Oxygen-induced Retinopathy Mice Research Articles

MicroRNA-145 Regulates Pathological Retinal Angiogenesis by Suppression of TMOD3

Pathological angiogenesis is a hallmark of various vascular diseases, including vascular eye disorders. Dysregulation of microRNAs (miRNAs), a group of small regulatory RNAs, has been implicated in the regulation of ocular neovascularization. This study investigated the specific role of microRNA-145 (miR-145) in regulating vascular endothelial cell (EC) function and pathological ocular angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Expression of miR-145 was significantly upregulated in OIR mouse retinas compared with room air controls. Treatment with synthetic miR-145 inhibitors drastically decreased levels of pathological neovascularization in OIR, without substantially affecting normal developmental angiogenesis. In cultured human retinal ECs, treatment with miR-145 mimics significantly increased the EC angiogenic function, including proliferation, migration, and tubular formation, whereas miR-145 inhibitors attenuated in vitro angiogenesis. Tropomodulin3 (TMOD3), an actin-capping protein, is a direct miR-145 target and is downregulated in OIR retinas. Treatment with miR-145 mimic led to TMOD3 inhibition, altered actin cytoskeletal architecture, and elongation of ECs. Moreover, inhibition of TMOD3 promoted EC angiogenic function and pathological neovascularization in OIR and abolished the vascular effects of miR-145 inhibitors in vitro and in vivo. Overall, our findings indicate that miR-145 is a novel regulator of TMOD3-dependent cytoskeletal architecture and pathological angiogenesis and a potential target for development of treatments for neovascular eye disorders.

The vasoreparative potential of endothelial colony-forming cells in the ischemic retina is enhanced by cibinetide, a non-hematopoietic erythropoietin mimetic

PurposeRetinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina. MethodsThe effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation. ResultsARA290 activated pro-survival signaling and enhanced cell viability in response to H2O2-mediated oxidative stress in ECFCs in vitro. Preconditioning of ECFCs with EPO or ARA290 prior to delivery to the ischemic retina did not enhance vasoreparative function. ARA290 delivered systemically to OIR mice reduced pro-inflammatory expression of IL-1β and TNF-α in the mouse retina. Following intravitreal transplantation, ECFCs incorporated into the damaged retinal vasculature and significantly reduced avascular area. The vasoreparative function of ECFCs was enhanced in the presence of ARA290 but not EPO. DiscussionRegulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.

Attenuation of periostin in retinal Müller glia by TNF-α and IFN-γ.

To investigate the regulation and mechanisms of periostin expression in retinal Müller glia, and to explore the relevance to retinal neovascularization. The oxygen-induced retinopathy (OIR) mouse model and the human Moorfield/Institute of Ophthalmology-Müller 1 (MIO-M1) cell line were used in the study. Immunofluorescence staining was used to determine the distribution and expression of periostin and a Müller glial cell marker glutamine synthetase (GS). Cytokines TNF-α and IFN-γ were added to stimulate the MIO-M1 cells. ShRNA was used to knockdown periostin expression in MIO-M1 cells. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was conducted to assess the mRNA expression of periostin. Immunofluorescence staining showed that periostin was expressed by MIO-M1 Müller glia. GS-positive Müller glia and periostin increased in OIR retinas, and were partially overlaid. The stimulation of TNF-α and IFN-γ reduced the mRNA expression of periostin significantly and dose-dependently in MIO-M1 cells. Knockdown of periostin reduced mRNA expression of vascular endothelial growth factor A (VEGFA) in MIO-M1 cells, while VEGFA expression was not changed in periostin knock-out OIR retinas. Müller glia could be one of the main sources of periostin in the retina, and might contribute to the pathogenesis of retinal neovascularization. Proinflammatory cytokines TNF-α and IFN-γ attenuate the periostin expression in retinal Müller glia, which provides a potential and novel method in treating retinal neovascular diseases.

β‑elemene inhibits oxygen‑induced retinal neovascularization via promoting miR‑27a and reducing VEGF expression.

