Role In Retinal Neovascularization Research Articles

Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.

YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2

BackgroundOcular neovascularization is a leading cause of blindness. Retinal microglia have been implicated in hypoxia-induced angiogenesis and vasculopathy, but the underlying mechanisms are not entirely clear. Lactylation is a novel lactate-derived posttranslational modification that plays key roles in multiple cellular processes. Since hypoxia in ischemic retinopathy is a precipitating factor for retinal neovascularization, lactylation is very likely to be involved in this process. The present study aimed to explore the role of lactylation in retinal neovascularization and identify new therapeutic targets for retinal neovascular diseases.ResultsMicroglial depletion by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 suppresses retinal neovascularization in oxygen-induced retinopathy. Hypoxia increased lactylation in microglia and accelerates FGF2 expression, promoting retinal neovascularization. We identify 77 sites of 67 proteins with increased lactylation in the context of increased lactate under hypoxia. Our results show that the nonhistone protein Yin Yang-1 (YY1), a transcription factor, is lactylated at lysine 183 (K183), which is regulated by p300. Hyperlactylated YY1 directly enhances FGF2 transcription and promotes angiogenesis. YY1 mutation at K183 eliminates these effects. Overexpression of p300 increases YY1 lactylation and enhances angiogenesis in vitro and administration of the p300 inhibitor A485 greatly suppresses vascularization in vivo and in vitro.ConclusionsOur results suggest that YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression. Targeting the lactate/p300/YY1 lactylation/FGF2 axis may provide new therapeutic targets for proliferative retinopathies.

An Integrative Multi-Omics Analysis Reveals MicroRNA-143 as a Potential Therapeutic to Attenuate Retinal Angiogenesis.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.

N6-methyladenosine modifications of mRNAs and long noncoding RNAs in oxygen-induced retinopathy in mice

Retinal neovascular diseases are major causes of blindness worldwide. As a common epitranscriptomic modification of eukaryotic RNAs, N6-methyladenosine (m6A) is associated with the pathogenesis of many diseases, including angiogenesis, through the regulation of RNA metabolism and functions. The aim of this study was to identify m6A modifications of mRNAs and long noncoding RNAs (lncRNAs) and determine their potential roles in retinal neovascularization. The transcriptome-wide m6A profiles of mRNAs and lncRNAs in the retinal tissues of mice with oxygen-induced retinopathy (OIR) and controls were identified by microarray analysis of immunoprecipitated methylated RNAs. The m6A methylation levels of mRNAs and lncRNAs identified in the microarray data were validated by MeRIP-qPCR. A total of 1321 mRNAs (151 hypermethylated and 1170 hypomethylated) and 192 lncRNAs (15 hypermethylated and 177 hypomethylated) were differentially methylated with the m6A modification in OIR and control mice. Gene ontology analysis showed that hypermethylated mRNAs were enriched in the regulation of multicellular organismal process, intracellular organelle, and protein binding, while hypomethylated mRNAs were enriched in cellular metabolic process, intracellular process, and binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that hypermethylated mRNAs were involved in dopaminergic synapses, glutamatergic synapse, and PI3K-Akt signaling pathway, while hypomethylated mRNAs were involved in autophagy, ubiquitin-mediated proteolysis, and spliceosome. Moreover, the altered levels of m6A methylation of ANGPT2, GNG12, ROBO4, and ENSMUST00000153785 were validated by MeRIP-qPCR. The results revealed an altered m6A epitranscriptome in OIR retinas. These methylated RNAs may act as novel modulators and targets in retinal neovascularization.

