Human Retinal Endothelial Cells Research Articles

12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR.

Development of dual drug loaded-hydrogel scaffold combining microfluidics and coaxial 3D-printing for intravitreal implantation

Treating diabetic retinopathy (DR) effectively is challenging, aiming for high efficacy with minimal discomfort. While intravitreal injection is the current standard, it has several disadvantages. Implantable systems offer an alternative, less invasive, with long-lasting effects drug delivery system (DDS). The current study aims to develop a soft, minimally invasive, biodegradable, and bioadhesive material-based hydrogel scaffold to prevent common issues with implants. A grid-shaped scaffold was created using coaxial 3D printing (3DP) to extrude two bioinks in a single filament. The scaffold comprises an inner core of curcumin-loaded liposomes (CUR-LPs) that prepared by microfluidics (MFs) embedded in a hydrogel of hydroxyethyl cellulose (HEC), and an outer layer of hyaluronic acid-chitosan matrix with free resveratrol (RSV), delivering two Sirt1 agonists synergistically activating Sirt1 downregulated in DR. Optimized liposomes, prepared via MFs, exhibit suitable properties for retinal delivery in terms of size (<200 nm), polydispersity index (PDI) (<0.3), neutral zeta potential (ZP), encapsulation efficiency (∼97 %), and stability up to 4 weeks. Mechanical studies confirm scaffold elasticity for easy implantation. The release profiles show sustained release of both molecules, with different patterns related to different localization of the molecules. RSV released initially after 30 min with a total release more than 90 % at 336 h. CUR release starts after 24 h with only 4.78 % of CUR released before and gradually released thanks to its internal localization in the scaffold. Liposomes and hydrogels can generate dual drug-loaded 3D structures with sustained release. Microscopic analysis confirms optimal distribution of liposomes within the hydrogel scaffold. The latter resulted compatible in vitro with human retinal microvascular endothelial cells up to 72 h of exposition. The hydrogel scaffold, composed of hyaluronic acid and chitosan, shows promise for prolonged treatment and minimally invasive surgery.

Dysfunction of Calcyphosine-Like gene impairs retinal angiogenesis through the MYC axis and is associated with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a severe genetic disorder characterized by incomplete vascularization of the peripheral retina and associated symptoms that can lead to vision loss. However, the underlying genetic causes of approximately 50% of FEVR cases remain unknown. Here, we report two heterozygous variants in calcyphosine-like gene (CAPSL) that is associated with FEVR. Both variants exhibited compromised CAPSL protein expression. Vascular endothelial cell (EC)-specific inactivation of Capsl resulted in delayed radial/vertical vascular progression, compromised endothelial proliferation/migration, recapitulating the human FEVR phenotypes. CAPSL-depleted human retinal microvascular endothelial cells (HRECs) exhibited impaired tube formation, decreased cell proliferation, disrupted cell polarity establishment, and filopodia/lamellipodia formation, as well as disrupted collective cell migration. Transcriptomic and proteomic profiling revealed that CAPSL abolition inhibited the MYC signaling axis, in which the expression of core MYC targeted genes were profoundly decreased. Furthermore, a combined analysis of CAPSL-depleted HRECs and c-MYC-depleted human umbilical vein endothelial cells uncovered similar transcription patterns. Collectively, this study reports a novel FEVR-associated candidate gene, CAPSL, which provides valuable information for genetic counseling of FEVR. This study also reveals that compromised CAPSL function may cause FEVR through MYC axis, shedding light on the potential involvement of MYC signaling in the pathogenesis of FEVR.

LncRNA-MM2P regulates retinal neovascularization through M2 macrophage polarization

