Identification of key lncRNAs in age-related macular degeneration through integrated bioinformatics and experimental validation.

This study aimed to identify key long noncoding RNAs (lncRNAs) in age-related macular degeneration (AMD) patients and to identify relevant pathological mechanisms of AMD development. We identified 407 differentially expressed mRNAs and 429 differentially expressed lncRNAs in retinal pigment epithelium (RPE) and retina in the macular region of AMD patients versus controls (P < 0.05 and |log2FC| > 0.585) from GSE135092. A total of 14 key differentially expressed mRNAs were obtained through external data validation from GSE115828. A miRNA-mRNA and miRNA-lncRNA network containing 52 lncRNA nodes, 49 miRNA nodes, 14 mRNA nodes and 351 edges was constructed via integrated analysis of these components. Finally, the LINC00276-miR-619-5p-IFIT3 axis was identified via protein-protein network analysis. In the t-BH-induced ARPE-19 senescent cell model, LINC00276 and IFIT3 were downregulated. Overexpression of LINC00276 could accelerate cell migration in combination with IFIT3 upregulation. This compelling finding suggests that LINC00276 plays an influential role in the progression of AMD, potentially through modulating senescence processes, thereby setting a foundation for future investigative efforts to verify this relationship.

Downregulation of SIRT6 and NMNAT2 is associated with proliferative diabetic retinopathy.

Purpose: To determine the expression levels of SIRT6 and NMNAT2 in diabetic retinopathy (DR). Methods: We obtained peripheral blood mononuclear cells (PBMCs) and vitreous samples from 77 patients with type 2 diabetes mellitus: 52 with DR and 25 without DR, and 27 healthy control subjects. Western blot analysis and qRT-PCR were performed to evaluate the expression of SIRT6 and NMNAT2 in their PBMCs. The levels of IL-1ß, IL-6, and TNF-α in the vitreous fluid were determined by ELISA. Immunohistochemistry was performed to detect the expression of SIRT6 and NMNAT2 in proliferative DR (PDR) and the control subjects. Results: The expression of SIRT6 and NMNAT2 was markedly downregulated in DR patients, which was negatively correlated with the increased expression of IL-1ß, IL-6 and TNF-α. Additionally, we observed decreased expression of SIRT6 and NMNAT2 in the fibrovascular membranes of PDR patients. Conclusions: The downregulated expression of SIRT6 and NMNAT2 in PDR patients reveals a potential pathogenic association; more extended studies could verify them as potential therapeutic targets.

Decreased expression of Glucagon-like peptide-1 receptor and Sodium-glucose co-transporter 2 in patients with proliferative diabetic retinopathy.

Purpose: To investigate the expression of Glucagon-like peptide-1 receptor (GLP-1R), sodium-glucose co-transporter (SGLT) 1, SGLT2, Glucose transporter type 1 (GLUT1) and GLUT2 in patients with diabetic retinopathy (DR). Methods: We obtained peripheral blood mononuclear cells (PBMCs) and vitreous samples from 26 proliferative DR (PDR) patients, 25 non-proliferative DR (NPDR) patients, 25 non-DR (NDR) patients, and 26 nondiabetic patients with idiopathic epiretinal membranes (ERMs, control). The protein level and mRNA expression level of GLP-1R were quantified by immunoblot and qRT-PCR and the levels of SGLT1, SGLT2, GLUT1, and GLUT2 expression were determined by PCR. Their association with clinical parameters and PBMCs/vitreous cytokine was analyzed. Furthermore, immunofluorescence staining of GLP-1R and SGLT2 was carried out on samples of fibrovascular membranes (FVMs) retrieved from 26 patients with PDR and 26 patients with ERMs. Results: The transcriptional levels of GLP-1R and SGLT2 in PBMCs were significantly more decreased in PDR patients than in patients without DR and controls, which was simultaneously associated with an increased level of expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The expression levels of GLUT1 and GLUT2 were tightly correlated with their SGLT partners, respectively. Further, Immunofluorescence staining showed no positive staining of GLP-1R and SGLT2 was detected in the FVMs from PDR. Conclusions: GLP-1R and SGLT2 were significantly decreased in PDR patients which was associated with an increased level of expression of TNF-α and IFN-γ. These findings implicate that defective GLP-1R and SGLT2 signaling may potentially correlate with immune response cytokines in patients with PDR.

