Retinal Capillary Endothelial Cells Research Articles

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.

Pipecolic acid mitigates ferroptosis in diabetic retinopathy by regulating GPX4-YAP signaling

Diabetic retinopathy (DR) is currently recognized as the leading cause of end-stage eye disease. Pipecolic acid, a metabolite, has a significant regulatory effect on several pathological processes. However, the exact mechanism by which it causes damage in diabetic retinopathy is unknown. Between September 2021 and December 2022, 40 patients were retrospectively examined and divided into two groups: the healthy group (n = 20) and the DR group (n = 20). Metabolomic analysis found that pipecolic acid plays an important role in this process. Streptozotocin-induced diabetic mice and high-glucose cultured human retinal capillary endothelial cells (HRCECs) were then treated with pipecolic acid. Several oxidative stress measurements and RNA sequencing of retinal cells were tested. A gene interaction study was conducted using bioinformatics. Comparison of serological metabolites between healthy volunteers and DR patients showed that pipecolic acid was significantly lower in DR patients, and there was a negative correlation between the level of pipecolic acid with blood glucose and glycated hemoglobin. Yes-associated protein (YAP) mRNA, Malondialdehyde (MDA), and reactive oxygen species (ROS) levels were significantly higher in diabetic mice, but glutathione peroxidase (GSH-Px) levels were significantly lower. Pipecolic acid significantly alleviated oxidative stress and YAP expression. The number of vascular tubes was significantly higher in the DR group, and pipecolic acid treatment significantly reduced tube formation. RNA-Sequencing analysis revealed that YAP and glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) expression was reduced, and functional enrichment analysis revealed that ferroptosis and Hippo signaling pathways play an important role in this process. Additionally, pipecolic acid's ability to improve DR is diminished after YAP and GPX4 ablation. This study found that pipecolic acid, as a metabolite, may impede the progression of DR by inhibiting the YAP-GPX4 signaling pathway.

SIRT2 inhibition attenuates the vasculopathy and vision impairment via Akt signaling in retinopathy of prematurity

Despite decades of researches, the underlying mechanism of retinopathy of prematurity (ROP) remains unclear. The role of Sirt2, which is involved in both angiogenesis and inflammation, both pivotal in ROP, was investigated in an animal model of ROP known as oxygen-induced retinopathy (OIR). Our study found that Sirt2 was overexpressed and colocalized with microglia in OIR. Furthermore, it demonstrated that the level of Sirt2 was upregulated in hypoxia microglia BV-2 in vitro. Subsequently, our results elucidated that administration of the Sirt2 antagonist AGK2 attenuated the avascular and neovascular area and downregulated the expression of IGF-1. The phosphorylation of Akt and the expression of IGF-1 were upregulated in hypoxia BV-2 and conditional media collected from BV-2 under hypoxia promoted the migration and tube formation of retinal capillary endothelial cells, which were suppressed with AGK2. Notably, our findings are the first to demonstrate the deleterious role of Sirt2 in ROP, as Sirt2 inhibition led to the downregulation of Akt/IGF-1 and ameliorated vasculopathy, ultimately improving visual function. These results suggest that Sirt2 may be a promising therapeutic target for ROP.

Alpha 7-nicotinic cholinergic regulation of pericyte-containing retinal capillaries.

Local blood flow regulation relies mostly on the coordination between neurons and pericyte-containing capillaries. Pericyte relaxation and contraction are influenced by various vasoactive substances and regulated by neurotransmitters. α7 nicotinic acetylcholine receptors (α7-nAChRs), involved in the regulation of vascular function and inhibitory GABA systems, have neuroprotective effects against central nervous system diseases. Although α7-nAChRs are found throughout the retina, their contribution to the retinal capillary tone remains unknown. Here we investigate the neurovascular coupling mechanism underlying α7-nAChR-mediated retinal capillary tone regulation. Changes in capillary diameter and transverse diameter of pericytes during drug perfusion were observed using differential interference contrast microscopy (DIC) to elucidate signaling pathways underlying α7-nAChR-mediated regulation of capillary blood flow at the whole retinal level. Patch clamp technique was used to investigate α7-nAChRs-mediated regulation of the γ-aminobutyric acid (GABA) synaptic circuit. Immunofluorescence was used to explore the expression of α7-nAChRs and GABA receptors. Activating α7-nAChRs on the endothelial cell membrane caused perinuclear accumulation of eNOS. This resulted in dilated retinal capillaries and pericytes via the NOS/NO-cGMP signaling pathway. Neuronal α7-nAChRs activation relaxed retinal capillaries and pericytes via a neurovascular coupling mechanism. α7-nAChR increased the vesicular release of GABA, possibly promoting the release of NO by binding to GABAA receptors in retinal ganglionic cells (RGCs), and relaxation of blood vessels via eNOS-NO by binding to GABAB receptors on retinal capillary endothelial cells. α7-nAChR activation causes vasorelaxation of retinal capillaries.

