Astrocytic expression of Yes-associated protein (YAP) regulates retinal neovascularization in a mouse model of oxygen-induced retinopathy.

Pathological neovascularization is the hallmark of many vascular oculopathies. There is still a great deal of uncertainty surrounding retinal neovascularization research. A working hypothesis that astrocytic Yes-associated protein (YAP) act as a key factor in retinal neovascularization was proposed. And our study was conducted to verified this hypothesis. In vivo, we successfully generated mice deficient in YAP in astrocytes (YAPf/f GFAP-Cre mice) and set up oxygen-induced retinopathy (OIR) model. Pathological neovascularization was evaluated by immunofluorescence staining and western blotting. In vitro, cultured retinal astrocytes were transfected with YAP siRNA. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to determine the proteins in the supernatants and cells. The results showed that YAP was upregulated and activated in the OIR mice retinas. Conditional ablation of YAP aggravated pathological neovascularization, along with the upregulation of vascular endothelial growth factor A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1). Studies in vitro confirmed that the knockdown of YAP in astrocytes lead to increases in VEGF-A and MCP-1 levels, thus enhancing pro-angiogenic capability of YAP-deficit astrocytes. In conclusion, astrocytic YAP alleviates retinal pathological angiogenesis by inhibiting the over-activation of astrocytes, which suppresses excessive VEGF-A production and neuroinflammation.

Simultaneous interference of SP1 and HIF1α retarding the proliferation, migration, and invasion of human microvascular endothelial cells (HMEC-1) under hypoxia.

OBJECTIVE: To investigate the regulation of special protein 1 (SP1) and hypoxia-inducible factor-1α (HIF1α) on human microvascular endothelial cells (HMEC-1) under hypoxic conditions. METHODS: The expression of SP1 and HIF1α under normoxia and hypoxic conditions were assessed by Western blot. SP1 and HIF1α were knocked down by small interfering RNA (siRNA) under hypoxic conditions. The proliferation, migration, and invasion of HMEC-1 were measured by cell counting kit 8, 5-ethynyl-2'-deoxyuridine and Transwell coculture system. Western blot analysis and Immunofluorescence were carried out to study the mechanisms of simultaneously inhibiting the adenosine triphosphatase (CD39), 5'-nucleotidase (CD73), adenosine, and vascular endothelial growth factor (VEGF). We compared the inhibitory effects between groups concurrently interfering SP1, HIF-1α, and ranibizumab under hypoxic conditions. RESULTS: Under hypoxic conditions, the protein expression of SP1 and HIF1α was increased in HMEC-1, contrarily, SP1 siRNA and HIF1α siRNA downregulated the expression. Simultaneous inhibition of SP1 and HIF1α demonstrated a much greater restraint of proliferation, migration, and invasion characteristics on HMEC-1 than respectively knocking down SP1 or HIF1α and anti-VEGF drugs (0.5 mg/mL ranibizumab) (siRNA and the VEGF inhibitor were applied separately in different groups). Meanwhile, simultaneous inhibition of SP1 and HIF1α effectively reduced the expression of CD39, CD73, adenosine, and VEGF on HMEC-1 under hypoxic conditions. CONCLUSIONS: Our study demonstrated that both SP1 and HIF1α played important roles in HMEC-1 under hypoxia condition. Simultaneous inhibition of SP1 and HIF1α effectively decreased the activity of HMEC-1 under hypoxic conditions through the CD39-CD73-adenosine and VEGF angiogenesis pathways. Our study may provide a new approach to the treatment of retinal neovascular diseases.

Clinicohistopathological implications of MMP/VEGF expression in retinoblastoma: a combined meta-analysis and bioinformatics analysis.

BACKGROUND: No in-depth systematic evidence is available for assessing retinoblastoma malignancy and eligibility for subsequent treatment. METHODS: The Cochrane Library, EMBASE, PubMed, Web of Science, and China Biology Medicine databases were searched, and 16 studies comprising 718 retinoblastoma patients were included. Pooled odds ratios (ORs) and summary correlation coefficients (r) with 95% confidence intervals (CIs) in random-effects, fixed-effects or quality-effects models were calculated using Review Manager 5.3 and MetaXL. GO functional annotation and KEGG pathway analysis were performed using the GO and STRING databases. RESULTS: We observed significant associations between high levels of MMP-1 (OR, 4.21; 95% CI 1.86-9.54), MMP-2 (OR, 11.18; 95% CI 4.26-29.30), MMP-9 (OR, 10.41, 95% CI 4.26-25.47), and VEGF (OR, 8.09; 95% CI 4.03-16.20) with tumor invasion; high levels of MMP-1 (OR, 3.58; 95% CI 1.48-8.71), MMP-2 (OR, 2.96; 95% CI 1.32-6.64), MMP-9 (OR, 5.49; 95% CI 3.55-8.48) and VEGF (OR, 5.30; 95% CI 2.93-9.60) with poor differentiation; and overexpression of MMP-9 (OR, 5.17; 95% CI 2.85-9.38) with advanced clinical stages. Moreover, MMP-9 and VEGF expression were positively correlated (r, 0.61; 95% CI 0.38-0.77). Multiple GO terms were enriched associated with MMP-1, MMP-2, MMP-9 and VEGF, and they are closely associated with pathways, proteoglycans and microRNAs related to cancer. CONCLUSIONS: MMP-1, MMP-2, MMP-9 and VEGF play important roles in the development and progression of retinoblastoma. High levels of MMP-1, MMP-2, MMP-9 and VEGF are credible implications for increased malignancy, thus the need for more aggressive treatments.

NLRP3 Deficiency Attenuates Secondary Degeneration of Visual Cortical Neurons Following Optic Nerve Injury.

In the visual pathway, optic nerve (ON) injury may cause secondary degeneration of neurons in distal regions, such as the visual cortex. However, the role of the neuroinflammatory response in regulating secondary impairment in the visual cortex after ON injury remains unclear. The NOD-like receptor family pyrin domain containing 3 (NLRP3) is an important regulator of neuroinflammation. In this study, we established a mouse model of unilateral ON crush (ONC) and showed that the expression of NLRP3 was significantly increased in the primary visual cortex (V1) as a response to ONC and that the NLRP3 inflammasome was activated in the contralateral V1 1 days-14 days after ONC. Ablation of the NLRP3 gene significantly decreased the trans-neuronal degeneration within 14 days. Visual electrophysiological function was improved in NLRP3-/- mice. Taken together, these findings suggest that NLRP3 is a potential therapeutic target for protecting visual cortical neurons against degeneration after ON injury.

Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

PURPOSE: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. METHODS: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. RESULTS: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. CONCLUSIONS: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.

Endothelial Yes-Associated Protein 1 Promotes Astrocyte Proliferation and Maturation via Cytoplasmic Leukemia Inhibitory Factor Secretion in Oxygen-Induced Retinopathy.

Purpose: Purpose The role of endothelial Yes-associated protein 1 (YAP) in the pathogenesis of retinal angiogenesis and the astrocyte network in the mouse oxygen-induced retinopathy (OIR) model is unknown. Methods: For in vivo studies, OIR was induced in conditional endothelial YAP knockout mice and their wild-type littermates. Retinal vascularization and the astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro experiments were performed in astrocytes cultured with human microvascular endothelial cell-1-conditioned medium to analyze the mechanisms underlying the effect of endothelial YAP on astrocytes.

Retinal blood vessel-origin yes-associated protein (YAP) governs astrocytic maturation via leukaemia inhibitory factor (LIF).

OBJECTIVES: To testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion. MATERIALS AND METHODS: In vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek-Cre) to finally obtain the following three genotypes: YAPf/f , Tek-Cre; YAPf/w , Tek-Cre; and YAPf/f . Retinal vascularization and astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC-1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes. RESULTS: In vivo, YAPf/f ;Tek-Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet-derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC-1-astrocyte coculture. The effect of YAP overexpression on LIF-LIFR axis in HMEC-1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs. CONCLUSIONS: We show that EC yes-associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production.

Exploration of the glutamate-mediated retinal excitotoxic damage: a rat model of retinal neurodegeneration.

AIM: To explore the more suitable concentration of glutamate or N-methyl-D-aspartic acid (NMDA) for intravitreal injection to establish a rat model of retinal neurodegeneration. METHODS: We injected different doses of glutamate (20 or 50 nmol) or NMDA (40 nmol) into the vitreous chambers of rats, then measured the concentration of glutamate and retinal thickness, quantified apoptotic cells and determined the degree of tau hyperphosphorylation at different time points. T-test was used for comparison of two groups. One-way ANOVA and Turkey's multiple comparisons test were used for comparisons of different groups, and P values below 0.05 were considered statistically significant. RESULTS: The glutamate level in the rats treated with 50 nmol of glutamate was twice that of the control group and persisted two weeks. Seven days after intravitreal injection of 50 nmol of glutamate, three parameters [inner retinal thickness (IRT), retinal thickness (RT) and ganglion cell layer (GCL) cell number] were reduced significantly. Furthermore, numerous TUNEL-positive cells were observed in the GCL one day after intravitreal injection of 50 nmol of glutamate, the expression of the apoptosis-related factor cleaved casepase-3 was markedly increased compared with the expression levels in the other treatment groups, and the expression levels of tau s396 and tau s404 were significantly increased compared with those in the control group. CONCLUSION: This study demonstrates that the intravitreal injection of 50 nmol of glutamate can establish the more effective retinal neurodegeneration animal model relative to other treatment groups.