VCAM1 regulates Fra1/JunB mediated IL‐8 expression and pathological retinal neovascularization in a murine model of oxygen‐induced retinopathy

PurposePathological retinal neovascularization is a clinical manifestation of various proliferative retinopathies, including retinopathy of prematurity (ROP), diabetic retinopathy, and the wet form of macular degeneration. Over the last decade, numerous studies have reported an increased infiltration of hematopoietic stem cells (HSCs), macrophage/microglia in patients with intraocular angiogenic diseases.MethodsUsing a murine model of oxygen‐induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we are trying to understand the significance of IL‐33/Hypoxic induced VCAM1 expression in retinal neovascularization.ResultsHere, using a murine model of oxygen‐induced retinopathy, we found that NFkB and VCAM1 levels were highly up regulated in retina when new vessels were developing in the retina. Genetic deletion of IL‐33 resulted in reduced NFkB activation and VCAM1 levels in hypoxic retina, resulting in reduced neovascularization. These findings suggest a role for IL‐33‐NFkB‐VCAM1 signaling in pathological neovascularization. Our results demonstrate that IL‐33 induce tube formation in human retinal microvascular endothelial cells and depletion of VCAM1 levels significantly blocked it. Protein profiler analysis showed that IL‐33 regulates VCAM1 dependent IL‐8 expression in HRMVECs. In order, to understand how IL‐33 induced VCAM1 regulates the expression of IL‐8, we did sequence analysis of IL‐8 promoter and observed that IL‐8 promoter has AP1 binding site. We observed that IL‐33 induced the expression of Fra1 and JunB significantly in HRMVECs. Cloning and site directed mutagenesis of IL‐8 promoter revealed that the AP1 binds to IL‐8 promoter in response to IL‐33 and regulate the expression of IL‐8 in HRMVECs. Consequently, down regulation of VCAM1 levels significantly reduced IL‐33 induced Fra1 and JunB expression as well as IL‐8 expression.ConclusionsThese results strongly suggest that IL‐33/ST2L‐NFkB‐VCAM1‐AP1 regulates IL‐8 expression, playing a key role in retinal neovascularization and thus represents a new pharmacological target for the treatment of diseases where excessive neovascularization is the underlying pathology.

Novel Role of Prereplication Complex Component Cell Division Cycle 6 in Retinal Neovascularization.

The major aim of this study is to investigate whether CDC6 (cell division cycle 6), a replication origin recognition complex component, plays a role in retinal neovascularization, and if so, to explore the underlying mechanisms. In this study, we used a variety of approaches including cellular and moleculer biological methodologies as well as global and tissue-specific knockout mice in combination with an oxygen-induced retinopathy model to study the role of CDC6 in retinal neovascularization. VEGFA (vascular endothelial growth factor A)-induced CDC6 expression in a time-dependent manner in human retinal microvascular endothelial cells. In addition, VEGFA-induced CDC6 expression was dependent on PLCβ3 (phospholipase Cβ3)-mediated NFATc1 (nuclear factor of activated T cells c1) activation. Furthermore, while siRNA-mediated depletion of PLCβ3, NFATc1, or CDC6 levels blunted VEGFA-induced human retinal microvascular endothelial cell angiogenic events such as proliferation, migration, sprouting, and tube formation, CDC6 overexpression rescued these effects in NFATc1-deficient mouse retinal microvascular endothelial cells. In accordance with these observations, global knockdown of PLCβ3 or endothelial cell-specific deletion of NFATc1 or siRNA-mediated depletion of CDC6 levels substantially inhibited oxygen-induced retinopathy-induced retinal sprouting and neovascularization. In addition, retroviral-mediated overexpression of CDC6 rescued oxygen-induced retinopathy-induced retinal neovascularization from inhibition in PLCβ3 knockout mice and in endothelial cell-specific NFATc1-deficient mice. The above observations clearly reveal that PLCβ3-mediated NFATc1 activation-dependent CDC6 expression plays a crucial role in VEGFA/oxygen-induced retinopathy-induced retinal neovascularization.

Annexin A2 Promotes Development of Retinal Neovascularization through PI3K/ AKT Signaling Pathway

