Analysis and validation of potential ICD-related biomarkers in development of myopia using machine learning.

PURPOSE: We aimed to identify and verify potential biomarkers in the development of myopia associated with immunogenic cell death (ICD). METHODS: We download high myopia (HM) dataset GSE136701 from Gene Expression Omnibus. Differentially expressed genes in HM were identified to overlapped with ICD-related genes. Least absolute shrinkage and selection operator were used to select the Hub genes. Furthermore, the correlation between the hub genes and immune infiltration, immune response activities, and hub genes Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was investigated using Spearman's rank correlation. Prediction of the miRNAs upstream of the Hub genes was based on the TargetScan database. We used guinea pig lens-induced myopia model's scleral tissues performed quantitative real-time polymerase chain reaction. RESULTS: We identified overlapped with ICD-related genes (LY96, IL1A, IL33, and AGER) and two genes (LY96 and AGER) as hub genes. Single sample gene set enrichment analysis and Spearman's rank correlation revealed that hub gene expression levels in HM were significantly correlated with the infiltration percentages of CD56dim natural killer cells, macrophages, immature B cells, and the immune response activities of APC co-stimulation and Kyoto Encyclopedia of Genes and Genomes pathways, such as terpenoid backbone biosynthesis, aminoacyl-trna biosynthesis, Huntington's disease, oxidative phosphorylation; there were a few additional signaling pathways compared to normal samples. Additionally, several miRNA were predicted as upstream regulators of LY96 and AGER. LY96 was identified as a significantly differentially expressed biomarker in myopia guinea pig's scleral tissues, as verified by qPCR. CONCLUSION: LY96 was identified and verified as a ICD-related potential myopia biomarker. Molecular mechanisms or pathways involved in myopia development by LY96 requires further research.

Mechanisms of MALAT1 regulating proliferative diabetic retinopathy via targeting miR-126-5p.

OBJECTIVE: Diabetic retinopathy (DR) is the primary reason for blindness among the middle-aged and elderly. It can progress to proliferative diabetic retinopathy (PDR), a condition that is accompanied by retinal neovascularization as the disease worsens. Understanding the pathogenesis of PDR can facilitate the development of treatments. In this study, we aimed to investigate the involvement in the lncRNA MALAT1 (MALAT1)/miR-126-5p axis in modulating PDR progression. METHODS: Rat retinal endothelial cells (RECs) was induced with 30 mM glucose to build an in vitro PDR model. MALAT1 was down-regulated using siRNA sequences, and miR-126-5p was up-regulated with miRNA mimics. Dual-luciferase reporter assay and RNA immunoprecipitation assay were carried out to identify and validate the targeting relationship between MALAT1 and miR-126-5p. Angiogenesis, cell proliferation and cell migration were detected using tubule formation, CCK-8, and scratch assays, respectively. Western blots quantified angiogenesis- and migration-associated genes vascular endothelial growth factor (VEGF), MMP2 and MMP9, while qPCR measured MALAT1 and miR-126-5p levels. RESULTS: In high-glucose induced RECs, MALAT1 was up-regulated while miR-126-5p was down-regulated. The angiogenesis as well as the proliferation and migration capacities of high glucose-induced RECs were suppressed when MALAT1 was down-regulated or miR-126-5p was up-regulated, accompanied by reductions in VEGF, MMP-2 and MMP9. RNA immunoprecipitation assay confirmed that miR-126-5p could be enriched in MALAT1 sequences. Dual-luciferase reporter assay confirmed the targeted inhibition of miR-126-5p by MALAT1. Downregulating miR-126-5p counteracted the effect of MALAT1 downregulation on RECs induced by high glucose. CONCLUSIONS: MALAT1 promotes PDR by inhibiting miR126-5p and inducing REC proliferation, migration and angiogenesis.

