Protective roles of the TIR/BB-loop mimetic AS-1 in alkali-induced corneal neovascularization by inhibiting ERK phosphorylation.

Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 µL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45+ immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45+ cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn.

Prostaglandin E2 promotes pathological retinal neovascularisation via EP4R-EGFR-Gab1-AKT signaling pathway.

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E2 (PGE2) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP4R). The aim of this study was to investigate the role of PGE2/EP4R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE2, cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE2 or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE2-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE2 or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE2-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE2/EP4R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.

Prostaglandin E2/EP2 receptor signalling pathway promotes diabetic retinopathy in a rat model of diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a common microvascular complication of diabetes mellitus and is initiated by inflammation and apoptosis-associated retinal endothelial cell damage. Prostaglandin E2 (PGE2) has emerged as a critical regulator of these biological processes. We hypothesised that modulating PGE2 and its E-prostanoid receptor (EP2R) would prevent diabetes mellitus-induced inflammation and microvascular dysfunction. METHODS: In a streptozotocin (STZ)-induced rat model of diabetes, rats received intravitreal injection of PGE2, butaprost (a PGE2/EP2R agonist) or AH6809 (an EP2R antagonist). Retinal histology, optical coherence tomography, ultrastructure of the retinal vascular and biochemical markers were assessed. RESULTS: Intravitreal injection of PGE2 and butaprost significantly accelerated retinal vascular leakage, leucostasis and endothelial cell apoptosis in STZ-induced diabetic rats. This response was ameliorated in diabetic rats pre-treated with AH6809. In addition, pre-treatment of human retinal microvascular endothelial cells with AH6809 attenuated PGE2- and butaprost-induced activation of caspase 1, activation of the complex containing nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment domain (ASC), and activation of the EP2R-coupled cAMP/protein kinase A/cAMP response element-binding protein signalling pathway. CONCLUSIONS/INTERPRETATION: The PGE2/EP2R signalling pathway is involved in STZ-induced diabetic retinopathy and could be considered as a potential target for diabetic retinopathy prevention and treatment.

Differentially expressed genes in orbital adipose/connective tissue of thyroid-associated orbitopathy.

Background: Thyroid-associated orbitopathy (TAO) is a disease associated with autoimmune thyroid disorders and it can lead to proptosis, diplopia, and vision-threatening compressive optic neuropathy. To comprehensively understand the molecular mechanisms underlying orbital adipogenesis in TAO, we characterize the intrinsic molecular properties of orbital adipose/connective tissue from patients with TAO and control individuals. Methods: RNA sequencing analysis (RNA-seq) was performed to measure the gene expression of orbital adipose/connective tissues of TAO patients. Differentially expressed genes (DEGs) were detected and analyzed through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA). The protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified by the Cytoscape plug-in, cytoHubba. We validated several top DEGs through quantitative real-time polymerase chain reaction (qRT-PCR). Results: We identified 183 DEGs in adipose tissue between TAO patients (n = 3) and control patients (n = 3) through RNA sequencing, including 114 upregulated genes and 69 downregulated genes. The PPI network of these DEGs had 202 nodes and 743 edges. PCR-based validation results of orbital adipose tissue showed multiple top-ranked genes in TAO patients (n = 4) are immune and inflammatory response genes compared with the control individual (n = 4). They include ceruloplasmin isoform x3 (CP), alkaline tissue-nonspecific isozyme isoform x1 (ALPL), and angiotensinogen (AGT), which were overrepresented by 2.27- to 6.40-fold. Meanwhile, protein mab-21-like 1 (MAB21L1), phosphoinositide 3-kinase gamma-subunit (PIK3C2G), and clavesin-2 (CLVS2) decreased by 2.6% to 32.8%. R-spondin 1 (RSPO1), which is related to oogonia differentiation and developmental angiogenesis, was significantly downregulated in the orbital muscle tissues of patients with TAO compared with the control groups (P = 0.024). Conclusions: Our results suggest that there are genetic differences in orbital adipose-connective tissues derived from TAO patients. The upregulation of the inflammatory response in orbital fat of TAO may be consistent with the clinical phenotype like eyelid edema, exophthalmos, and excess tearing. Downregulation of MAB21L1, PIK3C2G, and CLVS2 in TAO tissue demonstrates dysregulation of differentiation, oxidative stress, and developmental pathways.

Long Noncoding RNA-GAS5: A Novel Regulator of Hypertension-Induced Vascular Remodeling.

Vascular remodeling is an important pathological feature of hypertension, leading to increased vascular resistance and reduced compliance. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of EC and VSMC function. Herein, we determined whether long noncoding RNA-growth arrest-specific 5 (GAS5) is involved in hypertension-related vascular remodeling. We revealed that GAS5 knockdown aggravated hypertension-induced microvascular dysfunction as shown by increased retinal neovascularization and capillary leakage. GAS5 regulated the remodeling of arteries, including caudal arteries, carotid arteries, renal arteries, and thoracic arteries. GAS5 was mainly expressed in ECs and VSMCs, and its expression was significantly downregulated in hypertension. GAS5 knockdown affected endothelial activation, endothelial proliferation, VSMC phenotypic conversion, and EC-VSMC communication in vivo and in vitro. Mechanistically, GAS5 regulated EC and VSMC function through ß-catenin signaling. This study identified GAS5 as a critical regulator in hypertension and demonstrated the potential of gene therapy and drug development for treating hypertension.

Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling.

The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non-coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up-regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro MALAT1-CREB binding maintains CREB phosphorylation by inhibiting PP2A-mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling.