Retinal Vascular Development Research Articles

Symposium 3: Inflammation: a key driving force in disease progression S3-1 - Control of endothelial function and angiogenesis by PGC-1α relies on ROS control of vascular stability

Peroxisome proliferator activated receptor g co-activator 1alpha (PGC-1α) is a regulator of oxidative metabolism and reactive oxygen species (ROS) homeostasis that has been show to play a relevant role in angiogenesis. PGC-1α KO mice show reduced vascular density in the retinas and KO primary vascular endothelial cells (ECs) migrate faster than the wild type, an effect that can be rescued by antioxidants, suggesting that excessive ROS levels might be relevant in PGC-1 α role in angiogenesis. This study aims to investigate the role of ROS homeostasis on the regulation by PGC-1 α of angiogenesis. We found that endothelial cells (ECs) from mice deleted for PGC-1 α display attenuated adhesion to the extracellular matrix, together with slower spreading, reduced formation of cellular junctions, a disorganized cytoskeleton and random motility, and a enhanced tip phenotype. Aditionally, PGC-1 α -deleted ECs exhibit an altered response to vascular endothelial growth factor-A (VEGF-A). In vivo, deletion of PGC-1 α results in addition to reduced retinal vascular density, sparse pericyte coverage. Exposure of PGC-1 α deleted mice to hyperoxia during retinal vascular development exacerbates these vascular abnormalities and mice show extensive retinal hemorrhaging, with highly unstructured areas and very poor perfusion, compared with wild-type mice. Structural analysis demonstrates a reduction of endothelial VE-cadherin, suggesting defective inter-cellular junctions. Interestingly, this hyperoxia-induced phenotype is partially reversed by antioxidant administration, indicating that elevated production of mitochondrial reactive oxygen species (ROS) in the absence of PGC-1 α is functionally important. Finally, in vitro studies show that antioxidant treatment improves VEGF-A signaling, suggesting that toxic effect of ROS may be caused by the alteration of the VEGF-A signaling pathway. In summary, our findings indicate that PGC-1 α control of ROS homeostasis plays an important role in the control of de novo angiogenesis, and is required for vascular stability.

Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis.

Neurons have an important role in retinal vascular development. Here we show that the G protein-coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1, previously known as Par2) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation interfere with Vegfa but not Ang1 expression. Complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.

Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(−/−) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(−/−) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(−/−) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

Plasmalogens, astrocytes, and retinal vascular development

AbstractSufficient oxygen and nutrient supplies to the retina are dependent on a correct development of blood vessels. Polyunsaturated fatty acids (PUFAs) were shown to modulate factors involved in tissue vascularization. A congenital deficiency in ether‐linked phospholipids also termed as “plasmalogens” was shown to lead to abnormal ocular vascularization. Because plasmalogens are considered as reservoirs of PUFAs, we investigated whether they may influence retinal vascular development through the release of fatty acids by a specific phospholipase A2 (PLA2). Studies on mouse models of plasmalogen deficiency and of retinal PLA2 inhibition have shown that plasmalogens regulate retinal angiogenesis during early phases of retinal vascular development by influencing glial activity and primary astrocyte template formation. Subsequent modifications in the expression of genes involved in retinal angiogenesis impacted endothelial cell proliferation and retinal vessel outgrowth. These early defects in retinal vascular development led to a disorganized and dysfunctional retinal vascular network at adult age. These data suggest a crucial role of plasmalogens in retinal vascularization processes through a signalization involving PUFAs and PLA2.

Plasmalogens in the retina: from occurrence in retinal cell membranes to potential involvement in pathophysiology of retinal diseases.

Plasmalogens (Pls) represent a specific subclass of glycerophospholipids characterized by the presence of a vinyl-ether bond at the sn-1 position of glycerol. Pls are quantitatively important in membranes of neuronal tissues, including the brain and the retina, where they can represent until almost two-third of ethanolamine glycerophospholipids. They are considered as reservoirs of polyunsaturated fatty acids as several studies have shown that arachidonic and docosahexaenoic acids are preferentially esterified on Pls when compared to other glycerophospholipids. Reduced levels of Pls were observed in a number of neurodegenerative disorders such as glaucoma, the second leading cause of blindness worldwide. In a mouse model of Pls deficiency, "glaucoma-like" optic nerve abnormalities were observed as well as developmental defects in the eye. These included microphthalmia, dysgenesis of the anterior segment of the eye, and abnormalities in retinal vessel architecture. Several data from animal and invitro studies suggest that Pls may be involved in the regulation of retinal vascular development through the release of polyunsaturated fatty acids by a calcium-independent phospholipase A2.

Abstract 282: Functional Analysis Of Micrornas In Vascular Disease

microRNAs (miRNA, miR) are emerging as pivotal modulators of vascular development and disease. Research is my lab is focused on elucidating the functional mechanism and exploring therapeutic potential of microRNAs in vascular diseases. Our recent studies have shown that miR-23 and miR-27 in miR-23~27~24 family are required for proper angiogenesis and neovascularization in a laser-induced vascular injury model. Here we extend our study and provide evidence that miR-24 regulates actin dynamics in ECs through targeting multiple members downstream of Rho signaling, including Pak4, Limk2 and Diaph1 proteins. Consistent with the critical role for actin cytoskeleton in cell motility and proliferation, overexpression of miR-24 in ECs blocks stress fiber and lamellipodia formation, represses EC migration, proliferation and tube formation in vitro, as well as angiogenesis in an ex vivo aortic ring assay. Overexpression of miR-24 in transgenic mice represses postnatal retinal vascular development. Moreover, subretinal delivery of miR-24 mimics represses laser-induced CNV in vivo. Mechanistically, knockdown of miR-24 target protein LIMK2 or PAK4 inhibits stress fiber formation and tube formation in vitro, mimicking miR-24 overexpression phenotype in angiogenesis. Taken together, these findings demonstrate that miR-24 represses angiogenesis by simultaneously regulating multiple components in the actin cytoskeleton pathways, suggesting distinct function of miR-23~27~24 family members in angiogenesis. Manipulation of actin cytoskeleton pathways by miR-24 may represent an attractive therapeutic solution for numerous vascular diseases.

Age-Dependent Changes in the Severity of Capillary Degeneration in Rat Retina Following N-Methyl-D-Aspartate-Induced Neurotoxicity

Purpose: Previous studies have shown that injury to the retinal vasculature, including capillary degeneration, occurs following N-methyl-d-aspartate (NMDA)-induced neuronal cell loss, but it is unclear whether there are age-dependent differences in the severity of vascular damage. The purpose of the present study was to determine age-related changes in retinal capillary degeneration in NMDA-induced retinal damage rat model.Materials and Methods: A single intravitreal injection of NMDA (200 nmol/eye) was performed in 1-, 2-, 3-, 7- and 15-week-old male Sprague–Dawley rats. Seven days after NMDA injection, damage to retinal neurons and blood vessels were assessed by measuring the number of cells in the ganglion cell layer and the length of empty basement membrane sleeves, left behind by regressing endothelial cells, respectively.Results: At all ages examined, the NMDA-induced neurotoxicity exhibited similar levels of ganglion cell death. However, the degeneration of capillaries in NMDA-treated retina was severe during the early stages of retinal vascular development, and the degree of capillary degeneration decreased with age.Conclusion: The degree of retinal capillary degeneration in NMDA-induced retinal damage rat model decreases in an age-dependent manner. Retinal capillaries may be resistant to retinal neuronal damage in young adults.

Involvement of plasmalogens in post-natal retinal vascular development.

ObjectiveProper development of retinal blood vessels is essential to ensure sufficient oxygen and nutrient supplies to the retina. It was shown that polyunsaturated fatty acids (PUFAs) could modulate factors involved in tissue vascularization. A congenital deficiency in ether-phospholipids, also termed “plasmalogens”, was shown to lead to abnormal ocular vascularization. Because plasmalogens are considered to be reservoirs of PUFAs, we wished to improve our understanding of the mechanisms by which plasmalogens regulate retinal vascular development and whether the release of PUFAs by calcium-independent phospholipase A2 (iPLA2) could be involved.Methods and ResultsBy characterizing the cellular and molecular steps of retinal vascular development in a mouse model of plasmalogen deficiency, we demonstrated that plasmalogens modulate angiogenic processes during the early phases of retinal vascularization. They influence glial activity and primary astrocyte template formation, endothelial cell proliferation and retinal vessel outgrowth, and impact the expression of the genes involved in angiogenesis in the retina. These early defects led to a disorganized and dysfunctional retinal vascular network at adult age. By comparing these data to those obtained on a mouse model of retinal iPLA2 inhibition, we suggest that these processes may be mediated by PUFAs released from plasmalogens and further signalling through the angiopoietin/tie pathways.ConclusionsThese data suggest that plasmalogens play a crucial role in retinal vascularization processes.

Up-regulation of VEGF by retinoic acid during hyperoxia prevents retinal neovascularization and retinopathy.

Retinopathy of prematurity (ROP) is directly associated with abnormal expression of retinal vascular endothelial growth factor (VEGF) in premature neonates. This study was to investigate whether the systemic administration of retinoic acid (RA) regulates retinal VEGF expression and prevents retinal neovascularization and retinopathy in the oxygen-induced retinopathy (OIR) mouse model. C57BL/6 mice were subjected to OIR by exposure to 75% oxygen from postnatal day (P) 7 to 12 of age. RA was intraperitoneally injected daily to pups from P6 to P9. Retinal whole mount staining and image analysis, immunostaining, Western blotting, quantitative RT-PCR, TUNEL assay, and electroretinography were performed to evaluate the effects of RA on VEGF expression, retinal neovascularization, and retinal neuron functions. Systemic administration of RA in OIR mice promoted retinal VEGF mRNA and protein expression in phase I; the stabilized level of VEGF in phase I supported retinal vascular development and counteracted vaso-obliteration in OIR mice. Subsequently, the excessive generation of VEGF in phase II was attenuated; the retinal vascular leakage and apoptotic cells were significantly ameliorated. As a result, RA significantly prevented the development of hypoxia-induced retinal neovascularization and retinopathy in OIR mice and improved the functional recovery of retinal neurons downstream of photoreceptor cells as measured by focal electroretinography. Systemic administration of RA regulates retinal VEGF expression and supports retinal vascular development in OIR mouse model. We propose that systemic administration of RA to extremely low birth weight, preterm infants during oxygen therapy could potentially be an effective therapeutic approach for the prevention of ROP.

Treatment of mid-pregnant mice with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, induces abnormal retinal vascular patterning in their newborn pups.

We previously reported that treatment of mid-pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid-pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633-treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633-treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633-treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network.

Bcl‐2 expression in endothelial cells impacts postnatal retinal vascular development without affecting retinal neovascularization (679.2)

Bcl‐2 plays an important role in retinal vascular homeostasis. Global lack of Bcl‐2 expression results in attenuation of postnatal retinal vascular development and neovascularization during Oxygen‐induced Ischemic Retinopathy (OIR).To determine the impact lack of Bcl‐2 expression in endothelial cells has during retinal vascular development, remodeling, and neovascularization, we generated mice carrying a conditional Bcl‐2 allele (Bcl‐2Flox/Flox) and VE‐cadherin‐cre called Bcl‐2EC mice. Bcl‐2EC mice displayed decreased numbers of endothelial cells at both 3 and 6 weeks of age compared to wild‐type controls. However, the remodeling of the retinal vasculature proceeded with similar percentage loss of endothelial cells in both wild‐type and Bcl‐2EC mice. In addition, Bcl‐2EC mice had increased pericyte numbers at 6 weeks of age contributing to a lower EC/PC ratio. Retinal arteriogenesis was also decreased in Bcl‐2EC mice similar to what we observed in global Bcl‐2 knockouts. However, unlike the global knockouts the migrating front of the retinal vasculature proceeded normally and neovascularization during OIR occurred at levels similar to wild‐type mice. Therefore, our data suggests that Bcl‐2 expression modulates endothelial cell survival impacting postnatal retinal vascular development with minimal impact on retinal neovascularization during OIR.Grant Funding Source: Supported by R21EY023024

Alteration of retinal vascular development by postnatal systemic lipopolysaccharide administration: a novel model of retinopathy of prematurity (832.6)

Purpose: ROP is the leading cause of blindness in the children. Inflammation is being highlighted as the major factor for the morbidity of prematurity. We intended to investigate the effect of neonatal systemic inflammation on the retinal angiogenesis in newborn rats and to show the role of inflammation in the retinopathy of prematurity.Method: Systemic inflammation was induced by the pre‐ and post‐natal intraperitoneal injections of lipopolysaccharide (LPS) in neonatal rats. Enucleation and retinal separation was performed at 1 and 2 post‐natal weeks. Analyses on morphology of retinal vessels, recruited immune cells, change of inflammatory cytokines were assessed with immunofluorescence, FACS and RT‐PCR.Result: Administration of LPS postnatlly, not prenatally, induced inflammation in the retina with recruitment of CD11c positive cells and increased expression of a set of inflammatory cytokine genes. Postnatal LPS administration also suppressed retinal vessel development and deranged the retinal vascular structures combined with abnormal vascular tuft formation that resembles features of retinopathy of prematurity.Conclusion: Neonatal systemic inflammation alters the normal development of retinal vessels by inducing retinal inflammation. Our model of systemic inflammation‐induced abnormal development of retinal vasculature can be a novel experimental model of retinopathy of prematurity.

An interesting case of Coats' disease

Coats' disease is an idiopathic exudative retinopathy characterized by abnormal retinal vascular development (telangiectasia) that results in massive intraretinal and subretinal lipid accumulation. This disease entity was first described by a Scottish ophthalmologist, George Coats, in 1908.1 It is a relatively rare disease, with a reported incidence of 0.09 per 100,000 of the population.2 Coats' disease is classically a unilateral process, usually affecting young males in (85%) cases, with a mean age of diagnosis at 6–16 years of age,3 without any racial predominance. It is sporadic and nonhereditary.4 An adult variant of the disease is less commonly seen and is frequently associated with hypercholesterolemia. Coats' disease mimics retinoblastoma, and may present with leucocoria and strabismus, which makes it that much more important for an early and accurate diagnosis.5 If left untreated the disease shows progressive worsening which can lead to neovascular glaucoma and phthisis bulbi, sometimes even requiring enucleation.3 Here we report an interesting case of Coats' disease, presenting to us with a severe diminution of vision. He was managed aggressively with intra-vitreal long acting steroids (triamcinolone), laser photocoagulation and finally with intra-vitreal anti-vascular endothelial growth factor (VEGF) agents, with a successful restoration of useful functional vision.

Egfl7 Is Differentially Expressed in Arteries and Veins during Retinal Vascular Development

The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRβ blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.

Quantitative Analyses of Retinal Vascular Area and Density After Different Methods to Reduce VEGF in a Rat Model of Retinopathy of Prematurity

Targeted inhibition of Müller cell (MC)-produced VEGF or broad inhibition of VEGF with an intravitreal anti-VEGF antibody reduces intravitreal neovascularization in a rat model of retinopathy of prematurity (ROP). In this study, we compared the effects of these two approaches on retinal vascular development and capillary density in the inner and deep plexi in the rat ROP model. In the rat model of ROP, pups received 1 μL of (1) subretinal lentivector-driven short hairpin RNA (shRNA) to knockdown MC-VEGFA (VEGFA.shRNA) or control luciferase shRNA, or (2) intravitreal anti-VEGF antibody (anti-VEGF) or control isotype goat immunoglobulin G (IgG). Analyses of lectin-stained flat mounts at postnatal day 18 (p18) included: vascular/total retinal areas (retinal vascular coverage) and pixels of fluorescence/total retinal area (capillary density) of the inner and deep plexi determined with the Syncroscan microscope, and angles between cleavage planes of mitotic vascular figures labeled with anti-phosphohistone H3 and vessel length. Retinal vascular coverage and density increased in both plexi between p8 and p18 in room air (RA) pups. Compared with RA, p18 ROP pups had reduced vascular coverage and density of both plexi. Compared with respective controls, VEGFA.shRNA treatment significantly increased vascular density in the deep plexus, whereas anti-VEGF reduced vascular density in the inner and deep plexi. Vascular endothelial growth factor-A.shRNA caused more cleavage angles predicting vessel elongation and fewer mitotic figures, whereas anti-VEGF treatment led to patterns of pathologic angiogenesis. Targeted treatment with lentivector-driven VEGFA.shRNA permitted physiologic vascularization of the vascular plexi and restored normal orientation of dividing vascular cells, suggesting that regulation of VEGF signaling by targeted treatment may be beneficial.

Hypoxia Inducible Factor-2α Regulates the Development of Retinal Astrocytic Network by Maintaining Adequate Supply of Astrocyte Progenitors

Here we investigate the role of hypoxia inducible factor (HIF)-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26CreERT2, Tie2Cre, and GFAPCre. Global Hif-2α disruption by Rosa26CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAPCre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP+ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2+ and PDGFRα+ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other's development, mediated at least in part by HIF-2α.

Effects of pre- and post-natal treatment with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, on retinal vascular development and patterning in mice

The impaired function of angiogenic factors, including vascular endothelial growth factor (VEGF), during pregnancy is associated with preeclampsia and intrauterine growth restriction. To determine how the attenuation of VEGF signals during retinal vascular development affects retinal vascular growth and patterns, we examined the effects of pre- and post-natal treatment of mice with KRN633, a VEGF receptor tyrosine kinase inhibitor, on retinal vascular development and structure. Delays in retinal vascular development were observed in the pups of mother mice that were treated daily with KRN633 (5 mg/kg/day) from embryonic day 13.5 until the day of delivery. A more marked delay was seen in pups treated with the inhibitor (5 mg/kg/day) on the day of birth and on the following day. Pups treated postnatally with KRN633 showed abnormal retinal vascular patterns, such as highly dense capillary networks and decreased numbers of central arteries and veins. The high-density vascular networks in KRN633-treated pups showed a greater sensitivity to VEGF signaling inhibition than the normal vascular networks in vehicle-treated pups. Compared to vehicle-treated pups, more severe hypoxia and stronger VEGF mRNA expression were observed in avascular areas in KRN633-treated pups. These results suggest that a short-term loss of VEGF function at the earliest stages of vascular development suppresses vascular growth, leading to abnormal vascular patterning, at least in part via mechanisms involving VEGF in the mouse retina.

Cytochrome P450 2C8 ω3-Long-Chain Polyunsaturated Fatty Acid Metabolites Increase Mouse Retinal Pathologic Neovascularization—Brief Report

Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown. The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8-overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays. Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.

Oxidative stress retards vascular development before neural degeneration occurs in retinal degeneration rd1 mice

To investigate the role of reactive oxygen species (ROS) in retinal development during the early postnatal stage of rd1 mice. Development of the three retinal vascular layers of C57BL/6J (WT) and C3H/HeN (rd1) mice was evaluated from 9th postnatal day (P9) to P21. Retinal ROS production was semi-quantitatively measured using dihydroethidium fluorescence. Mice were treated with intraperitoneal injections of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) at a dose of 275mg/kg body weight, and PBS as the control from P3 to P8. Rd1 mice showed retardation of retinal vascular development in the deep layer at P9. No significant difference was observed in the outer nuclear layer thickness of rd1 and WT mice. ROS production in the outer nuclear layer of rd1 mice was significantly higher than that in the outer nuclear layer of WT mice at P9, P13, and P17 (P < .05). TEMPOL facilitated the development of the deep vascular layer when compared with injection of PBS. Retardation of retinal vascular development is observed in rd1 mice; ROS is partially responsible for this finding. When using rd1 mice, we should be aware of this difference in comparison to other retinal degeneration animal models and human pathophysiological changes.

In Vivo Retinal Vascular Oxygen Tension Imaging and Fluorescein Angiography in the Mouse Model of Oxygen-Induced Retinopathy

Oxygenation abnormalities are implicated in the development of retinopathy of prematurity (ROP). The purpose of this study is to report in vivo retinal vascular oxygen tension (PO2) measurements and fluorescein angiography (FA) findings in the mouse model of oxygen-induced retinopathy (OIR). We exposed 19 neonatal mice to 77% oxygen from postnatal day 7 (P7) to P12 (OIR), while 11 neonatal mice were kept under room air (control). Using phosphorescence lifetime imaging, retinal vascular PO2 was measured followed by FA. Repeated measures ANOVA was performed to determine the effects of blood vessel type (artery and vein) and group (OIR and control) on PO2. Avascular retinal areas were measured from FA images in OIR mice. There was a significant effect of vessel type on PO2 (P < 0.001). The effect of group on PO2 was not significant (P = 0.3), indicating similar PO2 between OIR and control mice. The interaction between group and vessel type was significant (P = 0.03), indicating a larger arteriovenous PO2 difference in OIR mice than control mice. In control mice, FA displayed normal vascularization, while FA of OIR mice showed abnormalities, including dilation and tortuosity of major retinal blood vessels, and avascular regions. In OIR mice, the mean percent avascular retinal area was 33% ± 18%. In vivo assessment of retinal vascular oxygen tension and vascularization patterns demonstrated abnormalities in the mouse model of OIR. This approach has the potential to improve understanding of retinal vascular development and oxygenation alterations due to ROP and other ischemic retinal diseases.