Transcription factor FOXP1 mediates vascular endothelial dysfunction in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is still the fastest growing cause of blindness in working aged adults, and its typical characteristics are endothelial cell dysfunction and pericytes loss. Transcription factor fork head box P1 (FOXP1) is a member of FOX family involved in diabetes progression and is expressed in endothelial cells. The purpose of this study was to investigate the role and mechanism of FOXP1 in DR. METHODS: The vitreous of DR patients and non-DR patients were collected, and the expression of FOXP1 was detected by real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) cultured in high glucose simulated DR environment, and the expressions of FOXP1, vascular endothelial growth factor (VEGF), and pigment epithelium derived factor (PEDF) were detected by RT-qPCR and western blot (WB) after transfection of small interfering RNA (siRNA) to knock out FOXP1. At the same time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), 5-ethynyl-2'-deoxyuridine assay (EDU), flow cytometry, Transwell assay, and tube-forming experiment were performed to determine cell proliferation, migration, and tube-forming ability. RESULTS: We found that FOXP1 was highly expressed in the vitreous of DR patients and HUVECs under high glucose condition. After FOXP1 was decreased, the activation of VEGF expression and inhibition of PEDF expression in HUVECs induced by high glucose were reversed; meanwhile, cell proliferation, migration, and tube formation decreased, and apoptosis was promoted. CONCLUSION: Generally, FOXP1 is highly expressed in the vitreous of DR patients, and its silence prevented VEGF/PEDF signaling pathway stimulated by high glucose and also reduced the proliferation, migration, and tube formation of endothelial cell, thus improving vascular endothelial dysfunction caused by DR. The results indicate that FOXP1 may be a therapeutic target of DR.

Urolithin A ameliorates diabetic retinopathy via activation of the Nrf2/HO-1 pathway.

Diabetic retinopathy (DR) is a progressive microvascular complication of diabetes mellitus and is characterised by excessive inflammation and oxidative stress. Urolithin A (UA), a major metabolite of ellagic acid, exerts anti-inflammatory and antioxidant functions in various human diseases. This study, for the first time, uncovered the role of UA in DR pathogenesis. Streptozotocin-induced diabetic rats were used to determine the effects of UA on blood glucose levels, retinal structures, inflammation, and oxidative stress. High glucose (HG)-induced human retinal endothelial cells (HRECs) were used to elucidate the anti-inflammatory and antioxidant mechanisms of UA in DR in vitro. The in vivo experiments demonstrated that UA injection reduced blood glucose levels, decreased albumin and vascular endothelial growth factor concentrations, and ameliorated the injured retinal structures caused by DR. UA administration also inhibited inflammation and oxidative damage in the retinal tissues of diabetic rats. Similar anti-inflammatory and antioxidant effects of UA were observed in HRECs induced by HG. Furthermore, we found that UA elevated the levels of nuclear Nrf2 and HO-1 both in vivo and in vitro. Nrf2 silencing reversed the inhibitory effects of UA on inflammation and oxidative stress during DR progression. Together, our findings indicate that UA can ameliorate DR by repressing inflammation and oxidative stress via the Nrf2/HO-1 pathway, which suggests that UA could be an effective drug for clinical DR treatment.

Extracellular vesicles secreted from mesenchymal stem cells exert anti-apoptotic and anti-inflammatory effects via transmitting microRNA-18b in rats with diabetic retinopathy.

Diabetic retinopathy (DR) is a major cause of visual deficits and blindness in the working-age population and inflammatory response is a key event during DR. In this study, we investigated the anti-inflammatory properties of small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells (hUCMSCs) in a diabetic rat model and human retinal microvascular endothelial cells. After development of DR in rats subjected to diabetes induction with streptozotocin (STZ), the DR rats were treated with different concentrations of hUCMSC-sEVs. Our results showed that the treatment of the retinas of DR rats with hUCMSC-sEVs not only reduced the level of vascular leakage in the retinas of rats but also decreased the retinal thickness as well as the associated inflammation. Further, our in vitro evidences suggest that hUCMSC-sEVs repress high glucose (HG)-induced cell inflammation and apoptosis. Subsequently, we analyzed the differentially expressed microRNAs (miRNAs) in the hUCMSC-sEVs by microarray and performed in silico studies to predict the target mRNA of miR-18b. Our findings also revealed that the expression of miR-18b was significantly elevated in the retina of diabetic rats after sEV treatment. In addition, miR-18b was found to target mitogen-activated protein kinase kinase kinase 1 (MAP3K1), thereby inhibiting NF-κB p65 phosphorylation to alleviate DR. Overall, this study highlights the potential of hUCMSCs-sEVs as biomaterials for anti-inflammatory and anti-apoptotic effects in DR by transferring miR-18b.