Ageing fundus degenerations of Macaca fascicularis on multi-modal imaging and histopathology: Similarities and differences compared to human.

To characterize the ageing fundus degenerations in Macaca fascicularis, we used multimodal imaging including color fundus photograph, spectral domain optical coherence tomography, fundus autofluorescence, fundus fluorescence angiography, and indocyanine green angiography (ICGA) to survey and track fundus changes of 84 Macaca fascicularis, ranging from 5 to 24 years old over 2 years, and followed by hematoxylin-eosin (HE) and immunofluorescence (IF) staining. The Macaca fascicularis in our cohort showed ageing characteristics different from human, including the more common yellow dot maculopathy, the unique appearance of patchy hyperautoflurescence, and the absence of subretinal drusenoid deposit, basal laminar deposit, geographic atrophy or choroidal neovascularization. Same with human, hard drusen, soft drusen, atherosclerosis, tessellated retina, staining of vessels in peripheral choroid on late-phase ICGA, and peripheral hard drusen were detected. HE and IF staining suggested the patchy hyperautoflurescence to be drusenoid deposits. BMI were significantly higher in the Macaca fascicularis with yellow dot maculopathy and hard drusen, compared to the ones without (p < 0.05). Our study reveals fundus degenerations that develop with ageing in the nonhuman primate of Macaca fascicularis. Their differences and similarities compared to human worth notice by future translational research in degenerative fundus diseases, especially age-related macular degeneration.

CORRELATION BETWEEN FOCAL CHOROIDAL EXCAVATION AND UNDERLYING RETINOCHOROIDAL DISEASE: A Pathological Hypothesis From Clinical Observation.

PURPOSE: To evaluate focal choroidal excavation (FCE) in eyes with various diseases using multimodal imaging modalities and to investigate the correlation of FCE and underlying chorioretinal diseases. METHODS: This retrospective observational study included 62 eyes from 56 patients who were identified by optical coherence tomography as having FCE. All included patients underwent comprehensive clinical examinations and multimodal imaging to identify and detect the characteristics of FCE and its correlation with underlying chorioretinal diseases. RESULTS: All included patients were of Chinese descent, and the median age at diagnosis was 43 years (range: 15-66). Seventy-three FCEs appeared in these included eyes. Most FCEs were formed at sites with anatomical changes caused by various chorioretinal diseases. Choroidal osteoma, punctate inner choroidopathy, and central serous chorioretinopathy were the most common etiologies of FCE. During follow-up, 14 eyes (22.58%) exhibited a pattern change and three eyes (4.84%) developed new-onset choroid neovascularization. CONCLUSION: Focal choroidal excavation is a common sign found in a variety of chorioretinal diseases. Processes that involve impairment or tissue loss of the outer retina and inner choroid and disrupt the balance of intraocular pressure and choroidal pressure because of mechanical disturbance may play a role in FCE formation.

Patchy hyperautofluorescence as a predictive factor for the recurrence of punctate inner choroidopathy.

PURPOSE: To investigate baseline clinical and imaging factors that may correlate with risk of recurrence of punctate inner choroidopathy (PIC). METHODS: In this retrospective observational study, charts and multimodal imaging of forty-five patients diagnosed with PIC during the active inflammatory phase were reviewed. MMI examinations, including fundus photography, shortwave fundus autofluorescence(SW-FAF), fluorescein angiography(FFA), indocyanine green angiography(ICGA), and spectral domain optical coherence tomography(SD-OCT_), were conducted to diagnose PIC, and MMI parameters at baseline were assessed as potential biomarkers indicating the recurrence of inflammation. Statistical analysis was performed to determine the clinical and imaging factors associated with recurrence of PIC. RESULTS: Among the 45 recruited patients, 18 (40 %) had at least one episode of recurrence during a mean follow-up period of 23.66 ± 12.65 months (range, 12-50 months). Best corrected visual acuity (BCVA) at the final visit during the follow-up was significantly different between the recurrence and nonrecurrence groups. Patchy hyperautofluorescence at baseline appeared in 77.78 % of the patients with recurrence, and the incidence of patchy hyperautofluorescence was significantly different between the patients with recurrence and those without recurrence (P＜0.001). CONCLUSIONS: Recurrence is not rare among PIC patients and leads to a worse visual acuity outcome. Patchy hyperautofluorescence at baseline is a risk factor for recurrence of PIC. Patchy hyperautofluorescent areas in PIC patients may indicate a need for close follow-up even though PIC-related inflammation regresses.

Identification of NLRP3 Inflammation-Related Gene Promoter Hypomethylation in Diabetic Retinopathy.

Purpose: To identify and validate key genes that could provide a new perspective for genetic marker screening of diabetic retinopathy (DR). Methods: The gene expression and DNA methylation profiles were obtained from the Gene Expression Omnibus. Differential expression analysis was conducted using the limma package, and then the functions of the differentially expressed genes (DEGs) were analyzed using the DAVID database, followed by protein-protein interaction (PPI) networks using Cytoscape software. We employed the Sequenom MassARRAY system to detect the promoter methylation levels of the candidate genes in peripheral blood mononuclear cells from 32 healthy individuals and 94 patients with type 2 diabetes mellitus (T2D; 64 with DR and 30 without DR) and in fibrovascular membranes (FVMs) from three proliferative DR patients and three controls with idiopathic epiretinal membranes. The mRNA levels of candidate genes were further confirmed via real-time polymerase chain reaction. Results: A significant enrichment of 5906 DEGs was found in immune and inflammatory responses. TGFB1, CCL2, and TNFSF2 were identified as the top three core genes associated with NLRP3 inflammation in PPI networks. These genes have relatively low levels of promoter methylation, which have been validated in peripheral blood mononuclear cells and FVMs from DR patients, and the methylation levels were found to be negative correlated with the mRNA levels and HbA1c levels in T2D patients. Conclusions: Overall, these data indicate that promoter hypomethylation of NLRP3, TGFB1, CCL2, and TNFSF2 may increase the risk of DR in the Chinese Han population, indicating that these genes might serve as potential targets for the detection and treatment of DR.

Age-Related Scattered Hypofluorescent Spots on Late-Phase Indocyanine Green Angiography as Precursor Lesions of Polypoidal Choroidal Vasculopathy.

Purpose: Age-related scattered hypofluorescent spots on late-phase indocyanine green angiography (ASHS-LIA) might represent lipid accumulation in Bruch's membrane in the form of basal linear deposits (BlinD). The present study was conducted to describe the clinical characteristics of polypoidal choroidal vasculopathy (PCV) associated with ASHS-LIA. Methods: Consecutive patients with treatment-naïve PCV who underwent color fundus photography (FP), fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), and spectral-domain optical coherence tomography (SD-OCT) at the Zhongshan Ophthalmic Center from June 2016 through May 2018, were reviewed. ASHS-LIA and choroidal vascular hyperpermeability (CVH) were evaluated by ICGA. Subfoveal choroidal thickness (SFCT) was assessed by SD-OCT. Results: A total of 187 patients were eligible for inclusion in this study (mean, 63.2 ± 7.6 years; range, 41-85 years). Of these patients, 117 (62.6%) showed ASHS-LIA, 57 (30.5%) had bilateral lesions and 70 (37.4%) showed CVH. Moreover, compared with patients without ASHS-LIA, PCV patients with ASHS-LIA were older (P = 0.001), more frequently had bilateral lesions (P = 0.001), and less frequently showed CVH (P = 0.006). SFCT in eyes with ASHS-LIA was significantly greater than that in eyes without ASHS-LIA after adjusting for age, sex, and CVH (P = 0.026). Nevertheless, there was no significant difference in best-corrected visual acuity or lesion characteristics between the two groups. Conclusions: ASHS-LIA, which is very common in PCV patients, might be involved in the pathogenesis of PCV. PCV with ASHS-LIA was more frequently associated with bilateral involvement, less CVH, and a thicker choroid than PCV without ASHS-LIA.

Enhanced Expression of NLRP3 Inflammasome-Related Inflammation in Diabetic Retinopathy.

Purpose: The aim of this study was to determine the association between nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) family, pyrin domain-containing 3 (NLRP3) inflammasome-induced inflammation and disease severity in diabetic retinopathy (DR). Methods: Blood samples were collected from 64 patients with diabetes (DR, 43; without DR, 21) and 25 healthy controls. The protein and mRNA expression levels of NLRP3 inflammasomes in peripheral blood mononuclear cells were determined using western blotting and quantitative real-time reverse transcription-PCR. A total of 82 vitreous samples were obtained from patients with DR (n = 60) and nondiabetic controls (n = 22). All patients were candidates for vitrectomy. Interleukin (IL)-1ß and IL-18 in the peripheral blood mononuclear cell culture medium and vitreous fluid were detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence staining for apoptosis-associated speck-like protein with a caspase recruitment domain (ASC) and NLRP3 was performed in fibrovascular membranes from 21 proliferative DR patients and 22 controls with idiopathic epiretinal membranes. Results: We observed increased gene and protein expression of NLRP3, ASC, and caspase-1 in peripheral blood mononuclear cells of adults with DR compared with that in normal controls. Furthermore, the elevated expressions of NLRP3 and ASC were observed in the fibrovascular membranes from 21 adults with proliferative DR when compared with the 22 controls. IL-1ß and IL-18 in the peripheral blood mononuclear cells and vitreous fluid were elevated in the DR patients when compared with controls. Conclusions: These outcomes suggested that NLRP3 inflammasomes are upregulated in adults with DR and may play a key role in the pathogenesis and progression of DR.

Assessment of biomarkers using multiplex assays in aqueous humor of patients with diabetic retinopathy.

BACKGROUND: With the high prevalence of type 2 diabetes, diabetic retinopathy (DR) has become a leading health problem worldwide. The pathogenesis of DR is complex and several vascular, inflammatory, and neuronal mechanisms are involved. The purpose of this study was to assess the levels of immune and inflammatory biomarkers in the aqueous humor of patients with different severities of DR and to analyze the correlations between Interleukin-6 (IL-6) and these biomarkers, and between IL-6 and the severity of the disease. METHODS: Aqueous humor samples were obtained from 51 non-diabetic patients and 151 diabetic patients. Levels of 45 different cytokines, chemokines, and growth factors were measured using a multiplex bead immunoassay. RESULTS: IL-6, IL-8, Inducible Protein-10 (IP-10), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF)-A were significantly higher (p < 0.05) in the aqueous humor of the DR patients compared to the non-diabetic patients, while the concentrations of IL-1α, IL-4, IL-9, IL-21, IL-23, IL-27, IL-31, RANTES, interferon-α, growth regulated oncogene (GRO), and tumor necrosis factor (TNF)-α were significantly lower (p < 0.05) in the DR patients. The IL-6 levels increased as the severity of DR increased. In addition, the IL-6 level positively correlated with the IL-8, HGF and LIF levels, while negatively with the IL-31and GRO levels. CONCLUSIONS: These findings suggest that inflammation and immune response may contribute to the pathogenesis of DR, and these biomarkers may potentially be new therapeutic targets for DR.

Analysis of the Serum Lipid Profile in Polypoidal Choroidal Vasculopathy.