The study aims to investigate the effects and potential mechanisms of lncRNA-MM2P on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). The OIR model was established in C57BL/6J mice. RAW264.7 cell line and bone marrow-derived macrophages (BMDMs) from mice were used for in vitro studies. RT-qPCR was used to analyze the expressions of lncRNA and mRNAs. The protein expression levels were determined by western blotting. The size of avascular areas and neovascular tufts were assessed based on isolectin B4 immunofluorescence staining images. The human retinal endothelial cells (HRECs) were used to evaluate the proliferation, migration, and tube formation of endothelial cells. The expression of lncRNA-MM2P was significantly upregulated from P17 to P25 in OIR retinas. Knockdown of lncRNA-MM2P levels in vivo led to a significant reduction in the neovascular tufts and avascular areas in the retinas of OIR mice. Knockdown of lncRNA-MM2P levels in vitro suppressed the expression of M2 markers in macrophages. Moreover, we found a significant inhibition of avascular areas and neovascular tufts in OIR mice injected intravitreally with M2 macrophages treated by shRNA-MM2P. The cellular functions of proliferation, migration, and tube formation were significantly attenuated in HRECs cultured with a supernatant of shRNA-MM2P-treated M2 macrophages. Our results indicate that lncRNA-MM2P regulates retinal neovascularization by inducing M2 polarization of macrophages in OIR mice. Therefore, lncRNA-MM2P may be a potential molecular target for immunoregulation of retinal neovascularization.

Knockdown of HCK promotes HREC cell viability and inner blood-retinal barrier integrity by regulating the AMPK signaling pathway.

Diabetic retinopathy (DR), a major complication of diabetes causing blindness, is characterized by retinal damage due to capillary degeneration and vascular leakage. Current treatments are not fully effective, highlighting the need for searching new therapeutic targets. Hematopoietic cell kinase (HCK), a protein involved in various diseases, has been identified as a potential biomarker in DR, but its role in disease progression requires further investigation. Here we investigated the role of HCK in DR and its potential mechanism. We found the expression of HCK increased under the stimulation of high glucose (HG) in human retinal capillary endothelial cells (HRECs). Knockdown of HCK can improve HREC cell viability and the integrity of the internal blood-retinal barrier. HCK depletion suppressed the AMPK pathway in HG-induced HRECs. In summary, HCK may be a potential target for the treatment of DR, which provides a theoretical basis for the development of new treatment strategies.

VEGF-B prevents chronic hyperglycemia-induced retinal vascular leakage by regulating the CDC42-ZO1/VE-cadherin pathway.

Non-proliferative diabetic retinopathy (NPDR) is the early stage of diabetic retinopathy (DR) and is a chronic oxidative stress-related ocular disease. Few treatments are approved for early DR. This study aimed to investigate the pathogenic mechanisms underlying the retinal micro-vasculopathy induced by diabetes and to explore an early potential for treating early DR in a mouse model. The mouse model of type 1 diabetes was established by intraperitoneal injection of streptozotocin (STZ, 180 mg/kg), which was used as the early DR model. The body weight and blood glucose mice were measured regularly; The retinal vascular leakage in the early DR mice was determined by whole-mount staining; Label-free quantitative proteomic analysis and bioinformatics were used to explore the target proteins and signaling pathways associated with the retinal tissues of early DR mice; To detect the effects of target protein on endothelial cell proliferation, migration, and tube formation, knockdown and overexpression of VEGF-B were performed in human retinal vascular endothelial cells (HRECs); Western blotting was used to detect the expression of target proteins invitro and invivo; Meanwhile, the therapeutic effect of VEGF-B on vascular leakage has also been evaluated invitro and invivo. The protein expressions of vascular endothelial growth factor (VEGF)-B and the Rho GTPases family member CDC42 were reduced in the retinal tissues of early DR. VEGF-B upregulated the expression of CDC42/ZO1/VE-cadherin and prevented hyperglycemia-induced vascular leakage in HRECs. Standard intravitreal VEGF-B injections improved the retinal vascular leakage and neurovascular response in early DR mice. Our findings demonstrated, for the first time, that in diabetes, the retinal vessels are damaged due to decreased VEGF-B expression through downregulation of CDC42/ZO1/VE-cadherin expression. Therefore, VEGF-B could be used as a novel therapy for early DR.

Chlorogenic acid ameliorates non-proliferative diabetic retinopathy via alleviating retinal inflammation through targeting TNFR1 in retinal endothelial cells