PDLIM1 inhibits cell migration and invasion in diabetic retinopathy via negatively regulating Wnt3a

The injury of vascular endothelial cells is a crucial factor in the development of diabetic retinopathy (DR). PDLIM1 (a member of the PDZ and LIM protein family) has been reported to exert an essential function in vascular diseases. This study aimed to elucidate the role of PDLIM1 on retinal vascular endothelial cells in DR. Immunofluorescence staining was used to localize the expression of PDLIM1 in the mouse retina. In some tumor diseases, PDLIM1 has been reported to play a key role in regulating the Wnt pathway. However, no in-depth reports have been found in DR. Retinal capillary endothelial cells (RCECs) were treated with high-glucose and high-lipid (HG/HL) culture medium, and siRNA transfection to investigate the role of PDLIM1 in DR. PDLIM1 and Wnt3a expression was confirmed by qRT-PCR and western blotting. Flow cytometry, Transwell assay, and scratch assay were used to test the ability of cell apoptosis, migration, and invasion. PDLIM1 was mainly expressed in the retinal pigment epithelium (RPE), ganglion cell layer (GCL), inner plexus layer (IPL), and outer plexus layer (OPL). HG/HL increased Wnt3a levels and promoted cell’s ability of apoptosis, migration, and invasion, which were reversed by the knockdown of PDLIM1. PDLIM1 was found to play a protective role in diabetic retinopathy by counter-regulating Wnt3a. PDLIM1 ameliorates cell apoptosis, migration, and invasion by negatively regulating Wnt3a in RCECs of DR, which suggests that PDLIM1 might be a promising therapeutic target for DR treatment.

The process of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats.

Methylglyoxal, a highly reactive dicarbonyl compound, is increased and accumulated in patients with diabetic mellitus. Methylglyoxal forms advanced glycation end products (AGE), contributing to the pathogenesis of diabetic complications, including diabetic retinopathy. Recent studies have shown that methylglyoxal induces diabetic retinopathy-like abnormalities in retinal vasculature. In this study, we investigated the processes and mechanisms of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes) were assessed in the retinas 2, 7, and 14days after intravitreal injection of methylglyoxal. Intravitreal methylglyoxal injection induced retinal capillary endothelial cell degeneration in a dose- and time-dependent manner. Changes in the shape and distribution of pericytes occurred before the initiation of capillary regression in the retinas of methylglyoxal-injected eyes. The receptor for AGEs (RAGEs) antagonist FPS-ZM1, and the matrix metalloproteinase (MMP) inhibitor GM6001 significantly attenuated methylglyoxal-induced capillary endothelial cell degeneration. FPS-ZM1 failed to prevent pathological changes in pericytes in methylglyoxal-injected eyes. In situ zymography revealed that MMP activity was enhanced at sites of blood vessels with reduced pericyte coverage in methylglyoxal-injected eyes. These results suggest that intravitreal methylglyoxal injection induces pathological changes in pericytes before the initiation of capillary endothelial cell degeneration via an AGE-RAGE-independent pathway. The capillary endothelial cell degeneration is mediated by activating the AGE-RAGE pathway and increasing MMP activity in endothelial cells by impairing pericyte function in the retina.

TFEB attenuates hyperglycemia-induced retinal capillary endothelial cells injury via autophagy regulation.

