Silencing the long noncoding RNA MALAT1 inhibits vitreous-induced epithelial-mesenchymal transition in RPE cells by regulating the PDGFRs/AKT axis.

PURPOSE: Epithelial-mesenchymal transition (EMT) is a crucial pathological process that contributes to proliferative vitreoretinopathy (PVR), and research indicates that factors present in the vitreous that target cells play pivotal roles in regulating EMT. Experimental studies have confirmed that rabbit vitreous (RV) promotes EMT in human retinal pigment epithelial (RPE) cells. The long noncoding RNA (lncRNA) MALAT1 has been implicated in EMT in various diseases. Thus, this study aimed to investigate the involvement of lncRNA MALAT1 in vitreous-induced EMT in RPE cells. METHODS: MALAT1 was knocked down in ARPE-19 cells by short hairpin RNA (shRNA) transfection. Reverse transcription PCR (RT‒PCR) was used to evaluate MALAT1 expression, and Western blotting analysis was used to measure the expression of EMT-related proteins. Wound-healing, Transwell, and cell contraction assays were conducted to assess cell migration, invasion, and contraction, respectively. Additionally, cell proliferation was assessed using the CCK-8 assay, and cytoskeletal changes were examined by immunofluorescence. RESULTS: MALAT1 expression was significantly increased in ARPE-19 cells cultured with RV. Silencing MALAT1 effectively suppressed EMT and downregulated the associated factors snail1 and E-cadherin. Furthermore, silencing MALAT1 inhibited the RV-induced migration, invasion, proliferation, and contraction of ARPE-19 cells. Silencing MALAT1 also decreased RV-induced AKT and P53 phosphorylation. CONCLUSIONS: In conclusion, lncRNA MALAT1 participates in regulating vitreous-induced EMT in human RPE cells; these results provide new insight into the pathogenesis of PVR and offer a potential direction for the development of antiproliferative drugs.

Exploitation of enhanced prime editing for blocking aberrant angiogenesis.

INTRODUCTION: Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders. OBJECTIVES: The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy. METHODS: An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis. RESULTS: NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology. CONCLUSION: DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.

Association between Axial Length to Corneal Curvature Radius Ratio and Myopia in Adult Patients.

Purpose: To analyze the distribution characteristics of axial length to corneal curvature radius ratio (AL/CR) and other ocular biometric parameters in adult myopia patients and their association with myopia. Methods: A cross-sectional study was conducted among patients with no eye diseases except ametropia who attended the optometry clinic of the First Affiliated Hospital of Hainan Medical College from January 2022 to June 2022. In total, 187 eyes (right eye) of 187 myopic patients aged 18-35 years were selected by random sampling. Based on the results of spherical equivalent (SE, (D)) obtained by postdilation optometry, all subjects were divided into three groups: mild myopia (≤-0.50D and >-3.00D, 42 eyes), moderate myopia (≤-3.00D and >-6.00D, 80 eyes), and high myopia (≤-6.00D, 65 eyes). The axial length (AL), corneal curvature radius (CR), and AL/CR were measured and compared between the three groups. The association between AL and AL/CR of the eye and SE was analyzed by multiple linear regression. Also, the predictive ability of AL/CR for high myopia was investigated by ROC curve. Results: There were no statistically significant differences in age, gender, or intraocular pressure between the three groups. The mean values of AL/CR in mild, moderate, and high myopia groups were 3.17 ± 0.06, 3.31 ± 0.08, and 3.43 ± 0.10, respectively, and the difference between the groups was statistically significant (P < 0.001). Linear regression analysis showed that both AL and AL/CR were strongly negatively correlated with SE (P < 0.05), while CR had a weak positive correlation with SE without statistically significant differences (P > 0.05). The adjusted linear regression equation shows that for every 0.1 unit increase in AL/CR, SE increases by 1.54 D. Compared with 0.830 (95% confidence interval: 0.769 to 0.900) for AL, the area under ROC curve of AL/CR was 0.896 (95% confidence interval: 0.851 to 0.941), indicating that the diagnostic value of AL/CR for high myopia was higher than that of AL (P < 0.01). When the Youden index reached its maximum (0.626), the AL/CR cutoff point was 3.309, and the sensitivity and specificity were 0.954 and 0.672, respectively. Conclusion: This study showed that AL and AL/CR in adult myopia patients were significantly negatively correlated with SE, and the corralation between AL/CR and SE is greater than that between AL and SE. Therefore, AL/CR can be used to analyze the dynamic changes of SE in the development of adult myopia independently of optometry on a certain basis, and it is especially suitable for the diagnosis of high myopia in adults. This trial is registered with ChiCTR2300069070.

