Publications by authors named "Ana I Arroba"

38 Publications

Unravelling the Inflammatory Processes in the Early Stages of Diabetic Nephropathy and the Potential Effect of ()-DS-ONJ.

Int J Mol Sci 2022 Jul 30;23(15). Epub 2022 Jul 30.

Unidad de Investigación, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), 11009 Cádiz, Spain.

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. The authors demonstrate that the glycolipid mimetic ()-DS-ONJ is able to abolish inflammation via the induction of autophagy flux and provokes the inhibition of inflammasome complex in ex vivo and in vitro models, using adult kidney explants from BB rats. The contribution of ()-DS-ONJ to reducing inflammatory events is mediated by the inhibition of classical stress kinase pathways and the blocking of inflammasome complex activation. The (-DS-ONJ treatment is able to inhibit the epithelial-to-mesenchymal transition (EMT) progression, but only when the IL18 levels are reduced by the treatment. These findings suggest that ()-DS-ONJ could be a novel, and multifactorial treatment for DN.
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http://dx.doi.org/10.3390/ijms23158450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9368839PMC
July 2022

A New Perspective on Huntington's Disease: How a Neurological Disorder Influences the Peripheral Tissues.

Int J Mol Sci 2022 May 29;23(11). Epub 2022 May 29.

Undad de Investigación, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), 11002 Cádiz, Spain.

Huntington's disease (HD) is a neurodegenerative disorder caused by a toxic, aggregation-prone expansion of CAG repeats in the HTT gene with an age-dependent progression that leads to behavioral, cognitive and motor symptoms. Principally affecting the frontal cortex and the striatum, mHTT disrupts many cellular functions. In fact, increasing evidence shows that peripheral tissues are affected by neurodegenerative diseases. It establishes an active crosstalk between peripheral tissues and the brain in different neurodegenerative diseases. This review focuses on the current knowledge of peripheral tissue effects in HD animal and cell experimental models and identifies biomarkers and mechanisms involved or affected in the progression of the disease as new therapeutic or early diagnostic options. The particular changes in serum/plasma, blood cells such as lymphocytes, immune blood cells, the pancreas, the heart, the retina, the liver, the kidney and pericytes as a part of the blood-brain barrier are described. It is important to note that several changes in different mouse models of HD present differences between them and between the different ages analyzed. The understanding of the impact of peripheral organ inflammation in HD may open new avenues for the development of novel therapeutic targets.
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http://dx.doi.org/10.3390/ijms23116089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9181740PMC
May 2022

Adult kidney explants is a physiologic model for studying diabetic nephropathy.

Life Sci 2022 Jul 25;300:120575. Epub 2022 Apr 25.

Unidad de Investigación, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), Cádiz, Spain; Departamento de Endocrinología y Nutrición, Hospital Universitario Puerta del Mar, Universidad de Cádiz, Cádiz, Spain. Electronic address:

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. In vitro approach using cell lines help to understand the mechanisms involves and allow the molecular and biochemical processes. Adult kidney (AK) explants remain an essential instrument for advancing our understanding of the molecular and cellular regulation of signalling pathways from an organotipic view with physiological system interaction integrated. AK explants from T1DM animal model (BB rat) are obtained by slicing central kidney area preserving the organ's cytoarchitecture and reproduce the classical events detected during the DN in an in vivo model such as inflammation, epithelial-mesenchymal transition (EMT) processes by the modulation of a-SMA and e-Cadherin among others which have been determined by qRT-PCR, western-blot and immunohistochemistry. In this regard, AK explants reproduce the signalling pathways involve in DN progression (proinflammatory NFkB and inflammasome complex). This work demonstrates AK explants is a physiological experimental approach for studying the development and progression of DN. Furthermore, the inflammatory processes in AK explants under a diabetic environment and/or BB rats could be modulated by potential treatments for DN.
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http://dx.doi.org/10.1016/j.lfs.2022.120575DOI Listing
July 2022

Angiogenic Imbalance and Inflammatory Biomarkers in the Prediction of Hypertension as Well as Obstetric and Perinatal Complications in Women with Gestational Diabetes Mellitus.

J Clin Med 2022 Mar 10;11(6). Epub 2022 Mar 10.

Department of Endocrinology and Nutrition, Puerta del Mar Hospital, 11009 Cádiz, Spain.

Gestational diabetes mellitus (GDM) increases the risk of hypertensive disorders of pregnancy (HDP). We aimed to analyze the altered inflammatory markers and angiogenic factors among women with GDM to identify pregnant women at higher risk of developing HDP.

Methods: This was a prospective study of 149 women without hypertension diagnosed in the third trimester with GDM. Inflammatory markers and angiogenic factors were measured at 28-32 weeks of pregnancy. Obstetric and perinatal outcomes were evaluated.

Results: More than eight percent of the women developed HDP. Higher levels of the soluble fms-like tyrosine kinase-1/placental growth factor (sFlt-1/PIGF) ratio (4.9 ± 2.6 versus 2.3 ± 1.3, respectively; < 0.001) and leptin (10.9 ± 0.8 versus 10.08 ± 1.1, respectively; = 0.038), as well as lower levels of adiponectin (10.5 ± 1.3 versus 12.9 ± 2.7, respectively; = 0.031), were seen in women who developed HDP versus normotensive women with GDM. A multivariable logistic regression analysis showed that adiponectin had a protective effect with 0.45-fold odds (0.23-0.83; = 0.012), and that the sFlt-1/PIGF ratio was associated with 2.70-fold odds of developing HDP (CI 95%: 1.24-5.86; = 0.012).

Conclusion: An increase in angiogenic imbalance in the sFlt-1/PIGF ratio in women with GDM was detected and may be an indicator of developing HDP in addition to any subsequent obstetric and perinatal complications.
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http://dx.doi.org/10.3390/jcm11061514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8953606PMC
March 2022

Blood Pressure Monitoring and Perinatal Outcomes in Normotensive Women with Gestational Diabetes Mellitus.

J Clin Med 2022 Mar 5;11(5). Epub 2022 Mar 5.

Endocrinology and Nutrition Department, Hospital Universitario Puerta del Mar, 11009 Cadiz, Spain.