Transient Ischemic Attack and Hypoventilation 12 Hours After Intra-vitreal Aflibercept Injection.

Given the role the vascular endothelial growth factor (VEGF) plays in controlling and preserving the integrity of the vascular endothelium intravitreal administration of anti-VEGF agents may affect the risk of thromboembolic events. This is particularly noticeable in patients who are at risk for atherosclerosis. Here, we present one of the first case reports of transient ischemic attack (TIA) together with hypoventilation secondary to aflibercept injection. A 63-year-old female suffered TIA together with hypoventilation about 12 hours after the third administration of intravitreal aflibercept, which is a VEGF inhibitor for diabetic macular edema (DME). Upon presentation, she was confused, had right-sided weakness and her respiratory rate was six breaths per minute, all of these resolved within the next 24 hours. The serum tests, cerebrospinal fluid (CSF) analysis, brain imaging, and carotid Doppler ultrasound were unremarkable. An ophthalmic exam revealed signs of bilateral proliferative diabetic retinopathy with left macular edema. Detailed reports of similar cases are lacking in the literature. Hypoventilation and thromboembolic could be possible side effects of aflibercept that necessitate more investigation.

MicroRNA­126 suppresses the proliferation and migration of endothelial cells in experimental diabetic retinopathy by targeting polo­like kinase 4.

As a specific microvascular complication of diabetes, diabetic retinopathy (DR) causes severe visual impairment in patients with diabetes. The expression of microRNA­126 (miRNA/miR­126) has previously been found to be significantly decreased in the serum of patients with DR. In the present study, the functions of miR­126 and its mechanisms of action in experimental diabetic retinopathy were examined in rats with streptozotocin (STZ)­induced diabetes and in high glucose (HG)­induced human retinal capillary endothelial cells (HRCECs). In vivo, diabetic rat models were established and the rats were intravitreally injected with lentivirus expressing rno­miR­126 (lenti­miR­126) or negative control (lenti­NC). RT­qPCR was used to determine the miR­126 level in the serum and retina. Paraffin sections and retinal vasculature were used to determine the extent of retinopathy. The protein content of vascular endothelial growth factor (VEGF) and pigment epithelium­derived factor (PEDF) in the retina was used as an auxiliary measurement of retinopathy. Western blot analysis and immunofluorescence staining were used to measure the expression of polo­like kinase 4 (PLK4) in rat retinal tissue. In vitro, the cells were transfected with miR­126 inhibitor or mimic and treated with the PLK4 inhibitor, CFI­400945 fumarate. RT­qPCR and western blot analysis were used to detect the miR­126 level and PLK4 expression. Cell proliferation and migration were measured by EdU and Transwell assays. The diabetic rats were found to exhibit downregulated serum and retinal miR­126 levels compared with the non­diabetic rats. The intravitreal delivery of miR­126 alleviated retinopathy and reduced the diabetes­induced upregulation of PLK4 in retinal tissues. Luciferase reporter assays confirmed that PLK4 mRNA was the target of miR­126. In HG­induced HRCECs, transfection with miR­126 mimic increased the miR­126 level, whereas it downregulated that of its downstream target, PLK4, which was opposite to the effects exerted by the miR­126 inhibitor. Furthermore, miR­126 mimic and CFI­400945 fumarate reduced the HG­induced upregulation of PLK4 expression, as well as cell proliferation and migration. On the whole, the findings of the present study demonstrate that miR­126 reduces experimental diabetic retinopathy and suppresses endothelial cell proliferation and migration by targeting PLK4. Thus, miR­126 and CFI­400945 fumarate may be therapeutic targets for DR.

Choroidal Changes in Diabetic Patients With Different Stages of Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the long-term microvascular complications of diabetes mellitus (DM) and is considered a leading cause of vision loss worldwide. Chronic hyperglycemia can cause microvascular abnormalities to the retina and the choroid as well. The vascular tissue of the choroid supplies blood to the outer retina, photoreceptors, and retinal pigment epithelium. It plays an important role in the metabolic exchange of the retina. Many experimental studies reported that choroidal pathology in diabetic patients might play a role in developing DR. Choroidal thickness (CT) can reflect changes in the vasculature of the choroid and can be used to assess the vascularity of the choroid itself. CT differs between healthy and diseased states of the eye as well as with the aging process. This means that thinner or thicker choroid may indicate an ocular disease. Choroidal vascularity index (CVI) is also used as a marker for choroidal vascularity assessment and indirectly measures choroidal vascularity quantitatively. Many studies have been conducted to evaluate the choroid in many different ocular diseases. However, the results regarding CT in DM, especially in patients with DR, are various as thickened, thinned, or no changes. Thus, the status of the choroid in patients with DM with or without DR remains controversial between researchers. In this systematic review, we reviewed 18 articles that were done to investigate the relationship between structural choroidal changes in diabetic patients with different stages of DR, focusing on CT, CVI, and some other parameters evaluating choroidal changes.

The expression and significance of mTORC1 in diabetic retinopathy.

BACKGROUND: To investigate the expression and significance of mechanistic target of rapamycin complex 1(mTORC1) in diabetic retinopathy (DR), and to find new targets and new methods for the treatment of DR. METHODS: A DR rat model was prepared by general feeding combined with intraperitoneal injection of 10% streptozotocin (60 mg/kg). The rats were randomly divided into a control group (NDM group) and a diabetes group (DM group). Three months later, the degrees of retinopathy was determined using hematoxylin and eosin staining, and the levels of p-S6, VEGF, and PEDF proteins were detected by immunohistochemistry and western blotting. Human retinal capillary endothelial cells (HRCECs) were cultured in high glucose (HG) conditions, then treated with rapamycin or transfected with siTSC1.The protein levels of p-S6 were assessed by western blotting. The 5-ethynyl-2'-deoxyuridine assay was used to detect cell proliferation, and the Transwell assay was used to detect cell migration. RESULTS: A DM rat model was successfully developed. The expressions of p-S6 and VEGF proteins were significantly increased in the DM group (p < 0.05), and the expression of PEDF protein was significantly decreased compared with the NDM group (p < 0.05). In vitro, the p-S6 protein, as well as cell proliferation and migration, in HG induced HRCECs were increased (p < 0.05) compared with the control (normal glucose) group (p < 0.05). After transfection with siTSC1 to activate mTORC1, the expression of p-S6, as well as cell proliferation and migration, were increased. In contrast, rapamycin decreased p-S6 expression, as well as proliferation and migration, in HG induced HRCECs compared to the control group (p < 0.05). CONCLUSION: mTORC1 plays an important role in DR. After activation, mTORC1 induced expression of the p-S6 protein, regulated the expressions of VEGF and PEDF proteins, and changed the proliferation and migration of endothelial cells. The mTORC1 can therefore be used as a new target,as well as in the treatment of DR.

Sensitized heat shock protein 27 induces retinal ganglion cells apoptosis in rat glaucoma model.

AIM: To investigate the relationships between the changes of heat shock protein 27 antibody (anti-HSP27) in serum/cerebrospinal fluid (CSF), intraocular pressure (IOP), retinal ganglion cell (RGC) apoptosis in a rat glaucoma model and disclose the underlying pathogenesis of glaucoma. METHODS: A total of 115 Wistar rats were randomly divided into 4 groups. Group 1 was the ocular hypertension group by condensing 3 episcleral & limbal veins or episcleral area of right eye (HP group, n=25) and sham operation group with conjunctiva incision without coagulation (n=25). Group 2: HSP27 or dose-matched PBS was injected into the vitreous (V-HSP27 group, n=15; V-PBS group, n=15). Group 3: HSP27 and complete Freund's adjuvant or dose-matched PBS was injected subcutaneously into the hind limb accompanied intraperitoneal injection of pertussis toxin [sensitized group (I-HSP27 group), n=15; I-PBS group, n=15)]. Group 4 was normal group without any treatment (n=5). IOPs of the rats were measured before, day 3, weeks 1, 2, 4, 6, and 8 after treatment. Paraffin-embedded sections were prepared for HE staining and RGCs apoptosis were detected by TUNEL. Anti-HSP27 level in serum and CSF were examined by ELISA. RESULTS: IOPs were elevated significantly in HP and V-HSP27, V-PBS groups (P<0.01) and positively related to anti-HSP27 levels in serum and CSFs. Anti-HSP27 levels in serum and CSF were elevated significantly in I-HSP27 group compared to other groups (P<0.05). However, the IOPs did not show any relationship with the high-level anti-HSP27 in serum and CSFs. RGC apoptosis were all elevated significantly in the HP, V-HSP27, V-PBS and I-HSP27 groups and also positively relative with anti-HSP27 level in serum and CSFs except that high-level of anti-HSP27 in the serum of I-HSP group. CONCLUSION: The increases of anti-HSP27 levels in serum and CSFs both promote IOP escalation and the increase of RGC apoptosis in retina when anti-HSP27 is at low level. The case of high-level anti-HSP27 is opposite and shows protective function in preventing IOP increase and RGC apoptosis.

Protective factors for diabetic retinopathy in Type 2 diabetes mellitus patients: Long duration of no less than 10 years.

AIM: To study the factors protecting against diabetic retinopathy (DR) in patients with over a decade-long history of type 2 diabetes mellitus. METHODS: A total of 490 patients with type 2 diabetes mellitus lasting for ≥10 years were divided into DR and no diabetic retinopathy (no DR) groups. Their basic information was collected, including age, sex, and duration of diabetes mellitus, as well as pertinent laboratory data. Potential correlations between these factors and DR were evaluated using multivariate analysis. RESULTS: Overall, 208 patients met the diagnostic criteria for DR. Multivariate logistic regression was used to evaluate factors with P < 0.10 after univariate analysis. Age, total bilirubin, and total cholesterol were found to be protective factors against DR. Presence of diabetic kidney disease and diabetic peripheral neuropathy, duration of diabetes mellitus, apolipoprotein B, blood urea nitrogen, and prothrombin time were found to be risk factors for DR. CONCLUSIONS: We conclude that total cholesterol is a protective factor against DR. Specifically, it was confirmed that high levels of total cholesterol reduce the risk of DR. These findings may provide a basis for new diet and lifestyle guidelines for patients with diabetes mellitus.

mTORC1 regulates apoptosis and cell proliferation in pterygium via targeting autophagy and FGFR3.

Pterygium is one of the most common ocular surface diseases. During the initiation of pterygium, resting epithelial cells are activated and exhibit aberrant apoptosis and cell proliferation. Mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, cell proliferation, protein synthesis, autophagy and transcription. However, the effect of mTORC1 activation in epithelial cells on pterygium development has not yet been reported. Additionally, the roles of mTORC1 in aberrant apoptosis and cell proliferation during the initiation of pterygium, and the underlying mechanisms, are not known. Herein, we evaluated mTOR signalling in pterygium growth and development. The results revealed that mTOR signalling, especially mTORC1 signaling, is highly activated, and aberrant apoptosis and cell proliferation were observed in pterygium. mTORC1 activation inhibits apoptosis in pterygium by regulating Beclin 1-dependent autophagy via targeting Bcl-2. mTORC1 also negatively regulates fibroblast growth factor receptor 3 (FGFR3) through inhibition of p73, thereby stimulating cell proliferation in pterygium. These data demonstrate that mTORC1 signalling is highly activated in pterygium and provide new insights into the pathogenesis and progression of pterygium. Hence, mTORC1 may be a novel therapeutic target for the treatment of pterygium.

MicroRNA-126: a promising novel biomarker in peripheral blood for diabetic retinopathy.

AIM: To investigate the content of serum microRNA-126 (miR-126) and its role in screening retinal endothelial injury and early diagnosis of proliferative diabetic retinopathy. METHODS: The study included 184 serum samples, 59 samples from healthy individuals, 44 samples from diabetes mellitus (DM) patients without diabetic retinopathy (NDR), 42 from non-proliferative diabetic retinopathy (NPDR) patients and 39 samples from proliferative diabetic retinopathy (PDR) patients. The expression of miR-126 was evaluated using a real-time quantitative polymerase chain reaction. RESULTS: The serum content of miR-126 declined as the damage degree in the retina. There was significant difference between the two retinopathy groups (P<0.001). No difference was observed in miR-126 content between healthy individuals and NDR patients (P>0.05). Receiver operating characteristic curve (ROC) analyses indicated that serum miR-126 had significant diagnostic value for PDR. It yielded an area under the curve (AUC) of ROC of 0.976 with 81.21% sensitivity and 90.34% specificity in discriminating PDR from healthy controls, and an AUC of ROC of 0.919 with 84.75% sensitivity and 94.41% specificity in discriminating NDR and NPDR from healthy controls. When the diagnostic threshold was greater than or equal to 8.43, there was an increase in the possibility of NPDR. When the content of miR-126 was less than or equal to 5.02, the possibility of the occurrence of PDR increased. CONCLUSION: Serum miR-126 can serve as a non-invasive biomarker for screening retinal endothelial injury and early diagnosis PDR.

Detection and comparison of matrix metalloproteinase in primary and recurrent pterygium fibroblasts.

AIM: To detect and compare the levels of matrix metalloproteinases (MMPs) secreted by primary and recurrent human pterygium fibroblasts (HPFs). METHODS: Primary and recurrent HPFs as well as human conjunctival fibroblasts (HCF) were cultured in RPMI 1640 medium at the same conditions. The protein levels of MMP-1, MMP-3 and MMP-9 were determined by enzyme-linked immune sorbent assay (ELISA), respectively. RESULTS: 1) The protein level of MMP-1 in serum-free supernatant from cultured primary and recurrent HPFs was higher than that in normal HCFs (P<0.05); similarly, the protein level of MMP-1 in serum-free supernatant from cultured primary HPFs was higher than that in recurrent HCFs (P<0.05). 2) The protein level of MMP-3 in serum-free supernatant from cultured primary HPFs was higher than that in normal HCFs (P<0.05); meanwhile, the protein level of MMP-3 in serum-free supernatant from cultured recurrent HPFs was lower when compared with that in primary HPFs and normal HCFs (P<0.05). 3) MMP-9 was not detected in primary and recurrent HPFs in the conditioned medium. CONCLUSION: The protein levels of MMP-1 and MMP-3 in supernatant secreted by primary HPFs are different from recurrent HPFs. Different pathological mechanisms may exist between primary and recurrent pterygia.

[Oxidative damage and photoreceptor cell apoptosis in contusion injury of the rabbit retina].

OBJECTIVE: To study the mechanism of retina injury and photoreceptor apoptosis after contusion as well as their triggering factor. METHODS: The rabbit retina was injured by a 3 J energy contusion caused by a free falling iron bar hitting the cornea. The morphologic changes of the retina were observed by light and electron microscopes. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique was used to assess apoptosis. The amount of the malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the retina were assayed and analyzed statistically. RESULTS: Apoptosis of photoreceptors was present in retina contusion caused by a 3 J energy injury. After contusion, the MDA level increased gradually. The activity of SOD increased reflectively at first, then decreased markedly 3 days after the contusion, which was parallel with the appearance of apoptotic photoreceptors. CONCLUSIONS: Apoptosis is an important mechanism of retina contusion. Free oxygen radicals are the important triggering factors for the apoptosis of photoreceptors in the retina contusion of the rabbits.