Mouse Model Of Retinopathy Of Prematurity Research Articles

Transcription Factor EGR1 Facilitates Neovascularization in Mice with Retinopathy of Prematurity by Regulating the miR-182-5p/EFNA5 Axis.

Neovascularization is the hallmark of retinopathy of prematurity (ROP). Early growth response 1 (EGR1) has been reported as an angiogenic factor. This study was conducted to probe the regulatory mechanism of EGR1 in neovascularization in ROP model mice. The ROP mouse model was established, followed by determination of EGR1 expression and assessment of neovascularization [vascular endothelial growth factor-A (VEGF-A) and pigment epithelium-derived factor (PEDF)]. Retinal vascular endothelial cells were cultured and treated with hypoxia, followed by the tube formation assay. The state of oxygen induction was assessed by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay to determine hypoxia-inducible factor 1-alpha (HIF-1A). The levels of microRNA (miRNA)-182-5p and ephrin-A5 (EFNA5) in tissues and cells were determined by RT-qPCR. Chromatin immunoprecipitation and dual-luciferase assay were used to validate gene interaction. EGR1 and EFNA5 were upregulated in the retina of ROP mice while miR-182-5p was downregulated. EGR1 knockdown decreased VEGF-A and HIF-1A expression and increased PEDF expression in the retina of ROP mice. In vitro, EGR1 knockdown also reduced neovascularization. EGR1 binding to the miR-182-5p promoter inhibited miR-182-5p transcription and further promoted EFNA5 transcription. miR-182-5p downregulation or EFNA5 overexpression averted the inhibition of neovascularization caused by EGR1 downregulation. Overall, EGR1 bound to the miR-182-5p promoter to inhibit miR-182-5p transcription and further promoted EFNA5 transcription, thus promoting retinal neovascularization in ROP mice.

A single intravenous injection of cyclosporin A-loaded lipid nanocapsules prevents retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a retinal disease that threatens the vision of prematurely born infants. Severe visual impairment up to complete blindness is caused by neovascularization and inflammation, progressively destroying the immature retina. ROP primarily affects newborns in middle- and low-income countries with limited access to current standard treatments such as intraocular drug injections and laser- or cryotherapy. To overcome these limitations, we developed a nanotherapeutic that effectively prevents ROP development with one simple intravenous injection. Its lipid nanocapsules transport the antiangiogenic and anti-inflammatory cyclosporin A efficiently into disease-driving retinal pigment epithelium cells. In a mouse model of ROP, a single intravenous injection of the nanotherapeutic prevented ROP and led to normal retinal development by counteracting neovascularization and inflammation. This nanotherapeutic approach has the potential to bring about a change of paradigm in ROP therapy and prevent millions of preterm born infants from developing ROP.

Competing endogenous RNA network associated with oxygen-induced retinopathy: Expression of the network and identification of the MALAT1/miR-124–3p/EGR1 regulatory axis

Retinopathy of prematurity (ROP) is a severe retinal dysfunction in prematurely born babies. The relationship between non-coding RNAs and retinopathy of prematurity (ROP) remain unclear. Microarray analysis of lncRNAs, miRNAs, and mRNAs was conducted in a mouse model of ROP. A competing endogenous RNA (ceRNA) network was constructed. The relationship among MALAT1, miR-124–3p, and Early growth response protein 1 (EGR1) was assessed in hypoxia-induced primary human umbilical vein endothelial cells (HUVECs) and ROP mouse model. In the study, we found 2252 lncRNAs, 1239 mRNAs, and 36 miRNAs were differentially regulated. ceRNA network consisting of 21 lncRNAs, 10 miRNAs, and 19 mRNAs was established. Of the most down-regulated miRNAs, miR-124–3p was selected for additional study. miR-124–3p ceased the migration and proliferation of primary HUVECs in hypoxic conditions, and directly suppressed EGR1. Additionally, MALAT1 directly sponged miR-124–3p. Knockdown of MALAT1 decreased EGR1 expression and inhibited the migration and proliferation of primary HUVECs in hypoxia. Furthermore, these changes were rescued by depletion of miR-124–3p. In vivo, intravitreal injection of miR-124–3p, shMALAT1 decreased EGR1 expression and markedly suppressed retinal neovascularization in OIR models. Intravitreal injection of shMALAT1 and miR-124–3p antagomir at the same time can promote retinal neovascularization, which reversed the suppression of retinal neovascularization functioned by shMALAT1. In conclusion, the expression profiles of lncRNAs and miRNAs and the ceRNA network in a mouse model of ROP may be indicative of the underlying mechanisms of retinal angiogenesis and neural activity. The MALAT1/miR-124–3p/EGR1 regulatory axis is partly responsible for retinal neovascularization, which may provide a novel theoretical basis for the pathogenesis of ROP.