Alterations in ambulatory blood pressure detected by monitoring (ABPM) have been associated with perinatal complications in hypertensive pregnant women.

Aim: To establish the relationships between the blood pressure (BP) profiles detected by ABPM and adverse perinatal outcomes in normotensive women with gestational diabetes mellitus (GDM).

Methods: A prospective study of normotensive women in whom 24 h ABPM was performed at 28-32 weeks of pregnancy. The obstetric and perinatal outcomes were evaluated.

Results: Two hundred patients were included. Thirty-seven women with GDM and obesity had significantly higher mean systolic BP (SBP) and nocturnal SBP and diastolic BP (DBP) compared to women with only GDM ( = 86). Nocturnal SBP (OR = 1.077; = 0.015) and obesity (OR = 1.131; = 0.035) were risk factors for the development of hypertensive disorders of pregnancy (HDPs). Mothers of newborns with neonatal complications ( = 27) had higher nocturnal SBP (103.8 vs. 100 mmHg; = 0.047) and DBP (62.7 vs. 59.4; = 0.016). Women who delivered preterm ( = 10) had higher BP and a non-dipper pattern ( = 0.005).

Conclusions: Nocturnal SBP was a predictor of HDPs in normotensive women with obesity or GDM. Alterations in ABPM in these patients were associated with poor obstetric and perinatal outcomes.
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http://dx.doi.org/10.3390/jcm11051435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8911501PMC
March 2022

IL-1β Implications in Type 1 Diabetes Mellitus Progression: Systematic Review and Meta-Analysis.

J Clin Med 2022 Feb 27;11(5). Epub 2022 Feb 27.

Research Unit, Biomedical Research and Innovation Institute of Cadiz (INiBICA), Puerta del Mar University Hospital, 11009 Cadiz, Spain.

During Type 1 Diabetes Mellitus (T1DM) progression, there is chronic and low-grade inflammation that could be related to the evolution of the disease. We carried out a systematic review and meta-analysis to evaluate whether peripheral levels of pro-inflammatory markers such as interleukin-1 beta (IL-1β) is significantly different among patients with or without T1DM, in gender, management of the T1DM, detection in several biological fluids, study design, age range, and glycated hemoglobin. We searched PubMed, Embase, Web of Science, and Scopus databases, and 26 relevant studies (2186 with T1DM, 2047 controls) were included. We evaluated the studies' quality using the Newcastle-Ottawa scale. Meta-analyses were conducted, and heterogeneity and publication bias were examined. Compared with controls, IL-1β determined by immunoassays (pooled standardized mean difference (SMD): 2.45, 95% CI = 1.73 to 3.17; < 0.001) was significantly elevated in T1DM. The compared IL-1β levels in patients <18 years (SMD = 2.81, 95% CI = 1.88-3.74) was significantly elevated. The hemoglobin-glycated (Hbg) levels in patients <18 years were compared (Hbg > 7: SMD = 5.43, 95% CI = 3.31-7.56; = 0.001). Compared with the study design, IL-1β evaluated by ELISA (pooled SMD = 3.29, 95% CI = 2.27 to 4.30, < 0.001) was significantly elevated in T1DM patients. IL-1β remained significantly higher in patients with a worse management of T1DM and in the early stage of T1DM. IL-1β levels determine the inflammatory environment during T1DM.
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http://dx.doi.org/10.3390/jcm11051303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8910979PMC
February 2022

Synthesis of sp-Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties.

Molecules 2021 Dec 11;26(24). Epub 2021 Dec 11.

Department of Organic Chemistry, Faculty of Chemistry, University of Seville, C/Profesor García González 1, 41012 Seville, Spain.

sp-Iminosugar glycolipids (sp-IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp-hybridized N-atom imparts chemical and enzymatic stability to sp-IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of -, -, - and -pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp-IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5,6-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure-activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp-IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C vs. C), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.
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http://dx.doi.org/10.3390/molecules26247501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8705409PMC
December 2021

Diterpenoids from the Brown Alga and Their Anti-Inflammatory Activity.

Mar Drugs 2021 Nov 27;19(12). Epub 2021 Nov 27.

Departamento de Química Orgánica, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real (Cádiz), Spain.

Brown algae of the Family Dictyotaceae produce an array of structurally diverse terpenoids, whose biomedical potential in the anti-inflammatory area has been scarcely explored. Herein, the chemical study of the alga has led to the isolation of ten new diterpenoids: rugukadiol A (), rugukamurals A-C (-), and ruguloptones A-F (-). The structures of the new compounds were established by spectroscopic means. Compound exhibits an unprecedented diterpenoid skeleton featuring a bridged tricyclic undecane system. Compounds - belong to the secospatane class of diterpenoids and differ by the oxygenated functions that they contain. In anti-inflammatory assays, the new diterpenoid and the secospatanes and significantly inhibited the production of the inflammatory mediator NO in LPS-stimulated microglial cells Bv.2 and macrophage cells RAW 264.7. Moreover, compounds and were found to strongly inhibit the expression of and the pro-inflammatory cytokine in both immune cell lines.
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http://dx.doi.org/10.3390/md19120677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8704470PMC
November 2021

Anti-Inflammatory (M2) Response Is Induced by a sp-Iminosugar Glycolipid Sulfoxide in Diabetic Retinopathy.

Front Immunol 2021 18;12:632132. Epub 2021 Mar 18.

Research Unit, Instituto de Investigación e Innovación en Ciencias Biomédicas de la Provincia de Cádiz (INiBICA), Hospital Universitario Puerta del Mar, Cádiz, Spain.