As a prominent complication of diabetes mellitus (DM) affecting microvasculature, diabetic retinopathy (DR) originates from blood-retinal barrier (BRB) damage. Natural polyphenolic compound chlorogenic acid (CGA) has already been reported to alleviate DR. This study delves into the concrete mechanism of the CGA-supplied protection against DR and elucidates its key target in retinal endothelial cells. DM in mice was induced using streptozotocin (STZ). CGA mitigated BRB dysfunction, leukocytes adhesion and the formation of acellular vessels in vivo. CGA suppressed retinal inflammation and the release of tumor necrosis factor-α (TNFα) by inhibiting nuclear factor kappa-B (NFκB). Furthermore, CGA reduced the TNFα-initiated adhesion of peripheral blood mononuclear cell (PBMC) to human retinal endothelial cell (HREC). CGA obviously decreased the TNFα-upregulated expression of vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), and abrogated the TNFα-induced NFκB activation in HRECs. All these phenomena were reversed by overexpressing type 1 TNF receptor (TNFR1) in HRECs. The CGA-provided improvement on leukocytes adhesion and retinal inflammation was disappeared in mice injected with an endothelial-specific TNFR1 overexpression adeno-associated virus (AAV). CGA reduced the interaction between TNFα and TNFR1 through binding to TNFR1 in retinal endothelial cells. In summary, excepting reducing TNFα expression via inhibiting retinal inflammation, CGA also reduced the adhesion of leukocytes to retinal vessels through decreasing VCAM1 and ICAM1 expression via blocking the TNFα-initiated NFκB activation by targeting TNFR1 in retinal endothelial cells. All of those mitigated retinal inflammation, ultimately alleviating BRB breakdown in DR.

Novel Small Molecules with Anti-Inflammatory and Anti-Angiogenic Activity in a Mouse Model of Oxygen-Induced Retinopathy.

Retinopathy of prematurity (ROP) has a dual-phase disease pathology; in phase 1, hyperoxia-induced vaso-obliteration occurs in the retinal vasculature due to increased oxidative stress (OS) and inflammation, followed by phase 2, where hypoxia increases the overproduction of growth factors, inducing retinal neovascularization. Toll-like receptor 2 and -4 (TLR2 and TLR4) overactivation, hyper-inflammation, macrophages, and neutrophil infiltration contribute to the developing ROP. AVR-121 and AVR-123 are novel classes of small-molecule dual inhibitors of TLR2/4 tested in a human leukemia monocytic cell line (THP-1) and cord-blood-derived mononuclear cells (CBMCs). Both compounds inhibited TLR2/4 signaling-related inflammatory cytokines in THP-1 cells and inhibited VEGF-induced neovascularization in human retinal endothelial cells (HRECs), which are hallmarks of ROP. In an oxygen-induced retinopathy (OIR) murine model, the intraperitoneal injection of AVR-123 in the hyperoxia phase (P7-P12) or a nanosuspension eyedrop of AVR-123 in the hypoxic phase (P12-P17) significantly reduced vaso-obliteration, angiogenesis, and inflammatory cytokine profiles while not inhibiting the necessary growth factor VEGF in the juvenile mouse eyes. The results are consistent with our hypothesis that targeting the dual TLR2/4 pathway will reduce inflammation, angiogenesis, and vaso-obliteration in vitro and in vivo and reduce cytotoxic immune cells. AVR-123 has the potential to be developed as a therapy for ROP.

CST3 alleviates retinal vascular leakage by regulating the Rap1 signaling pathway

Retinal vascular leakage is a major event in several retinal diseases, including diabetic retinopathy (DR). In a previous study, we demonstrated that the aqueous humor concentration of Cystatin C (CST3), a physiological inhibitor of cysteine protease, is negatively correlated with the severity of diabetic macular edema. However, its function in the retina has not been clearly elucidated. In this study, we found a significant decrease in the aqueous humor concentration of CST3 with DR progression. Furthermore, we found that CST3 was expressed in retinal endothelial cells and that its expression was significantly downregulated in high glucose-treated human retinal microvascular endothelial cells (HRMECs) and the retinal vessels of oxygen-induced retinopathy (OIR) mice. Silencing CST3 expression resulted in decreased HRMEC migration and tubule formation ability. Exogenous addition of the CST3 protein significantly improved HRMEC migration and tubular formation. In-vivo experiments demonstrated that CST3 silencing induced retinal vascular leakage in WT mice, while its intravitreal injection significantly reduced retinal leakage in OIR mice. Mechanistically, CST3 promoted the expression of the downstream adhesion molecules, claudin5, VE-cadherin, and ZO-1, in retinal vascular cells by regulating the Rap1 signaling pathway. Therefore, this study revealed a novel mechanism by which CST3 improves retinal vascular function and provided evidence that it is a potential therapeutic target for retinal vascular leakage.