Purpose Retinal Neovascularization (RNV) is a pathological characteristic of ocular diseases. Annexin A2 (ANXA2) plays important roles in RNV while the mechanism remains unclear. The study aimed to explore relationship between ANXA2 and PI3K/AKT signaling pathway in RNV. Methods We used human retinal vascular endothelial cells (HRECs) and oxygen-induced retinopathy (OIR) mice model to show ANXA2 can promote the development of RNV through PI3K/AKT signaling pathway. We divided HRECs into 6 groups by infecting lentivirus containing appropriate plasmid and adding corresponding solution. Assays showing ability of HRECs were performed in vitro. Mice were randomly divided into 3 groups and treated accordingly. Results Expression of ANXA2 and activity of PI3K/AKT signaling pathway in HRECs were detected. RNV and expression of ANXA2 in mice retinas were detected. Results showed that ANXA2 expression is positively related with RNV-forming ability of HRECs in vitro and development of RNV in vivo while low activity of PI3K/AKT signaling pathway could attenuate the role of ANXA2. Conclusions We can make ANXA2 and PI3K/ AKT signaling pathway as a promising target for the regulation of pathological neovascularization of the retina, which also provides a novel idea for effective prevention and treatment of diseases related to RNV in future.

IL‐33/ST2L mediated ADAMTS10 activation regulate retinal endothelial cell sprouting and neovascularization in a murine model of hypoxia‐induced retinopathy

Pathological retinal neovascularization is a clinical manifestation of various proliferative retinopathies, including retinopathy of prematurity (ROP), diabetic retinopathy, and the wet form of macular degeneration. A characteristic feature of these conditions is the presence of hypoxic areas and over‐expression of the proangiogenic vascular endothelial growth factor (VEGF). Treatments of these neovascular diseases using laser or anti‐VEGF therapies not only induce various local and systemic side effects but also are only partially effective. Here, using a murine model of oxygen‐induced retinopathy, we found that either intravitreal injections of IL‐33 siRNA or genetic deletion of IL‐33 resulted in reduced neovascularization with no significant changes in the avascular area. These findings suggest a role for IL‐33 in pathological neovascularization with little or no effect on the normal retinal repair. Interestingly, we observed that along with IL‐33 activation, hypoxia also induces expression of ST2 transmembrane receptor (ST2L), both at mRNA and protein levels, with little or no effect on secretory ST2 (sST2) levels. Our in vitro experiments using human retinal microvascular endothelial cells as a model suggest the importance of IL‐33/ST2L‐signaling on endothelial cell migration, tube formation, and sprouting. Besides it, our in vitro and in vivo findings provide evidence that IL‐33/ST2L‐signaling via ADAMTS10 activation facilitates sprout formation and invasion of connective tissue through controlled degradation of extracellular matrix proteins (ECMs). These results strongly suggest that IL‐33/ST2L‐signaling plays a key role in retinal neovascularization and thus represents a new pharmacological target for the treatment of diseases where excessive neovascularization is the underlying pathology.Support or Funding InformationThe research is supported by NIH EY029709 grant..

Neutralization of Bombina variegata peptide 8 suppresses retinal neovascularization in two different murine models: The oxygen-induced retinopathy model and the rhodopsin promoter/VEGF transgenic mouse model

Bombina variegata 8 (Bv8), also known as prokineticin-2 (PK-2), is a potent pro-angiogenic factor. However, its role in retinal neovascularization (RNV) remains unknown. In this study, we explored the role of Bv8 in the pathogenesis of RNV. We found that the expression of Bv8 was significantly increased in two different models of retinal neovascularization: the oxygen-induced retinopathy (OIR) mouse model and the rhodopsin promoter (rho)/VEGF transgenic mouse model. Neutralization of Bv8 by intravitreal injections of its antibody, not only inhibited retinal and subretinal neovascularization but also decreased the mRNA and protein levels of several pro-angiogenic factors. Our in vitro assay showed that recombinant human Bv8 (RhBv8) protein promoted human retinal microvascular endothelial cells (HRECs) tube-formation, cell proliferation and vascular endothelial growth factor receptor 1 (VEGFR1) and receptor 2 (VEGFR2) expression. Our findings suggest that Bv8 could be used as a novel target for the treatment of RNV-related ocular diseases.

