ABSTRACT
Purpose: In response to hypoxia, sympathetic fibers to the retina activate ß-adrenoceptors (ß-ARs) that play an important role in the regulation of vascular and neuronal functions. We investigated the role of ß3-AR using the mouse model of oxygen-induced retinopathy (OIR). Methods: Mouse pups were exposed to 75% oxygen at postnatal day 7 (PD7) followed by a return to room air at PD12. The ß3-AR preferential agonist BRL37344 was subcutaneously administered once daily at different times after the return to room air. At PD17, the OIR mice underwent flash and pattern electroretinogram. After sacrifice, retinal wholemounts were used for vessel staining or immunohistochemistry for astrocytes, Müller cells, or retinal ganglion cells (RGCs). In retinal homogenates, the levels of markers associated with neovascularization (NV), the blood-retinal barrier (BRB), or astrocytes were determined by western blot, and quantitative reverse-transcription polymerase chain reaction was used to assess ß3-AR messenger. Administration of the ß3-AR antagonist SR59230A was performed to verify BRL37344 selectivity. Results: ß3-AR expression is upregulated in response to hypoxia, but its increase is prevented by BRL37344, which counteracts NV by inhibiting the pro-angiogenic pathway, activating the anti-angiogenic pathway, recovering BRB-associated markers, triggering nitric oxide production, and favoring revascularization of the central retina through recovered density of astrocytes that ultimately counteracts NV in the midperiphery. Vasculature rescue prevents dysfunctional retinal activity and counteracts OIR-associated retinal ganglion cell loss. Conclusions: ß3-AR has emerged as a crucial intermediary in hypoxia-dependent NV, suggesting a role of ß3-AR agonists in the treatment of proliferative retinopathies.
Subject(s)
Adrenergic beta-3 Receptor Agonists , Disease Models, Animal , Electroretinography , Mice, Inbred C57BL , Oxygen , Receptors, Adrenergic, beta-3 , Retinal Neovascularization , Animals , Mice , Retinal Neovascularization/metabolism , Retinal Neovascularization/prevention & control , Retinal Neovascularization/pathology , Oxygen/toxicity , Adrenergic beta-3 Receptor Agonists/pharmacology , Receptors, Adrenergic, beta-3/metabolism , Animals, Newborn , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Ethanolamines/pharmacology , Retinal Vessels/drug effects , Retinopathy of Prematurity/metabolism , Retinopathy of Prematurity/drug therapy , Astrocytes/metabolism , Astrocytes/drug effects , Immunohistochemistry , AngiogenesisABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Efferent sympathetic nerve fibers regulate several renal functions activating norepinephrine receptors on tubular epithelial cells. Of the beta-adrenoceptors (ß-ARs), we previously demonstrated the renal expression of ß3-AR in the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the collecting duct (CD), where it participates in salt and water reabsorption. Here, for the first time, we reported ß3-AR expression in the CD intercalated cells (ICCs), where it regulates acid-base homeostasis. Co-localization of ß3-AR with either proton pump H+-ATPase or Cl-/HCO3 - exchanger pendrin revealed ß3-AR expression in type A, type B, non-A, and non-B ICCs in the mouse kidney. We aimed to unveil the possible regulatory role of ß3-AR in renal acid-base homeostasis, in particular in modulating the expression, subcellular localization, and activity of the renal H+-ATPase, a key player in this process. The abundance of H+-ATPase was significantly decreased in the kidneys of ß3-AR-/- compared with those of ß3-AR+/+ mice. In particular, H+-ATPase reduction was observed not only in the CD but also in the TAL and DCT, which contribute to acid-base transport in the kidney. Interestingly, we found that in in vivo, the absence of ß3-AR reduced the kidneys' ability to excrete excess proton in the urine during an acid challenge. Using ex vivo stimulation of mouse kidney slices, we proved that the ß3-AR activation promoted H+-ATPase apical expression in the epithelial cells of ß3-AR-expressing nephron segments, and this was prevented by ß3-AR antagonism or PKA inhibition. Moreover, we assessed the effect of ß3-AR stimulation on H+-ATPase activity by measuring the intracellular pH recovery after an acid load in ß3-AR-expressing mouse renal cells. Importantly, ß3-AR agonism induced a 2.5-fold increase in H+-ATPase activity, and this effect was effectively prevented by ß3-AR antagonism or by inhibiting either H+-ATPase or PKA. Of note, in urine samples from patients treated with a ß3-AR agonist, we found that ß3-AR stimulation increased the urinary excretion of H+-ATPase, likely indicating its apical accumulation in tubular cells. These findings demonstrate that ß3-AR activity positively regulates the expression, plasma membrane localization, and activity of H+-ATPase, elucidating a novel physiological role of ß3-AR in the sympathetic control of renal acid-base homeostasis.