Inter-tissue differences in oxidative stress susceptibility reveal a less stable endothelial barrier in the brain than in the retina

Oxidative stress can impair the endothelial barrier and thereby enable autoantibody migration in Neuromyelitis optica spectrum disorder (NMOSD). Tissue-specific vulnerability to autoantibody-mediated damage could be explained by a differential, tissue-dependent endothelial susceptibility to oxidative stress. In this study, we aim to investigate the barrier integrity and complement profiles of brain and retinal endothelial cells under oxygen-induced oxidative stress to address the question of whether the pathomechanism of NMOSD preferentially affects the brain or the retina.Primary human brain microvascular endothelial cells (HBMEC) and primary human retinal endothelial cells (HREC) were cultivated at different cell densities (2.5*104 to 2*105 cells/cm2) for real-time cell analysis. Both cell types were exposed to 100, 500 and 2500 μM H2O2. Immunostaining (CD31, VE-cadherin, ZO-1) and Western blot, as well as complement protein secretion using multiplex ELISA were performed.HBMEC and HREC cell growth phases were cell type-specific. While HBMEC cell growth could be categorized into an initial peak, proliferation phase, plateau phase, and barrier breakdown phase, HREC showed no proliferation phase, but entered the plateau phase immediately after an initial peak. The plateau phase was 7 h shorter in HREC. Both cell types displayed a short-term, dose-dependent adaptive response to H2O2. Remarkably, at 100 μM H2O2, the transcellular resistance of HBMEC exceeded that of untreated cells. 500 μM H2O2 exerted a more disruptive effect on the HBMEC transcellular resistance than on HREC. Both cell types secreted complement factors H (FH) and I (FI), with FH secretion remaining stable after 2 h, but FI secretion decreasing at higher H2O2 concentrations.The observed differences in resistance to oxidative stress between primary brain and retinal endothelial cells may have implications for further studies of NMOSD and other autoimmune diseases affecting the eye and brain. These findings may open novel perspectives for the understanding and treatment of such diseases.

MicroRNA-2861 regulates the proliferation and apoptosis of human retinal vascular endothelial cells treated with high glucose by targeting NDUFB7

ObjectivesAlthough anti-VEGF and retinal laser photocoagulation are two therapeutic modalities that have been used in the clinical treatment of diabetic retinopathy (DR), it is unknown how these modalities target vascular endothelial function in DR. MethodsWe first downloaded and analyzed the differential genes in two DR-related datasets, GSE60436 and GSE53257. The differential gene expression was then verified using RT-qPCR, and the most upregulated gene, NDUFB7, was selected for subsequent experiments. Subsequently, the role of NDUFB7 silencing and enforced expression on the proliferation and apoptosis of HRVECs was explored using CCK-8 assay, EDU proliferation assay and apoptotic TUNEL staining. In addition, the upstream potential miRNAs of NDUFB7 were predicted online using the Targetscan website. RT-qPCR, Western blotting (WB), and dual luciferase gene reporter assay were used to confirm the targeting connection between miR-2861 and NDUFB7. Finally, miR-2861 expression after high glucose (HG) treatment and its effect on proliferation and apoptosis of HRVECs under HG were investigated. ResultsIn this study, we first downloaded and analyzed the differential genes in two DR-related datasets, GSE60436 and GSE53257. We found that TUFM, PRELID1, MRPL32, NDUFB7, MRPL4, MRPL40, HSD17B10 and SLC25A13 were upregulated in DR, and RT-qPCR showed that NDUFB7 was most upregulated. Subsequent CCK-8 assay, EDU proliferation assay and TUNEL staining showed that up-picked NDUFB7 promotes proliferation and inhibits apoptosis of HRVECs. In addition, the upstream potential miRNAs of NDUFB7 were predicted online using the Targetscan website. RT-qPCR, Western blotting (WB), and dual luciferase gene reporter assay confirmed the targeting connection between miR-2861 and NDUFB7. Finally, it was observed that miR-2861 can inhibit the proliferation and promote the apoptosis of HRVECs by targeting NDUFB7. ConclusionsOur findings showed that upregulated NDUFB7 in DR promotes proliferation and inhibits apoptosis of HRVECs, and miR-2861 can rescue the pathogenic effect of NDUFB7 upregulation by targeting NDUFB7.