The present study aimed to investigate the significant role of β-elemene in mouse models of oxygen-induced retinopathy (OIR). C57BL/6J neonatal mice were used to establish OIR models. They were divided into four groups: Normoxia, OIR, OIR control and OIR-treated. Mice in the OIR group were exposed to 75±5% oxygen for 5 days and returned to a normal oxygen environment on postnatal day 12 (P12). The OIR treated group was intravitreally injected with 1 µl β-elemene on P12 and subsequently returned to a normal oxygen environment for 5 days (P12-P17). Retinas were obtained on P17. Retinal neovascularization (RNV) was detected using adenosine diphosphatase staining and analyzed by counting the nuclei of neovascular endothelial cells. Vascular endothelial growth factor (VEGF) expression was determined by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. MicroRNA (miRNA/miR) microarrays were used to screen out differentially expressed miRNAs between the OIR and β-elemene-treated groups. Binding the 3′-untranslated region (UTR) of VEGF and miR-27a was confirmed using luciferase assays. It was found that high oxygen concentrations accelerated RNV and increased the number of preretinal neovascular cells; β-elemene treatment reduced these effects. VEGF mRNA and protein expression was higher in the OIR and OIR control groups, compared with the normoxia and OIR-treated groups. Further, it was shown that miR-22, miR-181a-1, miR-335-5p, miR-669n, miR-190b, miR-27a and miR-93 were upregulated in the OIR-treated group, and downregulated in the OIR group. The prediction websites TargetScan and miRanda revealed that VEGF contained a potential miR-27a binding site in its 3′-untranslated region (UTR). Luciferase assays demonstrated that miR-27a directly bound to the 3′-UTR of VEGF. In vitro experiments demonstrated that miR-27a inhibited VEGF expression. In addition, β-elemene treatment upregulate miR-27a expression in vivo and in vitro. When miR-27a expression was depleted by miR-27a inhibitor, the protective effect of β-elemene on RNV was eliminated. The present study demonstrated that β-elemene reduced RNV in mouse OIR models via miR-27a upregulation, leading to reduced VEGF expression. This finding may contribute to the development of novel therapeutic strategies for human retinopathy.

Anti-secretogranin III therapy of oxygen-induced retinopathy with optimal safety.

Retinopathy of prematurity (ROP) with pathological retinal neovascularization is the most common cause of blindness in children. ROP is currently treated with laser therapy or cryotherapy, both of which may adversely affect the peripheral vision with limited efficacy. Owing to the susceptibility of the developing retina and vasculatures to pharmacological intervention, there is currently no approved drug therapy for ROP in preterm infants. Secretogranin III (Scg3) was recently discovered as a highly disease-restricted angiogenic factor, and a Scg3-neutralizing monoclonal antibody (mAb) was reported with high efficacy to alleviate oxygen-induced retinopathy (OIR) in mice, a surrogate model of ROP. Herein we independently investigated the efficacy of anti-Scg3 mAb in OIR mice and characterized its safety in neonatal mice. We developed a new Scg3-neutralizing mAb recognizing a distinct epitope and independently established the therapeutic activity of anti-Scg3 therapy to alleviate OIR-induced pathological retinal neovascularization in mice. Importantly, anti-Scg3 mAb showed no detectable adverse effects on electroretinography and developing retinal vasculature. Furthermore, systemic anti-Scg3 mAb induced no renal tubular injury or abnormality in kidney vessel development and body weight gain of neonatal mice. In contrast, anti-vascular endothelial growth factor drug aflibercept showed significant side effects in neonatal mice. These results suggest that anti-Scg3 mAb may have the safety and efficacy profiles required for ROP therapy.

Secretogranin III as a novel target for the therapy of choroidal neovascularization

Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV.

Effect of intravitreal and intraperitoneal cyanidin-3-glucoside injection in oxygen-induced retinopathy mouse model.

