Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina.

(Poly)phenol-derived metabolites are small molecules resulting from (poly)phenol metabolization after ingestion that can be found in circulation. In the last decade, studies on the impact of (poly)phenol properties in health and cellular metabolism accumulated evidence that (poly)phenols are beneficial against human diseases. Diabetic retinopathy (DR) is characterized by inflammation and neovascularization and targeting these is of therapeutic interest. We aimed to study the effect of pyrogallol-O-sulfate (Pyr-s) metabolite in the expression of proteins involved in retinal glial activation, neovascularization, and glucose transport. The expression of PEDF, VEGF, and GLUT-1 were analyzed upon pyrogallol-O-sulfate treatment in RPE cells under high glucose and hypoxia. To test its effect on a diabetic mouse model, Ins2Akita mice were subjected to a single intraocular injection of the metabolite and the expression of PEDF, VEGF, GLUT-1, Iba1, or GFAP measured in the neural retina and/or retinal pigment epithelium (RPE), two weeks after treatment. We observed a significant decrease in the expression of pro-angiogenic VEGF in RPE cells. Moreover, pyrogallol-O-sulfate significantly decreased the expression of microglial marker Iba1 in the diabetic retina at different stages of disease progression. These results highlight the potential pyrogallol-O-sulfate metabolite as a preventive approach towards DR progression, targeting molecules involved in both inflammation and neovascularization.

Dysregulation of trophic factors contributes to diabetic retinopathy in the Ins2Akita mouse.

Diabetic retinopathy (DR) is considered as a diabetes-related complication that can lead to severe visual impairments. By 2030, it is expected that 1 in 5 adults will suffer from the disease. Suitable animal models for chronic DR are essential for a better understanding of the pathophysiology and to further develop new treatments. The Ins2Akita mouse is a type 1 diabetes model that shows signs of both early and late stages of DR, including pericyte loss, increased vascular permeability, increased acellular capillaries and neovascularization. To further characterize DR in the Ins2Akita mouse model, we have evaluated the protein levels of the angiogenesis inducers vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and the angiogenesis inhibitor pigment epithelium-derived factor (PEDF). Additionally, we have analyzed the protein expression profile of the glial markers ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) as well as of the chemokine monocyte chemoattractant protein 1 (MCP-1). In this study we demonstrate that, with disease progression, there is the development of an inflammatory response and an unbalanced expression of pro- and antiangiogenic factors in the neural retina and in the retinal pigment epithelium (RPE) of Ins2Akita mice. Therefore, our data provide support for the diabetic retinopathy features detected in the Ins2Akita retina, reflecting what is observed in the human pathology.

PlGF silencing combined with PEDF overexpression: Modeling RPE secretion as potential therapy for retinal neovascularization.

Ocular neovascularization is a defining feature of several blinding diseases. We have previously described the effectiveness of long-term pigment epithelium-derived factor (PEDF) expression in the retina of diabetic mice in ameliorating some diabetic retinopathy hallmarks. In this study, we aimed to investigate if the antiangiogenic potential of PEDF overexpression was enhanced in combination with placental growth factor (PlGF) silencing. Human RPE cells were transfected with a self-replicating episomal vector (pEPito) for PEDF overexpression and/or a siRNA targeting PlGF gene. Conditioned media from PEDF overexpression, from PlGF inhibition and from their combination thereof were used to culture human umbilical vein endothelial cells, and their proliferation rate, migration capacity, apoptosis and ability to form tube-like structures were analyzed in vitro. We here demonstrate that pEPito-driven PEDF overexpression in combination with PlGF silencing in RPE cells does not affect their viability and results in an enhanced antiangiogenic activity in vitro. We observed a significant decrease in the migration and proliferation of endothelial cells, and an increase in apoptosis induction as well as a significant inhibitory effect on tube formation. Our findings demonstrate that simultaneous PEDF overexpression and PlGF silencing strongly impairs angiogenesis compared with the single approaches, providing a rationale for combining these therapies as a new treatment for retinal neovascularization.

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2Akita.

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

Accuracy and Psychometric Properties of the Brazilian Version of the Montreal Cognitive Assessment as a Brief Screening Tool for Mild Cognitive Impairment and Alzheimer's Disease in the Initial Stages in the Elderly.

OBJECTIVE: To evaluate the applicability and the psychometric properties of Montreal Cognitive Assessment Brazilian Version (MoCA-BR) in the elderly, as well as comparing its accuracy as a tracking test for mild cognitive impairment (MCI) and mild Alzheimer's disease (AD) with the accuracy of Mini-Mental State Examination (MMSE). METHOD: A transversal study was performed in 4 reference medical centers that care for the elderly. In all, 229 elderly participated in the study. To select the sample, the clinical history of the elderly, Pfeffer Functional Activities Questionnaire, and neuropsychological battery, apart from MMSE and MoCA-BR cognitive tests, were selected. The elderly were classified into control, MCI, and mild AD groups. RESULTS: There was a significant statistical difference between the MoCA-BR scores of the elderly and the control group, MCI, and mild AD (p < 0.001). The Cronbach alpha for MoCA-BR was 0.77, indicating a good internal consistency. The test-retest reliability was elevated, with intraclass correlation coefficient (ICC) 0.91. The inter-examiner reliability was excellent (ICC 0.96). The area under curve of the receiver operating characteristics curve was 0.95, when evaluating the ability of MoCA-BR to discriminate between the elderly with cognitive impairment and cognitively healthy elderly. CONCLUSIONS: The results of the study show that the Brazilian version of MoCA is a reliable cognitive tracking tool and is accurate for the detection of MCI and early stage AD, with good applicability on the elderly with education equal to or more than 4 years and adequate to discriminate between cognitively healthy elderly, and those with MCI and mild, proving to be superior to MMSE in tracking MCI and similar to this test when tracking mild AD.

Aliskiren decreases oxidative stress and angiogenic markers in retinal pigment epithelium cells.

There is growing evidence on the role of ocular renin-angiotensin system (RAS) in the development of diabetic retinopathy (DR), particularly due to the trigger of oxidative stress and angiogenesis. Despite this there is no effective RAS-based therapy in DR capable of preventing retinal damage induced by RAS activation. We recently described that retinal pigment epithelium (RPE) cells express the main components of the RAS. We here propose to investigate the role of glucose upon the retinal RAS and whether aliskiren, a direct renin inhibitor, protects RPE cells from angiogenesis and oxidative stress. RPE cells were chosen as target since one of the first events in DR is the dysfunction of the RPE retinal layer, which as a key function in maintaining the integrity of the retina. We found that the RAS present in the RPE cells was deregulated by hyperglycemic glucose concentrations. Exposure of RPE cells to angiotensin II increased the levels of the main pro-angiogenic factor, vascular endothelial growth factor (VEGF) in a concentration-dependent manner. Additionally, angiotensin II also stimulated the production of reactive oxygen species in RPE cells. Treatment of RPE cells with aliskiren decreased the levels of oxidative stress and promoted the expression of anti-angiogenic factors such as the pigment epithelium-derived factor and the VEGF165b isoform. Our findings demonstrate that the RAS is deregulated in hyperglycemic conditions and that aliskiren successfully protected RPE cells from RAS over activation. These anti-angiogenic and antioxidant properties described for aliskiren over RPE cells suggest that this drug has potential to be used in the treatment of diabetic retinopathy.

GLUT1 activity contributes to the impairment of PEDF secretion by the RPE.

PURPOSE: In this study, we aimed to understand whether glucose transporter 1 (GLUT1) activity affects the secretion capacity of antiangiogenic factor pigment epithelium-derived factor (PEDF) by the RPE cells, thus explaining the reduction in PEDF levels observed in patients with diabetic retinopathy (DR). METHODS: Analysis of GLUT1 expression, localization, and function was performed in vitro in RPE cells (D407) cultured with different glucose concentrations, corresponding to non-diabetic (5 mM of glucose) and diabetic (25 mM of glucose) conditions, further subjected to normoxia or hypoxia. The expression of PEDF was also evaluated in the secretome of the cells cultured in these conditions. Analysis of GLUT1 and PEDF expression was also performed in vivo in the RPE of Ins2(Akita) diabetic mice and age-matched wild-type (WT) controls. RESULTS: We observed an increase in GLUT1 under hypoxia in a glucose-dependent manner, which we found to be directly associated with the translocation and stabilization of GLUT1 in the cell membrane. This stabilization led to an increase in glucose uptake by RPE cells. This increase was followed by a decrease in PEDF expression in RPE cells cultured in conditions that simulated DR. Compared with non-diabetic WT mice, the RPE of Ins2(Akita) mice showed increased GLUT1 overexpression with a concomitant decrease in PEDF expression. CONCLUSIONS: Collectively, our data show that expression of GLUT1 is stimulated by hyperglycemia and low oxygen supply, and this overexpression was associated with increased activity of GLUT1 in the cell membrane that contributes to the impairment of the RPE secretory function of PEDF.

Graphene Biosensors-A Molecular Approach.

Graphene is the material elected to study molecules and monolayers at the molecular scale due to its chemical stability and electrical properties. The invention of scanning tunneling microscopy has deepened our knowledge on molecular systems through imaging at an atomic resolution, and new possibilities have been investigated at this scale. Interest on studies on biomolecules has been demonstrated due to the possibility of mimicking biological systems, providing several applications in nanomedicine: drug delivery systems, biosensors, nanostructured scaffolds, and biodevices. A breakthrough came with the synthesis of molecular systems by stepwise methods with control at the atomic/molecular level. This article presents a review on self-assembled monolayers of biomolecules on top of graphite with applications in biodevices. Special attention is given to porphyrin systems adsorbed on top of graphite that are able to anchor other biomolecules.

Graphene Oxide Thin Films with Drug Delivery Function.

Graphene oxide has been used in different fields of nanomedicine as a manager of drug delivery due to its inherent physical and chemical properties that allow its use in thin films with biomedical applications. Several studies demonstrated its efficacy in the control of the amount and the timely delivery of drugs when it is incorporated in multilayer films. It has been demonstrated that oxide graphene layers are able to work as drug delivery or just to delay consecutive drug dosage, allowing the operation of time-controlled systems. This review presents the latest research developments of biomedical applications using graphene oxide as the main component of a drug delivery system, with focus on the production and characterization of films, in vitro and in vivo assays, main applications of graphene oxide biomedical devices, and its biocompatibility properties.

A positive HPV test with positive p16/Ki-67 double staining in self-sampled vaginal material is an accurate tool to detect women at risk for cervical cancer.

BACKGROUND: The development of efficient strategies for managing high-risk human papillomavirus (HR-HPV)-positive women is a major challenge when human papillomavirus-based primary screening is being performed. The objectives of this study were to evaluate the comparative effectiveness of HR-HPV testing based on self-collection (SC) and HR-HPV testing based on collection by a health professional (HP) and to assess the potential usefulness of HR-HPV testing combined with testing with the biomarkers p16/Ki-67, α-mannosidase, and superoxide dismutase 2 (SOD2). METHODS: This was a cross-sectional study of 232 women admitted for colposcopy because of an abnormal Papanicolaou smear. The collected material underwent liquid-based cytology, HR-HPV detection, and immunocytochemical testing (p16/Ki-67, α-mannosidase, and SOD2). The gold standard was the histopathological result; the positive reference was CIN2+. RESULTS: The overall accuracy of HR-HPV testing was 76.6%; the results for the SC group (78.1%) and the HP group (75.2%) were similar. The positive predictive values (HP, 76.5%; SC, 80.0%), the negative predictive values (HP, 66.7%; SC, 64.3%), the positive likelihood values (HP, 1.35; SC, 1.36), and the negative likelihood values (HP, 0.21; SC, 0.19) were also similar. p16/Ki-67 showed higher sensitivity than the other 2 biomarkers: 78.1% versus 45.8% for α-mannosidase and 44.5% for SOD2. The specificities of the biomarkers were equivalent: 71.4% for p16/Ki-67, 77.8% for α-mannosidase, and 71.2% for SOD2. In the HP group, accuracy also leaned more heavily toward the final score (using α-mannosidase and SOD2) without statistical significance (80.8% vs 77.9%). The contrast with the SC group yielded the same level of accuracy. CONCLUSIONS: SC, when associated with testing with biomarkers, is as accurate as collection by HPs in the detection of women at risk for cervical cancer.

Development of strategies to modulate gene expression of angiogenesis-related molecules in the retina.