The systemic antiangiogenic effect of intravitreal aflibercept injection in a mouse model of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness and intravitreal anti-vascular endothelial growth factor (VEGF) injection is becoming a first-line choice for treatment of ROP. However, there is a major concern that intravitreally injected anti-VEGF agents could escape from the eye into the systemic circulation and impair systemic development. Moreover, escaped anti-VEGF agents could have an effect on the retina of the fellow eye. In this study, we investigated the hematogenous effect of a single intravitreal anti-VEGF injection in a mouse model of ROP. Here, we showed that single intravitreal aflibercept injection to one eye can affect body weight gain, the fellow eye, and renal vessels, although no apparent effect was observed in brain vessels. Furthermore, this hematogenous effect was dose-dependent. Our results provide very important insights into the clinical use of anti-VEGF agents for ROP treatment.

Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow-derived CD34+ cells and vascular wall-derived endothelial colony-forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability, we investigated the role of sEH in a mouse model of ROP. In WT mice, hyperoxia elicited tyrosine nitration and inhibition of sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP). Correspondingly, in a murine model of ROP, sEH-/- mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than did their WT littermates. Astrocytes were the cells most affected by sEH deletion, and hyperoxia increased astrocyte apoptosis. In rescue experiments, 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 and presenilin-1-associated protein to attenuate poly ADP-ribose polymerase activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss, but also reduced pathological vascular tuft formation in sEH-/- mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement for preventing retinopathy in preterm infants.

Oxygen-induced circRNA profiles and coregulatory networks in a retinopathy of prematurity mouse model.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. At present, the molecular mechanisms underlying ROP are still far from being clearly understood. Circular RNAs (circRNAs), a novel class of noncoding RNAs, have been reported to serve vital regulatory roles in several human diseases. However, it is still unclear how circRNAs are involved in ROP. In the present study, oxygen-induced retinopathy (OIR) murine retinal samples and paired normal tissues were chosen for high-throughput transcriptome RNA sequencing and bioinformatic analyses. As a result, a total of 236 differentially expressed circRNAs, 14 differentially expressed miRNAs, and 9,756 differentially expressed mRNAs were identified in the OIR samples. Gene ontology analysis showed that angiogenesis ranked in the top five upregulated biological processes associated with differential mRNA expression. Then, 66 co-expression pairs of circRNA-mRNA were predicted according to the mRNAs that were enriched in angiogenesis. Furthermore, coregulation prediction was separately performed to identify the differentially expressed miRNAs that targeted angiogenesis-associated circRNAs or mRNAs. Finally, nine differentially expressed circRNAs were predicted to be competing endogenous RNAs by constructing a circRNA-miRNA-mRNA network followed by reverse transcription-quantitative PCR validation. The results of the present study suggest that the identified set of circRNA transcripts and the potential regulatory mechanisms for the development of ROP are worthy of functional studies.

Different effects of various anti-angiogenic treatments in an experimental mouse model of retinopathy of prematurity.