Purpose:To evaluate the effect of cyanidin-3-glucoside (C3G) in oxygen-induced retinopathy (OIR) mouse model.Methods:In this experimental study, 10 C57BL / 6J type mice exposed to room air comprised two control groups (n = 5 each; a negative control and a group receiving intravitreal sterile dimethyl sulfoxide [IVS DMSO]). Thirty C57BL / 6J type mice exposed to 75% ± 2% oxygen from postnatal day 7 to postnatal day 12 comprised the OIR groups. On postnatal day 12, these mice were randomized into six groups (n = 5 each): two OIR control groups (negative control and IVS DMSO), two intravitreal C3G groups (300 and 600 ng/μL), and two intraperitoneal C3G groups (0.05 and 0.1 mg/kg). We quantified neovascularization by counting endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina and examined histological and ultrastructural changes via light and electron microscopy and apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling.Results:The intravitreal C3G groups yielded lower endothelial cell counts compared with the intravitreal DMSO group. The intraperitoneal high-dose group had lower cell counts compared with the OIR control groups. Electron microscopy revealed significantly less mitochondrial dysmorphology in intravitreal groups and the high-dose intraperitoneal mice. We noted no difference in apoptotic cell count between the controls, low-dose intravitreal, and both intraperitoneal groups. However, apoptotic cell count was significantly higher in the high-dose intravitreal group.Conclusion:C3G suppresses endothelial cell proliferation in an OIR mouse model, leads to a reduced hyperoxia-induced mitochondrial dysmorphology, but increases apoptotic cell death in high concentrations.

Microarray Analysis of Long Non-Coding RNAs and Messenger RNAs in a Mouse Model of Oxygen-Induced Retinopathy.

Objective: Retinal neovascularization is a severe complication of many ocular diseases. To clarify the possible functions and therapeutic potential of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in retinal neovascularization, we assessed their expression profile in a mouse model of oxygen-induced retinopathy (OIR).Methods: Microarray analysis was performed to identify altered lncRNA and mRNA expressions between OIR and control mice. The microarray results were validated by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to determine biological functions and signaling pathways of the altered or interacted mRNAs. A coding-non-coding gene co-expression (CNC) network was constructed to identify the interaction of lncRNAs and mRNAs.Results: We identified 198 up-regulated and 175 down-regulated lncRNAs (fold change≥2.0, P<0.05), respectively in OIR mice compared to control mice. We also identified 412 up-regulated and 127 down-regulated mRNAs (fold change≥2.0, P<0.05), respectively in OIR mice compared to control mice. GO and KEGG analyses suggested that altered mRNAs were enriched in immune system process, exopeptidase activity, ECM-receptor interaction and protein digestion and absorption. Four validated lncRNAs (ENSMUST00000165968, ENSMUST00000153785, ENSMUST00000134409, and ENSMUST00000154285) and the nearby coding gene pairs were analyzed. A CNC network profile based on those validated altered lncRNAs as well as 410 interacted mRNAs was composed of 509 connections. Moreover, the GO and KEGG analyses demonstrated that these interacted mRNAs mainly enriched in blood vessel development, angiogenesis, cell adhesion molecules and leukocyte transendothelial migration pathways.Conclusion: Our data highlight the utility of altered lncRNA and mRNA profiling in understanding the pathogenesis of ischemia-induced retinal neovascularization and further suggest that therapeutic potential of altered lncRNA for retinal neovascularization.

Identifying circRNA-associated-ceRNA networks in retinal neovascularization in mice.

Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.

Oxidative stress, autophagy and pyroptosis in the neovascularization of oxygen‑induced retinopathy in mice.

Retinal neovascularization (RNV) is a principal cause of visual impairment and blindness worldwide. The present study aimed to investigate how oxidative stress, autophagy and pyroptosis alter in RNV. The oxygen-induced retinopathy (OIR) model was established in C57BL/6J mice by exposing them to a high concentration of oxygen. RNV was clearly visible in the fundus images and was qualitatively analyzed by counting the number of neovascular endothelial cell nuclei at postnatal day 17. Subsequently, the expression of vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α (HIF-1α) at the protein level were measured. Furthermore, oxidative stress was examined using dihydroethidium (DHE) staining, and NADPH oxidase (NOX) 1 and 4 in the retinas were detected using reverse transcription-quantitative polymerase chain reaction analysis. Additionally, immunostaining of microtubule associated protein 1 light chain 3α (LC3) was performed and the expression levels of the LC3, p62, autophagy protein (Atg)5, Atg7, Atg12, Beclin1, NOD-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, interleukin (IL)-1β, pro-caspase-1 and pro-IL-1β proteins were determined using western blotting in order to detect pyroptosis and autophagic flux. Autophagosomes were also detected using transmission electron microscopy. The results revealed that VEGF-A and HIF-1α protein expression levels, the DHE-positive area, and NOX1 and NOX4 mRNA expression levels were significantly increased in the OIR mice. Furthermore, increased levels of NLRP3, caspase-1, IL-1β, pro-caspase-1 and pro-IL-1β proteins demonstrated that pyroptosis was activated. However, an accumulation of p62 and a reduction in the levels of LC3II/I and autophagosomes indicated that autophagic flux was compromised. Therefore, elevated levels of reactive oxygen species and pyroptosis along with attenuated autophagy were demonstrated in the OIR mice. The combination of oxidative stress, pyroptosis and impaired autophagy may serve an important role in the pathophysiology of RNV and may be a potential target to prevent RNV.