Serum and vitreous resistin levels in patıents with proliferative diabetic retinopathy

AimThe aim of the study was to investigate the serum and vitreous levels of resistin in patients with the proliferative diabetic retinopathy (PDR) and to compare those with age-matched control subjects. MethodsThe study included 45 eyes with PDR (group 1) and a control group of 22 (group 2). All eyes underwent vitrectomy surgery. The lipid profile, fasting blood glucose (FBG), HbA1c and resistin levels were investigated in blood samples of all subjects. Complete ophthalmological examinations were evaluated. Vitreous samples were collected from both groups during vitrectomy surgery and resistin levels were investigated in those samples. The results were evaluated using SPSS 9.0 software. ResultsThe demographic characteristics of the diabetic group and the control group were similar (p > 0.05). There was no significant difference between the groups in respect of mean visual acuity (VA), body mass index (BMI) values, or lipid profiles (p ˃ 0.05). There was no measurable value of resistin in the vitreous samples of all the eyes. The mean blood resistin level was 367 ng/ml in the control group and 387 ng/ml in the study group and the difference was not statistically significant (p > 0.05). ConclusionsIn the light of the findings of this study, it can be assumed that resistin did not pass through the vitreous at measurable levels. However, the serum resistin levels of the diabetic patients were higher than those of the control group although not statistically significant. Therefore, it can be considered that resistin does not play a major role in retinal neovascularization.

Blockade of TREM-1 prevents vitreoretinal neovascularization in mice with oxygen-induced retinopathy

In pathological retinal neovascularization (RNV) disorders, the retina is infiltrated by activated leukocytes and macrophages. Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, activates monocytes and macrophages and plays an important role in cancer, autoimmune and other inflammation-associated disorders. Hypoxia-inducible TREM-1 is involved in cancer angiogenesis but its role in RNV remains unclear. Here, to close this gap, we evaluated the role of TREM-1 in RNV using a mouse model of oxygen-induced retinopathy (OIR). We found that hypoxia induced overexpression of TREM-1 in the OIR retinas compared to that of the room air group. TREM-1 was observed specifically in areas of pathological RNV, largely colocalizing with macrophage colony-stimulating factor (M-CSF) and CD45- and Iba-1-positive cells. TREM-1 blockade using systemically administered first-in-class ligand-independent TREM-1 inhibitory peptides rationally designed using the signaling chain homooligomerization (SCHOOL) strategy significantly (up to 95%) reduced vitreoretinal neovascularization. The peptides were well-tolerated when formulated into lipopeptide complexes for peptide half-life extension and targeted delivery. TREM-1 inhibition substantially downregulated retinal protein levels of TREM-1 and M-CSF suggesting that TREM-1-dependent suppression of pathological angiogenesis involves M-CSF. Targeting TREM-1 using TREM-1-specific SCHOOL peptide inhibitors represents a novel strategy to treat retinal diseases that are accompanied by neovascularization including retinopathy of prematurity.

Bevacizumab and Treatment Regimes in Diabetic Retinopathy and Diabetic Macular Edema

Diabetic macular edema and proliferative diabetic retinopathy is the most important cause of vision loss in diabetic patients. Vascular endothelial growth factor has been shown to play a major role in retinal neovascularization and vascular hyperpermeability in eyes with diabetic patients. Recent studies have demonstrated the usefulness of an intravitreal injection of bevacizumab in the reduction of diabetic macular edema, vascular permeability, and fibrovascular proliferation in retinal neovascularization secondary to proliferative diabetic retinopathy. This review aimed to evaluate the intravitreal bevacizumab in diabetic retinopathy and diabetic macular edema.

Therapeutic Effect of Novel Single-Stranded RNAi Agent Targeting Periostin in Eyes with Retinal Neovascularization.

Retinal neovascularization (NV) due to retinal ischemia remains one of the principal causes of vision impairment in patients with ischemic retinal diseases. We recently reported that periostin (POSTN) may play a role in the development of preretinal fibrovascular membranes, but its role in retinal NV has not been determined. The purpose of this study was to examine the expression of POSTN in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of POSTN on retinal NV using Postn KO mice and human retinal endothelial cells (HRECs) in culture. In addition, we used a novel RNAi agent, NK0144, which targets POSTN to determine its effect on the development of retinal NV. Our results showed that the expression of POSTN was increased in the vascular endothelial cells, pericytes, and M2 macrophages in ischemic retinas. POSTN promoted the ischemia-induced retinal NV by Akt phosphorylation through integrin αvβ3. NK0144 had a greater inhibitory effect than canonical double-stranded siRNA on preretinal pathological NV in vivo and in vitro. These findings suggest a causal relationship between POSTN and retinal NV, and indicate a potential therapeutic role of intravitreal injection of NK0144 for retinal neovascular diseases.

