "Cinderella" steps out of its family shadow.

In the retina, hypoxic condition leads to overgrowing leaky vessels resulting in altered metabolic supply that may cause impaired visual function. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the retinal response to hypoxia by activating the transcription of numerous target genes, including vascular endothelium growth factor, which acts as a major player in retinal angiogenesis. In the present review, oxygen urge by the retina and its oxygen sensing systems including HIF-1 are discussed in respect to the role of the beta-adrenergic receptors (ß-ARs) and their pharmacologic manipulation in the vascular response to hypoxia. In the ß-AR family, ß1- and ß2-AR have long been attracting attention because their pharmacology is intensely used for human health, while ß3-AR, the third and last cloned receptor is no longer increasingly emerging as an attractive target for drug discovery. Here, ß3-AR, a main character in several organs including the heart, the adipose tissue and the urinary bladder, but so far a supporting actor in the retina, has been thoroughly examined in respect to its function in retinal response to hypoxia. In particular, its oxygen dependence has been taken as a key indicator of ß3-AR involvement in HIF-1-mediated responses to oxygen. Hence, the possibility of ß3-AR transcription by HIF-1 has been discussed from early circumstantial evidence to the recent demonstration that ß3-AR acts as a novel HIF-1 target gene by playing like a putative intermediary between oxygen levels and retinal vessel proliferation. Thus, targeting ß3-AR may implement the therapeutic armamentarium against neovascular pathologies of the eye.

Fetal Oxygenation from the 23rd to the 36th Week of Gestation Evaluated through the Umbilical Cord Blood Gas Analysis.

The embryo and fetus grow in a hypoxic environment. Intrauterine oxygen levels fluctuate throughout the pregnancy, allowing the oxygen to modulate apparently contradictory functions, such as the expansion of stemness but also differentiation. We have recently demonstrated that in the last weeks of pregnancy, oxygenation progressively increases, but the trend of oxygen levels during the previous weeks remains to be clarified. In the present retrospective study, umbilical venous and arterial oxygen levels, fetal oxygen extraction, oxygen content, CO2, and lactate were evaluated in a cohort of healthy newborns with gestational age < 37 weeks. A progressive decrease in pO2 levels associated with a concomitant increase in pCO2 and reduction in pH has been observed starting from the 23rd week until approximately the 33-34th week of gestation. Over this period, despite the increased hypoxemia, oxygen content remains stable thanks to increasing hemoglobin concentration, which allows the fetus to become more hypoxemic but not more hypoxic. Starting from the 33-34th week, fetal oxygenation increases and ideally continues following the trend recently described in term fetuses. The present study confirms that oxygenation during intrauterine life continues to vary even after placenta development, showing a clear biphasic trend. Fetuses, in fact, from mid-gestation to near-term, become progressively more hypoxemic. However, starting from the 33-34th week, oxygenation progressively increases until birth. In this regard, our data suggest that the placenta is the hub that ensures this variable oxygen availability to the fetus, and we speculate that this biphasic trend is functional for the promotion, in specific tissues and at specific times, of stemness and intrauterine differentiation.

Pharmacotherapy and Nutritional Supplements for Neovascular Eye Diseases.

In this review, we aim to provide an overview of the recent findings about the treatment of neovascular retinal diseases. The use of conventional drugs and nutraceuticals endowed with antioxidant and anti-inflammatory properties that may support conventional therapies will be considered, with the final aim of achieving risk reduction (prevention) and outcome improvement (cooperation between treatments) of such sight-threatening proliferative retinopathies. For this purpose, we consider a medicinal product one that contains well-defined compound(s) with proven pharmacological and therapeutic effects, usually given for the treatment of full-blown diseases. Rarely are prescription drugs given for preventive purposes. A dietary supplement refers to a compound (often an extract or a mixture) used in the prevention or co-adjuvant treatment of a given pathology. However, it must be kept in mind that drug-supplement interactions may exist and might affect the efficacy of certain drug treatments. Moreover, the distinction between medicinal products and dietary supplements is not always straightforward. For instance, melatonin is formulated as a medicinal product for the treatment of sleep and behavioral problems; at low doses (usually below 1 mg), it is considered a nutraceutical, while at higher doses, it is sold as a psychotropic drug. Despite their lower status with respect to drugs, increasing evidence supports the notion of the beneficial effects of dietary supplements on proliferative retinopathies, a major cause of vision loss in the elderly. Therefore, we believe that, on a patient-by-patient basis, the administration of nutraceuticals, either alone or in association, could benefit many patients, delaying the progression of their disease and likely improving the efficacy of pharmaceutical drugs.

