Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic ex vivo model.

Rubeosis Iridis (RI) is characterized by an increase in neovascularization and inflammation factors in the iris. During angiogenesis, the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a pivotal role in extracellular matrix remodeling, where uPAR regulates endothelial cell migration and proliferation through assembly with transmembrane receptors. Here, in the context of hypoxia-induced angiogenesis, the uPA/uPAR system blockage was investigated by using UPARANT in a novel ex vivo human iris organotypic angiogenesis assay. The effects of uPA/uPAR system antagonism in the humanized model of ocular pathologic angiogenesis were analyzed by sprouting angiogenesis and protein assays (western, dot blots, and co-immunoprecipitation) and correlated to vascular endothelial growth factor (VEGF) inhibition. Phosphoprotein and co-immunoprecipitation assay illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low-density lipoprotein receptor-related protein-1 (LRP-1), resulting in inhibition of ß-catenin-mediated angiogenesis in this model. The effects of uPA/uPAR system inhibition were focal to endothelial cells ex vivo. Comparison between human iris endothelial cells and human retinal endothelial revealed an endothelial-specific mechanism of ß-catenin-mediated angiogenesis inhibited by uPA/uPAR system blockage and not by VEGF inhibition. Collectively, these findings broaden the understanding of the effects of the uPA/uPAR system antagonism in the context of angiogenesis, revealing non-canonical ß-catenin downstream effects mediated by LRP-1/uPAR interaction.

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.

ß-Adrenoceptors in Cancer: Old Players and New Perspectives.

Distress, or negative stress, is known to considerably increase the incidence of several diseases, including cancer. There is indeed evidence from pre-clinical models that distress causes a catecholaminergic overdrive that, mainly through the activation of ß-adrenoceptors (ß-ARs), results in cancer cell growth and cancer progression. In addition, clinical studies have evidenced a role of negative stress in cancer progression. Moreover, plenty of data demonstrates that ß-blockers have positive effects in reducing the pro-tumorigenic activity of catecholamines, correlating with better outcomes in some type of cancers as evidenced by several clinical trials. Among ß-ARs, ß2-AR seems to be the main ß-AR subtype involved in tumor development and progression. However, there are data indicating that also ß1-AR and ß3-AR may be involved in certain tumors. In this chapter, we will review current knowledge on the role of the three ß-AR isoforms in carcinogenesis as well as in cancer growth and progression, with particular emphasis on recent studies that are opening new avenues in the use of ß-ARs as therapeutic targets in treating tumors.

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.

Morpho-functional analysis of the early changes induced in retinal ganglion cells by the onset of diabetic retinopathy: The effects of a neuroprotective strategy.

PURPOSE: Retinal ganglion cells (RGCs) are highly susceptible to diabetes-induced metabolic stress. This study describes the early responses of RGCs to hyperglycemia and examines the effects of the neuroprotective somatostatin analog octreotide (OCT). METHODS: Thy1-green fluorescent protein (GFP)-M transgenic mice were used, which express GFP in a number of RGCs. OCT was intravitreally injected in mice with streptozotocin (STZ)-induced diabetes. A longitudinal electroretinography (ERG) analysis was performed up to 2 weeks from STZ treatment. RGC density was measured and extensive morphometric analyses were performed on identified RGC subtypes. RESULTS: STZ treatment caused a decline of RGC function, which was counteracted by OCT. No differences in RGC density were recorded, indicating that impaired activity was unlikely to be related to RGC death. Different GFP-labeled RGC subtypes were identified and analyzed. Overall, large RGCs were mostly affected by diabetes and responded to OCT treatment, while those with smaller dendritic arborizations were less involved. Interestingly, depending on the complexity of the dendritic tree, OCT could completely rescue RGC morphometric parameters or increase the effects of hyperglycemia. CONCLUSIONS: There is an early response of RGCs to diabetes, which involves specific morpho-functional deficits but not overt cell death. OCT induces adaptive changes that, although different among RGC subtypes, contribute to RGC functionality in the presence of metabolic stress. These results highlight the importance of neuronal protection in the early phases of diabetic retinopathy, when cell loss has not yet started and RGC morphology can be preserved or adjusted to maintain RGC physiology.

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy.

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.

Endothelial Progenitor Cells: An Appraisal of Relevant Data from Bench to Bedside.

The mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow is well known to be present in several clinical settings, including acute coronary syndrome, heart failure, diabetes and peripheral vascular disease. The aim of this review was to explore the current literature focusing on the great opportunity that EPCs can have in terms of regenerative medicine.

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.

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.

Autophagy-mediated neuroprotection induced by octreotide in an ex vivo model of early diabetic retinopathy.

Neuronal injury plays a major role in diabetic retinopathy (DR). Our hypothesis was that the balance between neuronal death and survival may depend on a similar equilibrium between apoptosis and autophagy and that a neuroprotectant may act by influencing this equilibrium. Ex vivo mouse retinal explants were treated with high glucose (HG) for 10days and the somatostatin analog octreotide (OCT) was used as a neuroprotectant. Chloroquine (CQ) was used as an autophagy inhibitor. Apoptotic and autophagic markers were evaluated using western blot and immunohistochemistry. HG-treated explants displayed a significant increase of apoptosis paralleled by a significant decrease of the autophagic flux, which was likely to be due to increased activity of the autophagy regulator mTOR (mammalian target of rapamycin). Treatment with OCT rescued HG-treated retinal explants from apoptosis and determined an increase of autophagic activity with concomitant mTOR inhibition. Blocking the autophagic flux with CQ completely abolished the anti-apoptotic effect of OCT. Immunohistochemical observations showed that OCT-induced autophagy is localized to populations of bipolar and amacrine cells and to ganglion cells. These observations revealed the antithetic role of apoptosis and autophagy, highlighting their equilibrium from which neuronal survival is likely to depend. These data suggest the crucial role covered by autophagy, which could be considered as a molecular target for DR neuroprotective treatment strategies.

In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases.

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.

Association between polymorphisms of TAS2R16 and susceptibility to colorectal cancer.

BACKGROUND: Genetics plays an important role in the susceptibility to sporadic colorectal cancer (CRC). In the last 10 years genome-wide association studies (GWAS) have identified over 40 independent low penetrance polymorphic variants. However, these loci only explain around 1­4% of CRC heritability, highlighting the dire need of identifying novel risk loci. In this study, we focused our attention on the genetic variability of the TAS2R16 gene, encoding for one of the bitter taste receptors that selectively binds to salicin, a natural antipyretic that resembles aspirin. Given the importance of inflammation in CRC, we tested whether polymorphic variants in this gene could affect the risk of developing this neoplasia hypothesizing a role of TAS2R16 in modulating chronic inflammation within the gut. METHODS: We performed an association study using 6 tagging SNPs, (rs860170, rs978739, rs1357949, rs1525489, rs6466849, rs10268496) that cover all TAS2R16 genetic variability. The study was carried out on 1902 CRC cases and 1532 control individuals from four European countries. RESULTS: We did not find any statistically significant association between risk of developing CRC and selected SNPs. However, after stratification by histology (colon vs. rectum) we found that rs1525489 was associated with increased risk of rectal cancer with a (Ptrend of = 0.0071). CONCLUSIONS: Our data suggest that polymorphisms within TAS2R16 gene do not have a strong influence on colon cancer susceptibility, but a possible role in rectal cancer should be further evaluated in larger cohorts.

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.