Diabetic retinopathy is a common microvascular complication of diabetes mellitus. The maintenance of retinal capillary endothelial cell homeostasis requires a complete and unobtrusive flow of autophagy because it may help combat the inflammatory response, apoptosis, and oxidative stress damage of cells in diabetes mellitus. The transcription factor EB is a master regulator of autophagy and lysosomal biogenesis, but its role in diabetic retinopathy remains unknown. This study aimed to confirm the involvement of transcription factor EB in diabetic retinopathy and explore the role of transcription factor EB in hyperglycemia-linked endothelial injury in vitro. First, the expression levels, including the nuclear location of transcription factor EB and autophagy, were reduced in diabetic retinal tissues and high glucose-treated human retinal capillary endothelial cells. Subsequently, autophagy was mediated by transcription factor EB in vitro. Moreover, transcription factor EB overexpression reversed high glucose-induced autophagy inhibition and lysosomal dysfunction and protected human retinal capillary endothelial cells from inflammation, apoptosis, and oxidative stress damage caused by high glucose treatment. Additionally, under high-glucose stimulation, the autophagy inhibitor chloroquine attenuated transcription factor EB overexpression-mediated protection, and the autophagy agonist Torin1 rescued transcription factor EB knockdown-induced damage effects. Taken together, these results suggest that transcription factor EB is involved in the development of diabetic retinopathy. In addition, transcription factor EB protects human retinal capillary endothelial cells from high glucose-induced endothelial damage via autophagy.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRβ. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Identification of potential ferroptosis-related biomarkers and a pharmacological compound in diabetic retinopathy based on machine learning and molecular docking.

Diabetic retinopathy (DR), a neurovascular disease, is a leading cause of visual loss worldwide and severely affects quality of life. Several studies have shown that ferroptosis plays an important role in the pathogenesis of DR; however, its molecule mechanism remains incompletely elucidated. Hence, this study aimed to investigate the pathogenesis of ferroptosis and explore potential ferroptosis-related gene biomarkers and a pharmacological compound for treating DR. Ferroptosis-related differentially expressed genes (DEGs) were identified in the GSE102485 dataset. Functional enrichment analyses were then performed and a protein-protein interaction (PPI) network was constructed to screen candidates of ferroptosis-related hub genes (FRHGs). FRHGs were further screened based on least absolute shrinkage and selection operator (LASSO) regression and random forest algorithms, and were then validated with the GSE60436 dataset and previous studies. A receiver operating characteristic (ROC) curve monofactor analysis was conducted to evaluate the diagnostic performance of the FRHGs, and immune infiltration analysis was performed. Moreover, the pharmacological compound targeting the FRHGs were verified by molecular docking. Finally, the FRHGs were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The 40 ferroptosis-related DEGs were extracted, and functional enrichment analyses mainly implicated apoptotic signaling, response to oxidative stress, ferroptosis, and lipid and atherosclerosis pathways. By integrating the PPI, LASSO regression, and random forest analyses to screen the FRHGs, and through validation, we identified five FRHGs that performed well in the diagnosis (CAV1, CD44, NOX4, TLR4, and TP53). Immune infiltration analysis revealed that immune microenvironment changes in DR patients may be related to these five FRHGs. Molecular docking also showed that glutathione strongly bound the CAV1 and TLR4 proteins. Finally, the upregulated expression of FRHGs (CD44, NOX4, TLR4, and TP53) was validated by qRT-PCR analysis in human retinal capillary endothelial cells cultured under high-glucose environment. CAV1, CD44, NOX4, TLR4, and TP53 are potential biomarkers for DR and may be involved in its occurrence and progression by regulating ferroptosis and the immune microenvironment. Further, glutathione exhibits potential therapeutic efficacy on DR by targeting ferroptosis. Our study provides new insights into the ferroptosis-related pathogenesis of DR, as well as its diagnosis and treatment.

Newly-established in vitro inner BRB spheroids to elucidate retinal Ang2-linked substance transfer.