Diabetic retinopathy (DR) is one of the most common complications of Diabetes Mellitus (DM) and is directly associated with inflammatory processes. Currently, neuro-inflammation is considered an early event in DR and proceeds microglia polarization. A hallmark of DR is the presence of retinal reactive gliosis. Here we report the beneficial effect of ( ,1)-1-docecylsulfiny-5,6-oxomethylidenenojirimycin (()-DS-ONJ), a member of the sp-iminosugar glycolipid (sp-IGL) family, by decreasing iNOS and inflammasome activation in Bv.2 microglial cells exposed to pro-inflammatory stimuli. Moreover, pretreatment with ()-DS-ONJ increased Heme-oxygenase (HO)-1 as well as interleukin 10 (IL10) expression in LPS-stimulated microglial cells, thereby promoting M2 (anti-inflammatory) response by the induction of Arginase-1. The results strongly suggest that this is the likely molecular mechanism involved in the anti-inflammatory effects of ( )-DS-ONJ in microglia. ( )-DS-ONJ further reduced gliosis in retinal explants from type 1 diabetic BB rats, which is consistent with the enhanced M2 response. In conclusion, targeting microglia polarization dynamics in M2 status by compounds with anti-inflammatory activities offers promising therapeutic interventions at early stages of DR.
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http://dx.doi.org/10.3389/fimmu.2021.632132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013727PMC
September 2021

Effect of Topical Administration of Somatostatin on Retinal Inflammation and Neurodegeneration in an Experimental Model of Diabetes.

J Clin Med 2020 Aug 10;9(8). Epub 2020 Aug 10.

Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERdem), ISCIII, 28029 Madrid, Spain.

Somatostatin (SST) is a neuroprotective peptide but little is known regarding the potential role of its anti-inflammatory effects on retinal neuroprotection. In a previous study, we provided the first evidence that topical (eye drops) administration of SST prevents retinal neurodegeneration in streptozotocin (STZ)-induced diabetic rats. However, STZ by itself could cause neurotoxicity, thus acting as a confounding factor. The aims of the present study were: (1) to test the effect of topical administration of SST in the db/db mouse model, a spontaneous model of type 2 diabetes, thus avoiding the confounding effect of STZ on neurodegeneration; (2) to further explore the anti-inflammatory mechanisms of SST in glial cells. This task was performed by using mouse retinal explants and cell cultures. In summary, we confirm that SST topically administered was able to prevent retinal neurodysfunction and neurodegeneration in db/db mice. Furthermore, we found that SST prevented the activation of the classical M1 response of Bv.2 microglial cells upon Lipopolysaccharide (LPS) stimulation as a potent pro-inflammatory trigger. The anti-inflammatory effect of SST in Bv.2 cells was also observed in response to hypoxia. In conclusion, we provide evidence that the neuroprotective effect of SST in diabetic retinas can be largely attributed to anti-inflammatory mechanisms.
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http://dx.doi.org/10.3390/jcm9082579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463891PMC
August 2020

Synthesis of polyfluoroalkyl sp-iminosugar glycolipids and evaluation of their immunomodulatory properties towards anti-tumor, anti-leishmanial and anti-inflammatory therapies.

Eur J Med Chem 2019 Nov 8;182:111604. Epub 2019 Aug 8.

Department of Organic Chemistry, Faculty of Chemistry, University of Seville, C/ Profesor García González 1, 41012, Seville, Spain. Electronic address:

Immunomodulatory glycolipids, among which α-galactosylceramide (KRN7000) is an iconic example, have shown strong therapeutic potential in a variety of conditions ranging from cancer and infection to autoimmune or neurodegenerative diseases. A main difficulty for those channels is that they often provoke a cytokine storm comprising both pro- and anti-inflammatory mediators that antagonize each other and negatively affect the immune response. The synthesis of analogues with narrower cytokine secretion-inducing capabilities is hampered by the intrinsic difficulty at controlling the stereochemical outcome in glycosidation reactions, particularly if targeting the α-anomer, which seriously hampers drug optimization strategies. Here we show that replacing the monosaccharide glycone by a sp-iminosugar glycomimetic moiety allows accessing N-linked sp-iminosugar glycolipids (sp-IGLs) with total α-stereocontrol in a single step with no need of protecting groups or glycosidation promotors. The lipid tail has been then readily tailored by incorporating polyfluoroalkyl segments of varied lengths in view of favouring binding to the lipid binding site of the master p38 mitogen activated protein kinase (p38 MAPK), thereby polarizing the immune response in a cell-context dependent manner. The compounds have been evaluated for their antiproliferative, anti-leishmanial and anti-inflammatory activities in different cell assays. The size of the fluorous segment was found to be critical for the biological activity, probably by regulating the aggregation and membrane-crossing properties, whereas the hydroxylation profile (gluco or galacto-like) was less relevant. Biochemical and computational data further support a mechanism of action implying binding to the allosteric lipid binding site of p38 MAPK and subsequent activation of the noncanonical autophosphorylation route. The ensemble of results provide a proof of concept of the potential of sp-IGLs as immunoregulators.
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http://dx.doi.org/10.1016/j.ejmech.2019.111604DOI Listing
November 2019

Insulin receptor substrate 2 (IRS2) deficiency delays liver fibrosis associated with cholestatic injury.

Dis Model Mech 2019 07 16;12(7). Epub 2019 Jul 16.

Unidad de Investigación Hepática, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria del Hospital Universitario de La Princesa, 28009 Madrid, Spain

Insulin receptor substrate 2 (IRS2) is a key downstream mediator of insulin and insulin-like growth factor 1 (IGF1) signalling pathways and plays a major role in liver metabolism. The aim of this study was to investigate whether IRS2 had an impact on the hepatic fibrotic process associated with cholestatic injury. Bile duct ligation (BDL) was performed in wild-type (WT) and -deficient (IRS2KO) female mice. Histological and biochemical analyses, together with fibrogenic and inflammatory responses were evaluated in livers from mice at 3, 7 and 28 days following BDL. We also explored whether activation of human hepatic stellate cells (HSCs) induced by IGF1 was modulated by IRS2. IRS2KO mice displayed reduced disruption of liver histology, such hepatocyte damage and excess deposition of extracellular matrix components, compared with WT mice at 3 and 7 days post-BDL. However, no histological differences between genotypes were found at 28 days post-BDL. The less pro-inflammatory profile of bile acids accumulated in the gallbladder of IRS2KO mice after BDL corresponded with the reduced expression of pro-inflammatory markers in these mice. Stable silencing of or inhibition of ERK1/2 reduced the activation of human LX2 cells and also reduced induction of MMP9 upon IGF1 stimulation. Furthermore, hepatic MMP9 expression was strongly induced after BDL in WT mice, but only a slight increase was found in mice lacking IRS2. Our results have unravelled the signalling pathway mediated by IGF1R-IRS2-ERK1/2-MMP9 as a key axis in regulating HSC activation, which might be therapeutically relevant for targeting liver fibrosis.
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http://dx.doi.org/10.1242/dmm.038810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679376PMC
July 2019

IGF-1, Inflammation and Retinal Degeneration: A Close Network.