Silencing of S100A4, a metastasis-associated protein, inhibits retinal neovascularization via the downregulation of BDNF in oxygen-induced ischaemic retinopathy.

BackgroundTo investigate the underlying mechanism of S100A4 function and whether it has a role in retinal neovascularization (RNV) in a mouse model of oxygen-induced retinopathy (OIR).MethodsRetinas from a mouse model of OIR were treated with and without an intravitreous injection of adenoviral-S100A4-RNAi or adenoviral green fluorescence protein (GFP) at postnatal day 12 (P12). At P17, the efficacy of adenoviral gene transfer was assessed using fluorescence microscopy and western blot analysis. RNV was evaluated by whole-mount immunofluorescence staining of the mouse retina and by counting the number of pre-retinal neovascular cells. Protein and mRNA expression levels of S100A4, brain-derived growth factor (BDNF), and vascular endothelial growth factor (VEGF) were measured using western blot analysis and real-time PCR.ResultsRetinal S100A4 levels were positively correlated with the progression of RNV. In the OIR-S100A4-RNAi group, both protein and mRNA expression levels of S100A4 in the retina significantly decreased at P17 compared with those in the OIR group. Ad-S100A4-RNAi transfer was clearly demonstrated by GFP fluorescence in many layers of the retina 5 days after the Ad-S100A4-RNAi transfer. Whole-mount immunofluorescence staining of the retina and quantification of the pre-retinal neovascular cells demonstrated that RNV was significantly inhibited. Meanwhile, the levels of the transcription and translation of BDNF, VEGF, and hypoxia-inducible factor-1α (HIF-1α) significantly decreased in the OIR-S100A4-RNAi group.ConclusionsAd-S100A4-RNAi transfer ameliorates RNV. The related mechanism may involve silencing S100A4 to decrease the activation of BDNF, which downregulates VEGF expression via HIF-1α. This finding could provide a new therapeutic target for the treatment of ocular neovascularization diseases.

Effects of intravitreal injection of netrin-1 in retinal neovascularization of streptozotocin-induced diabetic rats.

BackgroundIn a previous study, we confirmed that netrin-1 acts as an antiangiogenic factor by inhibiting alkali burn-induced corneal neovascularization in rats. Here, we continue working on the role of netrin-1 in retinal neovascularization.MethodsUsing an in vitro angiogenesis assay, we detected the effects of netrin-1 on human umbilical vein endothelial cell tube formation, viability and proliferation, migration, and invasion at concentrations of 0.1 μg/mL or 5 μg/mL. We intravitreally injected 0.1 μg/mL or 5 μg/mL netrin-1 into streptozotocin-induced rats to assess retinal neovascularization using retinal electrophysiology and electroretinography, enzyme-linked immunosorbent assay, fundus fluoresce in angiography, measurement of inner blood retinal barrier, retinal hematoxylin-eosin staining, and retinal flat-mount fluorescence assays.ResultsHuman umbilical vein endothelial cell tube formation, viability and proliferation, migration, and invasion were upregulated by netrin-1 at a concentration of 0.1 μg/mL (P<0.05), while 5 μg/mL netrin-1 had an opposite effect (P<0.05) in our in vitro angiogenesis assay. Retinal electrophysiology testing revealed that intravitreal injection of netrin-1 affected the amplitude of a- and b-waves (a-wave: 0.1 μg/mL netrin-1 =17.67±3.39 μm, 5 μg/mL netrin-1 =28.50±1.31 μm, phosphate-buffered saline [PBS]-treated =17.67±3.39 μm; b-wave: 0.1 μg/mL netrin-1 =44.67±4.80 μm, 5 μg/mL netrin-1 =97.17±9.63 μm, PBS-treated =44.67±4.80 μm) and the expression of VEGF-A (no-treatment rats, 9.29±0.80 pg/mL; PBS-treated rats, 19.64±3.77 pg/mL; 0.1 μg/mL netrin-1 treated rats, 21.37±3.64 pg/mL; 5 μg/mL netrin-1 treated rats, 9.85±0.54 pg/mL, at 6 weeks after induction). By comparing fluoresce in angiography, level of inner blood retinal barrier breakdown (% of control), retinal hematoxylin-eosin staining, and collagen-IV fluorescence assays in the retinas of PBS-treated rats, netrin-1 was found to suppress and reverse retinal neovascularization at a concentration of 5 μg/mL (P<0.05), while 0.1 μg/mL netrin-1 (P<0.05) led to an increase in the number of new retinal blood vessels, after 6 weeks’ injection.ConclusionNetrin-1 could play a significant role in retinal neovascularization by dual-direction regulating angiogenesis dependent on dosage.

CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy.

Retinal neovascularization (RNV) is a characteristic pathological finding of retinopathy of prematurity (ROP). Cysteine-rich 61 [Cyr61, also known as CCN family member 1 (CCN1)] has been reported to mediate angiogenesis. The aim of the present study was to investigate the mechanisms of CCN1/Cyr61-phosphoinositide 3-kinase (PI3K)/AKT signaling in ROP. The contribution of CCN1 to human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis under hypoxic conditions was determined using a cell counting kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining, respectively, as well as using siRNA targeting CCN1 (CCN1 siRNA). The cells exposed to hypoxia were also treated with the PI3K/AKT inhibitor, LY294002. In addition, mouse pups with oxygen-induced retinopathy (OIR) were administered an intravitreal injection of CCN1 siRNA. RNV was assessed by magnesium-activated adenosine diphosphatease (ADPase) staining. RT-qPCR, western blot analysis, immunofluorescence staining and immunohistochemistry were used to detect the distribution and expression of CCN1, PI3K and AKT. Exposure to hypoxia increased the neovascularization clock hour scores (from 1.23±0.49 to 5.60±0.73, P<0.05) and the number of preretinal neovascular cells, as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (all P<0.05). The injection of CCN1 siRNA decreased the neovascularization clock hour scores and the number of preretinal neovascular cells (1.53±0.72 vs. 4.76±1.04; 12.0±2.8 vs. 31.4±2.6, respectively, both P<0.05), as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (protein, −45.3, −22.5 and −28.4%; mRNA, −43.7, −58.7 and −42.9%, respectively, all P<0.05) compared to the administration of scrambled siRNA under hypoxic conditions. Treatment with LY294002 decreased the mRNA and protein expression levels of CCN1 in the cells exposed to hypoxia (both P<0.05). The administration of CCN1 siRNA resulted in less severe neovascularization in the eyes of the the mouse pups with OIR. Thus, out data suggest that CCN1 plays an important role in RNV in ROP, and may thus be a potential target for the prevention and treatment of ROP.

In vivo analysis of the time and spatial activation pattern of microglia in the retina following laser-induced choroidal neovascularization

Microglia play a major role in retinal neovascularization and degeneration and are thus potential targets for therapeutic intervention. In vivo assessment of microglia behavior in disease models can provide important information to understand patho-mechanisms and develop therapeutic strategies. Although scanning laser ophthalmoscope (SLO) permits the monitoring of microglia in transgenic mice with microglia-specific GFP expression, there are fundamental limitations in reliable identification and quantification of activated cells. Therefore, we aimed to improve the SLO-based analysis of microglia using enhanced image processing with subsequent testing in laser-induced neovascularization (CNV).CNV was induced by argon laser in MacGreen mice. Microglia was visualized in vivo by SLO in the fundus auto-fluorescence (FAF) mode and verified ex vivo using retinal preparations.Three image processing algorithms based on different analysis of sequences of images were tested. The amount of recorded frames was limiting the effectiveness of the different algorithms. Best results from short recordings were obtained with a pixel averaging algorithm, further used to quantify spatial and temporal distribution of activated microglia in CNV. Morphologically, different microglia populations were detected in the inner and outer retinal layers. In CNV, the peak of microglia activation occurred in the inner layer at day 4 after laser, lacking an acute reaction. Besides, the spatial distribution of the activation changed by the time over the inner retina. No significant time and spatial changes were observed in the outer layer. An increase in laser power did not increase number of activated microglia.The SLO, in conjunction with enhanced image processing, is suitable for in vivo quantification of microglia activation. This surprisingly revealed that laser damage at the outer retina led to more reactive microglia in the inner retina, shedding light upon a new perspective to approach the immune response in the retina in vivo.