The potential effect of human chorionic gonadotropin on vasoproliferative disorders of the immature retina.

Human chorionic gonadotropin (hCG) is known to be a powerful vascular endothelial growth factor (VEGF)-regulating hormone. It stimulates vascularization of the gravid uterus by upregulating VEGF expression. In the body, hCG activates the same receptor as luteinizing hormone (LH). Like hCG, LH is also strongly proangiogenic. Recently, it has been shown that LH/hCG receptors are present in the retina and that both LH and hCG are found in the eye. In fact, the human eye can synthesize its own hCG. We have previously shown that LH and VEGF are significantly correlated in mammalian eyes, potentially implicating LH-receptor/hCG-receptor activation in intraocular VEGF regulation. Given that elevated VEGF is associated with progression of two vasoproliferative pediatric retinal disorders, retinopathy of prematurity and retinoblastoma, our objective was to determine whether hCG may potentially affect VEGF production and pathologic retinal vascularization in vasoproliferative disorders affecting the immature retina. In this study, we used (a) oxygen-induced retinopathy mouse model (standard model for retinopathy of prematurity) and (b) Y79 retinoblastoma cells (a human cell line derived from immature retinal cells). In the oxygen-induced retinopathy model, number of preretinal nuclei (representing pathologic retinal neovascularization) significantly increases by 57% (P<0.05) in hCG-treated mice. In Y79 cells, VEGF production significantly increases by 37% (P<0.05) in hCG-treated cells. These findings suggest that hCG is potentially able to influence retinal vascularization and VEGF production and thus, the hCG receptor may potentially represent a therapeutic target for vasoproliferative retinal disorders affecting the young eye.

Role of TLR4-MAP4K4 signaling pathway in models of oxygen-induced retinopathy.

Retinopathy of prematurity is a vision-threatening condition, and therapies based on antagonizing VEGF may elicit serious side effects in premature infants. Mechanisms of retinal angiogenesis, particularly the signaling pathways independent of VEGF, remain elusive. The goals of our study were to explore TLR4-mediated signaling pathways in human retinal microvascular endothelial cells (HRMECs) and to examine the effects of TLR4 antagonists in models of oxygen-induced retinopathy (OIR). Our results show that intravitreal injection of the TLR4 antagonist TAK-242 reduced areas of nonperfusion, inhibited aberrant angiogenesis, and improved vascular density in the retina of OIR mice. The effects were further potentiated by the anti-VEGF antibody ranibizumab. In cultured HRMECs, the TLR4 agonist LPS up-regulated TLR4/MAPKK kinase kinase 4 (MAP4K4) signaling, and promoted cell proliferation and migration, and reduced barrier functions of the cells. Down-regulation of MAP4K4 in HRMECs abolished the proangiogenic effects by LPS. Our data suggest that the TLR4-MAP4K4 pathway can regulate retinal neovascularization via mechanisms independent of VEGF.-Chen, W., Zhang, J., Zhang, P., Hu, F., Jiang, T., Gu, J., Chang, Q. Role of TLR4-MAP4K4 signaling pathway in models of oxygen-induced retinopathy.

Overexpressing kringle 1 domain of hepatocyte growth factor with adeno-associated virus inhibits the pathological retinal neovascularization in an oxygen-induced retinopathy mouse model