ß3-Adrenoceptor, a novel player in the round-trip from neonatal diseases to cancer: Suggestive clues from embryo.

The role of the ß-adrenoceptors (ß-ARs) in hypoxia-driven diseases has gained visibility after the demonstration that propranolol promotes the regression of infantile hemangiomas and ameliorates the signs of retinopathy of prematurity (ROP). Besides the role of ß2-ARs, preclinical studies in ROP have also revealed that ß3-ARs are upregulated by hypoxia and that they are possibly involved in retinal angiogenesis. In a sort of figurative round trip, peculiarities typical of ROP, where hypoxia drives retinal neovascularization, have been then translated to cancer, a disease equally characterized by hypoxia-driven angiogenesis. In this step, investigating the role of ß3-ARs has taken advantage of the assumption that cancer growth uses a set of strategies in common with embryo development. The possibility that hypoxic induction of ß3-ARs may represent one of the mechanisms through which primarily embryo (and then cancer, as an astute imitator) adapts to grow in an otherwise hostile environment, has grown evidence. In both cancer and embryo, ß3-ARs exert similar functions by exploiting a metabolic shift known as the Warburg effect, by acquiring resistance against xenobiotics, and by inducing a local immune tolerance. An additional potential role of ß3-AR as a marker of stemness has been suggested by the finding that its antagonism induces cancer cell differentiation evoking that ß3-ARs may help cancer to grow in a nonhospital environment, a strategy also exploited by embryos. From cancer, the round trip goes back to neonatal diseases for which new possible interpretative keys and potential pharmacological perspectives have been suggested.

A Topical Formulation of Melatoninergic Compounds Exerts Strong Hypotensive and Neuroprotective Effects in a Rat Model of Hypertensive Glaucoma.

Melatonin is of great importance for regulating several eye processes, including pressure homeostasis. Melatonin in combination with agomelatine has been recently reported to reduce intraocular pressure (IOP) with higher efficacy than each compound alone. Here, we used the methylcellulose (MCE) rat model of hypertensive glaucoma, an optic neuropathy characterized by the apoptotic death of retinal ganglion cells (RGCs), to evaluate the hypotensive and neuroprotective efficacy of an eye drop nanomicellar formulation containing melatonin/agomelatine. Eye tissue distribution of melatonin/agomelatine in healthy rats was evaluated by HPLC/MS/MS. In the MCE model, we assessed by tonometry the hypotensive efficacy of melatonin/agomelatine. Neuroprotection was revealed by electroretinography; by levels of inflammatory and apoptotic markers; and by RGC density. The effects of melatonin/agomelatine were compared with those of timolol (a beta blocker with prevalent hypotensive activity) or brimonidine (an alpha 2 adrenergic agonist with potential neuroprotective efficacy), two drugs commonly used to treat glaucoma. Both melatonin and agomelatine penetrate the posterior segment of the eye. In the MCE model, IOP elevation was drastically reduced by melatonin/agomelatine with higher efficacy than that of timolol or brimonidine. Concomitantly, gliosis-related inflammation and the Bax-associated apoptosis were partially prevented, thus leading to RGC survival and recovered retinal dysfunction. We suggest that topical melatoninergic compounds might be beneficial for ocular health.

The urokinase-type plasminogen activator system as drug target in retinitis pigmentosa: New pre-clinical evidence in the rd10 mouse model.

Retinitis pigmentosa (RP) is characterized by progressive loss of vision due to photoreceptor degeneration leading to secondary inflammation. The urokinase-type plasminogen activator (uPA) system contributes to retinal inflammation, but its role in RP is unknown. In the rd10 mouse model of RP, we addressed this question with the use of the peptide UPARANT designed to interact with the uPA system. UPARANT was systemically administered from post-natal day (PD) 10 to PD30 when its efficacy in RP rescue was investigated using electroretinographic recordings, Western blot and immunocytochemistry. Temporal profile of protein expression in the uPA system was also investigated. UPARANT reduced both Müller cell gliosis and up-regulated levels of inflammatory markers and exerted major anti-apoptotic effects without influencing the autophagy cascade. Rescue from retinal cell degeneration was accompanied by improved retinal function. No scotopic phototransduction was rescued in the UPARANT-treated animals as determined by the kinetic analysis of rod-mediated a-waves and confirmed by rod photoreceptor markers. In contrast, the cone photopic b-wave was recovered and its rescue was confirmed in the whole mounts using cone arrestin antibody. Investigation of the uPA system regulation over RP progression revealed extremely low levels of uPA and its receptor uPAR both of which were recovered by HIF-1α stabilization indicating that HIF-1 regulates the expression of the uPA/uPAR gene in the retina. Ameliorative effects of UPARANT were likely to occur through an inhibitory action on up-regulated activity of the αvß3 integrin/Rac1 pathway that was suggested as a novel target for the development of therapeutic approaches against RP.

Inhibiting the urokinase-type plasminogen activator receptor system recovers STZ-induced diabetic nephropathy.

The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvß3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvß3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvß3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches.

Dynamics of blood circulation during diving in the bottlenose dolphin (Tursiops truncatus): the role of the retia mirabilia.

The retia mirabilia are vascular nets composed of small vessels dispersed among numerous veins, allowing blood storage, regulation of flow and pressure damping effects. Here, we investigated their potential role during the diving phase of the bottlenose dolphin (Tursiops truncatus). To this effect, the whole vertebral retia mirabilia of a series of dolphins were removed during post-mortem analysis and examined to assess vessel diameters, and estimate vascular volume and flow rate. We formulated a new hemodynamic model to help clarify vascular dynamics throughout the diving phase, based on the total blood volume of a bottlenose dolphin, and using data available about the perfusion of the main organs and body systems. We computed the minimum blood perfusion necessary to the internal organs, and the stroke volume and cardiac output during the surface state. We then simulated breath-holding conditions and perfusion of the internal organs under the diving-induced bradycardia and reduction of stroke volume and cardiac output, using 10 beats min-1 as the limit for the heart rate for an extended dive of over 3 min. Within these simulated conditions, the retia mirabilia play a vital role as reservoirs of oxygenated blood that permit functional performances and survival of the heart and brain. Our theoretical model, based on the actual blood capacity of the retia mirabilia and available data on organ perfusion, considers the dynamic trend of vasoconstriction during the diving phase and may represent a baseline for future studies on the diving physiology of dolphins and especially for the blood supply to their brain.

Molecular and Cellular Mechanisms Underlying Somatostatin-Based Signaling in Two Model Neural Networks, the Retina and the Hippocampus.

Neural inhibition plays a key role in determining the specific computational tasks of different brain circuitries. This functional "braking" activity is provided by inhibitory interneurons that use different neurochemicals for signaling. One of these substances, somatostatin, is found in several neural networks, raising questions about the significance of its widespread occurrence and usage. Here, we address this issue by analyzing the somatostatinergic system in two regions of the central nervous system: the retina and the hippocampus. By comparing the available information on these structures, we identify common motifs in the action of somatostatin that may explain its involvement in such diverse circuitries. The emerging concept is that somatostatin-based signaling, through conserved molecular and cellular mechanisms, allows neural networks to operate correctly.

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen-induced retinopathy: beneficial effect of the absence of AQP4.

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1α (HIF-1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1α to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia.

Propranolol 0.1% eye micro-drops in newborns with retinopathy of prematurity: a pilot clinical trial.

BACKGROUND: Oral propranolol reduces retinopathy of prematurity (ROP) progression, although not safely. This study evaluated safety and efficacy of propranolol eye micro-drops in preterm newborns with ROP. METHODS: A multicenter open-label trial, planned according to the Simon optimal two-stage design, was performed to analyze safety and efficacy of propranolol micro-drops in newborns with stage 2 ROP. To this end, hemodynamic and respiratory parameters were monitored, and blood samples were collected weekly, for 3 wk. Propranolol plasma levels were also monitored. The progression of the disease was evaluated with serial ophthalmologic examinations. RESULTS: Twenty-three newborns were enrolled. Since the fourth of the first 19 newborns enrolled in the first stage of the study showed a progression to stage 2 or 3 with plus, the second stage was prematurely discontinued. Even though the objective to complete the second stage was not achieved, the percentage of ROP progression (26%) was similar to that obtained previously with oral propranolol administration. However, no adverse effects were observed and propranolol plasma levels were significantly lower than those measured after oral administration. CONCLUSION: Propranolol 0.1% eye micro-drops are well tolerated, but not sufficiently effective. Further studies are required to identify the optimal dose and administration schedule.

Study protocol: safety and efficacy of propranolol 0.2% eye drops in newborns with a precocious stage of retinopathy of prematurity (DROP-ROP-0.2%): a multicenter, open-label, single arm, phase II trial.

BACKGROUND: Retinopathy of prematurity (ROP) still represents one of the leading causes of visual impairment in childhood. Systemic propranolol has proven to be effective in reducing ROP progression in preterm newborns, although safety was not sufficiently guaranteed. On the contrary, topical treatment with propranolol eye micro-drops at a concentration of 0.1% had an optimal safety profile in preterm newborns with ROP, but was not sufficiently effective in reducing the disease progression if administered at an advanced stage (during stage 2). The aim of the present protocol is to evaluate the safety and efficacy of propranolol 0.2% eye micro-drops in preterm newborns at a more precocious stage of ROP (stage 1). METHODS: A multicenter, open-label, phase II, clinical trial, planned according to the Simon optimal two-stage design, will be performed to analyze the safety and efficacy of propranolol 0.2% eye micro-drops in preterm newborns with stage 1 ROP. Preterm newborns with a gestational age of 23-32 weeks, with a stage 1 ROP will receive propranolol 0.2% eye micro-drops treatment until retinal vascularization has been completed, but for no longer than 90 days. Hemodynamic and respiratory parameters will be continuously monitored. Blood samplings checking metabolic, renal and liver functions, as well as electrocardiogram and echocardiogram, will be periodically performed to investigate treatment safety. Additionally, propranolol plasma levels will be measured at the steady state, on the 10th day of treatment. To assess the efficacy of topical treatment, the ROP progression from stage 1 ROP to stage 2 or 3 with plus will be evaluated by serial ophthalmologic examinations. DISCUSSION: Propranolol eye micro-drops could represent an ideal strategy in counteracting ROP, because it is definitely safer than oral administration, inexpensive and an easily affordable treatment. Establishing the optimal dosage and treatment schedule is to date a crucial issue. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02504944, registered on July 19, 2015, updated July 12, 2016. EudraCT Number 2014-005472-29.

ß3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of ß1- and ß2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of ß3-adrenergic receptor (ß3-AR) in mouse kidney. The ß3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that ß3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the ß3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of ß3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of ß3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of ß3-AR stimulation in the kidney. Hence, ß3-AR agonism might be useful to bypass AVPR2-inactivating mutations.

Glio-vascular modifications caused by Aquaporin-4 deletion in the mouse retina.

Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition. AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1ß and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role of AQP4 in the perivascular domain in the retina and its possible implications in the pathogenesis of retinal vascular diseases and of Neuromyelitis Optica, a human disease characterized by anti-AQP4 auto-antibodies.

Infantile hemangiomas, retinopathy of prematurity and cancer: a common pathogenetic role of the ß-adrenergic system.

The serendipitous demonstration that the nonselective ß-adrenergic receptor (ß-AR) antagonist propranolol promotes the regression of infantile hemangiomas (IHs) aroused interest around the involvement of the ß-adrenergic system in angiogenic processes. The efficacy of propranolol was related to the ß2-AR blockade and the consequent inhibition of the production of vascular endothelial growth factor (VEGF), suggesting the hypothesis that propranolol could also be effective in treating retinopathy of prematurity (ROP), a retinal pathology characterized by VEGF-induced neoangiogenesis. Consequent to the encouraging animal studies, a pilot clinical trial showed that oral propranolol protects newborns from ROP progression, even though this treatment is not sufficiently safe. Further, animal studies clarified the role of ß3-ARs in the development of ROP and, together with several preclinical studies demonstrating the key role of the ß-adrenergic system in tumor progression, vascularization, and metastasis, prompted us to also investigate the participation of ß3-ARs in tumor growth. The aim of this review is to gather the recent findings on the role of the ß-adrenergic system in IHs, ROP, and cancer, highlighting the fact that these different pathologies, triggered by different pathogenic noxae, share common pathogenic mechanisms characterized by the presence of hypoxia-induced angiogenesis, which may be contrasted by targeting the ß-adrenergic system. The mechanisms characterizing the pathogenesis of IHs, ROP, and cancer may also be active during the fetal-neonatal development, and a great contribution to the knowledge on the role of ß-ARs in diseases characterized by chronic hypoxia may come from research focusing on the fetal and neonatal period.

Effect of intermediate ZrO2-CaO coatings deposited by cold thermal spraying on the titanium-porcelain bond in dental restorations.

STATEMENT OF PROBLEM: Metal ceramic systems are used for the majority of dental crowns and fixed dental prostheses. However, problems with porcelain bonding are encountered when titanium is used as the substrate. PURPOSE: The purpose of this study was to evaluate the effect of intermediate calcium oxide-stabilized zirconia (ZrO2-CaO) coatings deposited by cold thermal spraying on the titanium-porcelain bonding in dental restorations. MATERIAL AND METHODS: Two different types of ZrO2-CaO coatings obtained by oxyacetylene cold thermal spraying deposition were applied on commercially pure titanium bars before adding the porcelain layer. Type 1 was obtained by directly spraying the ZrO2-CaO powder on the titanium substrate. Type 2 was obtained by spraying a bond coat of nickel-aluminum-molybdenum alloy before spraying the ZrO2-CaO powder. Three-point bend tests according to International Organization of Standardization 9693-1:2012 were carried out to evaluate the debonding strength for the ZrO2-CaO-coated specimens (types 1 and 2) in comparison with a noncoated group (control), which received a traditional bonder-based adhesive technique. The results were compared with ANOVA, followed by the Student-Newman-Keuls test for pairwise comparisons. Scanning electron microscopy and energy dispersion spectroscopy were used to examine the interfacial properties and the failure mode of each group. RESULTS: Mean (±standard deviation) debonding strength values for type 1 coating (25.97 ±2.53 MPa) and control (23.51 ±2.94 MPa) were near the acceptable lower limit of 25 MPa indicated by the International Organization of Standardization 9693-1:2012 and were not significantly different (Student-Newman-Keuls test, P>.05). Type 2 coating produced an improved titanium-porcelain bonding (debonding strength=39.47 ±4.12 MPa), significantly higher than both type 1 (Student-Newman-Keuls test, P<.05) and control (Student-Newman-Keuls test, P<.05). Scanning electron microscopy-energy dispersion spectroscopy analysis confirmed these findings, which revealed a predominant cohesive failure mode for type 2. CONCLUSIONS: An intermediate coating layer of ZrO2-CaO plus a substrate of bonding nickel-aluminum-molybdenum alloy applied by oxyacetylene cold thermal spraying deposition provided an improved titanium-porcelain bond.

A promising case of preclinical-clinical translation: ß-adrenoceptor blockade from the oxygen-induced retinopathy model to retinopathy of prematurity.

Although compartmentalization of the eye seems to promote its experimental manipulation, drug penetration to its posterior part is severely limited by hard barriers thus hindering drug development for eye diseases. In particular, angiogenesis-related retinal diseases share common mechanisms and are responsible for the majority of cases of blindness. Their prevalence is globally increasing mostly because of the increased incidence of systemic pathologies in the adult. Despite the number of preclinical findings demonstrating the efficacy of novel treatments, therapy of retinal neovascular diseases still remains confined to intravitreal anti-vascular endothelial growth factor treatments with some extension to anti-inflammatory therapy. In the mare magnum of preclinical findings aimed to develop novel avenues for future therapies, most compounds, despite their efficacy in experimental models, do not seem to meet the criteria for their therapeutic application. In particular, the groove between preclinical findings and their clinical application increases instead of decreasing and the attempt to bridging the gap between them creates intense frustration and a sense of defeat. In this complex scenario, we will discuss here the role that overactivation of the sympathetic system plays in retinal vessel proliferation in response to hypoxia using the oxygen-induced retinopathy (OIR) model. The potential application of the beta-adrenoceptor (ß-AR) blockade with propranolol to the treatment of retinopathy of prematurity will be also discussed in light of preclinical findings in the OIR model and clinical trials using propranolol in preterm infants either per os or as eye drops.

Propranolol: a new pharmacologic approach to counter retinopathy of prematurity progression.

Despite the evident progress in neonatal medicine, retinopathy of prematurity (ROP) remains a serious threat to the vision of premature infants, due to a still partial understanding of the mechanisms underlying the development of this disease and the lack of drugs capable of arresting its progression. Although ROP is a multifactorial disease, retinal vascularization is strictly dependent on oxygen concentration. The exposition of the retina of a preterm newborn, still incompletely vascularized, to an atmosphere relatively hyperoxic, as the extrauterine environment, induces the downregulation of proangiogenic factors and therefore the interruption of vascularization (first ischemic phase of ROP). However, over the following weeks, the growing metabolic requirement of this ischemic retina produces a progressive hypoxia that specularly promotes the surge of proangiogenic factors, finally leading to proliferative retinopathy (second proliferative phase of ROP). The demonstration that the noradrenergic system is actively involved in the coupling between hypoxia and the induction of vasculogenesis paved the way for a pharmacologic intervention aimed at counteracting the interaction of noradrenaline with specific receptors and consequently the progression of ROP. A similar trend has been observed in infantile hemangiomas, the most common vascular lesion of childhood induced by pre-existing hypoxia, which shares similar characteristics with ROP. The fact that propranolol, an unselective antagonist of ß1/2 adrenoceptors, counteracts the growth of infantile hemangiomas, suggested the idea of testing the efficacy of propranolol in infants with ROP. From preclinical studies, ongoing clinical trials demonstrated that topical administration of propranolol likely represents the optimal approach to reconcile its efficacy and maximum safety. Given the strict relationship between vessels and neurons, recovering retinal vascularization with propranolol may add further efficacy to prevent retinal dysfunction. In conclusion, the strategy of contrasting precociously the progression of the disease appears to be more advantageous than the current wait-and-see therapeutic approach, which instead is mainly focused on avoiding retinal detachment.

ß3-adREnoceptor Analysis in CORD Blood of Neonates (ß3 RECORD): Study Protocol of a Pilot Clinical Investigation.

Background and Objective: The embryo and the fetus develop in a physiologically hypoxic environment, where vascularization is sustained by HIF-1, VEGF, and the ß-adrenergic system. In animals, ß3-adrenoceptors (ß3-ARs), up-regulated by hypoxia, favor global fetal wellness to such an extent that most diseases related to prematurity are hypothesized to be induced or aggravated by a precocious ß3-AR down-regulation, due to premature exposure to a relatively hyperoxic environment. In animals, ß3-AR pharmacological agonism is currently investigated as a possible new therapeutic opportunity to counteract oxygen-induced damages. Our goal is to translate the knowledge acquired in animals to humans. Recently, we have demonstrated that fetuses become progressively more hypoxemic from mid-gestation to near-term, but starting from the 33rd-34th week, oxygenation progressively increases until birth. The present paper aims to describe a clinical research protocol, evaluating whether the expression level of HIF-1, ß3-ARs, and VEGF is modulated by oxygen during intrauterine and postnatal life, in a similar way to animals. Materials and Methods: In a prospective, non-profit, single-center observational study we will enroll 100 preterm (group A) and 100 full-term newborns (group B). We will collect cord blood samples (T0) and measure the RNA expression level of HIF-1, ß3-ARs, and VEGF by digital PCR. In preterms, we will also measure gene expression at 48-72h (T1), 14 days (T2), and 30 days (T3) of life and at 40 ± 3 weeks of post-menstrual age (T4), regardless of the day of life. We will compare group A (T0) vs. group B (T0) and identify any correlations between the values obtained from serial samples in group A and the clinical data of the patients. Our protocol has been approved by the Pediatric Ethical Committee for Clinical Research of the Tuscany region (number 291/2022). Expected Results: The observation that in infants, the HIF-1/ß3-ARs/VEGF axis shows similar modulation to that of animals could suggest that ß3-ARs also promote fetal well-being in humans.