Rat Retinal Cells Research Articles

SIRT3 mitigates high glucose-induced damage in retinal microvascular endothelial cells via OPA1-mediated mitochondrial dynamics

Oxidative stress in endothelial cells is pivotal in diabetic retinopathy (DR), with mitochondrial homeostasis being crucial to mitigate this stress. This study explored the roles of mitochondrial sirtuins (SIRTs) in high glucose (HG)-induced oxidative stress, related endothelial impairment, and mitochondrial homeostasis damage in rat retinal microvascular endothelial cells (RMECs). RMECs were cultured under HG or equivalent osmotic conditions. Cell viability was assessed using the Cell Counting Kit-8 assay, whereas cell death and survival were determined via calcein-AM/propidium iodide double staining. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescein fluorescence. Expression of mitochondrial SIRTs3-5 and key mitochondrial homeostasis molecules was quantified by the quantitative real-time polymerase chain reaction and confirmed by western blotting. Mitochondrial morphology was evaluated using electron microscopy and the MitoTracker fluorescent probe. A SIRT3-overexpressing RMEC line was constructed to assess the role of SIRT3 in oxidative stress and mitochondrial dynamics. After 48 h of HG exposure, cell viability was significantly reduced, with a concomitant increase in cell death and ROS levels, alongside a marked decrease in SIRT3 expression and molecules associated with mitochondrial dynamics. SIRT3 overexpression reversed these effects, particularly increasing the mitochondrial fusion-related molecule, optic atrophy 1 (OPA1). However, the OPA1 inhibitor, MYLS22, blocked the protective effect of SIRT3, leading to more dead cells, a higher ROS level, and intensified mitochondrial fragmentation. These results suggest that SIRT3 is involved in HG-induced imbalanced mitochondrial dynamics of endothelial cells in DR, potentially through the OPA1 pathway.

N-acetylcysteine amide and di- N-acetylcysteine amide protect retinal cells in culture via an antioxidant action

Reactive oxygen species (ROS) play a significant role in toxicity to the retina in a variety of diseases. N-acetylcysteine (NAC), N-acetylcysteine amide (NACA) and the dimeric di-N-acetylcysteine amide (diNACA) were evaluated in terms of protecting retinal cells, in vitro, in a variety of stress models.Three types of rat retinal cell cultures were utilized in the study: macroglial-only cell cultures, neuron-only retinal ganglion cell (RGC) cultures, and mixed cultures containing retinal glia and neurons. Ability of test agents to attenuate oxidative stress in all cultures was ascertained. In addition, capability of agents to protect against a variety of alternate clinically-relevant stressors, including excitotoxins and mitochondrial electron transport chain inhibitors, was also evaluated. Capacity of test agents to elevate cellular levels of reduced glutathione under normal and compromised conditions was also determined.NAC, NACA and diNACA demonstrated concentration-dependent cytoprotection against oxidative stress in all cultures. These three compounds, however, had differing effects against a variety of alternate insults to retinal cells. The most protective agent was NACA, which was most potent against the most stressors (including oxidative stress, mitochondrial impairment by antimycin A and azide, and glutamate-induced excitotoxicity). Similar to NAC, NACA increased glutathione levels in non-injured cells, although diNACA did not, suggesting a different, unknown mechanism of antioxidant activity for the latter. In support of this, diNACA was the only agent to attenuate rotenone-induced toxicity in mitochondria.NAC, NACA and diNACA exhibited varying degrees of antioxidant activity, i.e., protected cultured rat retinal cells from a variety of stressors which were designed to mimic aspects of the pathology of different retinal diseases. A general rank order of activity was observed: NACA ≥ diNACA > NAC. These results warrant further exploration of NACA and diNACA as antioxidant therapeutics for the treatment of retinal diseases, particularly those involving oxidative stress. Furthermore, we have defined the battery of tests carried out as the “Wood, Chidlow, Wall and Casson (WCWC) Retinal Antioxidant Indices”; we believe that these are of great value for screening molecules for potential to reduce retinal oxidative stress in a range of retinal diseases.

Amelioration of Photoreceptor Degeneration by Intravitreal Transplantation of Retinal Progenitor Cells in Rats.

Photoreceptor degeneration is a major cause of untreatable blindness worldwide and has recently been targeted by emerging technologies, including cell- and gene-based therapies. Cell types of neural lineage have shown promise for replacing either photoreceptors or retinal pigment epithelial cells following delivery to the subretinal space, while cells of bone marrow lineage have been tested for retinal trophic effects following delivery to the vitreous cavity. Here we explore an alternate approach in which cells from the immature neural retinal are delivered to the vitreous cavity with the goal of providing trophic support for degenerating photoreceptors. Rat and human retinal progenitor cells were transplanted to the vitreous of rats with a well-studied photoreceptor dystrophy, resulting in substantial anatomical preservation and functional rescue of vision. This work provides scientific proof-of-principle for a novel therapeutic approach to photoreceptor degeneration that is currently being evaluated in clinical trials.

Lnc-216 regulates the miR-143-5p /MMP2 signaling axis aggravates retinal endothelial cell dysfunction

PURPOSE: Diabetic retinopathy (DR) is a serious retinal vascular disease that affects many individuals in their prime working years. The present research aimed at whether and how LOC681216 (LNC-216) is involved in retinal vascular dysfunction under diabetic conditions. METHODS: Rat retinal microvascular endothelial cells (RRMECs) treated with high glucose (HG) were used for functional analysis. Gene expression analysis was conducted using the Clariom D Affymetrix platform. The wound healing, transwell, and vascular tube formation assays were used to identify the migration, invasion, and tube formation capability of RRMECs. The dual-luciferase reporter confirmed the binding interaction between miR-143-5p and LNC-216 or matrix metallopeptidase 2 (MMP2). RESULTS: Lnc-216 was upregulated in RRMECs treated with HG. Lnc-216 knockdown markedly suppressed the tube formation, cell migration, and wound healing of cultured RRMECs under HG conditions. Mechanistically, Lnc-216 acted as a miR-143-5p sponge to affect the biological activity of miR-143-5p, which led to increased expression of matrix metallopeptidase 2 (MMP2). CONCLUSIONS: Lnc-216 attenuates diabetic retinal vascular dysfunction through the miR-143-5p/MMP2 axis, providing a potential therapeutic strategy for DR.

PLK-3-mediated phosphorylation of BAP1 prevents diabetic retinopathy

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus, and its main clinical manifestation is retinal vascular dysfunction. DR causes blindness and is a problem with significant global health implications. However, treating DR is still challenging. In this study, we aimed to explore the role of polo-like kinase-3 (PLK-3) and the potential regulatory mechanism in DR. Sprague-Dawley rats were injected intraperitoneally with streptozotocin (STZ, 60 mg/kg) to induce a rat model of DR, and rat retinal microvascular endothelial cells (RRMECs) were treated with high glucose (HG, 25 mmol/L glucose) to develop a cell model of DR. We found that PLK-3 was significantly downregulated in the retinal tissues of STZ-induced diabetic rats and HG-induced RRMECs. Lentivirus-mediated PLK-3 overexpression alleviated the histological damages in DR rats. After HG stimulation, cell proliferation, migration, and angiogenesis in RRMECs were inhibited after PLK-3 upregulation. By using label-free proteomics, we identified 82 differentially expressed proteins downstream of PLK-3, including BRCA1-associated protein 1 (BAP1), which was significantly upregulated in PLK-3-overexpressed RRMECs compared to control cells under the HG condition. In vivo and in vitro assays indicated that the forced expression of PLK-3 increased the phosphorylation of BAP1 at serine 592 and caspase-8 expression. Detailed evidence showed that BAP1-shRNA-mediated knockdown restored the cell function in HG-treated RRMECs when PLK-3 was overexpressed. Collectively, this study shows that PLK-3 alleviates retinal vascular dysfunction in DR by inhibiting the phosphorylation of BAP1. Thus, PLK-3 may develop as a promising target for the therapy of DR.