Increased serum cystatin C levels in patients with idiopathic epiretinal membrane.

CLINICAL RELEVANCE: Increased serum cystatin C may play a role in the pathogenesis of idiopathic epiretinal membrane (IERM). Physicians should be aware of this relationship and should refer patients to the ophthalmology clinic for screening. BACKGROUND: To evaluate the serum cystatin C level in patients with IERM, and its associations with visual acuity. METHODS: Sixty-eight patients with IERM and sixty-nine controls were enrolled in this cross-sectional study. Based on the results of optical coherence tomography, patients with IERM were divided into four stages (I, II, III and IV). Serum cystatin C was measured in all participants. Serum cystatin C levels were compared between the control group and IERM group and between the IERM group with different optical coherence tomography stages. Multiple linear regression was used to evaluate the relationship between serum cystatin C and IERM stages and best corrected visual acuity. RESULTS: Serum cystatin C level was higher in the IERM group than in the control group (P < 0.001). There were statistically significant differences in serum cystatin C among different stages of IERM (PI vs II = 0.011, PI vs IV < 0.001 and PIII vs IV = 0.040, respectively). There were significant differences in best corrected visual acuity among different stages of IERM (PI vs III = 0.018, PI vs IV < 0.001, PII vs IV < 0.001 and PIII vs IV < 0.001, respectively). Regression analysis showed a positive correlation between serum cystatin C and best corrected visual acuity (t = 2.238 P = 0.029). The cut-off value of receiver operation characteristic curve of serum cystatin C for IERM was 0.775. CONCLUSION: This study revealed that serum cystatin C may be involved in the pathogenesis of IERM and can predict its occurrence. Elevated serum cystatin C appears to be associated with the severity of the disease and relatively poor vision acuity in IERM patients.

Visual impairment and blindness caused by retinal diseases: A nationwide register-based study.

Background: Retinal disorders cause substantial visual burden globally. Accurate estimates of the vision loss due to retinal diseases are pivotal to inform optimal eye health care planning and allocation of medical resources. The purpose of this study is to describe the proportion of visual impairment and blindness caused by major retinal diseases in China. Methods: A nationwide register-based study of vitreoretinal disease covering all 31 provinces (51 treating centres) of mainland China. A total of 28 320 adults diagnosed with retinal diseases were included. Participants underwent standardised ocular examinations, which included best-corrected visual acuity (BCVA), dilated-fundus assessments, and optical coherence tomography. Visual impairment and blindness are defined using BCVA according to the World Health Organization (WHO) (visual impairment: <20/63-≥20/400; blindness: <20/400) and the United States (visual impairment: <20/40-≥20/200; blindness: <20/200) definitions. The risk factors of vision loss were explored by logistic regression analyses. Results: Based on the WHO definitions, the proportions for unilateral visual impairment and blindness were 46% and 18%, respectively, whereas those for bilateral visual impairment and blindness were 31% and 3.3%, respectively. Diabetic retinopathy (DR) accounts for the largest proportion of patients with visual impairment (unilateral visual impairment: 32%, bilateral visual impairment: 60%) and blindness (unilateral blindness: 35%; bilateral blindness: 64%). Other retinal diseases that contributed significantly to vision loss included age-related macular degeneration, myopic maculopathy, retinal vein occlusion, and rhegmatogenous retinal detachment and other macular diseases. Women (bilateral vision loss: P = 0.011), aged patients (unilateral vision loss: 45-64 years: P < 0.001, ≥65 years: P < 0.001; bilateral vision loss: 45-64 years: P = 0.003, ≥65 years: P < 0.001 (reference: 18-44 years)) and those from Midwest China (unilateral and bilateral vision loss: both P < 0.001) were more likely to suffer from vision loss. Conclusions: Retinal disorders cause substantial visual burden among patients with retinal diseases in China. DR, the predominant retinal disease, is accountable for the most prevalent visual disabilities. Better control of diabetes and scaled-up screenings are warranted to prevent DR. Specific attention should be paid to women, aged patients, and less developed regions.

Comparison of Peripheral Blood Inflammatory Indices in Patients with Neovascular Age-related Macular Degeneration and Haemorrhagic Polypoidal Choroidal Vasculopathy.

PURPOSE: To compare the differences in peripheral blood inflammatory indices between patients with neovascular age-related macular degeneration (nAMD) and haemorrhagic polypoidal choroidal vasculopathy (PCV). METHODS: Retrospective, best corrected visual acuity (BCVA), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR), were analysed across the nAMD, PCV and normal control (NCG) groups of patients. The ratios' cut-off values for nAMD were calculated. RESULTS: nAMD had a significantly longer duration and better BCVA than PCV (all P < .05). The NLR, MLR and PLR were significantly higher in nAMD than in PCV and NCG (all P < .01), no significant differences between PCV and NCG (all P > .05). The ROC curve analysis revealed that the cut-off values for NLR and MLR were 1.98 and 0.24, respectively, for nAMD. CONCLUSION: NLR, MLR and PLR are significantly high in patients with nAMD. The ability of these inflammatory indicators to distinguish nAMD and PCV is unclear.

Retinal hemangioblastoma in a patient with Von Hippel-Lindau disease: A case report and literature review.

Background: Retinal hemangioblastoma (RH) is a rare benign tumor and a considerable number of which are caused by Von Hippel-Lindau disease (VHL). Herein, we described a case of VHL-associated RH with retinal detachment who underwent both laser photocoagulation and vitreoretinal surgery and received satisfactory visual recovery. In addition, we reviewed the current diagnosis, genotype-phenotype association, and treatment of VHL-associated RH. Case description: A 34-year-old woman presented with vision loss in the right eye at our hospital. Fundus photography and angiography showed retinal detachment and multiple large hemangiomas in the right eye. The visual acuity improved significantly after laser photocoagulation and vitreoretinal surgery. Genetic analyses showed a p.Asn78Ser (c.233A>G) heterozygous missense mutation in the VHL gene. Conclusion: We described a rare case of VHL-associated RH and may provide a new perspective towards diagnosis and treatment of this disease. RH is one of the most common manifestations of VHL and poses a serious threat to vision. Ophthalmic examination methods include fundus examination and fundus photography, etc. The management of the disease emphasizes timely follow-up, early detection of the lesion, and the decision of treatment options according to the size, location and complications of the lesion, including ablation therapy and vitreoretinal surgery. Clinicians should strengthen the understanding of this rare disease for early detection and treatment.

Conbercept improves macular microcirculation and retinal blood supply in the treatment of nonischemic branch retinal vein occlusion macular edema.