Anti-vascular endothelial growth factor (VEGF) drugs are an option for the treatment of retinopathy of prematurity (ROP). Blocking of other angiogenic factors is also of interest. We therefore investigated in which effects would result blocking of placental growth factor (PlGF). C57BL/6 mice were exposed to 75% oxygen from P7 to P12. Intravitreal injections were performed at P12. Mice of control groups remained untouched after oxygen treatment, or phosphate buffered saline or neutral IgG molecules were injected. In the treatment groups, antibodies against VEGF or PlGF, a mixture of anti-VEGF and anti-PlGF, aflibercept or sunitinib were injected. On P17, electroretinographic (ERG) measurements were performed. Avascular zones and neovascularization were evaluated in retinal flat-mounts. Results are expressed as percent of total retinal area (median with median absolute deviation, MAD). Eyes of control groups showed similar neovascularization (1.4-3.3%, MAD 0.4-0.9%). Neovascularization was significantly less (0.5-0.7%, MAD 0.1-0.3%) in all treatment groups. Avascular zones in the retinas of control groups showed similar values (18.3-25.7%, MAD 4.8-8.8%). Avascular zones were significantly reduced down to 3.6 ± 1.3% after anti-VEGF injection, but they were not reduced significantly in the other treatment groups (13.3-22%, MAD 3.6-6.1%). ERG measurements did not reveal significant differences between the controls and the treatment groups. Blocking of PlGF or injection of sunitinib results in a similar inhibition of neovascularization as by anti-VEGF treatment in the mouse model of ROP. However, physiological angiogenesis that occurs after anti-VEGF treatment is blocked by anti-PlGF or sunitinib treatment, indicating that pathological neovascularization may follow different pathways than physiological angiogenesis.

AB028. Mesenchymal stem cells repair retinal vascular damage in retinopathy of prematurity mouse model

Background: Retinopathy of prematurity is a leading cause of visual impairment and blindness in infants. Exposure of premature babies to the hyperoxic extrauterine environment leads to vaso-obliteration (VO), followed by ischemia, and subsequently pathological intravitreal neovascularization (NV) in the immature retina. Current treatments target only aberrant intravitreal vessel growth without repopulating the avascular regions of the retina. Thus, there is a dire need of new therapies that arrest pathological NV and promote normal retinal revascularization. Mesenchymal stem cells (MSCs) have shown the ability to migrate to the damaged tissue in different animal models and enhance vascularization. We, therefore, investigated whether MSCs can promote vascular repair in a mouse model of ROP.

Lack of the Antioxidant Glutathione Peroxidase-1 (GPx1) Exacerbates Retinopathy of Prematurity in Mice

Glutathione peroxidase-1 (GPx1) is highly expressed during normal retinal maturation; however, its role in retinopathy of prematurity (ROP) is not fully understood. We postulated that GPx1 plays an important role in protecting the premature retina from oxidative injury in a mouse model of ROP. ROP was induced in wild-type (WT) and GPx1 knockout (KO) mice by exposing neonatal mice to 75% oxygen from postnatal days 7 to 11, followed by 1 week of room air. Structural effects of ROP were evaluated by retinal histology, and gene expression of retinal pro-angiogenic factors was measured by qRT-PCR. Retinas from ROP GPx1 KO mice had a significantly larger central avascular area compared to those from ROP WT mice (P < 0.001), indicative of a more severe vaso-obliteration. In ROP GPx1 KO mice, retinas also displayed increased preretinal neovascularization (P = 0.05) with a concurrent increase in the expression of vascular endothelial growth factor (P < 0.05) compared to values in ROP WT mice. Elevated oxidative stress was observed in ROP GPx1 KO retinas as evidenced by increased nitrotyrosine immunolabeling (P < 0.01) and superoxide (P < 0.05) in vessels compared to ROP WT retinas. In contrast to these findings of exacerbated retinal vascular injury in GPx1 KO mice, Müller cell gliosis and microglial density were similar in ROP GPx1 KO and ROP WT mice. GPx1, an important antioxidant enzyme of the premature retina, afforded protection against oxidative stress and oxidative injury in ROP. Lack of GPx1 was associated with increased oxidative stress, an increase in retinal avascular area, upregulation of retinal VEGF, and increased neovascularization in a mouse model of ROP.