Intravitreal anti-vascular endothelial growth factor agents are the gold standard treatment of ocular neovascular diseases. However, their short-term efficacy implies frequent intravitreal injections. Gene therapy has the ability to provide longer duration of the therapeutic effect. We have previously described the effectiveness of the self-replicating episomal vector, pEPito, in long-term gene expression in mouse retina. In this study, we evaluated different constructs to overexpress pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor, and simultaneously, to silence placental growth factor (PlGF), a key player in neovascularization. We employed the human cytomegalovirus promoter to drive the expression of PEDF and PlGF shRNA, in conjunction with cis-acting ribozymes, using pEPito as expressing vector. Our results demonstrated that the non-viral systems were able to efficiently promote a sustained increase of the PEDF: PlGF ratio in the mice retina, decreased in pathological conditions. This innovative approach could open avenues for the development of new therapeutic strategies.

Correlation between hyperglycemia and glycated albumin with retinopathy of prematurity.

To determine the association between hyperglycemia, glycated albumin (GlyA) and retinopathy of prematurity (ROP). Prospective study of all infants under ROP screening from March 2017 to July 2019. All demographic, clinical and laboratory data were collected. Glucose was measured at birth and every 8 h for the first week and serum GlyA was evaluated at birth, 1st, 2nd and 4th weeks after birth. Reference range for GlyA was obtained. Univariate logistic regression was used to examine risk factors for ROP followed by multivariate regression. A total of 152 infants were included in the study. Median gestational age was 30 weeks and median birth weight 1240 g. Thirty-three infants (21.7%) had ROP. Hyperglycemia was present in 24 (72.7%) infants diagnosed with any ROP versus 6 (0.05%) in those without ROP. Median GlyA at birth, 1st, 2nd and 4th and respective reference ranges were 8.50% (6.00-12.65), 8.20% (5.32-11.67), 8.00% (5.32-10.00) and 7.90% (5.30-9.00) respectively. After multivariate logistic regression, hyperglycemia but not GlyA, remained a significant risk factor for ROP overpowering the other recognized risk factors (Exp (B) 28.062, 95% CI for Exp(B) 7.881-99.924 p < 0.001). In our cohort, hyperglycemia but not GlyA, remained a significant risk factor for ROP overpowering the other recognized risk factors.

Applicability and validation of the Reaction to Tests Scale (RTT) in a sample of Portuguese medical students.

BACKGROUND: Test anxiety is a crucial factor in determining academic outcomes, and it may lead to poor cognitive performance, academic underachievement, and psychological distress, interfering specifically with their ability to think and perform during tests. The main objective of this study was to explore the applicability and psychometric properties of a Portuguese version of the Reactions to Tests scale (RTT) in a sample of medical students. METHOD: A sample of 672 medical students completed the RTT. The sample was randomly split in half to allow for independent Exploratory Factor Analysis (EFA) and to test the best fit model-Confirmatory Factor Analysis (CFA). CFA was used to test both the first-order factor structure (four subscales) and second-order factor structure, in which the four subscales relate to a general factor, Test Anxiety. The internal consistency of the RTT was assessed through Cronbach's alpha, Composite reliability (CR) and Average Variance Extracted (AVE) for the total scale and each of the four subscales. Convergent validity was evaluated through the correlation between RTT and the State-Trait Anxiety Inventory (STAI-Y).To explore the comparability of measured attributes across subgroups of respondents, measurement invariance was also studied. RESULTS: Results from exploratory and confirmatory factor analyses showed acceptable fits for the Portuguese RTT version. Concerning internal consistency, results indicate that RTT was found to be reliable to measure test anxiety in this sample. Convergent validity of the RTT with both state and trait anxiety STAI-Y's subscales was also shown. Moreover, multigroup analyses showed metric invariance across gender and curriculum phase. CONCLUSION: Our results suggest that the RTT scale is a valid and reliable instrument for the measurement of test anxiety among Portuguese Medical Students.

Human-derived NLS enhance the gene transfer efficiency of chitosan.

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.

Dual-Acting Antiangiogenic Gene Therapy Reduces Inflammation and Regresses Neovascularization in Diabetic Mouse Retina.

Intravitreal injections of anti-vascular endothelial growth factor drugs have become the gold standard treatment for diabetic retinopathy (DR). However, several patients are classified as non-responders or poor responders to treatment. Therefore, it is essential to study alternative target molecules. We have previously shown that the progression of DR in the Ins2Akita mouse reflects the imbalance between pro- and anti-angiogenic molecules found in the human retina. We report, for the first time, the therapeutic potential of a dual-acting antiangiogenic non-viral gene therapy. We have used an expressing vector encoding both the pigment epithelium-derived factor gene and a short hairpin RNA (shRNA) targeted to the placental growth factor to restore the balance between these factors in the retina. Twenty-one days after a single subretinal injection, we observed a marked decrease in the inflammatory response in the neural retina and in the retinal pigment epithelium, together with reduced vascular retinal permeability in the treated diabetic mouse. These results were accompanied by the restoration of the retinal capillary network and regression of neovascularization, with significant improvement of DR hallmarks. Concomitant with the favorable therapeutic effects, this approach did not affect retinal ganglion cells. Hence our results provide evidence toward the use of this approach in DR treatment.

Self-Assembled Multilayer Films for Time-Controlled Ocular Drug Delivery.

The patient's compliance on the therapeutics to treat glaucoma is significantly low contributing for a fast evolution of the disease. This article presents an autonomous system with controlled release using an alpha2-adrenergic receptor agonist, brimonidine, usually used to treat glaucoma. More specifically, biocompatible and layer-by-layer drug delivery films containing monolayers with brimonidine encapsulated in polymer-ß-cyclodextrin were prepared with the objective to obtain a system able to release precise amounts of drug at specific times. To delay the erosion-controlled drug release, we included nanosheets of graphene oxide and layers of a biodegradable polymer (poly-ß-aminoester) between the drug-containing monolayers to obtain a time-controlled drug delivery system. An increase in the number of graphene oxide layers is proportional to the brimonidine release delay and its kinetic release can be tuned as a function of the number of layers. Two types of films with brimonidine encapsulated in ß-cyclodextrin were analyzed. One of them composed of barrier layers with PBAE and another with two types of barrier layers, PBAE and graphene oxide. The results indicate that one graphene oxide bilayer can delay the brimonidine release for more than 24 h. In vitro assays confirmed that the films have a cell viability of 100%.

Optimal Cutoff Scores for Dementia and Mild Cognitive Impairment in the Brazilian Version of the Montreal Cognitive Assessment among the Elderly.

OBJECTIVE: To propose cutoff scores for the Brazilian version of the Montreal Cognitive Assessment (MoCA-BR) stratified by education in order to detect mild cognitive impairment (MCI) and mild Alzheimer's disease (AD) in the elderly. METHOD: A transversal study in health centers was performed on 159 elderly people with 4-12 years of education and 70 of their peers with over 12 years of schooling. The MoCA-BR cutoff scores for screening cognitive impairment were determined based on an ROC curve analysis. RESULTS: The ROC curve analysis indicated that cutoff scores under 20 were good for screening elderly people with cognitive impairment with more than 12 years of education, and scores under 21 were good for screening those with 4-12 years of education. CONCLUSIONS: MoCA-BR scores under 21 points (after adding 1 point to the elderly with ≤12 years of education) indicate a need to continue the diagnostic investigation with regular follow-ups.

Molecular biology tools for the study and therapy of PDE6ß mutations.

Gene therapy has the potential for treating retinal diseases, and we have been developing delivery vehicles and expression vectors for this purpose. In this short communication, we describe the generation of tools for both in vitro studies of the disease mechanism and for in vivo testing of therapeutic approaches. We have cloned the PDE6ß gene and also recreated the same mutation present in the rd10 mouse using an optimized plasmid vector. To allow visual detection, we have also generated, through site-directed mutagenesis, plasmids expressing the normal and mutated PDE6ß gene fused with the GFP gene. Next, we have transfected retinal pigment epithelium cells with the different vectors and detected the protein expression of both the normal and mutated PDE6ß. With this work we have created gene therapy tools for in vitro and in vivo studies of retinal disease-causing mutations, namely for the PDE6ß, implicated in retinitis pigmentosa.

The role of the retinal pigment epithelium and Müller cells secretome in neovascular retinal pathologies.

Secreted trophic factors are key to maintain the structural and functional integrity of the retina, as they regulate cellular pathways responsible for survival, function, and response to injury. Nevertheless, these same factors can also be involved in retinal pathologies, as a consequence of the impairment of the secretory function of cells. The cells considered as major contributors to the retinal secretome are the retinal pigmented epithelium (RPE) and Müller cells. Their role in the pathophysiology of the most common neovascular pathologies in the retina - Age-related Macular Degeneration (AMD), Diabetic Retinopathy (DR), and Retinopathy of Prematurity (ROP) - is highlighted in this short review, together with current trophic factor-based therapies, which are mainly focused on controlling inflammation, cell survival, and angiogenesis.