Front Aging Neurosci 2018 5;10:203. Epub 2018 Jul 5.

Alberto Sols Biomedical Research Institute (IIBm) (CSIC/UAM), Madrid, Spain.

Retinal degenerative diseases are a group of heterogeneous diseases that include age-related macular degeneration (AMD), retinitis pigmentosa (RP), and diabetic retinopathy (DR). The progressive degeneration of the retinal neurons results in a severe deterioration of the visual function. Neuroinflammation is an early hallmark of many neurodegenerative disorders of the retina including AMD, RP and DR. Microglial cells, key components of the retinal immune defense system, are activated in retinal degenerative diseases. In the microglia the interplay between the proinflammatory/classically activated or antiinflammatory/alternatively activated phenotypes is a complex dynamic process that occurs during the course of disease due to the different environmental signals related to pathophysiological conditions. In this regard, an adequate transition from the proinflammatory to the anti-inflammatory response is necessary to counteract retinal neurodegeneration and its subsequent damage that leads to the loss of visual function. Insulin like-growth factor-1 (IGF-1) has been considered as a pleiotropic factor in the retina under health or disease conditions and several effects of IGF-1 in retinal immune modulation have been described. In this review, we provide recent insights of inflammation as a common feature of retinal diseases (AMD, RP and RD) highlighting the role of microglia, exosomes and IGF-1 in this process.
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http://dx.doi.org/10.3389/fnagi.2018.00203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041402PMC
July 2018

The sp-iminosugar glycolipid 1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO-ONJ) as selective anti-inflammatory agent by modulation of hemeoxygenase-1 in Bv.2 microglial cells and retinal explants.

Food Chem Toxicol 2018 Jan 27;111:454-466. Epub 2017 Nov 27.

Instituto de Investigaciones Biomédicas Alberto Sols (IIBm) (CSIC/UAM), C/ Arturo Duperier 4, 28029 Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem), ISCIII, 28029 Madrid, Spain. Electronic address:

Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp-iminosugar glycolipid (sp-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO-ONJ in microglia.
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http://dx.doi.org/10.1016/j.fct.2017.11.050DOI Listing
January 2018

Somatostatin protects human retinal pericytes from inflammation mediated by microglia.

Exp Eye Res 2017 11 20;164:46-54. Epub 2017 Jul 20.

Dept of Medical Sciences, University of Turin, Corso AM Dogliotti 14, 10126 Torino, Italy. Electronic address:

Diabetic retinopathy (DR) is usually considered a microvascular disease. However, involvement of the neuroretina in the early stages of DR has recently gained major credit. Inflammatory processes, leading to glial activation and neuronal apoptosis, develop early in the retina of diabetic subjects. Pericytes constitute a link between the vascular and the neural retina, play a central role in blood-retinal barrier maintenance, and may influence neuroinflammation. Somatostatin (SST) is a potent neuroprotective factor, which is down-regulated during early DR. In this paper, we have investigated the effects of the inflammatory signals triggered by the activation of microglia on inflammation and apoptosis/survival pathways in pericytes. Microglia cells (Bv-2) were stimulated with lipopolysaccharide (LPS) and/or SST. Human retinal pericytes (HRP) were exposed to conditioned media (CM) collected from Bv-2 cells in physiological conditions and in the settings described above. A panel of inflammation, apoptosis and survival mediators was analyzed. HRP treated with LPS-CM showed a significant increase of pro-inflammatory (iNos and TNFα) and pro-apoptotic mediators (FasL, active caspase-8, tBid and Bax), and a concomitant decrease in pro-survival factors (BclxL and pAkt). SST added to LPS was able to counteract these effects in all conditions. In conclusion, SST is able to modulate apoptosis/survival pathways in HRP during microglia-mediated inflammation. These results demonstrate a crosstalk between microglia and retinal pericytes, evidencing a possible defensive role of microglia in the early phases of DR.
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http://dx.doi.org/10.1016/j.exer.2017.07.011DOI Listing
November 2017

Modulation of microglia in the retina: new insights into diabetic retinopathy.

Acta Diabetol 2017 Jun 27;54(6):527-533. Epub 2017 Mar 27.

Alberto Sols Biomedical Research Institute (IIBm) (CSIC/UAM), 28029, Madrid, Spain.

During last decades, the diagnosis of diabetes has been associated with several chronic complications such as diabetic retinopathy (DR). Recent studies of DR have revealed an inflammatory component, which precedes the detection of alterations in the visual function. During DR, the inflammatory process presents two opposite roles depending on the polarization of resident immune cells of the retina triggering proinflammatory (M1) or antiinflammatory (M2) actions. In an early stage of DR, the M2 response concurs with the M1 and is able to ameliorate inflammation and delay the progression of the disease. However, during the progression of DR, the M1 response is maintained whereas the M2 declines and, in this scenario, the classical proinflammatory signaling pathways are chronically activated leading to retinal neurodegeneration and the loss of visual function. The M1/M2 responses are closely related to the activation and polarization of microglial cells. This review aims to offer an overview of the recent insights into the role of microglial cells during inflammation in DR. We have focused on the possibility of modulating microglia polarization as a new therapeutic strategy in DR treatments.
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http://dx.doi.org/10.1007/s00592-017-0984-zDOI Listing
June 2017

Imbalance between pro-apoptotic and pro-survival factors in human retinal pericytes in diabetic-like conditions.

Acta Ophthalmol 2018 Feb 27;96(1):e19-e26. Epub 2017 Jan 27.

Department of Medical Sciences, University of Turin, Torino, Italy.