PURPOSE: To investigate the effect of conbercept on macular microvascular system and retinal blood supply in the treatment of nonischemic branch retinal vein occlusion macular edema (BRVO-ME). METHODS: Patients were divided into three groups: group A (containing 12 nonischemic BRVO-ME eyes), group B (containing contralateral 12 healthy eyes), and group C (containing 30 cataract eyes to obtain normal aqueous humor cytokine levels). Group A received monthly intravitreal injections of conbercept for 3 months. General data and best-corrected visual acuity (BCVA) were compared among the three groups. Optical coherence tomography angiography (OCTA) results (including central macular thickness [CMT], retinal vascular density and perfusion, and foveal avascular zone [FAZ]) at baseline were compared among groups A and B. Aqueous humor cytokine levels (including VEGF, IL-8, PDGF-AA, TNF-α, and ANGPTL-4) at baseline were compared between groups A and C. Moreover, BCVA, OCTA results, and aqueous humor cytokine levels of group A before and after conbercept treatment were compared. RESULT: At baseline, group A had a significantly worse BCVA, lower retinal vascular density and perfusion, and numerically larger CMT and FAZ area comparing to the group B, and had a higher aqueous cytokine level (IL-8, VEGF, and ANGPTL-4) comparing to the group C (all ps < 0.05). After the injection of conbercept, group A presented a better BCVA (at initial diagnosis vs. after three conbercept injections: 1.16 ± 0.51 vs. 0.81 ± 0.30, logMAR, p < 0.05), higher retinal vascular density (11.56 ± 4.73 vs. 15.88 ± 2.31, mm-1 , p < 0.05) and perfusion (0.28 ± 0.12 vs. 0.39 ± 0.06, mm2 , p < 0.05), smaller CMT (504.92 ± 184.11 vs. 219.83 ± 46.63, mm2 , p < 0.05), as well as a lower levels of VEGF (before first injection vs. before third injection: 113.84 [70.81, 235.4] vs. 3.94 [3.56, 8.07], pg/ml, p < 0.05) and ANGPTL-4 (45,761 [7327.5, 81,402.5] vs. 25,015.5 [6690, 43,396], pg/ml, p < 0.05). However, the average FAZ area of group A expanded (at initial diagnosis vs. after three conbercept injections: 0.41 ± 0.14 vs. 0.62 ± 0.36, mm2 , p < 0.05). CONCLUSION: This study demonstrated that intraocular injection of conbercept could effectively improve macular microcirculation and increase retinal blood supply in the treatment of nonischemic BRVO-ME based on the combination of visual acuity, OCTA parameters, and aqueous humor cytokine assay results. However, further study with a larger sample size and longer observation period is still needed in the future.

The long noncoding RNA MALAT1/microRNA-598-3p axis regulates the proliferation and apoptosis of retinoblastoma cells through the PI3K/AKT pathway.

Purpose: This study was designed to dissect the role of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in retinoblastoma (RB) and its underlying mechanism. Methods: Gain- and loss-of-function experiments were adopted to explore the effects of MALAT1 and microRNA (miR)-598-3p on the biologic behaviors of RB cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of MALAT1 and miR-598-3p in Y79 and HXO-RB44 cells. The proliferation of RB cells was determined with the cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining. Flow cytometry was employed for the measurement of the apoptotic rate, western blotting for examination of the expression of apoptosis-related proteins (Bax and Bcl-2) and phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) pathway-related factors (PI3K, AKT, p-PI3K, and p-AKT), and the luciferase reporter assay for assessment of the interaction between MALAT1 and miR-598-3p. Results: High expression of MALAT1 and low expression of miR-598-3p were noticed in Y79 and HXO-RB44 cells. MALAT1 upregulation or miR-598-3p downregulation facilitated RB cell proliferation and inhibited cell apoptosis, as evidenced by the increased proliferation rate and Bcl-2 expression, as well as diminished Bax expression and apoptotic rate, in the RB cells after transfection with pcDNA3.1-MALAT1 or miR-598-3p inhibitor. MALAT1 bound to and negatively regulated miR-598-3p. The PI3K/AKT pathway activation occurred with MALAT1 overexpression. MALAT1 promoted RB cell proliferation and repressed cell apoptosis by repressing miR-598-3p to activate the PI3K/AKT pathway. Conclusions: MALAT1 repressed miR-598-3p to activate the PI3K/AKT pathway, thus facilitating cell proliferation and inhibiting cell apoptosis in RB.

Idelalisib inhibits experimental proliferative vitroretinopathy.

Proliferative vitreoretinopathy (PVR) is a fibrotic eye disease that develops after rhegmatogenous retinal detachment surgery and open-globe traumatic injury. Idelalisib is a specific inhibitor of phosphoinositide 3-kinase (PI3K) δ. While PI3Kδ is primarily expressed in leukocytes, its expression is also considerably high in retinal pigment epithelial (RPE) cells, which play a crucial part in the PVR pathogenesis. Herein we show that GeoMx Digital Spatial Profiling uncovered strong expression of fibronectin in RPE cells within epiretinal membranes from patients with PVR, and that idelalisib (10 µM) inhibited Akt activation, fibronectin expression and collagen gel contraction induced by transforming growth factor (TGF)-ß2 in human RPE cells. Furthermore, we discovered that idelalisib at a vitreal concentration of 10 µM, a non-toxic dose to the retina, prevented experimental PVR induced by intravitreally injected RPE cells in rabbits assessed by experienced ophthalmologists using an indirect ophthalmoscope plus a + 30 D fundus lens, electroretinography, optical coherence tomography and histological analysis. These data suggested idelalisib could be harnessed for preventing patients from PVR.

MicroRNA-199a-3p inhibits angiogenesis by targeting the VEGF/PI3K/AKT signalling pathway in an in vitro model of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major inducer of blindness and visual impairment. As a critical cause for DR, hyperglycaemia is able to trigger multiple biochemical alterations. MiRNAs, which contain various functions, can effectively regulate blood glucose levels. This research aims to confirm the roles of miRNA-199a-3p in the progression of angiogenesis in an in vitro model of DR. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried to determine the expression levels of miR-199a-3p and VEGF in both hRMECs and APRE-19 cells. The luciferase reporter assay was used to study the interaction between miR-199a-3p and VEGF. Western blot assay was conducted to examine the expression levels of VEGF and the PI3K/AKT signalling pathway. The cell proliferation capacity was detected via the CCK-8 test. The impact of miR-199a-3p on migration was determined using Transwell and wound healing assays. A Matrigel tube formation assay was employed to determine the vascular formation of hRMECs. Flow cytometry was used to determine cell apoptosis in the presence of LY294002 as a PI3K inhibitor. RESULTS: Our results showed that high glucose (HG) decreased the relative expression level of miR-199a-3p but increased VEGF expression in hRMECs and APRE-19 cells. MiR-199a-3p inhibitor augmented cell growth, migration and angiogenesis of hRMECs. Moreover, upregulation of miR-199a-3p evidently alleviated the increases in cell proliferation, migration and angiogenesis caused by HG. In addition, the luciferase reporter assay indicated that miR-199a-3p directly targeted VEGF. The overexpression of miR-199a-3p obviously restrained the HG-stimulated PI3K/AKT signalling pathway and angiogenesis, which could be further inhibited by LY294002. Moreover, LY294002 could slightly ameliorate the miR-199a-3p inhibitor-stimulated PI3K/AKT signalling pathway and angiogenesis. CONCLUSION: MiR-199a-3p upregulation ameliorated HG-stimulated angiogenesis of hRMECs by modulating the PI3K/AKT pathway through inhibiting VEGF. Although retinal neovascularization in vivo has not been studied, these in vitro findings provide more evidence for the role of miR-199a-3p upregulation against HG-induced angiogenesis.

PDGFRß plays an essential role in patient vitreous-stimulated contraction of retinal pigment epithelial cells from epiretinal membranes.

Platelet-derived growth factor (PDGF) is associated with clinical proliferative vitreoretinopathy (PVR), which is characterized by formation of sub- or epi-retinal membranes that consist of cells including retinal pigment epithelial （RPE） cells and extracellular matrix. RPE cells play an important role in PVR pathogenesis. Previous findings indicated that PDGF receptor (PDGFR)α was essential in experimental PVR induced by fibroblasts. In RPE cells derived from epiretinal membranes from patients with PVR (RPEMs)， Akt was activated by PDGF-B but not PDGF-A, which suggested that PDGFRß was the predominant PDGFR isoform expressed in RPEMs. Indeed, CRISPR/Cas9-mediated depletion of PDGFRß in RPEMs attenuated patient vitreous-induced Akt activation and cellular responses intrinsic to PVR including cell proliferation, migration, and contraction. We conclude that PDGFRß appears to be the PVR relevant PDGFR isoform in RPEMs.

PI3Kδ as a Novel Therapeutic Target in Pathological Angiogenesis.