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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.
Subject(s)
Endothelial Cells , Vascular Endothelial Growth Factor A , Humans , beta Catenin , Angiogenesis , IrisABSTRACT
Glaucoma is characterized by cupping of the optic disc, apoptotic degeneration of retinal ganglion cells (RGCs) and their axons, and thinning of the retinal nerve fiber layer, with patchy loss of vision. Elevated intraocular pressure (IOP) is a major risk factor for hypertensive glaucoma and the only modifiable one. There is a need to find novel compounds that counteract other risk factors contributing to RGC degeneration. The oil derived from the wild olive tree (Olea europaea var. sylvestris), also called Acebuche (ACE), shows powerful anti-inflammatory, antioxidant and retinoprotective effects. We evaluated whether ACE oil could counteract glaucoma-related detrimental effects. To this aim, we fed mice either a regular or an ACE oil-enriched diet and then induced IOP elevation through intraocular injection of methylcellulose. An ACE oil-enriched diet suppressed glaucoma-dependent retinal glia reactivity and inflammation. The redox status of the glaucomatous retinas was restored to a control-like situation, and ischemia was alleviated by an ACE oil-enriched diet. Notably, retinal apoptosis was suppressed in the glaucomatous animals fed ACE oil. Furthermore, as shown by electroretinogram analyses, RGC electrophysiological functions were almost completely preserved by the ACE oil-enriched diet. These ameliorative effects were IOP-independent and might depend on ACE oil's peculiar composition. Although additional studies are needed, nutritional supplementation with ACE oil might represent an adjuvant in the management of glaucoma.
Subject(s)
Antioxidants , Glaucoma , Mice , Animals , Antioxidants/pharmacology , Intraocular Pressure , Disease Models, Animal , Glaucoma/drug therapy , Anti-Inflammatory Agents/pharmacologyABSTRACT
Purpose: Glaucoma is an eye-brain axis disorder characterized by loss of retinal ganglion cells (RGCs). Although the role of intraocular pressure (IOP) elevation in glaucoma has been established, the reduction of oxidative stress and inflammation has emerged as a promising target for neuronal tissue-supporting glaucoma management. Therefore, we evaluated the effect of a proprietary spearmint extract (SPE) on RGC density, activity, and neuronal health markers in a rat model of hypertensive glaucoma. Methods: Animals were divided in four groups: untreated healthy control and three glaucomatous groups receiving orally administered vehicle, SPE-low dose, or SPE-high dose for 28 days. Ocular hypertension was induced through intracameral injection of methylcellulose at day 15. At day 29, rats underwent electroretinogram (ERG) recordings, and retinas were analyzed for RGC density and markers of neural trophism, oxidative stress, and inflammation. Results: SPE exerted dose-dependent response benefits on all markers except for IOP elevation. SPE significantly improved RGC-related ERG responses, cell density, neurotrophins, oxidative stress, and inflammation markers. Also, in SPE-high rats, most of the parameters were not statistically different from those of healthy controls. Conclusions: SPE, a plant-based, polyphenolic extract, could be an effective nutritional support for neuronal tissues. Translational Relevance: These results suggest that SPE not only may be a complementary approach in support to hypotensive treatments for the management of glaucoma but may also serve as nutritional support in other ocular conditions where antioxidant, anti-inflammatory, and neuroprotective mechanism are often disrupted.