Current clinical treatments for ocular neovascularization are characterized by high possibility of damaging healthy tissues and high recurrence rates. It is necessary to develop new treatment methods to control neovascularization with a stable and effective effect. Kringle1 domain of hepatocyte growth factor (HGFK1) has anti-angiogenesis activity. Here, we established oxygen-induced retinopathy (OIR) model to study if using adeno-associated virus (AAV) as a delivery system to overexpression HGFK1 in retinal cells could benefit retinal neovascularization. We show that, overexpressed exogenous gene was mainly expressed in the inner and outer nuclear layer of the retina. Compared with control mice, the mice pretreated with rAAV-HGFK1 at P3 showed relatively normal vascular branches examined by fluorescence fundus angiography. Subsequent H&E staining and immunohistochemical staining of CD31 of the eye tissue sections showed that the mice received rAAV-HGFK1 had a relatively normal distribution of vascular endothelial cells. Additionally, immunohistochemical staining indicated a lower expression of VEGF in the eye tissues of rAAV-HGFK1 treated OIR mice. Further in vitro studies showed that HGFK1 could inhibit the proliferation but promote the apoptosis of bovine retinal microvascular endothelial cells (BRECs) under the presence of VEGF. Moreover, HGFK1 could inhibit VEGF induced ERK activation but promote p38 activation in BRECs. Therefore, we propose that intravitreal injection of rAAV-HGFK1 might be used to improve the retinal neovascularization and HGFK1 may function through regulating VEGF signaling pathway to inhibit neovascularization.

SIRT1 Deletion Impairs Retinal Endothelial Cell Migration Through Downregulation of VEGF-A/VEGFR-2 and MMP14.

Silent information regulator protein 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase that is abundantly expressed in vascular endothelial cells (VECs), and it has an essential role in angiogenesis. However, its contribution to retinal vascular development remains unclear. Here we characterize its involvement in regulating this process under both physiological and pathologic conditions. Endothelium-specific Sirt1 knockout mice were established using the Cre-lox system. VECs were isolated using magnetic beads and identified by immunostaining. Retinal whole-mount staining analyzed the retinal vascular patterns. SIRT1 was knocked down or overexpressed in human retinal microvascular endothelial cells (HRMECs) using small interfering RNA (siRNA) or lentivirus infection, respectively. Scratch assay, Transwell, and Matrigel angiogenesis assay evaluated cell migration and tube formation, respectively. Quantitative RT-PCR analyzed genes regulating VEC migration. Western blotting determined protein expression. Coimmunoprecipitation detected the interaction of hypoxia-inducible factor 1α (HIF-1α) and SIRT1 as well as acetylation status of HIF-1α. Specific deletion of Sirt1 in VECs dramatically delayed retinal vessel expansion and reduced vessel density. In the oxygen-induced retinopathy (OIR) mouse model, Sirt1 ablation markedly suppressed retinal revascularization and consequently increased retinal avascularity. SIRT1 downregulation in HRMECs inhibited cell migration and tube formation, while overexpression of SIRT1 had the opposite effects. Vascular endothelial growth factor-A (VEGF-A)/VEGF receptor-2 (VEGFR-2), and matrix metalloproteinases 14 (MMP14) expression significantly declined in Sirt1-null VECs, as well as SIRT1 siRNA-transfected HRMECs. SIRT1 downregulation upregulated the HIF-1α acetylation status. Conversely, SIRT1 overexpression decreased this response. SIRT1 contributes to both physiological and pathologic retinal angiogenesis through promoting retinal VEC migration. Its underlying molecular mechanism involves SIRT1-mediated deacetylation of HIF-1α and subsequent upregulation of VEGF-A/VEGFR-2 and MMP14 expression.

Intravitreal Delivery of VEGF-A165-loaded PLGA Microparticles Reduces Retinal Vaso-Obliteration in an In Vivo Mouse Model of Retinopathy of Prematurity

ABSTRACTPurpose: Retinopathy of prematurity (ROP) is a condition of abnormal retinal vascularization with reduced levels of vascular endothelial growth factor (VEGF) causing vaso-obliteration (Phase I), followed by abnormal neovascularization from increased VEGF (Phase II). We hypothesized that intravitreal pro-angiogenic VEGF-A in microparticle form would promote earlier retinal revascularization in an oxygen-induced ischemic retinopathy (OIR) mouse model.Materials and Methods: Wildtype mice (39) were exposed to 77% oxygen from postnatal day 7 (P7) to P12. VEGF-A165-loaded poly(lactic-co-glycolic acid) (PLGA) (n = 15) or empty PLGA (n = 14) microparticles were fabricated using a water-in-oil-in-water double emulsion method, and injected intravitreally at P13 into mice right eyes (RE). Left eyes (LE) were untreated. At P20, after retinal fluorescein angiography, vascular parameters were quantified. Retinal VEGF levels at P13 and flatmounts at P20 were performed separately.Results: VEGF-A165-loaded microparticles had a mean diameter of 4.2 μm. with a loading level of 8.6 weight.%. Retinal avascular area was reduced in VEGF-treated RE (39.5 ± 9.0%) compared to untreated LE (52.6 ± 6.1%, p < 0.0001) or empty microparticle-treated RE (p < 0.001) and untreated LEs (p = 0.001). Retinal arteries in VEGF-treated RE were less tortuous than untreated LE (1.08 ± 0.05 vs. 1.18 ± 0.08, p < 0.001) or empty-microparticles-treated RE (p = 0.02). Retinal arterial tortuosity was similar in the LE of VEGF and empty microparticle-treated mice (P > 0.05). Retinal vein width was similar in VEGF-treated and empty microparticle-treated RE (P > 0.9), which were each less dilated than their contralateral LE (p < 0.01). VEGF levels were higher in P13 OIR mice than RA mice (p < 0.0001). Retinal flatmounts showed vaso-obliteration and neovascularization.Conclusions: Endogenous retinal VEGF is suppressed in OIR mice. Exogenous intravitreal VEGF-A165-loaded microparticles in OIR mice reduced retinal vaso-obliteration and accelerated recovery from vein dilation and arterial tortuosity. This may be beneficial in preventing Phase II ROP without systemic effects.

YAP via interacting with STAT3 regulates VEGF-induced angiogenesis in human retinal microvascular endothelial cells

Endothelial dysfunction is a main feature of retinal neovascular diseases which are the leading cause of blindness in developed countries. Yes-associated protein (YAP) and signal transducer and activator of transcription factor 3 (STAT3) participate in angiogenesis via vascular endothelial growth factor (VEGF) signaling. Additionally, YAP can bind STAT3 in endothelial cells. In the study, dimethyloxalylglycine (DMOG) stimulated human retinal microvascular endothelial cells (HRMECs) was used as retinal endothelial hypoxia model. The proliferation of HRMECs, as well as t-YAP, p-STAT3 (Tyr705) increased, while p-YAP (Ser127), p-YAP (Ser397) decreased following hypoxia. Meanwhile, YAP and STAT3 translocated to the nucleus. YAP knockdown inhibited the proliferation, migration and tube formation of HRMECs. YAP overexpression up-regulated phosphorylation of STAT3. The YAP overexpression-induced HRMECs proliferation, migration and tube formation were reversed by S3I-201, a selective STAT3 inhibitor. YAP interacted with STAT3 to promote STAT3 nuclear translocation. Additionally, YAP and STAT3 promoted the transcription of VEGF synergistically. Finally, inhibition of YAP alleviated retinal pathological neovascularization in mouse oxygen-induced retinopathy (OIR) model. In summary, activated YAP interacted with STAT3 to promote the activation and nuclear translocation of STAT3, hence boosted the proliferation, migration and tube formation of HRMECs via VEGF signaling following hypoxia. The data will further elucidate the mechanisms of retinal neovascular diseases.

Contribution of the clock gene DEC2 to VEGF mRNA upregulation by modulation of HIF1α protein levels in hypoxic MIO-M1 cells, a human cell line of retinal glial (Müller) cells.

Clock genes are components of the molecular clock. Their malfunction is thought to increase the risk of numerous diseases, including cancer. Vascular endothelial growth factor (VEGF) has a pivotal role in angiogenesis, and its expression levels are controlled by clock genes in tumor cells. Ophthalmic diseases such as age-related macular degeneration, proliferative diabetic retinopathy, and neovascular glaucoma are also associated with abnormal angiogenesis followed by upregulation of VEGF in the eye. In the present study, we aimed to uncover the relationship between clock genes and VEGF in the eye. Laboratory investigation METHODS: Oxygen-induced retinopathy (OIR) mice were prepared to mimic hypoxic conditions in the eye. Deferoxamine (DFO) was used to mimic hypoxic conditions in human Müller cell line MIO-M1 cells. Expression levels of mRNA and protein were quantified by quantitative reverse transcription polymerase chain reaction and Western blot analysis, respectively. In the retinas of OIR mice, the expression levels of Vegf and the clock gene Dec2 increased transiently, and their temporal profiles were correlated. Knockdown of DEC2 resulted in a significant (26.7%) reduction of VEGF expression in MIO-M1 cells under hypoxia-mimicking conditions induced by DFO (P < .05). Levels of HIF1α protein were also reduced significantly, by 60.2%, in MIO-M1 cells treated with siRNA against the DEC2 gene (P < .05). Moreover, HIF1α levels showed a significant (2.5-fold) increase in MIO-M1 cells overexpressing DEC2 (P < .05). DEC2 could upregulate retinal VEGF gene expression through modulation of HIF1α levels under hypoxic conditions.