The mechanism of CCN1-enhanced retinal neovascularization in oxygen-induced retinopathy through PI3K/Akt-VEGF signaling pathway.

BackgroundCCN1 (also called Cyr 61) is an extracellular matrix signaling molecule that has been implicated in neovascularization through its interactions with several endothelial integrin receptors. The roles of vascular endothelial growth factor (VEGF) in angiogenesis are well described. The aim of this study was to investigate the signal transduction mechanism of CCN1–PI3K/Akt–VEGF in retinopathy of prematurity (ROP), and the effects of CCN1 knockdown on ROP.MethodsThe oxygen-induced retinopathy (OIR) model was established in C57BL/6J mice exposed to a high concentration of oxygen. Retinas were obtained from the normoxia, OIR, OIR control (treated with scramble siRNA) and OIR treated (with CCN1 siRNA) groups. Retinal neovascularization (RNV) was qualitatively analyzed with ADPase staining and quantitatively analyzed by counting neovascular endothelial cell nuclei at postnatal day 17 when RNV reached a peak. mRNA level and protein expression of CCN1, p-Akt, and VEGF were measured by real-time PCR and Western blotting, and located with immunohistochemistry.ResultsCCN1 depletion resulted in less neovascularization clock hour scores in the number of preretinal neovascular cells compared with the OIR treated group (1.28±0.83 versus 4.80±0.82; and 7.12±2.50 versus 23.25±2.35, respectively, both P<0.05). Furthermore, CCN1, p-Akt and VEGF mRNA, and protein were significantly expressed in the retina of the OIR and OIR control groups. Intravitreal injection of CCN1 siRNA significantly reduced PI3K/Akt–VEGF pathway expression of the OIR mouse model (all P<0.05). CCN1 siRNA significantly enhanced the avascular area and avascular diameter of OIR model (P<0.05). CCN1 siRNA decreased the levels of IL-1β, IL-6, and TNF-α significantly compared to the OIR group (P<0.05).ConclusionThese results suggest that CCN1 plays an important role in RNV via the PI3K/Akt–VEGF signaling pathway. CCN1 may be a potential target for the prevention and treatment of ROP.

Diacylglycerol kinase (DGK) inhibitor II (R59949) could suppress retinal neovascularization and protect retinal astrocytes in an oxygen-induced retinopathy model.

Hypoxia-inducible factors (HIF) play a fundamental role in retinal neovascularization (NV) induced by low oxygen tension. In the presence of oxygen, the HIF-α subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases (PHD), which triggers HIF-α for degradation. In our present study, we examined the effect of R59949, the diacylglycerol kinase (DGK) inhibitor II, on the retinal NV and its potential mechanism in an oxygen-induced retinopathy (OIR) model. OIR model was induced by exposure of hyperoxia (75 % oxygen) to C57BL/6J mice from postnatal day 7 (P7) to P12 and then returned to room air. By intraperitoneal injection once a day (10 μg/g/day) from P12 to P17, R59949 not only effectively prevented pathologic NV but also preserved the astrocyte morphology. Furthermore, the expression of PHD-2 was upregulated and HIF-1α and vascular endothelial growth factor (VEGF) were downregulated in the retina of OIR mice following R59949 treatment. These findings suggested a potential possibility that R59949 suppressed retinal neovascular pathophysiology via PHD2/HIF-1α/VEGF pathway.

A hypoxia-responsive glial cell-specific gene therapy vector for targeting retinal neovascularization.

Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model. Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS. The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature. These findings suggest that the hypoxia-regulated, glial cell-specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy.