Comparative ligandomics implicates secretogranin III as a disease-restricted angiogenic factor in laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. All approved anti-angiogenic drug therapies for CNV target vascular endothelial growth factor (VEGF) but confer limited efficacy. Identification of other CNV-related angiogenic factors will facilitate the development of VEGF-independent alternative therapies. Here, we applied comparative ligandomics to live mice with or without laser-induced CNV for global mapping of CNV-selective endothelial ligands. Secretogranin III (Scg3) previously identified by the same approach as a diabetes-restricted angiogenic factor was mapped with a more than 935-fold increase in binding to CNV vessels compared to healthy choriocapillaris. A novel in vivo ligand binding assay independently confirmed a marked increase in Scg3 binding to CNV vessels, whereas VEGF showed no increase in CNV-selective binding. A new technique of functional immunohistochemistry allowed the visualization and confirmed the increase in in vivo Scg3 binding to CNV vasculatures, including CNV microcapillaries with detailed vascular structures, which was blocked by anti-Scg3 humanized antibody Fab fragment (hFab). The hFab effectively alleviated laser-induced CNV with an efficacy similar to the anti-VEGF drug aflibercept. Homozygous deletion of the Scg3 gene in mice significantly reduced the severity of CNV. Furthermore, the therapeutic activity of anti-Scg3 hFab, but not aflibercept, was abolished in Scg3-/- mice, suggesting the Scg3-dependent nature of the hFab-mediated therapy. These findings suggest that Scg3 plays an important role in CNV pathogenesis and is a promising disease-restricted angiogenic factor for ligand-guided disease-targeted anti-angiogenic therapy of CNV.

Selectively targeting disease-restricted secretogranin III to alleviate choroidal neovascularization.

Choroidal neovascularization (CNV), a leading cause of blindness in the elderly, is routinely treated with vascular endothelial growth factor (VEGF) inhibitors that have limited efficacy and potentially adverse side effects. An unmet clinical need is to develop novel therapies against other angiogenic factors for alternative or combination treatment to improve efficacy and safety. We recently described secretogranin III (Scg3) as a disease-selective angiogenic factor, causally linked to diabetic retinopathy and acting independently of the VEGF pathway. An important question is whether such a disease-selective Scg3 pathway contributes to other states of pathological angiogenesis beyond diabetic retinopathy. By applying a novel in vivo endothelial ligand binding assay, we found that the binding of Scg3 to CNV vessels in live mice was markedly increased over background binding to healthy choriocapillaris and blocked by an Scg3-neutralizing antibody, whereas VEGF showed no such differential binding. Intravitreal injection of anti-Scg3 humanized antibody Fab (hFab) inhibited Matrigel-induced CNV with similar efficacy to the anti-VEGF drug aflibercept. Importantly, a combination of anti-Scg3 hFab and aflibercept synergistically alleviated CNV. Homozygous deletion of the Scg3 gene markedly reduced CNV severity and abolished the therapeutic activity of anti-Scg3 hFab, but not aflibercept, suggesting a role for Scg3 in VEGF-independent CNV pathogenesis and therapy. Our work demonstrates the stringent disease selectivity of Scg3 binding and positions anti-Scg3 hFab as a next-generation disease-targeted anti-angiogenic therapy for CNV.

Asiatic acid attenuates diabetic retinopathy through TLR4/MyD88/NF-κB p65 mediated modulation of microglia polarization.

AIM: This study aimed to evaluate the effects of Asiatic acid (AA), a naturally occurring compound of pentacyclic triterpenoid, on the pathological processes of diabetic retinopathy (DR). METHODS: SD rats were induced to develop early DR by intraperitoneal injection of STZ (60 mg/kg). Four weeks after injection, the diabetic rats were orally administrated with 37.5 mg/kg or 75 mg/kg AA every day for four weeks. The integrity of blood-retinal barrier (BRB) was measured by Evans blue staining. The polarization of microglia was determined by real-time PCR, western blot, and ELISA assays. The inner BRB (iBRB) or outer BRB (oBRB) breakdown was induced in human retinal endothelial cells or APRE19 cells through co-culture with high glucose and LPS-stimulated microglia BV2 cells. The damage to the iBRB and oBRB was measured using transendothelial/transepithelial electrical resistance (TEER/TER) and FITC-conjugated dextran cell permeability assays. KEY FINDINGS: Results demonstrated that AA alleviated BRB breakdown, as evidenced by decreased protein expression of occludin, claudin-5, and ZO-1. Furthermore, AA treatment suppressed inflammation and M1 polarization, while it increased M2 polarization in the retina of DR rats. In vitro, the iBRB or oBRB breakdown was alleviated by AA. LPS-induced M1-polarization of BV2 cells under high glucose condition was also repressed through AA administration. Finally, we demonstrated that AA weakened the TLR4/MyD88/NF-κB p65 signaling pathway both in vivo and in vitro. SIGNIFICANCE: AA ameliorated early DR by regulating microglia polarization via the TLR4/MyD88/NF-κB p65 pathway. These data indicate that AA is a potential candidate for DR treatment.

Obstructive Sleep Apnea is Related with the Risk of Retinal Vein Occlusion.

BACKGROUND: Retinal vein occlusion (RVO) was a vision-threatening retinal vascular disorder, however, the relationship between obstructive sleep apnea (OSA) and RVO risk remained unclear. METHODS: A total of 45 RVO cases and 45 controls between April 2018 and April 2020 were included. All the participants underwent full-night polysomnography (PSG) and thus detected the severity of OSA. Besides, the relationship between the apnea-hypopnea index (AHI) and oxidative and inflammatory biomarkers, including 8-hydroxy-2 deoxyguanosine (8-OHdG), C-reactive protein (CRP), interleukin 1 beta (IL1ß), interleukin 6 (IL6) and tumor necrosis factor alpha (TNFα) were detected. The incidences of macular edema (ME) and neovascular glaucoma (NVG) were detected in a three-months follow-up. RESULTS: In this case-control study, it was found that OSA incidence was increased in the RVO cases comparing with the cataract controls. Advanced analyses about the RVO subtypes demonstrated that incidence of OSA was higher in the central RVO (CRVO) cases comparing with branch RVO (BRVO) cases. Plasma samples from OSA cases demonstrated relatively higher concentrations of oxidative stress parameters and inflammatory biomarkers, including 8-OHdG, CRP, IL1ß, and IL6, in the RVO cases. Significant linear correlations between AHI and oxidative/inflammatory biomarkers were detected, and advanced analyses on the OSA subtypes demonstrated that these biomarkers were significantly higher in cases with later stages of OSA. In a three months follow-up, an impaired visual activity improvement rate and increased ME incidence in the OSA group among all the RVO cases were detected. CONCLUSION: OSA was related with an increased incidence of RVO. Besides, OSA would lead to increased oxidative and inflammatory biomarkers concentrations in the RVO cases. OSA could be used as a harmful prognostic factor of visual activity improvement and ME incidences. These findings highlighted the role of OSA in the development of RVO.

MiR-25-3p promotes malignant phenotypes of retinoblastoma by regulating PTEN/Akt pathway.

Aberrant expression of microRNAs plays an important role in the pathogenesis and progression of retinoblastoma. MiR-25, a member of the miR-106b˜25 cluster, has been reported to be abnormally expressed in retinoblastoma, but the exact role of it remains unclear. In our study, we found that miR-25-3p was upregulated in retinoblastoma tissues and cell lines. Enforced expression of miR-25-3p in retinoblastoma cell line WERI-RB-1 increased cell growth, colony formation, anchorage-independent growth, cell migration and invasion in vitro and tumor xenograft growth in vivo. In contrast, inhibited miR-25-3p expression in retinoblastoma cell line Y79 suppressed cell growth, colony formation, anchorage-independent growth, cell migration and invasion. Through luciferase reporter assay, we found that phosphatase and tensin homolog (PTEN) was a direct target of miR-25-3p. This was verified by western blot that miR-25-3p overexpression suppressed PTEN and activated Akt signaling. In addition, miR-25-3p was found to promote epithelial-mesenchymal transition (EMT) of WERI-RB-1 cells through PTEN/Akt pathway. Western blot analysis revealed that miR-25-3p overexpression increased Vimentin and Snail expression, and suppressed E-cadherin expression, but this could be reversed by restoring PTEN. Moreover, LY294002 treatment or restoring PTEN expression abolished the effects of miR-25-3p on cell invasion, colony formation and anchorage-independent growth in vitro and tumor xenograft growth in vivo. Taken together, our results suggested that miR-25-3p promotes malignant transformation of retinoblastoma cells by suppressing PTEN.

Long noncoding RNA HAGLROS regulates apoptosis and autophagy in Parkinson's disease via regulating miR-100/ATG10 axis and PI3K/Akt/mTOR pathway activation.

Parkinson's disease (PD) is a common age-related neurodegenerative disease resulted from the progressive degeneration of dopaminergic neurons in the pars compacta region of substantia nigra. The goal of this study was to investigate the effects and mechanisms of long noncoding RNA (lncRNA) HAGLROS on the apoptosis and autophagy in PD. The MPTP-induced PD mouse model and MPP+-intoxicated SH-SY5Y cell model were established, and the expression levels of HAGLROS and miR-100 were determined. Subsequently, the effects of suppression of HAGLROS on apoptosis and autophagy in MPTP-induced PD mouse model and in MPP+-intoxicated SH-SY5Y cells were investigated. In addition, the association between HAGLROS and miR-100 as well as HAGLROS and activation of phosphoinositide-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in MPP+-intoxicated SH-SY5Y cells was explored. HAGLROS was increasingly expressed in MPTP-induced PD mouse model and MPP+-intoxicated SH-SY5Y cells and suppression of HAGLRO decreased apoptosis and autophagy in both in vivo and in vitro PD models. Further in vitro studies showed that HAGLRO negatively regulated miR-100 expression, and HAGLROS regulated apoptosis and autophagy of MPP+-intoxicated SH-SY5Y cells through sponging miR-100. Moreover, ATG10 was identified as a target of miR-100. Besides, suppression of HAGLROS alleviated MPP+-intoxicated SH-SY5Y cell injury by activating PI3K/AKT/mTOR pathway. Our findings reveal that upregulation of HAGLROS may contribute to the development of PD via inhibiting apoptosis and autophagy, which may be achieved by regulating miR-100/ATG10 axis and PI3K/AKT/mTOR pathway activation.

The Neurovascular Protective Effect of S14G-Humanin in a Murine MCAO Model and Brain Endothelial Cells.

Endothelial dysfunction is fundamental to ischemic stroke and brain injury. The humanin analogue S14G-humanin (HNG) has been shown to be a cytoprotective derivative. In this study, we investigated the neuroprotective effects of HNG in vivo and in vitro. In a murine middle cerebral artery occlusion (MCAO) stroke model, HNG ameliorates cerebral infarction and suppresses the production of TNF-α, IL-1ß, IL-6 and MCP-1 cytokines. HNG inhibits the expression of vascular adhesion molecules such as VCAM-1 and ICAM-1 in the cortex tissue. In mouse brain endothelial cells bEnd.3, HNG protects cell survival under oxygen deprivation (OGD) conditions. HNG suppresses ROS production as well as that of the same panel of cytokines and vascular adhesion molecules induced by OGD. HNG also reduces the numbers of THP-1 cells attached to bEnd.3 by OGD. Mechanistically, we show that HNG exerts its effect via inhibition of the NF- κB pathway factor IKKα, activation of IκBα and accumulation of p65 in the nucleus. Our data conclude that S14G-humanin serves as a neuroprotective factor, especially in brain vascular disorders. © 2018 IUBMB Life, 70(7):691-699, 2018.

Let-7a inhibits migration of melanoma cells via down-regulation of HMGA2 expression.

This study aimed to investigate the effects of exosomes derived from BM-MSCs transduced with let-7a on B16f10 cells and BM-MSCs. BM-MSCs were transduced with let-7a and the exosomes of them were isolated for further culture of B16f10 cells and BM-MSCs. The migration of B16f10 cells were detected by transwell, proliferation of B16f10 cells and BM-MSCs was examined by MTT method, HMGA2 expression was measured by western blot. In addition, the let-7a secreted level in exosomes and IGF level were measured by RT-PCR and ELISA respectively. Our results showed that the level of let-7a in exosomes derived from Let-7a-transducted BM-MSCs was increased after treated by exosomes. HMGA2 in B16f10 cells was down-regulated and cell survival rate of BM-MSCs was decreased. However, neither cell survival rate of B16f10 cells nor IGF-1 secreted by B16f10 cells in different groups had significant differences. In conclusion, Let-7a contained in exosomes can inhibit the migration of Melanoma cells and inhibit the proliferation of BM-MSCs.

Detection of Delta-like 1 ligand for the diagnosis of tuberculous meningitis: An effective and rapid diagnostic method.

OBJECTIVE: To investigate the diagnostic value of Delta-like 1 ligand (DLL1) in cerebrospinal fluid (CSF) and serum, in tuberculous meningitis (TBM). METHODS: Patients with a definite diagnosis of central nervous system infection (TBM, viral meningitis/encephalitis or bacterial meningitis) were prospectively enrolled alongside patients with intracranial metastatic tumour and patients with no diagnosis (who served as controls). DLL1 content in CSF and serum was measured quantitatively by enzyme-linked immunosorbent assay; analyses were blinded. RESULTS: A total of 173 patients were enrolled: 62 with TBM; 38 with viral meningitis/encephalitis; 26 with bacterial meningitis; 17 with intracranial metastatic tumour; 30 with no diagnosis. CSF DLL1 content was highest for TBM; there were no differences in CSF DLL1 between the other groups. Serum DLL1 content was highest for the TBM and intracranial metastatic tumour groups, with significant differences between the TBM group and the viral meningitis/encephalitis, bacterial meningitis and nondiagnosed groups. There were no differences in serum DLL1 between the viral meningitis/encephalitis, bacterial meningitis and nondiagnosed groups, or between the TBM group and the tumour group. CONCLUSION: As a new biomarker, DLL1 may be of great clinical importance in the diagnosis of TBM.