Conjugation of angiopep-2 (Ang2) with drugs/compounds is known to increase plasma membrane permeability across endothelial barriers. The inner blood-retinal barrier (BRB) regulates retinal drug distribution and is formed by retinal capillary endothelial cells, supported by Müller cells and retinal pericytes. To elucidate the potential of Ang2 conjugation in promoting retinal drug distribution after peripheral administration across the inner BRB, an in vivo administration study and in vitro transport experiments using newly developed multicellular inner BRB spheroids were performed. After intravenous administration of Ang2-linked green fluorescence protein (GFP-Ang2) in mice, GFP-derived signals were observed in the neural retina. In contrast, GFP-derived signals were not observed after intravenous GFP administration, suggesting the promotion of the retinal distribution of substances by Ang2 conjugation. To overcome the limitations of in vitro studies using cells cultured on dishes, inner BRB spheroids were established using conditionally immortalized rat retinal capillary endothelial cells, Müller cells, and retinal pericytes. Immunocytochemistry of marker molecules suggests that the central part of the spheroids is occupied by Müller cells, and encapsulated by retinal pericytes and capillary endothelial cells. Studies on the expression and functions of tight junctions suggest that tight junctions are formed on the surface of the inner BRB spheroids by retinal capillary endothelial cells. The functional expression of drug transporters, such as P-glycoprotein, was observed in the spheroids, implying that the inner side of the spheroids reflects the retinal side of the inner BRB. In the inner BRB spheroids, energy-dependent accumulation of GFP-Ang2 and Ang2-linked 5(6)-carboxyfluorescein (FAM-Ang2) was observed. Moreover, an endocytic inhibition study revealed that clathrin-dependent endocytosis/transcytosis was involved in the transcellular transport of Ang2-conjugated drugs/compounds across the inner BRB. Consequently, it is suggested that the Ang2 linkage is useful for promoting retinal drug distribution via clathrin-dependent transcytosis at the inner BRB.

Endothelial SIRT-1 has a critical role in the maintenance of capillarization in brown adipose tissue

Brown adipose tissue (BAT) has critical roles in thermogenesis and systemic metabolism. Capillary rarefaction was reported to develop in BAT with dietary obesity, and previous studies showed that suppression of vascular endothelial growth factor A (VEGF-A) reduced capillary density in BAT, promoting the functional decline of this organ. Capillarization is regulated through the balance between angiogenesis and vasculogenesis on the one hand and apoptosis of endothelial cells (ECs) on the other; however, the role of EC apoptosis in BAT remained to be explored. In studies testing the role of boysenberry polyphenols (BoyP) in BAT, we found that BoyP decreased EC apoptosis, enhanced capillarization in BAT, and ameliorated dietary BAT dysfunction, which was associated with the upregulation of nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin 1 (SIRT-1) in ECs. Our studies suggest that EC SIRT-1 would be one of the potential targets of BoyP that contributes to BAT capillarization and function.

Withaferin a Attenuates Retinal Ischemia-Reperfusion Injury via Akt-Dependent Inhibition of Oxidative Stress

Background: Retinal ischemia-reperfusion (I/R) injury often results in intractable visual impairments. The survival of retinal capillary endothelial cells is crucial for the treatment of retinal I/R injury. How to protect retinal endothelia from damage is a challenging work. Withaferin A, a small molecule derived from plants, has antibacterial and anti-inflammatory effects and has been used for about millennia in traditional medicine. The present study aimed to investigate the potential protective effect of withaferin A on retinal I/R injury. Methods: The drug-likeness of withaferin A was evaluated by the SwissADME web tool. The potential protective effect of withaferin A on the I/R-induced injury of human retinal microvascular endothelial cells (HRMECs) was investigated using multiple approaches. RNA sequencing was performed and associated mechanistic signaling pathways were analyzed based on the Kyoto Encyclopedia of Genes and Genomes data. The analytical results of RNA sequencing data were further validated by in vitro and in vivo experiments. Results: Withaferin A reduced the I/R injury-induced apoptotic death of HRMECs in vitro with a good drug-like property. RNA sequencing and experimental validation results indicated that withaferin A increased the production of the crucial antioxidant molecules heme oxygenase 1 (HO-1) and peroxiredoxin 1 (Prdx-1) during I/R. In addition, withaferin A activated the Akt signaling pathway and increased the expression of HO-1 and Prdx-1, thereby exerting an antioxidant effect, attenuated the retinal I/R injury, and decreased the apoptosis of HRMECs. The blockade of Akt completely abolished the effects of withaferin A. Conclusions: The study identified for the first time that withaferin A can protect against the I/R-induced apoptosis of human microvascular retinal endothelial cells via increasing the production of the antioxidants Prdx-1 and HO-1. Results suggest that withaferin A is a promising drug candidate for the treatment of retinal I/R injury.