Purpose: Loss of pericytes is one the key events in the pathogenesis of diabetic retinopathy. We have previously demonstrated that human retinal pericytes (HRP) are more vulnerable to intermittent than stable high glucose concentrations, with an increase in apoptosis. Our aim was to explore the expression of molecules involved in pro-apoptotic and survival pathways in pericytes cultured in stable/intermittent high glucose and/or hypoxia, to clarify the mechanisms of action of these diabetic-like stressing stimuli.

Methods: Human retinal pericytes (HRP) were exposed intermittently at 48-hr intervals to high/physiological glucose for 8 days (intHG) and/or hypoxia over the last 48 hr. Control cells were kept in stable physiological and high glucose. Cell proliferation and apoptosis were assessed. The expression of pro-apoptotic and pro-survival molecules was evaluated by Western blotting. Caspase-8 translocation from the cytoplasm into the nucleus was checked by Western blotting of nuclear versus cytoplasmic fractions and immunofluorescence.

Results: Hypoxia, alone and combined with intHG, increased HRP apoptosis and decreased proliferation. Pro-apoptotic molecules increased in HRP cultured in these conditions, while some survival markers decreased. Conversely, in stable HG, pro-apoptotic molecules were stable or even decreased, and survival factors increased. Translocation of caspase-8 from cytoplasm into nucleus indicates a primary role for this molecule in inducing apoptosis.

Conclusion: Diabetic-like conditions are able to stimulate pericyte apoptosis through activation of pro-apoptotic molecules, leading to an imbalance between pro-apoptotic and survival signalling pathways, with caspase-8 playing a pivotal role. Our identification of such intermediates could help finding new therapeutic approaches for the prevention of diabetic retinopathy.
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http://dx.doi.org/10.1111/aos.13377DOI Listing
February 2018

Somatostatin protects photoreceptor cells against high glucose-induced apoptosis.

Mol Vis 2016 30;22:1522-1531. Epub 2016 Dec 30.

Alberto Sols Biomedical Research Institute (IIBm) (CSIC/UAM), 28029 Madrid, Spain; Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERdem), ISCIII, Madrid, Spain.

Purpose: Many cellular and molecular studies in experimental animals and early retinal function tests in patients with diabetic retinopathy (DR) have shown that retinal neurodegeneration is an early event in the pathogenesis of the disease. Somatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina: SST levels are decreased in parallel to retinal neurodegeneration in early stages of DR. In this study, we characterized the induction of apoptosis (programmed cell death) in a 661W photoreceptor-like cell line cultured under high glucose (HG) conditions and the effect of SST.

Methods: A 661W photoreceptor-like cell line and retinal explants from 10-week-old male C57BL/6 mice were cultured under HG conditions and treated with SST.

Results: Hyperglycemia significantly reduced the cellular viability by increasing the percentage of apoptotic cells, and this effect was ameliorated by SST (p˂0.05). Activation of caspase-8 by hyperglycemia was found in the 661W cells and retinal explants and decreased in the presence of SST (p˂0.05). Moreover, we detected activation of calpain-2 associated with hyperglycemia-induced cell death, as well as increased protein tyrosine phosphatase 1B (PTP1B) protein levels; both had a pattern of cleavage that was absent in the presence of SST (p˂0.05). Treatment of the 661W cells and retinal explants with SST for 24 h increased the phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR; tyrosine 1165/1166) and protein kinase B (Akt; serine 473), suggesting this survival signaling is activated in the neuroretina by SST (p˂0.05).

Conclusions: This study has provided new mechanistic insights first into the involvement of calpain-2 and PTP1B in the loss of cell survival and increased caspase-8-dependent apoptosis induced by hyperglycemia in photoreceptor cells and second, on the protective effect of SST against apoptosis by the enhancement of IGF-IR-mediated Akt phosphorylation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204461PMC
January 2018

A novel glucagon-like peptide 1/glucagon receptor dual agonist improves steatohepatitis and liver regeneration in mice.

Hepatology 2017 03 30;65(3):950-968. Epub 2017 Jan 30.

Instituto de Investigaciones Biomédicas Alberto Sols (Centro Mixto CSIC-UAM), Madrid, Spain.

Because nonalcoholic steatohepatitis (NASH) is associated with impaired liver regeneration, we investigated the effects of G49, a dual glucagon-like peptide-1/glucagon receptor agonist, on NASH and hepatic regeneration. C57Bl/6 mice fed chow or a methionine and choline-deficient (MCD) diet for 1 week were divided into 4 groups: control (chow diet), MCD diet, chow diet plus G49, and M+G49 (MCD diet plus G49). Mice fed a high-fat diet (HFD) for 10 weeks were divided into groups: HFD and H+G49 (HFD plus G49). Following 2 (MCD groups) or 3 (HFD groups) weeks of treatment with G49, partial hepatectomy (PH) was performed, and all mice were maintained on the same treatment schedule for 2 additional weeks. Analysis of liver function, hepatic regeneration, and comprehensive genomic and metabolic profiling were conducted. NASH was ameliorated in the M+G49 group, manifested by reduced inflammation, steatosis, oxidative stress, and apoptosis and increased mitochondrial biogenesis. G49 treatment was also associated with replenishment of intrahepatic glucose due to enhanced gluconeogenesis and reduced glucose use through the pentose phosphate cycle and oxidative metabolism. Following PH, G49 treatment increased survival, restored the cytokine-mediated priming phase, and enhanced the proliferative capacity and hepatic regeneration ratio in mice on the MCD diet. NASH markers remained decreased in M+G49 mice after PH, and glucose use was shifted to the pentose phosphate cycle and oxidative metabolism. G49 administered immediately after PH was also effective at alleviating the pathological changes induced by the MCD diet. Benefits in terms of liver regeneration were also found in mice fed HFD and treated with G49.

Conclusion: Dual-acting glucagon-like peptide-1/glucagon receptor agonists such as G49 represent a novel therapeutic approach for patients with NASH and particularly those requiring PH. (Hepatology 2017;65:950-968).
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http://dx.doi.org/10.1002/hep.28962DOI Listing
March 2017

Rapamycin negatively impacts insulin signaling, glucose uptake and uncoupling protein-1 in brown adipocytes.