Polypoidal choroidal vasculopathy (PCV), the predominant subtype of neovascular age-related macular degeneration in the Asian population, is associated with genetic polymorphism of lipid metabolism. In this study, we performed the untargeted lipidomics approach of ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) to reveal the potential discriminating lipid profile of PCV patients in serum (21 PCV patients and 19 age-matched controls). Unsupervised principal component, supervised orthogonal partial least squares analysis, correlation analysis, and heatmap analysis were performed with the data obtained by UPLC-MS. Forty-one discriminating metabolites were identified. Receiver operating characteristic curve analysis, pathway analysis and functional analysis were performed subsequently, and platelet-activating factor (PAF) was further selected as the key indicator of the distinct lipid metabolism in PCV patients. Finally, the serum level of PAF was validated by enzyme-linked immunosorbent assay, which is significantly higher in PCV patients compared to controls (65 PCV patients and 63 age-matched controls, p < 0.0001), consistent with the UPLC-MS analysis. Our results suggested that PAF is considered as the major indicator of the distinct lipid metabolism in PCV patients.

Th17 cell frequency and IL-17A concentrations in peripheral blood mononuclear cells and vitreous fluid from patients with diabetic retinopathy.

Objective To quantify T helper (Th)17 cells and determine interleukin (IL)-17A levels in peripheral blood mononuclear cell (PBMC) culture and vitreous fluid from patients with type 2 diabetes mellitus (T2DM) with diabetic retinopathy (DR). Methods Th17 cell frequency and IL-17A concentrations in PBMCs from 60 patients with T2DM with DR, 30 without DR and 30 sex- and age-matched healthy individuals were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. IL-17A levels in vitreous fluid from 31 eyes with proliferative DR and diabetic macular oedema (DR group) and 32 eyes with an epiretinal membrane and macular hole (control group) that underwent vitrectomy were also examined by ELISA. Results Compared with the control group, the proportion of Th17 cells and IL-17A concentrations in PBMCs were significantly increased in patients without DR but decreased in those with DR. IL-17A concentrations and Th17 cell frequency in PBMCs tended to decrease with DR severity and were negatively correlated with body mass index, T2DM duration and glycated haemoglobin. Additionally, vitreous fluid IL-17A levels were significantly elevated in patients with DR compared with those of the control group. Conclusions We conclude that disturbances in Th17 cells and IL-17A levels are possibly associated with DR.

Increased levels of IL-6, sIL-6R, and sgp130 in the aqueous humor and serum of patients with diabetic retinopathy.

PURPOSE: Trans-signaling of interleukin (IL)-6 through its soluble receptor (sIL-6R) is critically involved in the promotion of chronic inflammatory diseases. The aim of the present study was to estimate IL-6, sIL-6R, and soluble gp130 (sgp130, a natural antagonist of IL-6 trans-signaling) concentrations in the serum and aqueous humor (AqH) of patients with diabetic retinopathy (DR). METHODS: Paired AqH and serum samples were collected from 152 consecutive diabetic patients (105 with DR and 47 without DR, NDR) and 51 healthy controls. The IL-6, sIL-6R, and sgp130 concentrations were measured with multiplex bead immunoassay. RESULTS: The sgp130 concentrations in the serum and AqH were statistically significantly elevated in patients with DR compared with the NDR patients and the healthy controls (p<0.001). The sgp130 concentrations in the serum and AqH increased as the DR severity increased (p = 0.008, p<0.001, respectively). Higher serum and AqH concentrations of IL-6 and sIL-6R were also observed in patients with DR when compared with the NDR patients and the healthy controls (p<0.001). The AqH concentration of sgp130 was found to be statistically significantly correlated with sIL-6R and IL-6. Similarly, the IL-6 concentration in the AqH was statistically significantly correlated with sIL-6R (p<0.001). Elevated sgp130, sIL-6R, and IL-6 concentrations in the AqH were associated with longer disease duration and higher body mass index, plasma glucose, and glycosylated hemoglobin (HbA1c). CONCLUSIONS: The sgp130, IL-6, and sIL-6R concentrations were statistically significantly elevated in patients with DR, suggesting a probable contributing role of the IL-6 trans-signaling pathway to the pathophysiology of DR.

Oral Chinese herbal medicine for kidney nourishment in Alzheimer's disease: a systematic review of the effect on MMSE index measures and safety.

OBJECTIVE: To evaluate the effectiveness and safety of the Chinese herbal medicine for kidney nourishment (CHMK) assessed with the Mini-Mental Status Examination (MMSE) index objective outcome measures in individuals with Alzheimer's disease. METHODS: Searches were conducted in 7 medical databases from their inceptions until July 19, 2014 for randomized controlled trials (RCTs) that compared the oral administration of CHMK plus conventional pharmacotherapy with the same conventional pharmacotherapy alone with MMSE index measures as outcomes. Relevant resources were also manually retrieved. Two reviewers screened the citations of the reports, assessed the risk of bias and extracted data independently. Data analysis was carried out with Cochrane Collaboration's RevMan5.2.6 software and evidence quality grading evaluation of the systematic review was conducted with Grades of Recommendations Assessment Development and Evaluation (GRADE) profiler software. RESULTS: A total of 20 studies involving 1682 participants were included in the meta-analysis. There were 15 trials that compared CHMK with conventional pharmacotherapy and 5 trials that compared CHMK plus conventional pharmacotherapy with conventional pharmacotherapy alone. The main meta-analysis results showed relative benefits in effective rates in five studies (odds ratio [OR] 2.74, 95% confidence interval [CI] 1.55-4.85) and cure rate/clinical-control rates in five studies (OR 1.91, 95% CI 1.27-2.88) in favor of the CHMK plus conventional pharmacotherapy group. As for CHMK compared with conventional pharmacotherapy, no significant differences were noted in the effective rate (OR 1.09, 95% CI 0.82-1.46; cure rate (OR 1.06, 95% CI 0.81-1.38) and detailed sub-group of MMSE scores from the onset time to 4 weeks (weighted mean difference [WMD] 0.31, 95% confidence interval [CI] -0.81 to 1.42, 8 weeks WMD 1.12, 95% CI -0.54 to 2.78, 12 weeks (WMD 0.43, 95% CI -1.62 to 2.48, or 24 weeks WMD 1.92, 95% CI -1.60 to 5.44) follow-up and the overall effect (WMD 0.79, 95% CI -0.11 to 1.69). Moreover, weaknesses in methodological quality were identified in most studies according to Cochrane Risk of Bias tool assessment, while the quality level of GRADE classification indicated "very low". The incidence of adverse events with CHMK (0.87%) was lower than in the conventional pharmacotherapy group (4.08%), which revealed use of CHMK was relatively safer than conventional pharmacotherapy alone. CONCLUSION: The effectiveness and safety of oral administration of CHMK cannot be currently determined because of publication bias and the low quality level of the included trials. Further studies on a larger scale and with more rigorous designs are required to define the role of CHMK in the treatment of AD.

[Simultaneous determination of eight derivatives of propranolol in cornea perfusate in vitro by high performance liquid chromatography].

A high performance liquid chromatographic method was developed for the simultaneous determination of eight derivatives of propranolol. Cassette dosing method was used in the epithelium side of cornea in vitro to get the effect of penetrant, and the perfusate was collected in the side of endothelium. The protein in the sample was precipitated and discarded by high speed centrifugation before injection. An Agilent Zorbax Extend column (150 mm x 3 mm, 5 microm) was used at 30 degrees C. The mobile phase system contained acetonitrile and 0.03% (v/v) phosphoric acid aqueous solution and the percentage of acetonitrile changed between 3% and 20% (v/v) in a linear gradient elution. The samples were detected by an ultraviolet (UV) detector at 205 nm. The results showed that the eight derivatives of propranolol were completely separated and determined in 31 min. The correlation coefficients were above 0.9970 and good linear relationships were obtained in the range of 0.2 (0.1)-40.0 micromol/L. Under the optimized conditions, the recoveries of the derivatives were in the range of 91.12%-105.73%. The intra-day relative standard deviations (RSDs) were in the range of 1.00%-11.63%, and the inter-day RSDs were in the range of 1.18%-18.58%. The sample showed stability under room temperature, freeze and three cycles of freeze-thaw conditions. This method is fast and accurate for the quantitative analysis of the derivatives of propranolol in transmembrane absorption such as cornea perfusion in vitro or transwell cell system.