Diabetic retinopathy is the most common microvascular complication of diabetes, and in the advanced diabetic retinopathy appear vitreal fibrovascular membranes that consist of a variety of cells, including vascular endothelial cells (ECs). New therapeutic approaches for this diabetic complication are urgently needed. Here, we report that in cultured human retinal microvascular ECs, high glucose induced expression of p110δ, which was also expressed in ECs of fibrovascular membranes from patients with diabetes. This catalytic subunit of a receptor-regulated PI3K isoform δ is known to be highly enriched in leukocytes. Using genetic and pharmacological approaches, we show that p110δ activity in cultured ECs controls Akt activation, cell proliferation, migration, and tube formation induced by vascular endothelial growth factor, basic fibroblast growth factor, and epidermal growth factor. Using a mouse model of oxygen-induced retinopathy, p110δ inactivation was found to attenuate pathological retinal angiogenesis. p110δ inhibitors have been approved for use in human B-cell malignancies. Our data suggest that antagonizing p110δ constitutes a previously unappreciated therapeutic opportunity for diabetic retinopathy.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

Phosphoinositide 3-kinase δ inactivation prevents vitreous-induced activation of AKT/MDM2/p53 and migration of retinal pigment epithelial cells.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kß, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.

An approach to revolutionize cataract treatment by enhancing drug probing through intraocular cell line.

The purpose of this study is to prepare and characterize solid lipid nanoparticles (SLN) of N-Acetyl Carnosine (NAC) to treat cataract since surgery necessitates equipments and professional help. Cataract is believed to be formed by the biochemical approach where the crystalline eye proteins lose solubility and forms high molecular weight masses. Added advantages of SLN of NAC (henceforth referred as SLN-NAC) in the study are reduced size, sustained release and better corneal penetration of drug. The method of preparation of SLN-NAC by Mill's method is unique in itself. The size of the SLN-NAC was 75 ± 10 nm in the range of ideal for penetration. The in-vitro release study and the SLN-NAC formulations prepared with Mill's method demonstrated sustained release up to 24 h following an initial burst after 1 h. The zeta potential of the prepared formulation was -22.1 ± 1 mV. Corneal permeation studies using goat corneas indicate that SLN-NAC penetration rate was higher than those from NAC eye drops. Corneal hydration studies indicated that the formulation caused no harm to the corneal cells. Therefore it may be concluded that SLN-NAC may revolutionize cataract treatment and reversal by improving drug permeation, reducing toxicity and no damage to corneal tissue.

Genome editing abrogates angiogenesis in vivo.

Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2. We further show that recombinant AAV serotype 1 (rAAV1) transduces ECs of pathologic vessels, and that editing of genomic VEGFR2 locus using rAAV1-mediated CRISPR/Cas9 abrogates angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization. This work establishes a strong foundation for genome editing as a strategy to treat angiogenesis-associated diseases.Abnormal angiogenesis causes many ocular diseases. Here the authors employ CRISPR/Cas9 gene editing technology to silence VEGFR2, a major regulator of angiogenesis, in retinal endothelium and abrogate angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization.

Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation.

Purpose: Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation. Methods: Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined. Results: In the VEGFR2-sgRNA/SpCas9-transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9-transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab. Conclusions: The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.

Prevention of Proliferative Vitreoretinopathy by Suppression of Phosphatidylinositol 5-Phosphate 4-Kinases.

PURPOSE: Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -ß) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -ß and whether suppression of PI5P4Kα and -ß would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). METHODS: PI5P4Kα and -ß encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -ß by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -ß knocked-down ARPE-19 cells. Expression of PI5P4Kα and -ß was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. RESULTS: We found that vitreous enhanced expression of PI5P4Kα and -ß in RPE cells and that knocking down PI5P4Kα and -ß abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -ß in the human PI5P4Kα and -ß knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -ß abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -ß was abundant in epiretinal membranes from PVR grade C patients. CONCLUSIONS: The findings from this study indicate that PI5P4Kα and -ß could be novel therapeutic targets for the treatment of PVR.