Glucose-Regulated Protein 78, via Releasing β-Catenin from Adherens Junctions, Facilitates Its Interaction with STAT3 in Mediating Retinal Neovascularization

Retinopathy due to neovascularization is one of the major causes of vision loss. To understand the mechanisms underlying retinal neovascularization, using the oxygen-induced retinopathy (OIR) model, we performed two-dimensional gel matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analysis of normoxic and 24-hour post-OIR mice pups' retinas. Two-dimensional gel analysis revealed that glucose-regulated protein 78 (GRP78) is one of the several molecules induced by OIR in the retinal endothelial cells (ECs). Vascular endothelial growth factor A (VEGFA) also induced GRP78 expression independent of endoplasmic reticulum stress response in human retinal microvascular endothelial cells, and depletion of its levels reduced VEGFA-induced EC angiogenic responses. Consistent with these observations, EC-specific deletion of GRP78 inhibited OIR-induced retinal neovascularization. In exploring the mechanisms, we found that GRP78 binds with vascular endothelial-cadherin and releases adherens junction- but not Wnt-mediated β-catenin and that β-catenin, in turn, via interacting with STAT3, triggers cyclin D1 expression. Furthermore, depletion of β-catenin or cyclin D1 levels negated VEGFA-induced EC angiogenic responses and OIR-induced retinal neovascularization. EC-specific deletion of GRP78 also suppressed OIR-induced vascular leakage. In elucidating the upstream signaling, we found that activating transcription factor 6 mediates GRP78 induction in the modulation of VEGFA-induced EC angiogenic responses and OIR-induced retinal neovascularization. Together, these observations reveal that GRP78, independent of its response to endoplasmic reticulum stress, is involved in mediating EC angiogenic responses by VEGFA and retinal neovascularization by OIR. In view of these findings, it appears that GRP78 could be a desirable target for drug development against diabetic retinopathy.

A neutrophil elastase-generated mature form of IL-33 is a potent regulator of endothelial cell activation and proliferative retinopathy

Human interleukin-33 (IL-33) is a 270 amino acid protein that belongs to the IL-1 cytokine family and plays an important role in various inflammatory disorders. Neutrophil proteases (Cathepsin G and Elastase) and mast cell proteases (tryptase and chymase) regulate the activity of IL-33 by processing full-length IL-33 into its mature form. There is little evidence on the role of these mature forms of IL-33 in retinal endothelial cell signaling and pathological retinal angiogenesis. Here, we cloned, expressed, and purified the various mature forms of human IL-33 and then evaluated the effects of IL-3395-270, IL-3399-270, IL-33109-270, and IL-33112-270 on angiogenesis in human retinal microvascular endothelial cells (HRMVECs). We observed that IL-3395-270, IL-3399-270, IL-33109-270, and IL-33112-270 significantly induced HRMVEC migration, tube formation and sprouting angiogenesis. However, only IL-3399-270 could induce HRMVEC proliferation. We used a murine model of oxygen-induced retinopathy (OIR) to assess the role of these mature forms of IL-33 in pathological retinal neovascularization. Our 3′-mRNA sequencing and signaling studies indicated that IL-3399-270 and IL-33109-270 were more potent at inducing endothelial cell activation and angiogenesis than the other mature forms. We found that genetic deletion of IL-33 significantly reduced OIR-induced retinal neovascularization in the mouse retina and that intraperitoneal administration of mature forms of IL-33, mainly IL-3399–270 and IL-33109–270, significantly restored ischemia-induced angiogenic sprouting and tuft formation in the hypoxic retinas of IL-33–/– mice. Thus, our study results suggest that blockade or inhibition of IL-33 cleavage by neutrophil proteases could help mitigate pathological angiogenesis in proliferative retinopathies.

Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis invivo.

Mitochondrial Genome-Encoded Long Noncoding RNA Cytochrome B (LncCytB) and Mitochondrial Ribonucleases in Diabetic Retinopathy.