Biochim Biophys Acta 2016 12 26;1861(12 Pt A):1929-1941. Epub 2016 Sep 26.

Institute of Biomedicine Alberto Sols (CSIC/UAM), E-28029 Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain. Electronic address:

New onset diabetes after transplantation (NODAT) is a metabolic disorder that affects 40% of patients on immunosuppressive agent (IA) treatment, such as rapamycin (also known as sirolimus). IAs negatively modulate insulin action in peripheral tissues including skeletal muscle, liver and white fat. However, the effects of IAs on insulin sensitivity and thermogenesis in brown adipose tissue (BAT) have not been investigated. We have analyzed the impact of rapamycin on insulin signaling, thermogenic gene-expression and mitochondrial respiration in BAT. Treatment of brown adipocytes with rapamycin for 16h significantly decreased insulin receptor substrate 1 (IRS1) protein expression and insulin-mediated protein kinase B (Akt) phosphorylation. Consequently, both insulin-induced glucose transporter 4 (GLUT4) translocation to the plasma membrane and glucose uptake were decreased. Early activation of the N-terminal Janus activated kinase (JNK) was also observed, thereby increasing IRS1 Ser 307 phosphorylation. These effects of rapamycin on insulin signaling in brown adipocytes were partly prevented by a JNK inhibitor. In vivo treatment of rats with rapamycin for three weeks abolished insulin-mediated Akt phosphorylation in BAT. Rapamycin also inhibited norepinephrine (NE)-induced lipolysis, the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and uncoupling protein (UCP)-1 in brown adipocytes. Importantly, basal mitochondrial respiration, proton leak and maximal respiratory capacity were significantly decreased in brown adipocytes treated with rapamycin. In conclusion, we demonstrate, for the first time the important role of brown adipocytes as target cells of rapamycin, suggesting that insulin resistance in BAT might play a major role in NODAT development.
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http://dx.doi.org/10.1016/j.bbalip.2016.09.016DOI Listing
December 2016

Friedelane-type triterpenoids as selective anti-inflammatory agents by regulation of differential signaling pathways in LPS-stimulated macrophages.

Toxicol Appl Pharmacol 2016 Dec 15;313:57-67. Epub 2016 Oct 15.

Instituto de Investigaciones Biomédicas Alberto Sols (IIBm) (CSIC/UAM), C/ Arturo Duperier 4, 28029 Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem), ISCIII, 28029 Madrid, Spain. Electronic address:

A series of 31 pentacyclic triterpenoids isolated from the root barks of Celastrus vulcanicola and Maytenus jelskii were tested for cytotoxicity and inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 18 (C18) and 25 (C25) exhibited significant inhibition of LPS-induced NO release at 50 and 25μM concentrations, respectively, and decreased mRNAs of pro-inflammatory cytokines. At the molecular level, C18 neither inhibited LPS-mediated phosphorylation of mitogen activated protein kinases (MAPKs) nor nuclear translocation of nuclear factor kappa beta (NFκB). Instead, C18 enhanced and prolonged nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and increased the expression of its target genes including hemeoxigenase 1 (HO1). C25 efficiently inhibited LPS-mediated phosphorylation of JNK, p38 and ERK, without affecting NFκB or Nrf2 signaling pathways. Both compounds reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1β (IL1β) proform, reflecting their ability to target the inflammasome. C25 also counteracted LPS effects on iNOS expression and pro-inflammatory cytokines mRNA levels in Bv-2 microglial cells. The anti-inflammatory effect of both compounds was also assessed in human macrophages. Our results suggest that triterpenoids C18 and C25 possess anti-inflammatory effects, which may be therapeutically relevant for diseases linked to inflammation.
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http://dx.doi.org/10.1016/j.taap.2016.10.004DOI Listing
December 2016

Effects of the neuroprotective drugs somatostatin and brimonidine on retinal cell models of diabetic retinopathy.

Acta Diabetol 2016 Dec 23;53(6):957-964. Epub 2016 Aug 23.

Department of Medical Sciences, University of Turin, Corso AM Dogliotti 14, 10126, Turin, Italy.

Aims: Diabetic retinopathy is considered a microvascular disease, but recent evidence has underlined early involvement of the neuroretina with interactions between microvascular and neural alterations. Topical administration of somatostatin (SST), a neuroprotective molecule with antiangiogenic properties, prevents diabetes-induced retinal neurodegeneration in animals. The α-adrenergic receptor agonist brimonidine (BRM) decreases vitreoretinal vascular endothelial growth factor and inhibits blood-retinal barrier breakdown in diabetic rats. However, SST and BRM effects on microvascular cells have not yet been studied. We investigated the behaviour of these drugs on the crosstalk between microvasculature and neuroretina.

Methods: Expression of SST receptors 1-5 in human retinal pericytes (HRP) was checked. We subsequently evaluated the effects of diabetic-like conditions (high glucose and/or hypoxia) with/without SST/BRM on HRP survival. Endothelial cells (EC) and photoreceptors were maintained in the above conditions and their conditioned media (CM) used to culture HRP. Vice versa, HRP-CM was used on EC and photoreceptors. Survival parameters were assessed.

Results: HRP express the SST receptor 1 (SSTR1). Glucose fluctuations mimicking those occurring in diabetic subjects are more damaging for pericytes and photoreceptors than stable high glucose and hypoxic conditions. SST/BRM added to HRP in diabetic-like conditions decrease EC apoptosis. However, neither SST nor BRM changed the response of pericytes and neuroretina-vascular crosstalk under diabetic-like conditions.

Conclusions: Retinal pericytes express SSTR1, indicating that they can be a target for SST. Exposure to SST/BRM had no adverse effects, direct or mediated by the neuroretina, suggesting that these molecules could be safely evaluated for the treatment of ocular diseases.
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http://dx.doi.org/10.1007/s00592-016-0895-4DOI Listing
December 2016

Autophagy resolves early retinal inflammation in Igf1-deficient mice.

Dis Model Mech 2016 09 21;9(9):965-74. Epub 2016 Jul 21.

Alberto Sols Biomedical Research Institute (IIBm) (CSIC/UAM), 28029, Madrid, Spain Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERdem), ISCIII, 28029, Madrid, Spain IdiPAZ Institute for Health Research, Madrid 28029, Spain

Insulin-like growth factor-1 (IGF-1) is a growth factor with differentiating, anti-apoptotic and metabolic functions in the periphery, and anti-inflammatory properties in the nervous system. Mice that have mutations in the Igf1 gene, rendering the gene product inactive (Igf1(-/-)), present with age-related visual loss accompanied by structural alterations in the first synapses of the retinal pathway. Recent advances have revealed a crucial role of autophagy in immunity and inflammation. Keeping in mind this close relationship, we aimed to decipher these processes in the context of the defects that occur during ageing in the retina of Igf1(-/-) mice. Tnfa and Il1b mRNAs, and phosphorylation of JNK and p38 MAPK were elevated in the retinas of 6- and 12-month old Igf1(-/-) mice compared to those in age-matched Igf1(+/+) controls. In 6-month-old Igf1(-/-) retinas, increased mRNA levels of the autophagy mediators Becn1, Atg9, Atg5 and Atg4, decreased p62 (also known as SQSTM1) protein expression together with an increased LC3-II:LC3-I ratio reflected active autophagic flux. However, in retinas from 12-month-old Igf1(-/-) mice, Nlrp3 mRNA, processing of the IL1β pro-form and immunostaining of active caspase-1 were elevated compared to those in age-matched Igf1(+/+) controls, suggesting activation of the inflammasome. This effect concurred with accumulation of autophagosomes and decreased autophagic flux in the retina. Microglia localization and status of activation in the retinas of 12-month-old Igf1(+/+) and Igf1(-/-) mice, analyzed by immunostaining of Cd11b and Iba-1, showed a specific distribution pattern in the outer plexiform layer (OPL), inner plexiform layer (IPL) and inner nuclear layer (INL), and revealed an increased number of activated microglia cells in the retina of 12-month-old blind Igf1(-/-) mice. Moreover, reactive gliosis was exclusively detected in the retinas from 12-month-old blind Igf1(-/-) mice. In conclusion, this study provides new evidence in a mouse model of IGF-1 deficiency that autophagy is an adaptive response that might confer protection against persistent inflammation in the retina during ageing.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047685PMC
http://dx.doi.org/10.1242/dmm.026344DOI Listing
September 2016

Modulation of microglia polarization dynamics during diabetic retinopathy in db/db mice.

Biochim Biophys Acta 2016 09 4;1862(9):1663-74. Epub 2016 Jun 4.

Alberto Sols Biomedical Research Institute (IIBm) (CSIC/UAM), 28029 Madrid, Spain; Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERdem), ISCIII, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.bbadis.2016.05.024DOI Listing
September 2016

Inhibition of Protein Tyrosine Phosphatase 1B Improves IGF-I Receptor Signaling and Protects Against Inflammation-Induced Gliosis in the Retina.

Invest Ophthalmol Vis Sci 2015 Dec;56(13):8031-44

Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, Spain 2Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC/UAM), Madrid, Spain.

Purpose: Insulin-like growth factor-I receptor (IGF-IR) signaling mediates retinal growth and survival and its failure may contribute to aggravate diabetic retinopathy (DR). Protein tyrosine phosphatase 1B (PTP1B) negatively modulates IGF-IR signaling, but its involvement in inflammation during DR remains unknown. We investigated whether PTP1B participates in the cross-talk between proinflammatory signaling pathways and IGF-IR-mediated signaling in the retina.

Methods: 661W photoreceptors or mouse retinal explants were treated with TNFα, IL6, and IL1β. Insulin-like growth factor-I receptor signaling cascade was evaluated in the absence or presence of PTP1B. db/db mice were used to test a PTP1B inhibitor in retinal gliosis.

Results: 661W retinal cells and retinal explants responded to IGF-I by inducing IGF-IR tyrosine (13-fold) and Akt phosphorylations (7- and 3-fold for serine 473 and threonine 308, respectively). Cytokines triggered early activation of stress kinases (c-jun [NH2] terminal kinase [JNK] and p38 MAPK), resulting in insulin receptor substrate 1 (IRS1) serine 307 phosphorylation that precedes its degradation. Pretreatment of 661W cells or retinal explants with cytokines upregulated PTP1B protein levels (1.45- and 4.5-fold, respectively), induced IRS1 degradation and decreased IGF-I-mediated IGF-IR/Akt phosphorylation. Silencing or deficiency in PTP1B ameliorated the negative effects of cytokines on IGF-IR signaling. Cytokines increased glial fibrillary acidic protein (GFAP) expression in retinal explants by 4.5-fold, this response being reduced by 2-fold with a PTP1B inhibitor. Protein tyrosine phosphatase 1B protein levels increased by 3-fold in retinas from db/db mice and its inhibition reduced gliosis.

Conclusions: Targeting PTP1B might be useful for modulating IGF-I effects in retinal cells during DR.
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http://dx.doi.org/10.1167/iovs.15-17234DOI Listing
December 2015

Topical Administration of GLP-1 Receptor Agonists Prevents Retinal Neurodegeneration in Experimental Diabetes.

Diabetes 2016 Jan 17;65(1):172-87. Epub 2015 Sep 17.

Diabetes and Metabolism Research Unit, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain CIBERDEM, Instituto de Salud Carlos III, Madrid, Spain.

Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Since glucagon-like peptide 1 (GLP-1) exerts neuroprotective effects in the central nervous system and the retina is ontogenically a brain-derived tissue, the aims of the current study were as follows: 1) to examine the expression and content of GLP-1 receptor (GLP-1R) in human and db/db mice retinas; 2) to determine the retinal neuroprotective effects of systemic and topical administration (eye drops) of GLP-1R agonists in db/db mice; and 3) to examine the underlying neuroprotective mechanisms. We have found abundant expression of GLP-1R in the human retina and retinas from db/db mice. Moreover, we have demonstrated that systemic administration of a GLP-1R agonist (liraglutide) prevents retinal neurodegeneration (glial activation, neural apoptosis, and electroretinographical abnormalities). This effect can be attributed to a significant reduction of extracellular glutamate and an increase of prosurvival signaling pathways. We have found a similar neuroprotective effect using topical administration of native GLP-1 and several GLP-1R agonists (liraglutide, lixisenatide, and exenatide). Notably, this neuroprotective action was observed without any reduction in blood glucose levels. These results suggest that GLP-1R activation itself prevents retinal neurodegeneration. Our results should open up a new approach in the treatment of the early stages of DR.
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http://dx.doi.org/10.2337/db15-0443DOI Listing
January 2016

Microglia-Müller glia crosstalk in the rd10 mouse model of retinitis pigmentosa.