TRIM46 aggravated high glucose-induced hyper permeability and inflammatory response in human retinal capillary endothelial cells by promoting IκBα ubiquitination

BackgroundDiabetic retinopathy (DR) as a severe diabetic complication contributes to blindness. The increased permeability of retinal capillary endothelial cells (RCECs) as well as the production of inflammatory markers are closely related to DR occurrence. We recently revealed that TRIM46 promotes high glucose (HG)-caused ferroptosis in human RCECs (HRCECs). The current study aims to explore the molecular mechanism of how TRIM46 plays its role in DR progression.MethodsWestern blot was utilized to determine protein expression. The cell counting kit-8 assay was used to observe cell viability. The permeability of the cell layer was determined by measuring the transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran leak. Enzyme-linked immunosorbent assay was used to quantify the protein level of pro-inflammatory cytokines and co-immunoprecipitation was employed to verify the relationship between TRIM46 and IκBα.ResultsHG dramatically upregulated TRIM46 protein expression in a dose-dependent way. Silencing TRIM46 effectively reversed HG-induced cell growth inhibition, cell cycle arrest, hyper permeability and pro-inflammatory cytokines secretion in HRCECs, while overexpression of TRIM46 exhibited an opposite effect. Furthermore, TRIM46 was able to interact with IκBα and promote the ubiquitination and degradation of IκBα. IκBα overexpression recovered the effects of TRIM46 overexpression in HRCECs. Furthermore, inhibiting the activation of NF-κB partially recovered HG-induced HRCEC injury, whereas TRIM46 overexpression reversed these effects.ConclusionThis study demonstrates that TRIM46 interacts with IκBα to activate the NF-κB signaling pathway, thereby enhancing cell proliferation inhibition, hyper permeability and the inflammatory response of HRCECs in a HG state.

Bie-Jia-Ruan-Mai-Tang, a Chinese Medicine Formula, Inhibits Retinal Neovascularization in Diabetic Mice Through Inducing the Apoptosis of Retinal Vascular Endothelial Cells.

Proliferative diabetic retinopathy (PDR) is one of the main complications of diabetes, mainly caused by the aberrant proliferation of retinal vascular endothelial cells and the formation of new blood vessels. Traditional Chinese medicines possess great potential in the prevention and treatment of PDR. Bie-Jia-Ruan-Mai-Tang (BJ), a Chinese medicine formula, has a good therapeutic effect on PDR clinically; however, the mechanism of action involved remains unclear. Therefore, we investigated the effect of BJ on PDR through in vitro and in vivo experiments. A diabetic mouse model with PDR was established by feeding a high-fat–high-glucose diet combined with an intraperitoneal injection of streptozotocin (STZ), while high-glucose-exposed human retinal capillary endothelial cells (HRCECs) were employed to mimic PDR in vitro. The in vivo experiments indicated that BJ inhibited the formation of acellular capillaries, decreased the expression of VEGF, and increased the level of ZO-1 in diabetic mice retina. In vitro experiments showed that high glucose significantly promoted cell viability and proliferation. However, BJ inhibited cell proliferation by cycle arrest in the S phase, thus leading to apoptosis; it also increased the production of ROS, decreased the mitochondrial membrane potential, reduced the ATP production, and also reduced the expressions of p-PI3K, p-AKT, and Bcl-xL, but increased the expressions of Bax and p-NF-κB. These results suggest that BJ induces the apoptosis of HRCECs exposed to high glucose through activating the mitochondrial death pathway by decreasing the PI3K/AKT signaling and increasing the NF-κB signaling to inhibit the formation of acellular capillaries in the retina, thus impeding the development of PDR.