Hyperglycemia damages mitochondria and downregulates transcription of mtDNA-encoded genes and the long noncoding RNA LncCytB, causing mitochondrial genomic instability. The genes encoded by mtDNA are transcribed as large polycistronic transcripts, and the 5' ends of precursor tRNAs are processed by mitochondrial-targeted ribonuclease P (MRPPs). Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy. Using human retinal endothelial cells incubated in 20 mM D-glucose for 96 h, the gene expression and mitochondrial localization (immunofluorescence) of MRPP1 and the interaction between MRPP1 and LncCytB (determined by RNA-FISH and RNA immunoprecipitation) were quantified. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. Compared to normal glucose, high glucose decreased mRNA and mitochondrial localization of MRPP1 and its interaction with LncCytB. While MRPP1 overexpression prevented glucose-induced decrease in MRPP1-LncCytB interaction, LncCytB expression and mitochondrial damage (reduction in protective nucleoids in mtDNA), MRPP1-siRNA further worsened them. Similar results were obtained from retinal microvessels from diabetic mice and from human donors with diabetic retinopathy. Downregulation of MRPP1 in diabetes suppresses LncCytB transcription, resulting in mitochondrial functional and genomic instability, ultimately leading to the development of diabetic retinopathy. Thus, preventing MRPP1 downregulation has the potential to inhibit retinopathy and prevent the fear of vision loss in diabetic patients.

Eucalyptol Ameliorates Retinal Microvascular Defects through Modulating ER Stress and Angiopoietin-Tie Signaling in Diabetic Eyes.

Loss of the inner blood-retinal barrier (BRB) integrity is a main feature of ocular diseases such as diabetic macular edema. However, there is a lack of clarity on how inner BRB function is modulated within the diabetic retina. The current study examined whether eucalyptol inhibited inner BRB destruction and aberrant retinal angiogenesis in 33 mM glucose-exposed human retinal microvascular endothelial (RVE) cells and db/db mice. This study further examined the molecular mechanisms underlying endothelial dysfunction including retinal endoplasmic reticulum (ER) stress and angiopoietin (Ang)/Tie axis in conjunction with vascular endothelial growth factor (VEGF). Eucalyptol is a naturally occurring monoterpenoid and an achiral aromatic component of many plants including eucalyptus leaves. Nontoxic eucalyptol reduced the production of amyloid-β (Aβ) protein in glucose-loaded RVE cells and in diabetic mice. This natural compound blocked apoptosis of Aβ-exposed RVE cells in diabetic mouse eyes by targeting ER stress via the inhibition of PERK-eIF2α-ATF4-CHOP signaling. Eucalyptol promoted activation of the Ang-1/Tie-2 pathway and dual inhibition of Ang-2/VEGF in Aβ-exposed RVE cells and in diabetic eyes. Supply of eucalyptol reversed the induction of junction proteins in glucose/Aβ-exposed RVE cells within the retina and reduced permeability. In addition, oral administration of eucalyptol reduced vascular leaks in diabetic retinal vessels. Taken together, these findings clearly show that eucalyptol inhibits glucose-induced Aβ-mediated ER stress and manipulates Ang signaling in diabetic retinal vessels, which ultimately blocks abnormal angiogenesis and loss of inner BRB integrity. Therefore, eucalyptol provides new treatment strategies for diabetes-associated RVE defects through modulating diverse therapeutic targets including ER stress, Ang-1/Tie-2 signaling, and Ang-2/VEGF.

CircZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

Severe hypoglycaemia-induced microglial inflammation damages microvascular endothelial cells, leading to retinal destruction.

Human microglia (HMC) are stress-induced inflammatory cells of the retina. It is unknown whether severe hypoglycaemia causes inflammation in microglia, affects the permeability of human retinal microvascular endothelial cells (HRMECs), and causes retinal damage. This study aimed to explore the effects of severe hypoglycaemia on retinal microglial inflammation and endothelial cell permeability and evaluate the damage caused by hypoglycaemia to the retina. The CCK-8 assay was used to measure cell viability. Western blotting was used to detect IL-1β, IL-6, TNF- α, claudin-1, and occludin expression. ELISA was used to detect IL-1β, IL-6, and TNF- α. Transmission electron microscopy (TEM) and haematoxylin and eosin staining were used to observe the retinal structure. Immunohistochemistry and immunofluorescence staining assays were also used to detect IL-1β, IL-6, TNF- α, claudin-1, and occludin expression. Severe hypoglycaemia promoted inflammation in HMC3 cells. Inflammation caused by hypoglycaemia leads to the decreased expression of tight junction proteins. In vivo, severe hypoglycaemia induced structural damage to the retina, increased the expression of inflammatory factors, and decreased the expression of tight junction proteins. Our results suggest that severe hypoglycaemia leads to acute retinal inflammation, affecting the permeability of HRMECs and causing retinal damage.