High Glucose Induces Oxidative Stress That Alters Glycocalyx Proteoglycan Levels in Primary Rat Retinal Microvascular Endothelial Cells and in Isolated Ophthalmic Arteries.

Our purpose in this study was to identify the role played by oxidative stress in the changes to proteoglycans that occur under hyperglycemic conditions, using primary rat retinal microvascular endothelial cells (RRMEC) and cultured ophthalmic arteries. The cells and blood vessels obtained from rats were cultured in normal glucose (5.6 mM) and high glucose (25 mM) with or without N-acetylcysteine (NAC), an antioxidant. Intracellular oxidative stress was determined by measuring dihydroethidium (DHE) fluorescence and malondialdehyde (MDA)-modified protein levels. mRNA and protein levels were evaluated using quantitative real-time polymerase chain reaction and immunoblot, respectively. High glucose increased levels of glypican-1 mRNA and protein. The level of syndecan-1 mRNA also was increased, but its protein level was decreased, by high glucose. Evaluation of DHE and MDA showed that high glucose increased oxidative stress. These changes caused by high glucose were significantly reversed by NAC treatment. Matrix metalloproteinase-9 (MMP-9) levels, which increased under high glucose conditions, were suppressed by NAC treatment. Oxidative stress caused by hyperglycemia may be responsible for significant changes to the ocular endothelial glycocalyx.

The mechanism of metallothionein MT1M-mediated PI3K/AKT signaling pathway in the regulation of diabetic retinopathy

Metallothionein (MT1M) is associated with tumors and autoimmune diseases. However, its role in DR has not yet been elucidated. DR and normal rat retinal endothelial cells (RECs) were isolated and cultured. DR Rat RECs achieved gene regulation by transfecting MT1M plasmid. PCR and MTT were used to detect MT1M expression, cell proliferation. Flow cytometry was used to detect cell apoptosis. Lactate dehydrogenase (LDH), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected by MTT. The expression of VEGF and PI3K/AKT signaling pathway was detected by Western blot. The levels of inflammatory factors TNF-α and IL-1β were detected by ELISA. The results showed that the expression of MT1M was reduced in the RECs of DRC rats compared to the normal control group, cell proliferation was enhanced, SOD activity was reduced, LDH and ROS levels were increased, TNF-α and IL-1β secretion increased, and vascular endothelial growth factor (VEGF), PI3K/AKT expression increased (P < 0.05). However, transfection with MT1M plasmid could significantly inhibit cell proliferation, increase SOD activity, reduce LDH and ROS levels, reduce TNF-α and IL-1β secretion, and reduce VEGF and PI3K/AKT expression (P <0.05). The expression of MT1M is reduced in RECs of DR rats. Up-regulation of MT1M can regulate DR3K/AKT signaling pathway and oxidative/antioxidant balance, alter VEGF expression, inhibit inflammation, regulate the growth and proliferation of RECs, and delay DR lesions.

Chitosan-Rapamycin Carbon Dots Alleviate Glaucomatous Retinal Injury by Inducing Autophagy to Promote M2 Microglial Polarization.

Glaucoma is a prevalent cause of irreversible vision impairment, characterized by progressive retinal ganglion cells (RGCs) loss, with no currently available effective treatment. Rapamycin (RAPA), an autophagy inducer, has been reported to treat glaucoma in rodent models by promoting RGC survival, but its limited water solubility, systemic toxicity, and pre-treatment requirements hinder its potential clinical applications. Chitosan (CS)-RAPA carbon dot (CRCD) was synthesized via hydrothermal carbonization of CS and RAPA and characterized by transmission electron microscopy, Fourier transform infrared spectra, and proton nuclear magnetic resonance. In vitro assays on human umbilical cord vein endothelial and rat retinal cell line examined its biocompatibility and anti-oxidative capabilities, while lipopolysaccharide-stimulated murine microglia (BV2) assays measured its effects on microglial polarization. In vivo, using a mouse retinal ischemia/reperfusion (I/R) model by acute intraocular pressure elevation, the effects of CRCD on visual function, RGC apoptosis, oxidative stress, and M2 microglial polarization were examined. CRCD exhibited good water solubility and anti-oxidative capabilities, in the form of free radical scavenging. In vitro, CRCD was bio-compatible and lowered oxidative stress, which was also found in vivo in the retinal I/R model. Additionally, both in vitro with lipopolysaccharide-stimulated BV2 cells and in vivo with the I/R model, CRCD was able to promote M2 microglial polarization by activating autophagy, which, in turn, down-regulated pro-inflammatory cytokines, such as IL-1β and TNF-α, as well as up-regulated anti-inflammatory cytokines, such as IL-4 and TGF-β. All these anti-oxidative and anti-inflammatory effects ultimately aided in preserving RGCs, and subsequently, improved visual function. CRCD could serve as a potential novel treatment strategy for glaucoma, via incorporating RAPA into CDs, in turn not only mitigating its toxic side effects but also enhancing its therapeutic efficacy.

Procyanidin B2 Protects TR-iBRB2 Cells Against Hyperglyc emia Stress by Attenuating Oxidative Stress and Inflammasome Activation via Regulation of Redoxosomes/NF-kB Signaling.

Microvascular dysfunction is a hallmark of diabetic retinopathy (DR), which may lead to visual impairment and blindness. Procyanidin B2 (PB2) is a subclass of flavonoids and is widely known due to its anti-oxidant and antiinflammatory effects. However, little is known about the effect of PB2 on hyperglycemia stress-induced retinal microvascular dysfunction. The purpose of this study was to investigate the effect of PB2 against hyperglycemia stress in rat retinal capillary endothelial cells (TR-iBRB2) as well as the underpinning mechanism. Cell viability was determined using MTT assay. ROS, NOX activity analysis, Western blot analysis, and immunofluorescence analysis were applied in the study. The results showed that PB2 pre-treatment significantly reduced high glucose- induced cytotoxicity in TR-iBRB2 cells by suppressing oxidative stress and inflammasome activation. Mechanistical study revealed that redoxosomes were formed and activated in TR-iBRB2 cells upon hyperglycemia stress, resulting in activation of NF- κB and thus induction of oxidative stress and inflammasomes activation. However, PB2 pre-treatment dose-dependently attenuated the above events, indicating the protective effect of PB2 against hyperglycemia stress was achieved by regulating redoxosomes/ NF-kB signaling. Our findings may contribute to the potential clinical use of PB2 in treating DR and suggest redoxosomes/NF-kB signaling may be a potential therapeutic target of this disease.

PPAR-γ promotes the polarization of rat retinal microglia to M2 phenotype by regulating the expression of CD200-CD200R1 under hypoxia.

Recent reports suggest that peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms that trigger PPAR-γ's anti-inflammatory ability in microglia are yet to be expounded. Thus, in this study, we aimed to explore the molecular mechanisms behind the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat retinal microglial cells. We used shRNA expressing lentivirus to knock down PPAR-γ and CD200 genes, and we assessed hypoxia-induced polarization markers release - M1 (iNOS, IL-1β, IL-6, and TNF-α) and M2 (Arg-1, YM1, IL-4, and IL-10) by RT-PCR. We also monitored PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200, and CD200Rs) by Western blot and RT-PCR. Our results showed that hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. Moreover, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, whereas sh-PPAR-γ had the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype. Our findings demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via the CD200-CD200R1 pathway.

Comparative analysis of co-culture and monoculture models in simulating diabetic neurovascular dysfunction: insights into diabetic retinopathy.

Interaction between retinal vascular endothelial cells and neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR). This study aims to compare an in vitro model over a monoculture model to simulate the neurovascular coupling under the hyperglycemic microenvironment of diabetes. Rat retinal vascular endothelial cells (RRMECs) and ganglion cells (RGCs) were seeded mono- or co-cultured in a normal (NG, 5.5 mM) and high (HG, 75 mM) glucose concentrations culture medium. Cell viability was detected by the cell counting kit-8 (CCK-8) assay. The ability of migration and lumen formation of RRMECs were determined by scratch wound, transwell migration, and lumen formation assays. The apoptosis index of cells was calculated and detected by propidium iodide (PI)/Hoechst staining. Quantitative and morphological analysis of RGCs was performed through the labeling of RGCs by brain-specific homeobox/POU domain protein 3A (BRN3A) and anti-beta-III tubulin (TUJ1). The gene and protein expression levels of occludin (OCLN) and zonula occludens-1 (ZO-1) were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The viability, migration, and lumen formation abilities of RRMECs in the HG group significantly increased (P<0.05) in both mono- and co-culture models. Migration and lumen formation abilities of RRMECs in the co-culture with HG were lower than that in the monoculture group (P<0.05). The viability of RGCs cells with HG significantly decreased in both mono- and co-culture models (Pmono<0.001, Pco<0.001), the apoptosis index of RGCs in the co-culture with HG was higher than that in the monoculture (P=0.010). The protein and gene expression of OCLN, and ZO-1 in RRMECs significantly decreased with HG culture medium in both culture models (P<0.05). In the HG group, the protein and gene expression level of the ZO-1 and OCLN of RRMECs significantly decreased in the co-culture model than that in the monoculture model (P<0.05). Compared with mono cell culture, the established co-culture in vitro system for diabetic neurovascular dysfunction can better stimulate the micro-environment of the retinal neurovascular unit.

Identification and Validation of Genes Related to RNA Methylation Modification in Diabetic Retinopathy

Purpose To identify and validate the differentially expressed genes related to RNA methylation modification in diabetic retinopathy. Methods The data sets GSE12610 and GSE111465 related to diabetic retinopathy in the Gene Expression Omnibus were selected. The R software package was used to identify differentially expressed genes related to RNA methylation modification in diabetic retinopathy. Protein-protein interaction network was constructed to explore the interactions between proteins and predict proteins. Then, Gene Ontology annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to analyze the potential enrichment pathways and clarify the biological functions of these genes. In addition, the correlation between them and immune cells was visualized, and receiver operating characteristic curves were drawn to evaluate the diagnostic performance of each one of them for diabetic retinopathy. To verify the differentially expressed genes, the mRNA expression of rat retinal vascular endothelial cells cultured in low and high glucose medium separately were detected by RT-qPCR. Results The expression of Lrpprc, Nsun4, Nsun6 and Trdmt1 were significantly up-regulated in diabetic retinopathy samples, while the expression of Cbll1, Hnrnpc, Mettl3 and Wtap were significantly down-regulated. Differentially expressed genes were mainly enriched in the RNA-methylation-medication pathways and biological function. The results of immune infiltration analysis proved that eosinophils aggregated more in diabetic group, while T cells follicular helper aggregated more in normal samples. These genes of Cbll1 (AUC = 0.986), Hnrnpc (AUC = 0.819), Lrpprc (AUC = 0.806), Mettl3 (AUC = 0.917), Nsun4 (AUC = 0.819), Nsun6 (AUC = 0.819), Trdmt1 (AUC = 0.972) and Wtap (AUC = 0.972) were respectively used as the diagnostic basis of diabetic retinopathy. According to the RT-qPCR results, the expression of Mettl3 was significantly down-regulated (p < 0.0005) in cells cultured in high glucose, while Trdmt1 (p < 0.05), Nsun4 (p < 0.05) and Nsun6 (p < 0.05) were significantly up-regulated. Conclusion Differentially expressed genes such as Mettl3, Nsun4, Nsun6, and Trdmt1 should be conducted to explore, and the role of RNA methylation in the process of diabetic retinopathy would be revealed in-depth.

A Dual‐Drug Nanohybrid System Incorporating Nimodipine and Brain‐Derived Neurotrophic Factor Promotes Retinal Ganglion Cells Survival

AbstractGlaucoma, a leading cause of irreversible visual impairments and even blindness, is characterized by elevated intraocular pressure and progressive loss of structure and function of neuronal systems. To effectively protect neuronal systems from damage caused by elevated intraocular pressure, a novel dual‐drug nanohybrid has been developed by incorporating brain‐derived neurotrophic factor (BDNF)‐loaded mesoporous silica nanoparticles (BDNF@MSN) into the thermogel matrix with nimodipine (NMD) (BDNF@MSN‐NMD@Thermogel). The carrier materials (i.e., silica and thermogel) in this nanohybrid do not show any cytotoxicity to human lens epithelial cells and rat retinal precursor cells. This nanohybrid regulates the in vitro release of BDNF and NMD in a sustainable way for weeks. In optic nerve crush rodent models, a single intravitreal injection of BDNF@MSN‐NMD@Thermogel inhibits retinal neuroinflammation, significantly promotes retinal ganglion cells survival and restores visual function for nearly three months. This nanohybrid is thus a promising alternative for effective neuroprotection treatment for neuroinflammation‐related neurodegenerative diseases.

A Multifunctional Hybrid Nanocarrier for Non-Invasive siRNA Delivery to the Retina.

Drug therapy for retinal diseases (e.g., age-related macular degeneration, the leading cause of blindness) is generally performed by invasive intravitreal injection because of poor drug delivery caused by the blood-retinal barrier (BRB). This study aimed to develop a nanocarrier for the non-invasive delivery of small interfering RNA (siRNA) to the posterior segment of the eye (i.e., the retina) by eyedrops. To this end, we prepared a hybrid nanocarrier based on a multifunctional peptide and liposomes, and the composition was optimized. A cytoplasm-responsive stearylated peptide (STR-CH2R4H2C) was used as the multifunctional peptide because of its superior ability to enhance the complexation, cell permeation, and intracellular dynamics of siRNA. By adding STR-CH2R4H2C to the surface of liposomes, intracellular uptake increased regardless of the liposome surface charge. The STR-CH2R4H2C-modified cationic nanocarrier demonstrated significant siRNA transfection efficiency with no cytotoxicity, enhanced siRNA release from endosomes, and effectively suppressed vascular endothelial growth factor expression in rat retinal pigment epithelium cells. The 2.0 mol% STR-CH2R4H2C-modified cationic nanocarrier enhanced intraocular migration into the retina after instillation into rat eyes.

Adaptive Response in Rat Retinal Cell Cultures Irradiated with γ-rays.

Retina can receive incidental γ-ray exposure from various sources. For example, although radiation therapy is a crucial tool for managing head and neck tumors, patients may develop ocular complications as collateral damage from accidental irradiation. Recently, there has been concern that retinal irradiation during space flight may compromise mission goals and long-term quality of life after space travel. Previously, in our in vitro model, we proved that immature retinal cells are more vulnerable to γ-radiation than differentiated neurons. Here, we investigate if a low-dose pre-irradiation (0.025 Gy), known to have a protective effect in various contexts, can affect DNA damage and oxidative stress in cells exposed to a high dose of γ-rays (2 Gy). Our results reveal that pre-irradiation reduces 2 Gy effects in apoptotic cell number, H2AX phosphorylation and oxidative stress. These defensive effects are also evident in glial cells (reduction in GFAP and ED1 levels) and antioxidant enzymes (catalase and CuZnSOD). Overall, our results confirm that rat retinal cultures can be an exciting tool to study γ-irradiation toxic effects on retinal tissue and speculate that low irradiation may enhance the skill of retinal cells to reduce damage induced by higher doses.

Autophagy and senescence of rat retinal precursor cells under high glucose.

Diabetic retinopathy (DR) is a common diabetic ocular disease characterized by retinal ganglion cell (RGC) changes. An abnormal environment, hyperglycemia, may progressively alter the structure and function of RGCs, which is a primary pathological feature of retinal neurodegeneration in DR. Accumulated studies confirmed autophagy and senescence play a vital role in DR; however, the underlying mechanisms need to be clarified. This study included the microarray expression profiling dataset GSE60436 from Gene Expression Omnibus (GEO) to conduct the bioinformatics analysis. The R software was used to identify autophagy-related genes (ARGs) that were differentially expressed in fibrovascular membranes (FVMs) and normal retinas. Co-expression and tissue-specific expression were elicited for the filtered genes. The genes were then analyzed by ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). R28 cells were cultured with high glucose, detected by reverse transcription-quantitative (RT-qPCR) and stained by apoptosis kit. In the retina, 31 differentially expressed ARGs (24 up-regulated genes) were discovered and enriched. The enrichment results revealed that differentially expressed ARGs were significantly enriched in autophagy, apoptosis, aging, and neural function. Four hub genes (i.e., TP53, CASP1, CCL2, and CASP1) were significantly up-regulated. Upregulation of cellular autophagy and apoptosis level was detected in the hyperglycemia model in vitro. Our results provide evidence for the autophagy and cellular senescence mechanisms involved in retinal hyperglycemia injury, and the protective function of autophagy is limited. Further study may favour understanding the disease progression and neuroprotection of DR.

Ferroptosis and glaucoma: implications in retinal ganglion cell damage and optic nerve survival.

Ferroptosis and glaucoma: implications in retinal ganglion cell damage and optic nerve survival.

The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation.

Microglia are resident immune cells in the central nervous system (CNS). Microglial activation plays a prominent role in neuroinflammation and CNS diseases. However, the underlying mechanisms of microglial activation are not well understood. Here, we report that the transcription factor interferon regulatory factor 1 (IRF1) plays critical roles in microglial activation and retinal inflammation by regulating pro- and anti-inflammatory gene expression. IRF1 expression was upregulated in activated retinal microglia compared to those at the steady state. IRF1 knockout (KO) in BV2 microglia cells (BV2ΔIRF1) created by CRISPR/Cas9 genome-editing technique causes decreased microglia proliferation, migration, and phagocytosis. IRF1-KO decreased pro-inflammatory M1 marker gene expression induced by lipopolysaccharides (LPS), such as IL-6, COX-2, and CCL5, but increased anti-inflammatory M2 marker gene expression by IL-4/13, such as Arg-1, CD206, and TGF-β. Compared to the wild-type cells, microglial-conditioned media (MCM) of activated BV2ΔIRF1 cell cultures reduced toxicity or death to several retinal cells, including mouse cone photoreceptor-like 661 W cells, rat retinal neuron precursor R28 cells, and human ARPE-19 cells. IRF1 knockdown by siRNA alleviated microglial activation and retinal inflammation induced by LPS in mice. Together, the findings suggest that IRF1 plays a vital role in regulating microglial activation and retinal inflammation and, therefore, may be targeted for treating inflammatory and degenerative retinal diseases.

The role of transforming growth factor- in hypertension-induced cerebrovascular remodeling

Background: Hypertension (HT) promotes structural and functional changes in the cerebral microcirculation that can provoke irreversible cerebrovascular injury, leading to neuronal loss and brain atrophy,1 cognitive impairment, vascular dementia,2 and Alzheimer’s disease.3 Currently, the mechanisms and consequences of such remodeling are not fully understood. Transforming growth factor (TGF) is a morphogen that regulates cellular differentiation, induces endothelial-to-mesenchymal transition (EndoMT), and works as a contractile-to-synthetic switch in vascular smooth muscle cells (VSMC) phenotype.4 Plasma levels of TGF are increased in HT patients, and in spontaneously hypertensive rats (SHR) that exhibit vascular fibrosis.5 Although coincidental, causal roles through which elevated TGF may drive cerebrovascular remodeling in the HT brain are suspected, but unproven. We hypothesize that HT-induced TGF drives cerebrovascular remodeling, which decreases blood-brain barrier and impairs cerebrovascular autoregulation.&#x0D; Methods: Brain cortices and penetrating cerebral microvessels (BMVs) were isolated from male and female SHR and Wistar-Kyoto rats (WKY, control), and subjected to western blotting and immunofluorescence labeling for endothelial cell (EC) and VSMC differentiation markers, TGF canonical pathway markers SMAD2, 3 and 4, as well as basement membrane protein expression, and glial fibrillary acidic protein (GFAP) as a marker of astrocyte inflammation. Additionally, TGF-treated rat retinal microvascular endothelial cells (RRMECs), and human brain microvascular endothelial cells (hCMEC/D3) ± the TGF inhibitor vactosertib, were also subjected to western blotting and immunofluorescence labeling to assess endothelial cell (EC) and VSMC differentiation markers, as well as basement membrane protein expression.&#x0D; Results: TGF was significantly increased in SHR BMVs. Moreover, GFAP levels were significantly increased in the SHR cortex. The EC junctional proteins platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin), were significantly decreased in SHR. In contrast, CRBP-1, a marker for synthetic VSMC, and collagen IV and fibronectin levels significantly increased in SHR. Interestingly, female SHR rats had significantly increased levels of SMAD2/3, which was not evident in male SHRs, indicating a possible role for the non-canonical TGF pathway in male SHRs. Additionally, TGF significantly increased -smooth muscle actin (-SMA) and decreased VE-cadherin expression in RRMECs and D3 cells, consistent with EndoMT. TGF inhibition with vactosertib reversed these effects and significantly suppressed -SMA, and maintained VE-Cadherin similar to control RRMECs and D3s.&#x0D; Conclusions: We now have strong evidence for HT-induced cerebrovascular remodeling, where TGF mediates loss of both smooth muscle and endothelial differentiated phenotypes. The reversal of these effects using TGF blockers suggests that therapy to modulate TGF may represent a means to control HT-induced cerebrovascular remodeling.

RIP140-Mediated NF-κB Inflammatory Pathway Promotes Metabolic Dysregulation in Retinal Pigment Epithelium Cells.

Metabolic dysregulation of the retinal pigment epithelium (RPE) has been implicated in age-related macular degeneration (AMD). However, the molecular regulation of RPE metabolism remains unclear. RIP140 is known to affect oxidative metabolism and mitochondrial biogenesis by negatively controlling mitochondrial pathways regulated by PPAR-γ co-activator-1 α(PGC-1α). This study aims to disclose the effect of RIP140 on the RPE metabolic program in vitro and in vivo. RIP140 protein levels were assayed by Western blotting. Gene expression was tested using quantitative real-time PCR (qRT-PCR), ATP production, and glycogen concentration assays, and the release of inflammatory factors was analyzed by commercial kits. Mice photoreceptor function was measured by electroretinography (ERG). In ARPE-19 cells, RIP140 overexpression changed the expression of the key metabolic genes and lipid processing genes, inhibited mitochondrial ATP production, and enhanced glycogenesis. Moreover, RIP140 overexpression promoted the translocation of NF-κB and increased the expression and production of IL-1β, IL-6, and TNF-α in ARPE-19 cells. Importantly, we also observed the overexpression of RIP140 through adenovirus delivery in rat retinal cells, which significantly decreased the amplitude of the a-wave and b-wave measured by ERG assay. Therapeutic strategies that modulate the activity of RIP140 could have clinical utility for the treatment of AMD in terms of preventing RPE degeneration.