Adv Exp Med Biol 2014 ;801:373-9

3D Lab (Development, Differentiation and Degeneration), Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu 9, E-28040, Madrid, Spain,

Retinitis pigmentosa refers to a large, genetically heterogeneous group of retinal dystrophies. This condition is characterized by the gradual onset of blindness due to progressive deterioration of the retina, a process that includes photoreceptor and retinal-pigmented-epithelium cell decay and death, microglial recruitment, reactive gliosis, and vascular disorganization and regression. We found that early in the degenerative process, the rd10 mouse retina exhibits high levels of photoreceptor cell death and reactive Müller gliosis. In explant cultures, both degenerative processes were abrogated by IGF-I treatment. Moreover, the beneficial effect of IGF-I was diminished by microglial depletion using clodronate-containing liposomes. Interestingly, in the absence of IGF-I, microglial depletion partially prevented cell death without affecting Müller gliosis. These findings strongly suggest a role for microglia-Müller glia crosstalk in neuroprotection. However, a subpopulation of microglial cells appears to promote neurodegeneration in the dystrophic retina. Our findings indicate that beneficial neuroprotective effects may be achieved through strategies that modulate microglial cell responses.
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http://dx.doi.org/10.1007/978-1-4614-3209-8_47DOI Listing
July 2014

Loss of protein tyrosine phosphatase 1B increases IGF-I receptor tyrosine phosphorylation but does not rescue retinal defects in IRS2-deficient mice.

Invest Ophthalmol Vis Sci 2013 Jun 19;54(6):4215-25. Epub 2013 Jun 19.

Instituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), Madrid, Spain.

Purpose: Mice with deletion of insulin receptor substrate (IRS) 2 develop type 2 diabetes and photoreceptor degeneration. Loss of protein tyrosine phosphatase 1B (PTP1B) in diabetic IRS2(-/-) mice restores insulin sensitivity and normalizes glucose homeostasis. Since insulin-like growth factor (IGF)-IR promotes survival of photoreceptors and is a substrate of PTP1B, we investigated IGF-IR-mediated survival signaling and visual function in PTP1B(-/-) and double mutant IRS2(-/-)/PTP1B(-/-) mice.

Methods: IGF-IR-mediated Akt signaling was evaluated in IGF-I-stimulated retinal explants. Histologic and electroretinogram analysis was performed in wild-type (WT), IRS2(-/-), PTP1B(-/-), and the double mutant IRS2(-/-)/PTP1B(-/-) mice.

Results: IGF-I stimulated the tyrosine phosphorylation of its receptor and Akt activation in retinal explants of WT mice. In PTP1B(-/-) retinal explants, these responses were enhanced. Conversely, in retinas from IRS2(-/-) mice, expression of PTP1B was increased, coincident with decreased IGF-I-mediated Akt serine 473 phosphorylation. PTP1B deletion in IRS2(-/-) mice also enhanced IGF-IR tyrosine phosphorylation but, unexpectedly, did not rescue Akt activation in response to IGF-I. One potential explanation is that PTEN was increased in retinas of IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice. Histologic evaluation revealed alterations in various structures of the retina in IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice, specifically in the outer nuclear layer (ONL) and retinal outer segments (ROS). Electroretinogram (ERG) analysis confirmed that PTP1B deficiency did not restore visual function in IRS2(-/-) mice.

Conclusions: Although loss of PTP1B enhances tyrosine phosphorylation of the IGF-IR in retinal explants of IRS2(-/-) mice, Akt activation remains defective owing to elevated PTEN levels and, thus, structural and functional visual defects persist in this model.
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http://dx.doi.org/10.1167/iovs.12-11438DOI Listing
June 2013

Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells.

Autophagy 2012 Feb 1;8(2):187-99. Epub 2012 Feb 1.

3D Lab (Development, Differentiation & Degeneration), Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.

Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.
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http://dx.doi.org/10.4161/auto.8.2.18535DOI Listing
February 2012

Microglia-mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmentosa.

Invest Ophthalmol Vis Sci 2011 Nov 25;52(12):9124-30. Epub 2011 Nov 25.

3D Lab (Development, Differentiation and Degeneration), Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

Purpose: To characterize the effect of IGF-I in the rd10 mouse model of retinitis pigmentosa at the cellular level, focusing on the role of microglia in the neurodegenerative process.

Methods: Both organotypic retinal explants and intravitreal injections were used to assess the effect of IGF-I on photoreceptor cell death in the Pde6b(rd10) mice. Cell death was determined by TUNEL in retinal sections and by ELISA of free nucleosomes in retinal extracts. The number and distribution of microglial cells was visualized by immunolabeling with Cd11b and Iba1 antibodies. Depletion of microglia in culture was achieved by treatment with liposomes containing clodronate.

Results: Both ex vivo and in vivo IGF-I treatment reduced the number of TUNEL-positive nuclei in rd10 mouse retinas. In addition, IGF-I treatment in explants increased the number of microglial cells in the ONL. Depletion of microglia in explants with liposomes containing clodronate diminished the neuroprotective effect of IGF-I but also moderately reduced photoreceptor cell death in rd10 retinas cultured in the absence of IGF-I.

Conclusions: IGF-I is able to attenuate photoreceptor cell death both ex vivo and in vivo in the rd10 mouse retina. Microglia is required for the neuroprotective effect of IGF-I in the dystrophic retina. In addition, microglial cells play a detrimental role, seemingly led to neuroprotection by IGF-I.
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http://dx.doi.org/10.1167/iovs.11-7736DOI Listing
November 2011
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