8. 670 NM red light: protection against retinopathy of prematurity and low developmental weight

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that can lead to blindness. Red light irradiation at 670 nm wavelength promotes cellular differentiation, proliferation and wound repair. 670 nm light is believed to stimulate mitochondrial function by increasing cytochrome oxidase efficiency and ATP production. In the retina, 670 nm light protects photoreceptors from the effects of a number of noxious stimuli including toxins, light-induced and oxygen-induced degeneration. Aims To determine whether treatment with 670 nm light would promote normal vessel development in a mouse model of ROP (oxygen induced retinopathy – OIR model) and whether it would affect organ development and growth. Method C57/Bl6j mice were treated as controls. The other groups were OIR mice (75% oxygen, p7–12 days; normoxia p12–17 days) and C57/Bl6j and OIR mice that were treated with 9 J/cm2 of 670 nm light daily from p7–17. At p17 animals were sacrificed and the retinal vasculature visualised by labelling with lectin. Analysis for the presence of neovascularisation and vaso-obliteration was performed using established protocols. Weight and length measurements were recorded daily, and at p17 their organs were harvested. All organs were examined macroscopically and microscopically. Results There was a significant reduction (p Conclusions Exposure to 670 nm red light may be a novel strategy to promote normal vessel development and protect against ROP. 670 nm treatment might also prove useful in assisting pre-term infants increase body weight and reduce lung damage.

Anti-Angiogenic Effect of Luteolin on Retinal Neovascularization via Blockade of Reactive Oxygen Species Production

Oxidative stress-induced vascular endothelial growth factor (VEGF) is thought to play a critical role in the pathogenesis of retinopathy of prematurity (ROP). This study was performed to investigate the anti-angiogenic effect of luteolin against reactive oxygen species (ROS)-induced retinal neovascularization. The toxicity of luteolin was evaluated through modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human retinal microvascular endothelial cells (HRMECs) as well as TUNEL staining in the retina of C57BL/6J mice. After intravitreal injection of luteolin in the mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting. Anti-angiogenic activity of luteolin was evaluated by VEGF-induced migration and tube formation assay. The effect of luteolin on tertiary-butylhydroperoxide (t-BH)-induced ROS production was measured with 2'7'-dichlorofluorescein diacetate. The effect of luteolin on t-BH-induced and hypoxia-induced VEGF transcription and expression were evaluated by RT-PCR and Western blot, respectively. Luteolin never affected the viability of HRMECs up to 10 μM, where luteolin never induced any structural change in all retinal layers. Luteolin inhibited retinal neovascularization in the mouse model of ROP. Moreover, VEGF-induced migration and tube formation were significantly decreased by cotreatment of luteolin. Luteolin attenuated VEGF transcription via blockade of t-BH-induced ROS production. Luteolin suppressed hypoxia-induced VEGF expression via attenuating hypoxia inducible factor 1 α expression. Our results suggest that luteolin could be a potent anti-angiogenic agent for retinal neovascularization, which is related to anti-oxidative activity to block ROS production and to subsequently suppress VEGF expression and the pro-angiogenic effect of VEGF.

Structural consequences after intravitreal bevacizumab injection without increasing apoptotic cell death in a retinopathy of prematurity mouse model

To evaluate the effect of different bevacizumab concentrations on retinal endothelial cell proliferation, retinal structures and apoptotic activity after intravitreal injection in a retinopathy of prematurity (ROP) mouse model. A total of 35 of C57BL/J6 mice were exposed to 75±2% oxygen from postnatal day 7 to postnatal day 12. On day 12, 10 mice (group C) were injected with 2.5 μg intravitreal bevacizumab (IVB), 11 mice (group D) were injected with 1.25 μg IVB, and 14 mice (group E) were injected with 0.625 μg IVB in one eye. The contralateral eyes were injected with isotonic saline (control group=group B). Four nonexposed mice served as negative controls (group A). Neovascularization was quantified by counting the endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina. Histological and ultrastructural changes were examined by light and electron microscopy. Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labelling (TUNEL) was used to detect apoptosis. The endothelial cell count per histological section was lower in groups C (p<0.0001), D (p<0.0001) and E (p<0.0001) compared with the control group B. Histological evaluation showed no retinal toxicity in any group. Electron microscopy revealed hyperoxia-induced mitochondrial dysmorphology in group B. Mitochondrial dysmorphology displayed dose-dependent gradual increase in IVB-injected eyes. Intravitreal bevacizumab induced no significant increase in apoptotic cell death. Bevacizumab suppresses endothelial cell proliferation in a ROP mouse model. In addition to hyperoxia-induced mitochondrial dysmorphology of C57BL/J6 retina, morphological findings implicate further mitochondrial vulnerability because of bevacizumab without increase in apoptotic cell death.

Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: An in-vivo study in rats and mice

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin® (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases.

Protective Role of Aldose Reductase Deletion in an Animal Model of Oxygen-Induced Retinopathy

Retinopathy of prematurity (ROP) is a common disease occurred in premature babies. Both vascular abnormality and neural dysfunction of the retina were reported, and oxidative stress was involved. Previously, it has been showed that deficiency of aldose reductase (AR), the rate-limiting enzyme in polyol pathway, lowered oxidative stress. Here, the effect of AR deletion on neonatal retinal injury was investigated by using a mouse model of ROP (oxygen-induced retinopathy, OIR). Seven-day-old pups were exposed to 75% oxygen for 5 days and then returned to room air. The vascular changes and neuronal/glial responses were examined and compared between wild-type and AR-deficient OIR mice. Significantly reduced vaso-obliterated area, blood vessel leakage, and early revascularization were observed in AR-deficient OIR mice. Moreover, reduced amacrine cells and less distorted strata were observed in AR-deficient OIR mice. Less astrocytic immunoreactivity and reduced Müller cell gliosis were also observed in AR-deficient mice. After OIR, nitrotyrosine immunoreactivity and poly (ADP-ribose) (PAR) translocation, which are two oxidative stress markers, were decreased in AR-deficient mice. Significant decrease in VEGF, pho-Erk1/2, pho-Akt, and pho-I?B expression was found in AR-deficient OIR retinae. Thus, these observations suggest that the deficiency of aldose reductase may protect the retina in the OIR model.

The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation

The pathological angiogenesis in the retina is the major cause of vision loss at all ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness in children. This study investigated whether gold nanoparticle (GNP) could inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection of GNP significantly inhibited retinal neovascularization in the mouse model of ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis of retinal microvascular endothelial cells including proliferation, migration and capillary-like networks formation. GNP blocked VEGF-induced auto-phosphorylation of VEGFR-2 to inhibit consequently ERK 1/2 activation. GNP never affected on the cellular viability of retinal microvascular endothelial cells and induced no retinal toxicity. Our data suggest that GNP could be a potent inhibitor to retinal neovascularization without retinal toxicity. Furthermore, GNP could be extensively applied to variable vaso-proliferative retinopathies mediated by VEGF.