Subject(s)
Glaucoma , Mentha spicata , Ocular Hypertension , Animals , Rats , Glaucoma/drug therapy , Ocular Hypertension/drug therapy , Retina , Inflammation/drug therapyABSTRACT
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.
ABSTRACT
Nutraceuticals are natural substances whose anti-oxidant and anti-inflammatory properties may be used to treat retinal pathologies. Their efficacy is limited by poor bioavailability, which could be improved using nanocarriers. Lisosan G (LG), a fermented powder from whole grains, protects the retina from diabetic retinopathy (DR)-induced damage. For this study, we tested whether the encapsulation of LG in liposomes (LipoLG) may increase its protective effects. Diabetes was induced in mice via streptozotocin administration, and the mice were allowed to freely drink water or a water dispersion of two different doses of LG or of LipoLG. Electroretinographic recordings after 6 weeks showed that only the highest dose of LG could partially protect the retina from diabetes-induced functional deficits, while both doses of LipoLG were effective. An evaluation of molecular markers of oxidative stress, inflammation, apoptosis, vascular endothelial growth factor, and the blood-retinal barrier confirmed that the highest dose of LG only partially protected the retina from DR-induced changes, while virtually complete prevention was obtained with either dose of LipoLG. These data indicate that the efficacy of LG in contrasting DR is greatly enhanced by its encapsulation in liposomes and may lay the ground for new dietary supplements with improved therapeutic effects against DR.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Retinopathy , Mice , Animals , Liposomes , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Vascular Endothelial Growth Factor A/metabolism , Diabetic Retinopathy/metabolism , WaterABSTRACT
Introduction: Much interest has been addressed to antioxidant dietary supplements that are known to lower the risk of developing glaucoma or delay its progression. Among them, niacin and citicoline protect retinal ganglion cells (RGCs) from degeneration by targeting mitochondria, though at different levels. A well-established mouse model of RGC degeneration induced by experimental intraocular pressure (IOP) elevation was used to investigate whether a novel combination of niacin/citicoline has better efficacy over each single component in preserving RGC health in response to IOP increase. Methods: Ocular hypertension was induced by an intracameral injection of methylcellulose that clogs the trabecular meshwork. Electroretinography and immunohistochemistry were used to evaluate RGC function and density. Oxidative, inflammatory and apoptotic markers were evaluated by Western blot analysis. Results: The present results support an optimal efficacy of niacin with citicoline at their best dosage in preventing RGC loss. In fact, about 50% of RGCs were spared from death leading to improved electroretinographic responses to flash and pattern stimulation. Upregulated levels of oxidative stress and inflammatory markers were also consistently reduced by almost 50% after niacin with citicoline thus providing a significant strength to the validity of their combination. Conclusion: Niacin combined with citicoline is highly effective in restoring RGC physiology but its therapeutic potential needs to be further explored. In fact, the translation of the present compound to humans is limited by several factors including the mouse modeling, the higher doses of the supplements that are necessary to demonstrate their efficacy over a short follow up period and the scarce knowledge of their transport to the bloodstream and to the eventual target tissues in the eye.
ABSTRACT
Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.
Subject(s)
Neuroprotection , Neuroprotective Agents , Retinal Ganglion Cells , PQQ Cofactor/pharmacology , Neuroprotective Agents/pharmacology , Adenosine TriphosphateABSTRACT
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.
Subject(s)
Receptors, Adrenergic, beta , Retinal Neovascularization , Humans , Receptors, Adrenergic, beta/metabolism , Retinal Neovascularization/metabolism , Retina/metabolism , Oxygen/metabolism , Hypoxia/metabolism , Receptors, Adrenergic, beta-3/metabolismABSTRACT
Age-related macular degeneration (AMD) is nowadays considered among the retinal diseases whose clinical management lacks established treatment approaches, mainly for its atrophic (dry) form. In this respect, the use of dietary patterns enriched in omega-3 and antioxidant xanthophylls has emerged as a promising approach to counteract dry AMD progression although the prophylactic potential of omega-3 of fish origin has been discussed. Whether enriched availability of omega-3 and xanthophylls may increase the effectiveness of diet supplementation in preventing dry AMD remains to be fully established. The present study aims at comparing the efficacy of an existing orally administered formulation based on lutein and fish oil, as a source of omega-3, with a novel formulation providing the combination of lutein and astaxanthin with Calanus oil (COil), which contains omega-3 together with their precursors policosanols. Using a mouse model of dry AMD based on subretinal injection of polyethylene glycol (PEG)-400, we assessed the comparative efficacy of both formulations on PEG-induced major hallmarks including oxidative stress, inflammation, glial reactivity and outer retinal thickness. Dietary supplementation with both mixtures has been found to exert a significant antioxidant and anti-inflammatory activity as reflected by the overall amelioration of the PEG-induced pathological hallmarks. Noteworthy, the formulation based on COil appeared to be more protective than the one based on fish oil, presumably because of the higher bioavailability of omega-3 in COil. These results support the use of dietary supplements combining omega-3 and xanthophylls in the prevention and treatment of AMD and suggest that the source of omega-3 might contribute to treatment efficacy.
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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.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Retinopathy , Hyperglycemia , Mice , Animals , Retinal Ganglion Cells , Diabetic Retinopathy/metabolism , Neuroprotection , Mice, Inbred C57BL , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Mice, Transgenic , Hyperglycemia/metabolism , Disease Models, AnimalABSTRACT
Retinopathy of prematurity (ROP) is one of the main blinding diseases affecting preterm newborns and is classically considered a vascular disorder. The premature exposure to the extrauterine environment, which is hyperoxic in respect to the intrauterine environment, triggers a cascade of events leading to retinal ischemia which, in turn, makes the retina hypoxic thus setting off angiogenic processes. However, many children with a history of ROP show persistent vision impairment, and there is evidence of an association between ROP and neurosensory disabilities. This is not surprising given the strict relationship between neuronal function and an adequate blood supply. In the present work, we revised literature data evidencing to what extent ROP can be considered a neurodegenerative disease, also taking advantage from data obtained in preclinical models of ROP. The involvement of different retinal cell populations in triggering the neuronal damage in ROP was described along with the neurological outcomes associated to ROP. The situation of ROP in Italy was assessed as well.
ABSTRACT
A major player in the homeostatic response to hypoxia is the hypoxia-inducible factor (HIF)-1 that transactivates a number of genes involved in neovessel proliferation in response to low oxygen tension. In the retina, hypoxia overstimulates ß-adrenoceptors (ß-ARs) which play a key role in the formation of pathogenic blood vessels. Among ß-ARs, ß3-AR expression is increased in proliferating vessels in concomitance with increased levels of HIF-1α and vascular endothelial growth factor (VEGF). Whether, similarly to VEGF, hypoxia-induced ß3-AR upregulation is driven by HIF-1 is still unknown. We used the mouse model of oxygen-induced retinopathy (OIR), an acknowledged model of retinal angiogenesis, to verify the hypothesis of ß3-AR transcriptional regulation by HIF-1. Investigation of ß3-AR regulation over OIR progression revealed that the expression profile of ß3-AR depends on oxygen tension, similar to VEGF. The additional evidence that HIF-1α stabilization decouples ß3-AR expression from oxygen levels further indicates that HIF-1 regulates the expression of the ß3-AR gene in the retina. Bioinformatics predicted the presence of six HIF-1 binding sites (HBS #1-6) upstream and inside the mouse ß3-AR gene. Among these, HBS #1 has been identified as the most suitable HBS for HIF-1 binding. Chromatin immunoprecipitation-qPCR demonstrated an effective binding of HIF-1 to HBS #1 indicating the existence of a physical interaction between HIF-1 and the ß3-AR gene. The additional finding that ß3-AR gene expression is concomitantly activated indicates the possibility that HIF-1 transactivates the ß3-AR gene. Our results are indicative of ß3-AR involvement in HIF-1-mediated response to hypoxia.
Subject(s)
Hypoxia-Inducible Factor 1 , Receptors, Adrenergic, beta-3 , Retinal Diseases , Vascular Endothelial Growth Factor A , Animals , Hypoxia/metabolism , Hypoxia-Inducible Factor 1/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Mice, Inbred C57BL , Oxygen/metabolism , Receptors, Adrenergic, beta-3/metabolism , Retina/metabolism , Retinal Diseases/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Retinopathy of prematurity (ROP) is an evolutive and potentially blinding eye disease that affects preterm newborns. Unfortunately, until now no conservative therapy of active ROP with proven efficacy is available. Although ROP is a multifactorial disease, premature exposition to oxygen concentrations higher than those intrauterine, represents the initial pathogenetic trigger. The increase of oxygenation in a retina still incompletely vascularized promotes the downregulation of proangiogenic factors and finally the interruption of vascularization (ischemic phase). However, the increasing metabolic requirement of the ischemic retina induces, over the following weeks, a progressive hypoxia that specularly increases the levels of proangiogenic factors finally leading to proliferative retinopathy (proliferative phase). Considering non-modifiable the coupling between oxygen levels and vascularization, so far, neonatologists and ophthalmologists have "played defense", meticulously searching the minimum necessary concentration of oxygen for individual newborns, refining their diagnostic ability, adopting a careful monitoring policy, ready to decisively intervene only in a very advanced stage of disease progression. However, recent advances have demonstrated the possibility to pharmacologically modulate the relationship between oxygen and vascularization, opening thus the perspective for new therapeutic or preventive opportunities. The perspective of a shift from a defensive towards an attack strategy is now at hand.
ABSTRACT
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.
Subject(s)
Infant, Newborn, Diseases , Neoplasms , Receptors, Adrenergic/metabolism , Humans , Infant, Newborn , Propranolol/pharmacology , Receptors, Adrenergic, beta/metabolism , Signal TransductionABSTRACT
Renin-angiotensin systems produce angiotensin II (Ang II) and angiotensin 1-7 (Ang 1-7), which are able to induce opposite effects on circulation. This study in vivo assessed the effects induced by Ang II or Ang 1-7 on rat pial microcirculation during hypoperfusion-reperfusion, clarifying the mechanisms causing the imbalance between Ang II and Ang 1-7. The fluorescence microscopy was used to quantify the microvascular parameters. Hypoperfusion and reperfusion caused vasoconstriction, disruption of blood-brain barrier, reduction of capillary perfusion and an increase in reactive oxygen species production. Rats treated with Ang II showed exacerbated microvascular damage with stronger vasoconstriction compared to hypoperfused rats, a further increase in leakage, higher decrease in capillary perfusion and marker oxidative stress. Candesartan cilexetil (specific Ang II type 1 receptor (AT1R) antagonist) administration prior to Ang II prevented the effects induced by Ang II, blunting the hypoperfusion-reperfusion injury. Ang 1-7 or ACE2 activator administration, preserved the pial microcirculation from hypoperfusion-reperfusion damage. These effects of Ang 1-7 were blunted by a Mas (Mas oncogene-encoded protein) receptor antagonist, while Ang II type 2 receptor antagonists did not affect Ang 1-7-induced changes. In conclusion, Ang II and Ang 1-7 triggered different mechanisms through AT1R or MAS receptors able to affect cerebral microvascular injury.