Model Of Retinal Neovascularization Research Articles

Method to Regulate Monocyte Function by Silencing HIF-1α mRNA in a Model of Retinal Neovascularization.

Circulating monocytes migrate into the retina in response to inflammation and neovascularization. Furthermore, under inflammatory conditions such as diabetes, healthy monocytes become activated in the circulation. However, the contribution of activated monocytes to neovascularization is largely unknown. HIF-1α has been shown to contribute to the pathogenesis of neovascularization. We describe here the synthesis of a hybrid nanomaterial for targeted delivery and gene silencing in activated monocytes that are associated with pathological neovascularization. To test the gene silencing ability of AS-shRNA-lipids in vitro, we used the probe to inhibit HIF-1α mRNA induced in mouse monocytes by exposing them to hypoxia. In addition, we tested AS-shRNA-lipids for inhibition of neovascularization in vivo using the mouse model of oxygen-induced retinopathy (OIR). Significant reduction of neovascularization was achieved in mouse OIR by targeting activated monocytes using intraperitoneal injections of AS-shRNA-lipids. Expression of HIF-1α and CD14 mRNA were both inhibited in circulating cells, suggesting normalization of the activated monocytes in P17 OIR animals treated with AS-shRNA-lipids. We hypothesized that inhibition of HIF-1α mRNA in activated monocytes may have a direct impact on VEGF expression in the retinal tissues in vivo. We observed that VEGF mRNA expression was inhibited in P17 retinal tissues after systemic treatment with HIF-1α-targeted AS-shRNA-lipids. These findings may provide a framework for a strategy to inhibit retinal neovascularization by targeting circulating activated monocytes.

Protective effect of ethyl ferulate against hypoxic injury in retinal cells and retinal neovascularization in an oxygen-induced retinopathy model

BackgroundPathological neovascularization is a major cause of visual impairment in hypoxia-induced retinopathy. Ethyl ferulate (EF), the natural ester derivative of ferulic acid commonly found in Ferula and Angelica Sinensis, has been shown to exert antioxidant, neuroprotective, and anti-inflammatory properties. However, whether EF exerts a protective effect on retinal neovascularization and the underlying mechanisms are not well known. PurposeThe aim of the study was to investigate the effect of EF on retinal neovascularization and explore its underlying molecular mechanisms. Study-design/methodsWe constructed hypoxia models induced by cobalt chloride (CoCl2) in ARPE-19 cells and Rhesus choroid-retinal vascular endothelial (RF/6A) cells in vitro, as well as a retinal neovascularization model in oxygen-induced retinopathy (OIR) mice in vivo. ResultsIn this work, we demonstrated that EF treatment inhibited hypoxia-induced vascular endothelial growth factor A (VEGFA) expression in ARPE-19 cells and abrogated hypoxia-induced tube formation in RF/6A cells. As expected, intravitreal injection of EF significantly suppressed retinal neovascularization in a dose-dependent manner in OIR retinas. We also found that hypoxia increased VEGFA expression by blocking autophagic flux, whereas EF treatment enhanced autophagic flux, thereby reducing VEGFA expression. Furthermore, EF activated the sequestosome 1 (p62) / nuclear factor E2-related factor 2 (Nrf-2) pathway via upregulating oxidative stress-induced growth inhibitor 1 (OSGIN1) expression, thus alleviating oxidative stress and reducing VEGFA expression. ConclusionAs a result of our findings, EF has an inhibitory effect on retinal neovascularization, implying a potential therapeutic strategy for hypoxia-induced retinopathy.

Delineating effects of angiopoietin-2 inhibition on vascular permeability and inflammation in models of retinal neovascularization and ischemia/reperfusion.

Clinical trials demonstrated that co-targeting angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF-A) with faricimab controls anatomic outcomes and maintains vision improvements, with strong durability, through 2 years in patients with neovascular age-related macular degeneration and diabetic macular edema. The mechanism(s) underlying these findings is incompletely understood and the specific role that Ang-2 inhibition plays requires further investigation. We examined the effects of single and dual Ang-2/VEGF-A inhibition in diseased vasculatures of JR5558 mice with spontaneous choroidal neovascularization (CNV) and in mice with retinal ischemia/reperfusion (I/R) injuries. In JR5558 mice, Ang-2, VEGF-A, and dual Ang-2/VEGF-A inhibition reduced CNV area after 1 week; only dual Ang-2/VEGF-A inhibition decreased neovascular leakage. Only Ang-2 and dual Ang-2/VEGF-A inhibition maintained reductions after 5 weeks. Dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 1 week. Both Ang-2 and dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 5 weeks. In the retinal I/R injury model, dual Ang-2/VEGF-A inhibition was statistically significantly more effective than Ang-2 or VEGF-A inhibition alone in preventing retinal vascular leakage and neurodegeneration. These data highlight the role of Ang-2 in dual Ang-2/VEGF-A inhibition and indicate that dual inhibition has complementary anti-inflammatory and neuroprotective effects, suggesting a mechanism for the durability and efficacy of faricimab in clinical trials.