Author Response: Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma

Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that binds to and inhibits the biological activity of human vascular endothelial growth factor (VEGF) in in vitro and in vivo assay systems. It contains human framework regions and the complementarity-determining regions of a murine antibody that binds to VEGF. When comparing the sequence of human VEGF protein with that of mouse VEGF protein, we found that 86% of the protein sequence in mouse VEGF164 is similar to that of human VEGF165.1 We believe that it is possible for bevacizumab to block human and mouse VEGF. A blocking antibody is defined as an antibody that does not have a reaction when combined with an antigen, but prevents other antibodies from combining with that antigen.2,3 We believe that bevacizumab fits this definition and explains why the neutralized VEGF is not detected by ELISA. The human or mouse VEGF immunoassay (Quantikine; R&D Systems, Minneapolis, MN) that we used in our experiment can detect human VEGF165 and VEGF121 or mouse VEGF164 and VEGF120, but does not detect VEGF189 and VEGF206. VEGF121 and VEGF165 are diffusible proteins that are secreted into the medium. VEGF189 and VEGF206 have high affinity for heparin and are mostly bound to heparin-containing proteoglycans in the extracellular matrix. Bevacizumab binds to and neutralizes all isoforms of VEGF-A and bioactive proteolytic fragments. Bock et al.4 performed three different experimental approaches using Western blot analysis, ELISA, and Biacore (surface plasmon resonance; GE Health Care, Piscataway, NJ) analysis to assess whether bevacizumab binds to murine VEGF-A. In the Western blot analyses, recombinant murine VEGF-A164, a 24-kDa protein consisting of 164 amino acid residues, and the 19.1-kDa homologous human VEGF165, which consists of 165 amino acid residues, were used. In ELISA and the Biacore analyses, the recombinant human and the homologous murine VEGF-A were used as well. Western blot analysis showed that bevacizumab bound murine and human VEGF-A. To confirm these results, Bock et al. performed two different experimental approaches for analysis of the binding of bevacizumab to murine VEGF-A. The first assay, an ELISA, analyzed the interaction of soluble bevacizumab with VEGF-A immobilized on a microtiter plate surface. In the second assay, the surface plasmon resonance technology (Biacore) was used for the analysis of interaction of soluble VEGF-A with bevacizumab immobilized on the sensor chip surface. Both assays allow a qualitative characterization of the interaction between bevacizumab and human or murine VEGF-A. In the ELISA binding assay, bevacizumab bound to both human and murine VEGF-A; however, a 1000-fold higher bevacizumab concentration was necessary to reach a similar level of binding to murine VEGF-A compared with that needed for binding to the human protein. There was no significant binding of isotype control IgG1k to murine VEGF-A at this concentration. During the Biacore surface plasmon resonance experiments, different concentrations of human and murine VEGF-A were injected, to analyze their binding to immobilized bevacizumab. A more than 10-fold higher concentration of murine VEGF-A than of human VEGF-A was necessary to reach similar resonance units. ELISA is more sensitive than Western blot analysis. It can detect a protein of at picogram concentrations; Western blot can only detect a protein at nanogram concentrations. Studies have also indicated that bevacizumab inhibits experimental choroidal and corneal neovascularization in a mouse model,5,6 endothelial cell proliferation in a retinopathy of prematurity mouse model,7 and tumor progression in a A/J Tsc2+/− C57BL/6 background mouse model of pancreatic tumor.8,9 When we initially used bevacizumab in a mouse model to treat mouse melanoma cells, we found that it slightly decreased the tumor size and number of metastases. We then performed bevacizumab experiments with human uveal melanoma cell lines. Our experiments showed that bevacizumab had much more effect on human melanoma than on mouse melanoma. We acknowledge point three of Sharma et al. and reply that we meant that 10 and 100 μg/mL of bevacizumab reduced VEGF levels by 8.51 and 15.99 pg/mL, respectively. Bevacizumab acts as a tumor suppressor by inhibiting angiogenesis and lymphangiogenesis. Lymphangiogenesis not only plays an important role in mediating immune reactions, but also facilitates tumor metastasis10. In our study, we investigated only the antiangiogensis function of bevacizumab.

367 Genomic profiles of single tumour cells in metastatic breast cancer patients

Retinopathy of prematurity (ROP) is a severe retinal dysfunction in prematurely born babies. The relationship between non-coding RNAs and retinopathy of prematurity (ROP) remain unclear. Microarray analysis of lncRNAs, miRNAs, and mRNAs was conducted in a mouse model of ROP. A competing endogenous RNA (ceRNA) network was constructed. The relationship among MALAT1, miR-124–3p, and Early growth response protein 1 (EGR1) was assessed in hypoxia-induced primary human umbilical vein endothelial cells (HUVECs) and ROP mouse model. In the study, we found 2252 lncRNAs, 1239 mRNAs, and 36 miRNAs were differentially regulated. ceRNA network consisting of 21 lncRNAs, 10 miRNAs, and 19 mRNAs was established. Of the most down-regulated miRNAs, miR-124–3p was selected for additional study. miR-124–3p ceased the migration and proliferation of primary HUVECs in hypoxic conditions, and directly suppressed EGR1. Additionally, MALAT1 directly sponged miR-124–3p. Knockdown of MALAT1 decreased EGR1 expression and inhibited the migration and proliferation of primary HUVECs in hypoxia. Furthermore, these changes were rescued by depletion of miR-124–3p. In vivo, intravitreal injection of miR-124–3p, shMALAT1 decreased EGR1 expression and markedly suppressed retinal neovascularization in OIR models. Intravitreal injection of shMALAT1 and miR-124–3p antagomir at the same time can promote retinal neovascularization, which reversed the suppression of retinal neovascularization functioned by shMALAT1. In conclusion, the expression profiles of lncRNAs and miRNAs and the ceRNA network in a mouse model of ROP may be indicative of the underlying mechanisms of retinal angiogenesis and neural activity. The MALAT1/miR-124–3p/EGR1 regulatory axis is partly responsible for retinal neovascularization, which may provide a novel theoretical basis for the pathogenesis of ROP.