Calycosin Alleviates Oxidative Stress and Pyroptosis Induced by High Glucose in Human Retinal Capillary Endothelial Cells Induced by High Glucose

Calycosin is an isoflavone isolated from the roots of Astragalus membranaceus. Due to its antioxidant and anti-inflammatory activities, it has been extensively used in traditional Chinese medicine formulations. Herein, we have evaluated the mechanism of action of calycosin using high glucose induced HRCEC cells as a model. The observed effects of calycosin include alleviation of oxidative stress induced by high glucose through promotion of SOD, GSH-Px and inhibition of ROS expression. Moreover, calycosin relieved HG-induced inflammation and pyroptosis by upregulating IL-1β, IL-18 and downregulating NLRP3, ASC, and cleaving caspase-1/procaspase-1. Calycosin also inhibited HMGB1/NF-κB pathway activation by promoting p-P65/P65, p-IκBα, HMGB1 and decreasing IκBα. All of these results support the notion that calycosin alleviates oxidative stress and pyroptosis of HRCEC cells induced by high glucose by inhibiting activation of the HMGB1/NF-κB pathway.

Secreted Protein Acidic and Rich in Cysteine Mediates the Development and Progression of Diabetic Retinopathy.

BackgroundsDiabetic retinopathy (DR) is one of the most severe microvascular complications of diabetes mellitus (DM). Secreted protein acidic and rich in cysteine (SPARC) has been found to play an important role in many diseases, but its role and mechanism in DR remain unknown.MethodsWe studied the role of SPARC and integrin β1 in vascular pathophysiology and identified potential therapeutic translation. The SPARC levels were tested in human serum and vitreous by ELISA assay, and then the Gene Expression Omnibus (GEO) dataset was used to understand the key role of the target gene in DR. In human retinal capillary endothelial cells (HRCECs), we analyzed the mRNA and protein level by RT-PCR, immunohistochemistry, and Western blotting. The cell apoptosis, cell viability, and angiogenesis were analyzed by flow cytometry, CCK-8, and tube formation.ResultsIn this study, we investigated the role of SPARC in the development and progression of human DR and high glucose-induced HRCEC cells and found that the SPARC-ITGB1 signaling pathway mimics early molecular and advanced neurovascular pathophysiology complications of DR. The result revealed that DR patients have a high-level SPARC expression in serum and vitreous. Knockdown of SPARC could decrease the expressions of inflammatory factors and VEGFR, inhibit cell apoptosis and angiogenesis, and increase cell viability by regulating integrin β1 in HRCECs.ConclusionSPARC promotes diabetic retinopathy via the regulation of integrin β1. The results of this study can provide a potential therapeutic application for the treatment of DR.

Prohibitin inhibits high glucose-induced apoptosis via maintaining mitochondrial function in human retinal capillary endothelial cells.

Mitochondrial dysfunction and excessive apoptosis of vascular endothelial cells play a critical role in the development of diabetic retinopathy (DR). Prohibitin (PHB), a significant regulator, maintains mitochondrial function and protects vascular endothelial cells against apoptosis. However, the mechanism underlying the protective effect of PHB on DR remains unclear. Since mitochondria are key regulators of vascular homeostasis, the present study aimed to investigate the molecular mechanism of PHB on maintaining mitochondrial function in human retinal capillary endothelial cells (HRCECs). To evaluate the role of PHB in cell apoptosis, HRCECs, transfected with or without PHB overexpression plasmid or small interfering RNA clones targeting PHB, were cultured in the presence of 5.5 mmol/l normal glucose (NG) or 30 mmol/l high glucose (HG). Subsequently, the apoptosis rate of HRCECs was determined using flow cytometry. The results showed that PHB was upregulated in HRCECs, while PHB knockdown promoted the generation of reactive oxygen species from mitochondria via inhibition of the activation of complex I. Additionally, the apoptosis rate of HRCECs in the HG group was notably enhanced compared with that in the NG group. Interestingly, PHB overexpression attenuated the increase in HG-mediated HRCEC apoptosis. Furthermore, treatment with HG upregulated expression of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in vitro. The present study indicated that PHB could be a key modulator of mitochondrial homeostasis and could protect HRCECs against HG-induced apoptosis. Overall, the aforementioned findings provided experimental evidence supporting the potential protective effects of PHB on DR.