Effective treatment of retinal neovascular leakage with fusogenic porous silicon nanoparticles delivering VEGF-siRNA.

Aim: To evaluate an intravitreally injected nanoparticle platform designed to deliver VEGF-A siRNA to inhibit retinal neovascular leakage as a new treatment for proliferative diabetic retinopathy and diabetic macular edema. Materials & methods: Fusogenic lipid-coated porous silicon nanoparticles loaded with VEGF-A siRNA, and pendant neovascular integrin-homing iRGD, were evaluated for efficacy by intravitrealinjectionin a rabbit model of retinal neovascularization. Results: For 12 weeks post-treatment, a reduction in vascular leakage was observed for treated diseased eyes versus control eyes(p=0.0137), with a corresponding reduction in vitreous VEGF-A. Conclusion: Fusogenic lipid-coated porous silicon nanoparticles siRNA delivery provides persistent knockdown of VEGF-A and reduced leakage in a rabbit model of retinal neovascularization as a potential new intraocular therapeutic.

Pulsatile tissue deformation dynamics of the murine retina and choroid mapped by 4D optical coherence tomography.

Irregular ocular pulsatility and altered mechanical tissue properties are associated with some of the most sight-threatening eye diseases. Here we present 4D optical coherence tomography (OCT) for the quantitative assessment and depth-resolved mapping of pulsatile dynamics in the murine retina and choroid. Through a pixel-wise analysis of phase changes of the complex OCT signal, we reveal spatiotemporal displacement characteristics across repeated frame acquisitions. We demonstrate in vivo fundus elastography (FUEL) imaging in wildtype mouse retinas and in a mouse model of retinal neovascularization and uncover subtle structural deformations related to ocular pulsation. Our data in mouse eyes hold promise for a powerful retinal elastography technique that may enable a new paradigm of OCT-based measurements and image contrast.

High-resolution, depth-resolved vascular leakage measurements using contrast-enhanced, correlation-gated optical coherence tomography in mice.

Vascular leakage plays a key role in vision-threatening retinal diseases such as diabetic retinopathy and age-related macular degeneration. Fluorescence angiography is the current gold standard for identification of leaky vasculature in vivo, however it lacks depth resolution, providing only 2D images that complicate precise identification and localization of pathological vessels. Optical coherence tomography (OCT) has been widely adopted for clinical ophthalmology due to its high, micron-scale resolution and rapid volumetric scanning capabilities. Nevertheless, OCT cannot currently identify leaky blood vessels. To address this need, we have developed a new method called exogenous contrast-enhanced leakage OCT (ExCEL-OCT) which identifies the diffusion of tracer particles around leaky vasculature following injection of a contrast agent. We apply this method to a mouse model of retinal neovascularization and demonstrate high-resolution 3D vascular leakage measurements in vivo for the first time.

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.

Analysis of miR‐21‐5p and ‐3p expression and function during pathological neovascularization

Retinal neovascularization (RNV) is a sight compromising complication of diabetes and prematurity. Previous studies from our laboratory have shown that the small noncoding microRNA, microRNA‐21‐5p (miR‐21‐5p), is an inducer of RNV. Emerging new evidence has shown that miRNA passenger strands, the 3p forms, which were thought to be degraded, possess biological functions. In this study, we have investigated the role of miR‐21‐3p in RNV. In particular, we have assessed the effect of miR‐21‐3p on the same molecular targets of miR‐21‐5p: the tissue inhibitor of matrix metalloproteinases 3 (TIMP3), peroxisome proliferator‐activated receptor alpha (PPAR‐α) and pigment epithelium‐derived factor (PEDF). Real‐time PCR (RT‐PCR) showed that the expression of miR‐21‐3p is significantly upregulated in retinas and blood spot samples of mice subjected to oxygen‐induced retinopathy (OIR), a model of RNV. Furthermore, we noted a significant upregulation of miR‐21‐3p levels, measured by RT‐PCR, in human retinal endothelial cells (HuREC) and pigment epithelial cells (ARPE‐19) when exposed to angiogenic stimuli such as 20ng/mL of vascular endothelial growth factor (VEGF) or to hypoxic conditions (pO2= 2+/−0.5) for different times (1, 3, 6, and 12 hours). Transfection of HuREC and ARPE‐19 with miR‐21‐3p mimic (50nM) for 24 and 48 hours had only marginal inhibition on the miR‐21‐5p target genes (TIMP3, PPARα, and PEDF). Additionally, blockade of miR‐21‐3p achieved by transfecting cells with a specific miR‐21‐3p inhibitor (50nM) did not rescue VEGF‐changes in TIMP3, PPARα, and PEDF expression. Our data demonstrated that the passenger strand, miR‐21‐3p, is co‐expressed with miR‐21‐5p in ischemic conditions, but targets different genes. Although further studies will be necessary to clarify miR‐21‐3p biological function, our studies suggest that, together with miR‐21‐5p, miR‐21‐3p may hold potential diagnostic and therapeutic value for treatment of RNV in ischemic retinopathies.Support or Funding InformationAll support for this research came from the Bartoli laboratory of the Ophthalmology Department of the Medical College of Georgia at Augusta University and the funding sources (EY0246, Knights Templar –GA Chapter).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IGF-1 regulates Ang II and VEGF signaling pathways in retinal neovascularization.

To investigate the effect of insulin-like growth factor (IGF)-1 on regulating the occurrence of retinal neovascularization and the expression mechanism of Angiotensin II (Ang II) and vascular endothelial factor (VEGF). A total of 40 C57BL/6J healthy mice of either gender were selected, and randomly divided into 4 groups: control group, model group, IGF-1 overexpression carrier intervention group and IGF-1 siRNA intervention group (10 in each group). Mice in control group were fed in the air without any further processing. The mice in other 3 groups were used to build high oxygen-induced retinal neovascularization model. The vitreous cavity of mice in the model group was injected with the equal amount of normal saline, while the mice in the IGF-1 overexpression carrier intervention group were injected with 1 μl IGF-1 liposomes mixture. At the same time, IGF-1 siRNA intervention group mice were continuously injected with 1 μl IGF-1 siRNA liposomes mixture for 3d. Stretched preparation of retina was used to observe the neovascularization form; HE staining tissue section were prepared to observe the amount of vascular endothelial nuclei out of the retina boundary; IGF-1, Ang II and VEGF expression levels of retinal tissue were detected by RT-PCR and Western blot. A large area of non-perfusion and tortuous expansion form of the blood vessel was found in the optic nerve surroundings of retinal serial sections in model group, while neovascularization plexus and fluorescence leakage were observed between perfusion and non-perfusion area. Non-perfusion and neovascularization have experienced a decrease in IGF-1 overexpression group but an increase in IGF-1 siRNA group. The number of neovascularization endothelial nuclei out of the internal limiting membrane in model group was remarkably higher than that in control group. This number in IGF-1 over-expression group decreases while in IGF-1 siRNA group it increases, with a significant statistical difference (p < 0.05). Histone IGF-1 expression level in the model group was lower than that in the control group, but Ang II and VEGF expression level were higher. IGF-1 in over-expression group was higher but with a lower expression level of Ang II and VEGF; however, the results in siRNA group were opposite. The difference was statistically significant (p < 0.05). The occurrence of retinal neovascularization is related to the reduction of IGF-1 expression and the increase of Ang II and VEGF.

Repurposing Griseofulvin as a Non‐Toxic Angiogenesis Inhibitor

BackgroundOcular neovascularization causes vision loss in major blinding diseases such as proliferative diabetic retinopathy, retinopathy of prematurity, and neovascular age‐related macular degeneration. Over 30% of patients are refractive to current therapies, hence developing small molecules targeting ocular neovascularization is critical. We previously identified the heme biosynthesis enzyme ferrochelatase (FECH) as an angiogenesis mediator. FECH is upregulated in human and mouse eyes undergoing neovascularization, and knockdown or mutation abolished angiogenesis in vitro and in vivo. FECH inhibition is therefore a promising therapeutic approach for blocking neovascularization. The FDA‐approved anti‐fungal drug, griseofulvin, inhibits FECH as an off‐target effect and blocks choroidal neovascularization ex‐vivo and in mice. Here, we investigated the anti‐angiogenic effect of griseofulvin in the mouse oxygen‐induced retinopathy (OIR) model of retinal neovascularization, and explored its ocular toxicity.MethodsIn the OIR model, newborn mice are exposed to 75% oxygen at postnatal day 7 (P7) for 5 days, causing retinal vascular regression. At P12, upon return to normoxia, the retina becomes ischemic, forming pre‐retinal neovascularization. Temporal expression of FECH was analyzed at various ages in mouse OIR retinas. Griseofulvin was intravitreally injected in OIR mice at P12 and retinal neovascularization assessed at P17 using Isolectin B4 staining. Tip cell filopodia and retinal sprouting were assessed by murine retinal ex‐vivo assays. For toxicity studies, adult mice were intravitreally injected with griseofulvin and retinal damage analyzed after 7 and 14 days. Structural and morphological analyses were performed using optical coherence tomography (OCT) and histology. Vascular leakage was assessed by fluorescein angiography (FA). Functional analysis of the retina was performed using electroretinogram (ERG). Apoptosis and immune cell activation were assessed by immunohistochemistry.ResultsTemporal FECH upregulation was observed in mouse OIR retinas. Griseofulvin inhibited retinal neovascularization in OIR and also decreased the avascular area in the OIR retina. Further, griseofulvin inhibited retinal tip cell filopodia and retinal sprouting ex‐vivo. OCT and FA revealed that retinal structure was maintained and no vascular leakage was found in griseofulvin injected eyes. ERG showed that retinal function was normal in griseofulvin treated mice, and histology showed normal retinal stratification in griseofulvin treated eyes. Immunostaining revealed no aberrant cell death, microglia activation or gliosis in griseofulvin treated eyes, suggesting no cellular toxicity or stress.ConclusionsGriseofulvin inhibits retinal neovascularization and also promotes revascularization, suggesting a dual effect on vascular repair. Griseofulvin lacks ocular toxicity. Together, these findings suggest that griseofulvin could be repurposed to treat neovascularization in ocular diseases.Support or Funding InformationNIH/NEI R01EY025641, Research to Prevent Blindness, Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppression of Retinal Neovascularization by Anti-CCR3 Treatment in an Oxygen-Induced Retinopathy Model in Mice

Purpose: To investigate the association between retinal neovascularization and the CC chemokine receptor-3 (CCR3) in a mouse model of oxygen-induced retinopathy (OIR). Methods: An OIR model in C57BL/6J mice was used as a retinal neovascularization model. An enzyme-linked immunosorbent assay was performed to evaluate the chronological change in vascular endothelial growth factor A (VEGF-A) and eotaxin expressions. CCR3 and VEGF subtype expression in the retina was examined using real-time RT-PCR, and CCR3, eotaxin, VEGF-A, and CD31 expression was examined immunohistochemically. A CCR3 neutralizing antibody (Ab) was injected into the vitreous humor on both postnatal days 12 (P12) and 14 (P14). Retinal neovascularizations were quantified by measurement of the percentages of neovascular area. Results: The mean eotaxin and VEGF-A protein level was significantly downregulated at P10 and P12 and was significantly upregulated at P14 and P17 (p < 0.05). CCR3 mRNA expression was significantly upregulated at P12 (p < 0.05). VEGF164 mRNA expression was significantly upregulated at P14 (p < 0.05). The areas of vaso-obliteration and neovascularization were significantly suppressed in anti-CCR3 Ab-treated eyes (p < 0.05). Anti-CCR3 Ab treatment suppressed VEGF and eotaxin but not monocyte chemoattractant protein-1. And VEGF 164 mRNA but not VEGF120 mRNA was suppressed by anti-CCR3 Ab treatment. Conclusions: The present data suggest that anti-CCR3 treatment can suppress retinal neovascularization. Anti-CCR3 treatment may have potential as a new therapy for retinopathies with retinal neovascularization such as diabetic retinopathy and retinopathy of prematurity.

Potential suppression of the high glucose and insulin-induced retinal neovascularization by Sirtuin 3 in the human retinal endothelial cells

Retinal neovascularization generally play roles in the formation of various severe eye diseases, such as age-related macular degeneration and diabetic retinopathy. The regulation of neovascularization-related pathways by Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, give us a cue that Sirt3 may participate in the retinal neovascularization. However, the mechanism remains unclear. Here, we established a retinal neovascularization model by using human retinal endothelial cells (HRECs) under the induction of high glucose and insulin (HGI). With this model, Sirt3-expressing lentivirus was constructed and then used to investigate the effect of Sirt3 overexpression on the expression of migration-, neovascularization- and autophagy-related genes. After the treatment of HGI on HRECs, the mRNA and protein levels of migration-related genes, including matrix metalloproteinase-2 (MMP-2) and MMP-9, were significantly upregulated. Meanwhile, angiogenesis-related genes, including vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor-1 (IGF-1) were promoted at both mRNA and protein levels. However, HGI had no clear effect on the mRNA and protein levels of microtubule associated protein 1 light chain 3 (LC3), an autophagy-related gene. When Sirt3 was overexpressed by lentivirus infection after HGI, the upregulation of MMP-2, MMP-9, VEGF, HIF-1α, and IGF-1 were suppressed at both transcription and translation levels. At the same time, LC3 mRNA and LC3-II protein increased. These results suggest that Sirt3 may inhibit retinal neovascularization by regulating the migration-, neovascularization- and autophagy-related factors expression. Thus we argue that Sirt3 may be a potential candidate drug for curing various eye diseases induced by retinal neovascularization.

VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis

During angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called podosomes contribute to this step. Invitro, VEGF-A/Notch signaling regulates the formation of functional podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble podosomes during physiological angiogenesis invivo. In the retina, podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in podosome areas. Moreover, Notch inhibition exacerbates podosome formation and collagen-IV loss. We propose that the localized proteolytic action of podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically.

Bioelectric impact of pathological angiogenesis on vascular function

Pathological angiogenesis, as seen in many inflammatory, immune, malignant, and ischemic disorders, remains an immense health burden despite new molecular therapies. It is likely that further therapeutic progress requires a better understanding of neovascular pathophysiology. Surprisingly, even though transmembrane voltage is well known to regulate vascular function, no previous bioelectric analysis of pathological angiogenesis has been reported. Using the perforated-patch technique to measure vascular voltages in human retinal neovascular specimens and rodent models of retinal neovascularization, we discovered that pathological neovessels generate extraordinarily high voltage. Electrophysiological experiments demonstrated that voltage from aberrantly located preretinal neovascular complexes is transmitted into the intraretinal vascular network. With extensive neovascularization, this voltage input is substantial and boosts the membrane potential of intraretinal blood vessels to a suprahyperpolarized level. Coincident with this suprahyperpolarization, the vasomotor response to hypoxia is fundamentally altered. Instead of the compensatory dilation observed in the normal retina, arterioles constrict in response to an oxygen deficiency. This anomalous vasoconstriction, which would potentiate hypoxia, raises the possibility that the bioelectric impact of neovascularization on vascular function is a previously unappreciated pathophysiological mechanism to sustain hypoxia-driven angiogenesis.

Proliferative retinopathies: animal models and therapeutic opportunities.

Proliferative retinopathies are the leading causes of blindness in Western societies. The development of new, more efficacious treatments that take advantage of recent advances in the fields of gene and cell therapy requires further investigations on the mechanisms underlying disease onset and progression, and adequate animal models that recapitulate the pathogenesis of human proliferative retinopathy and allow evaluation of the long-term therapeutic benefits that these therapies can offer. Unfortunately, most models of retinal neovascularization have short-term evolution and diabetic rodents show a very mild retinal phenotype, limited to non-proliferative changes, and do not develop proliferative retinopathy at all. Transgenic mice overexpressing Insulin-like Growth Factor-I (IGF-I) in the retina (TgIGF-I) constitute the only rodent model currently available that develops most of the retinal alterations observed in diabetic eyes, with a temporal evolution that resembles that of the human disease. TgIGF-I have retinal vascular alterations that progress as animals age from non-proliferative to proliferative disease, making these mice an excellent model of proliferative retinopathy that, due to its slow progression, allows long-term evaluation of novel antiangiogenic therapies. At the molecular level, transgenic retinas recapitulate a variety of changes that are also observed in diabetic retinas, which reinforces the validity of this model. In addition to vascular and glial alterations, Tg-IGF-I mice show progressive neurodegeneration that leads to blindness in old animals. Thus, TgIGF-I are a useful model for testing the long-term efficacy and safety of innovative antiangiogenic, glial-modulating and neuroprotective therapies for the treatment of diabetic retinopathy and other retinal proliferative disorders.

Dihydroartemisinin targets VEGFR2 via the NF-κB pathway in endothelial cells to inhibit angiogenesis

The anti-malarial agent dihydroartemisinin (DHA) has strong anti-angiogenic activity. This study aimed to investigate the molecular mechanism underlying this effect of DHA on angiogenesis. We found that DHA shows a dose-dependent inhibition of proliferation and migration of in HUVECs. DHA specifically down-regulates the mRNA and protein expression of VEGFR2 in endothelial cells. Treatment with DHA increases IκB-α protein and blocks nuclear translocation of NF-κB p65. In addition, DHA directly regulates VEGFR2 promoter activity through p65 binding motif, and decreases the binding activity of p65 and VEGFR2 promoter, suggesting defective NF-κB signaling may underlie the observed effects of DHA on VEGFR2 expression. In the presence of the NF-κB inhibitor PDTC, DHA could not further repress VEGFR2. Co-treatment with PDTC and DHA produced minimal changes compared to the effects of either drug alone in in vitro angiogenesis assays. Similar findings were found in vivo through a mouse retinal neovascularization model examining the effects of PDTC and DHA. Our data suggested that DHA inhibits angiogenesis largely through repression of the NF-κB pathway. DHA is well tolerated, and therefore may be an ideal candidate to use clinically as an angiogenesis inhibitor for cancer treatment.

Characterization of a spontaneous retinal neovascular mouse model.

BackgroundVision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.MethodsThe NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.ResultsWe found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.ConclusionsThe NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.

Endoglin promotes angiogenesis in cell- and animal-based models of retinal neovascularization.

We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization. Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies. Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats. Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease.

Regulation of Endothelial Progenitor Cell Release by Wnt Signaling in Bone Marrow

Endothelial progenitor cells (EPC) have been shown to participate in ischemia-induced retinal neovascularization (NV). Overactivation of Wnt signaling has a pathogenic role in ischemia-induced retinal NV. The purpose of this study is to determine whether Wnt signaling regulates EPC release. Oxygen-induced retinopathy (OIR) was used as a model of retinal NV and Wnt pathway activation. The EPC, marked as c-Kit(+)/Tie-2(+) cells in the peripheral blood and bone marrow, were quantified using flow cytometry following immunolabeling. The Wnt signaling activity was evaluated by measuring nonphosphorylated β-catenin levels and X-gal staining in the Wnt reporter mice (Bat-gal mice). The c-Kit(+)/Tie-2(+) cells were increased significantly in the peripheral blood and bone marrow of mice with OIR, compared to non-OIR mice. Overexpression of kallistatin, an endogenous inhibitor of the Wnt pathway, in kallistatin transgenic (kallistatin-TG) mice with OIR attenuated the increases of c-Kit(+)/Tie-2(+) cells in the peripheral blood and bone marrow, compared to WT mice with OIR. When the Bat-gal mice were crossed with kallistatin-TG mice, kallistatin overexpression suppressed the OIR-induced increases of X-gal-positive cells in the retinas and bone marrow, suggesting inhibition of Wnt signaling in these tissues. Furthermore, intraperitoneal injection of LiCl, a Wnt signaling activator, increased c-Kit(+)/Tie-2(+) cells in the peripheral blood of normal mice. Consistently, LiCl activated Wnt signaling in the retina and bone marrow cells in Bat-gal mice. The Wnt signaling pathway has an important role in EPC release during retinal NV in OIR.

Retinal Angiogenesis in the Ins2AkitaMouse Model of Diabetic Retinopathy

Purpose.: Diabetic retinopathy (DR) is the leading cause of blindness among working age adults and does not have any curative treatments. Although chemical- and injury-induced models of retinal neovascularization exist, the need for a genetic model that closely simulates the DR pathologic process is great. Methods.: Here we characterize the development of the retinal disease phenotype in a genetic model of type 1 diabetes, the Ins2Akita mouse, using structural, biochemical, molecular biological, and functional techniques. Results.: This model exhibits hyperglycemia by 2 months of age and by 6 months we detect retinal complications in Ins2Akita males, including early signs of vascular damage consistent with DR, specifically the appearance of pericyte ghosts, vascular leakage, and microaneurysm formation. By 9 months of age, these changes are accompanied by later vascular signs of DR, specifically retinal neovascularization, formation of new capillary beds, and the presence of new blood vessels abnormally localized in the outer plexiform layer. Consistent with the debilitating effects of such vasculopathy, we also observe increased retinal apoptosis and decreased retinal function measured by electroretinogram. Conclusions.: These data indicate that the Ins2Akita mouse is a good model for later-onset DR, modeling both early and some late disease signs. Furthermore, this work suggests that this model may be suitable for testing and development of targeted DR therapies.