Simultaneous assessment of aberrant retinal vascularization, thickness, and function in an in vivo mouse oxygen-induced retinopathy model.

Retinopathy of prematurity is a condition of abnormal retinal vascularization in premature infants. The effect of abnormal vascularization on retinal structure and function is unclear. In vivo studies of retinal vascularization, thickness, and function were performed in mice with oxygen-induced retinopathy (OIR mice). Eighteen mice were exposed to hyperoxia at postnatal day (P) 7, whereas 18 mice were raised in room air (RA). At P20 and 40, electroretinogram was performed for a-wave and b-wave amplitudes and peak times, followed by simultaneous fluorescein angiography for retinal avascular area, arterial tortuosity, and vein dilation assessments, and spectral domain optical coherence tomography for retinal thickness. Capillary density appeared sparser in OIR mice, but retinal avascular area similar to RA mice. Retinal artery tortuosity was higher at P20 and P40 (P = 0.0001) in OIR than RA mice. OIR mice had dilated retinal veins at P20 and thinner inner retinas at P40. Retinal vein width positively correlated with inner retinal thickness (P = 0.008). b-wave amplitude was decreased in avascular retinal areas, and correlated with inner retinal thinning. b-wave peak time was prolonged in adult OIR mice at high intensities (P = 0.03). Focal variations in retinal vascularization of OIR mice correlate with thickness and function. Adult OIR mice had increased retinal artery tortuosity, prolonged b-wave peak time, and decreased retinal vein width with inner retina attrition. These suggest abnormalities in inner retinal morphology or post-receptor signaling. Studying interactions between retinal vascular, structural, and functional changes could enhance knowledge of OIR pathogenesis and potential therapies.

ATN-161 as an Integrin α5β1 Antagonist Depresses Ocular Neovascularization by Promoting New Vascular Endothelial Cell Apoptosis.

BackgroundATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5β1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway.Material/MethodsAn oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin α5β1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling.ResultsIn OIR and CNV mice, the protein expression level of integrin α5β1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin α5β1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV.ConclusionsBlocking integrin α5β1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV.

Deletion of p75NTR prevents vaso-obliteration and retinal neovascularization via activation of Trk- A receptor in ischemic retinopathy model

Ischemic retinopathy is characterized by ischemia followed by retinal neovascularization (RNV) resulting in visual impairment. Given the role of neuron-secreted growth factors in regulating angiogenesis, we examined how genetic deletion of the neurotrophin receptor; p75NTR can overcome retinal ischemia using oxygen-induced retinopathy (OIR) mouse model. Wildtype (WT) or p75NTR−/− mice pups were subjected to hyperoxia (70% O2, p7-p12) then returned to normal air (relative hypoxia, p12-p17). Vascular alterations were assessed at p12 and p17 time-points. Deletion of p75NTR prevented hyperoxia-associated central vascular cell death (p12) and hypoxia-associated RNV and enhanced central vascular repair (p17). Decreased expression of apoptotic markers; preserved Akt survival signal decreased proNGF were also observed at p12. During hypoxia, deletion of p75NTR maintained VEGF and VEGFR2 activation and restored NGF/proNGF and BDNF/proBDNF levels. Deletion of p75NTR coincided with significant increases in expression and activation of NGF survival receptor, TrkA at basal and hyperoxic condition. Pharmacological inhibition of TrkA using compound K-252a (0.5 μg 1 μl−1/eye) resulted in 2-fold increase in pathological RNV and 1.34-fold increase in central vascular cell death in p75NTR−/− pups. In conclusion, deletion of p75NTR protected against retinal ischemia and prevented RNV, in part, through restoring neurotrophic support and activating TrkA receptor.