823 Pathway signatures in breast cancer progression − a genome-scale study based on integration of biology networks, DNA copy number, gene expression and mutations

Retinopathy of prematurity (ROP) is a severe retinal dysfunction in prematurely born babies. The relationship between non-coding RNAs and retinopathy of prematurity (ROP) remain unclear. Microarray analysis of lncRNAs, miRNAs, and mRNAs was conducted in a mouse model of ROP. A competing endogenous RNA (ceRNA) network was constructed. The relationship among MALAT1, miR-124–3p, and Early growth response protein 1 (EGR1) was assessed in hypoxia-induced primary human umbilical vein endothelial cells (HUVECs) and ROP mouse model. In the study, we found 2252 lncRNAs, 1239 mRNAs, and 36 miRNAs were differentially regulated. ceRNA network consisting of 21 lncRNAs, 10 miRNAs, and 19 mRNAs was established. Of the most down-regulated miRNAs, miR-124–3p was selected for additional study. miR-124–3p ceased the migration and proliferation of primary HUVECs in hypoxic conditions, and directly suppressed EGR1. Additionally, MALAT1 directly sponged miR-124–3p. Knockdown of MALAT1 decreased EGR1 expression and inhibited the migration and proliferation of primary HUVECs in hypoxia. Furthermore, these changes were rescued by depletion of miR-124–3p. In vivo, intravitreal injection of miR-124–3p, shMALAT1 decreased EGR1 expression and markedly suppressed retinal neovascularization in OIR models. Intravitreal injection of shMALAT1 and miR-124–3p antagomir at the same time can promote retinal neovascularization, which reversed the suppression of retinal neovascularization functioned by shMALAT1. In conclusion, the expression profiles of lncRNAs and miRNAs and the ceRNA network in a mouse model of ROP may be indicative of the underlying mechanisms of retinal angiogenesis and neural activity. The MALAT1/miR-124–3p/EGR1 regulatory axis is partly responsible for retinal neovascularization, which may provide a novel theoretical basis for the pathogenesis of ROP.

47 Molecular signatures of long non-coding RNAs in breast cancer patients

Retinopathy of prematurity (ROP) is a severe retinal dysfunction in prematurely born babies. The relationship between non-coding RNAs and retinopathy of prematurity (ROP) remain unclear. Microarray analysis of lncRNAs, miRNAs, and mRNAs was conducted in a mouse model of ROP. A competing endogenous RNA (ceRNA) network was constructed. The relationship among MALAT1, miR-124–3p, and Early growth response protein 1 (EGR1) was assessed in hypoxia-induced primary human umbilical vein endothelial cells (HUVECs) and ROP mouse model. In the study, we found 2252 lncRNAs, 1239 mRNAs, and 36 miRNAs were differentially regulated. ceRNA network consisting of 21 lncRNAs, 10 miRNAs, and 19 mRNAs was established. Of the most down-regulated miRNAs, miR-124–3p was selected for additional study. miR-124–3p ceased the migration and proliferation of primary HUVECs in hypoxic conditions, and directly suppressed EGR1. Additionally, MALAT1 directly sponged miR-124–3p. Knockdown of MALAT1 decreased EGR1 expression and inhibited the migration and proliferation of primary HUVECs in hypoxia. Furthermore, these changes were rescued by depletion of miR-124–3p. In vivo, intravitreal injection of miR-124–3p, shMALAT1 decreased EGR1 expression and markedly suppressed retinal neovascularization in OIR models. Intravitreal injection of shMALAT1 and miR-124–3p antagomir at the same time can promote retinal neovascularization, which reversed the suppression of retinal neovascularization functioned by shMALAT1. In conclusion, the expression profiles of lncRNAs and miRNAs and the ceRNA network in a mouse model of ROP may be indicative of the underlying mechanisms of retinal angiogenesis and neural activity. The MALAT1/miR-124–3p/EGR1 regulatory axis is partly responsible for retinal neovascularization, which may provide a novel theoretical basis for the pathogenesis of ROP.