Mitochondrial DNA transcription and mitochondrial genome-encoded long noncoding RNA in diabetic retinopathy

In diabetic retinopathy, mitochondrial DNA (mtDNA) is damaged and mtDNA-encoded genes and long noncoding RNA cytochrome B (LncCytB) are downregulated. LncRNAs lack an open reading frame, but they can regulate gene expression by associating with DNA/RNA/protein. Double stranded mtDNA has promoters on both heavy (HSP) and light (LSP) strands with binding sites for mitochondrial transcription factor A (TFAM) between them. The aim was to investigate the role of LncCytB in mtDNA transcription in diabetic retinopathy. Using human retinal endothelial cells incubated in high glucose, the effect of regulation of LncCytB on TFAM binding at mtDNA promoters was investigated by Chromatin immunoprecipitation, and binding of LncCytB at TFAM by RNA immunoprecipitation and RNA fluorescence in situ hybridization. High glucose decreased TFAM binding at both HSP and LSP, and binding of LncCytB at TFAM. While LncCytB overexpression ameliorated decrease in TFAM binding and transcription of genes encoded by both H- and L- strands, LncCytB-siRNA further downregulated them. Maintenance of mitochondrial homeostasis by overexpressing mitochondrial superoxide dismutase or Sirtuin-1 protected diabetes-induced decrease in TFAM binding at mtDNA and LncCytB binding at TFAM, and mtDNA transcription. Similar results were obtained from mouse retinal microvessels from streptozotocin-induced diabetic mice. Thus, LncCytB facilitates recruitment of TFAM at HSP and LSP, and its downregulation in diabetes compromises the binding, resulting in the downregulation of polypeptides encoded by mtDNA. Regulation of LncCytB, in addition to protecting mitochondrial genomic stability, should also help in maintaining the transcription of mtDNA encoded genes and electron transport chain integrity in diabetic retinopathy.

Preliminary research on LncRNA ATP2B2-IT2 in neovascularization of diabetic retinopathy

ObjectiveDiabetic retinopathy (DR) is a common complication of diabetes, and recent findings have shown that long noncoding RNAs (lncRNAs) may be involved in its pathogenesis. Through bioinformatics analysis, we found that lncRNA ATP2B2-IT2 may be involved in this process. This study primarily investigated the expression of the lncRNA ATP2B2-IT2 in human retinal microvascular endothelial cells (HRMECs) under high-glucose conditions and its effects on HRMEC proliferation, migration, and neovascularization.MethodsWe used RT‒PCR to assess the expression levels of lncRNA ATP2B2-IT2 and vascular endothelial growth factor (VEGF) in HRMECs under normal glucose (5.5 mmol/L) and high glucose (30 mmol/L) conditions. HRMECs were subsequently divided into four groups: the normal glucose (NG), high glucose (HG), high glucose with lncRNA ATP2B2-IT2 silencing (HG + si-lncRNA ATP2B2-IT2), and high glucose with silencing control (HG + si-NC) groups. The expression levels of the lncRNA ATP2B2-IT2 and VEGF in each group were determined using RT‒PCR. Thereafter, cell proliferation, migration, and neovascularization were assessed using CCK-8, Transwell, and tube formation assays, respectively.ResultsRT‒PCR revealed that the expression levels of the lncRNA ATP2B2-IT2 and VEGF were greater in the HG group than in the NG group (P < 0.05). After silencing of the lncRNA ATP2B2-IT2, the expression of VEGF decreased significantly (P < 0.05). Subsequent CCK-8, Transwell, and tube formation assays demonstrated that compared to those in the NG group, the HRMECs in the HG group exhibited significantly increased proliferation, migration, and neovascularization (P < 0.05). However, after silencing of the lncRNA ATP2B2-IT2, the proliferation, migration, and neovascularization of HRMECs were significantly decreased in the HG + si-lncRNA ATP2B2-IT2 group compared to those in the HG group (P < 0.05).ConclusionLncRNA ATP2B2-IT2 may promote the proliferation, migration and neovascularization of HRMECs under high-glucose conditions.