Paeoniflorin alleviates the progression of retinal vein occlusion via inhibiting hypoxia inducible factor-1α/vascular endothelial growth factor/STAT3 pathway

ABSTRACT Retinal vein occlusion (RVO) is a severe retinal vascular disease involving several complications, leading to weakening of vision and even blindness. Globally, over 16 million patients with RVO were found in the middle-aged population. Paeoniflorin (PF), a monomer of Taohong Siwu decoction, was reported to exhibit many pharmacological activities including anti-inflammatory, antioxidant, cardioprotective, and neuroprotective effects. However, the effect of PF on the progression of RVO remains unclear. In the current study, CCK8 assay was performed to investigate the cell viability. In addition, transwell assay and western blot were used to measure cell invasion and protein expression, respectively. Moreover, a mouse model of oxygen-induced dischemic retinopathy (OIR) was established. We found PF was able to inhibit the migration and angiogenesis of human retinal capillary endothelial cells under normoxia. Additionally, PF notably prevented hypoxia-induced angiogenesis of human retinal capillary endothelial cells via inhibiting hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/STAT3 pathway. Eventually, PF significantly alleviated the retinal lesions in the mouse with OIR. All in all, PF was able to alleviate the progression of retinal vein occlusion via inhibiting HIF-1α/VEGF/STAT3 pathway. These findings might provide some theoretical knowledge for exploring novel effective treatment for patients with RVO.

Glucocorticoids Promote Extracellular Matrix Component Remodeling by Activating YAP in Human Retinal Capillary Endothelial Cells.

Remodeling of extracellular matrix (ECM) components of endothelial cells is the main cause of retinal vascular basement membrane (BM) thickening, which leads to the initiation and perpetuation of microvasculopathy of diabetic retinopathy (DR). Excessive amounts of glucocorticoids (GCs) are related to the presence and severity of DR, however transcriptional effects of GCs on the biology of human retinal capillary endothelial cells (HRCECs) and its impacts on DR are still unclear. Here, we showed that GC (hydrocortisone) treatment induced ECM component [fibronectin (FN) and type IV collagen (Col IV)] expression and morphological changes in HRCECs via the glucocorticoid receptor (GR), which depended on the nuclear translocation of YAP coactivator. Mechanistically, GCs induced stress fiber formation in HRCECs, while blocking stress fiber formation inhibited GC-induced YAP nuclear translocation. Overexpression of FN, but not Col IV, activated YAP through the promotion of stress fiber formation via ECM-integrin signaling. Thus, a feedforward loop is established to sustain YAP activity. Using mRNA sequencing of HRCECs with overexpressed YAP or GC treatment, we found a similarity in Gene Ontology (GO) terms, differentially expressed genes (DEGs) and transcription factors (TFs) between the two RNA-seq datasets. In vivo, YAP was activated in retina vascular ECs of STZ-induced diabetic mice, and TF prediction analysis of published RNA-seq data of dermal vascular ECs from T2DM patients showed that GR and TEAD (the main transcription factor for YAP) were enriched. Together, GCs activate YAP and promote ECM component (FN and Col IV) remodeling in retinal capillary endothelial cells, and the underlying regulatory mechanism may provide new insights into the vascular BM thickening of the retina in the early pathogenesis of DR.

Role of the Blood-Retinal Barrier Transporters: Antiaging in Retina

Since the retina continuously receives light to enable vision, reactive oxygen species (ROS) are easily generated in neural retina. The oxidative stress induced by ROS may be involved in the onset and progression of blinding aging diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Although supply of antioxidants to the retina is important to maintain the redox homeostasis in neural retina, the blood-retinal barrier (BRB) is created by complex tight-junctions of retinal capillary endothelial cells and retinal pigment epithelial cells to prevent the free diffusion of substances. The BRB is equipped with several membrane transporters to supply nutrients and essential molecules including antioxidants and drugs which exhibit antiaging effect to the retina from the circulating blood. In this review, the transporter-mediated retinal distribution of key endogenous compounds and drugs, such as vitamin C, l-cystine and gabapentin, is introduced for antiaging of the retina.