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Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17β-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1−/−) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1−/− retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1−/− retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1−/− mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis. [ABSTRACT FROM AUTHOR]

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, mast cells, and vasculature in eyes of albino and pigmented mice using cell type-specific staining. We were able to visualize the choroidal arterial and venous systems. An arterial circle around the optic nerve was found in mice similar to the Zinn–Haller arterial circle that exists in humans and primates. Three different structural patterns of choriocapillaris were observed throughout the whole choroid: honeycomb-like, maze-like, and finger-like patterns. Choroidal mast cells were relatively few but dense around the optic nerve. Mast cell distribution in the middle and periphery was different among strains. Macrophages were found in all layers of the choroid. Thus, utilizing the simple and reliable methods described herein will allow the evaluation of transgenic and preclinical mouse models of ocular diseases that affect the choroid, including age-related macular degeneration (AMD), diabetic choroidopathy, and retinopathy of prematurity. These studies will advance our understanding of the pathophysiology, and molecular and cellular mechanisms that can be targeted therapeutically, in these diseases. [ABSTRACT FROM AUTHOR]

Retinopathy of prematurity (ROP) is one of the main causes of blindness in children worldwide. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), play critical protective roles in the development and function of neurons and vasculature. Lack of BDNF expression results in increased endothelial cell apoptosis and reduced endothelial cell-cell contact. Premature babies who develop ROP tend to have lower serum BDNF levels. BDNF expression is also significantly lower in mouse retinas following exposure to hyperoxia compared to those reared in room air. Specifically, BDNF promotes angiogenic tube formation of endothelial cells (EC), and it is considered an EC survival factor required for stabilization of intramyocardial vessels. We hypothesized that the activation of TrkB receptor protects retinal vasculature in the mice during oxygen-induced ischemic retinopathy (OIR), a model of ROP. To test this hypothesis, we treated neonatal mice with 7,8-dihydroxyflavone (DHF) (5 mg/kg body weight), a TrkB receptor agonist. We examined its potential protective effects on retinal vessel obliteration and neovascularization, two hallmarks of ROP and OIR. We found that retinas from DHF treated postnatal day 8 (P8) and P12 mice have similar levels of vessel obliteration as retinas from age-matched control mice subjected to OIR. Similarly, DHF showed no significant effect on mitigation of retinal neovascularization during OIR in P17 mice. Collectively, our studies demonstrate that the TrkB receptor agonist DHF provides no significant protective effects during OIR. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a major cause of long-term disabilities, including vision loss. Neuronal and blood vessel maturation can affect the susceptibility of and outcome after ischemic stroke. Although we recently reported that exposure of neonatal mice to hypoxia–ischemia (HI) severely compromises the integrity of the retinal neurovasculature, it is not known whether juvenile mice are similarly impacted. Here we examined the effect of HI injury in juvenile mice on retinal structure and function, in particular the susceptibility of retinal neurons and blood vessels to HI damage. Our studies demonstrated that the retina suffered from functional and structural injuries, including reduced b-wave, thinning of the inner retinal layers, macroglial remodeling, and deterioration of the vasculature. The degeneration of the retinal vasculature associated with HI resulted in a significant decrease in the numbers of pericytes and endothelial cells as well as an increase in capillary loss. Taken together, these findings suggest a need for juveniles suffering from ischemic stroke to be monitored for changes in retinal functional and structural integrity. Thus, there is an emergent need for developing therapeutic approaches to prevent and reverse retinal neurovascular dysfunction with exposure to ischemic stroke. [ABSTRACT FROM AUTHOR]

Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision. [ABSTRACT FROM AUTHOR]

Bcl–2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl–2 (Bcl–2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl–2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl–2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl–2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl–2 allele (Bcl-2Flox/Flox) and VE-cadherin-cre (Bcl-2EC mice). Bcl-2EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl–2 deficient mice. Bcl-2EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl–2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl–2 function in the endothelium. [ABSTRACT FROM AUTHOR]

Inflammation is increasingly recognized as an important modulator in the pathogenesis of neovascular age-related macular degeneration (nAMD). Although significant progress has been made in delineating the pathways that contribute to the recruitment of inflammatory cells and their contribution to nAMD, we know little about what drives the resolution of these inflammatory responses. Gaining a better understanding of how immune cells are cleared in the choroid will give a novel insight into how sustained inflammation could influence the pathogenesis of nAMD. The pro-apoptotic Bcl-2 family member Bim is a master regulator of immune cell homeostasis. In its absence, immune cell lifespan and numbers increase. Most therapeutic regimes that squelch inflammation do so by enhancing immune cell apoptosis through enhanced Bim expression and activity. To test the hypothesis that Bim expression tempers inflammation during the pathogenesis of nAMD, we used the mouse laser-induced choroidal neovascularization (CNV) model in which inflammation acts as a facilitator of CNV. Here, we showed minimal to no change in the recruitment of F4/80-, CD80-, CD11b-, and Iba1-positive myeloid-derived mononuclear phagocytes to the site of laser photocoagulation in the absence of Bim expression. However, the resolution of these cells from the choroid of Bim-deficient (Bim -/-) mice was significantly diminished following laser photocoagulation. With time, we noted increased scar formation, demonstrated by collagen I staining, in Bim -/- mice with no change in the resolution of neovascularization compared to wild-type littermates. We also noted that mice lacking Bim expression in mononuclear phagocytes (BimFlox/Flox; Lyz2-Cre (BimMP) mice) had delayed resolution of F4/80-, CD80-, CD11b-, and Iba1-positive cells, while those lacking Bim expression in endothelial cells (BimFlox/Flox; Cad5-Cre (BimEC) mice) had delayed resolution of only CD11b- and Iba1-positive cells. Both BimMP and BimEC mice demonstrated increased scar formation, albeit to differing degrees. Thus, our studies show that resolving inflammation plays an important role in moderating scar formation in nAMD, and it is impacted by Bim expression in both the endothelium and mononuclear phagocyte lineages. [ABSTRACT FROM AUTHOR]

Abstract: A total of 493 does from 50 herds were measured to identify the different Jordanian goat breeds based on morphostructural characteristics. Different discriminant analysis methods (simple, cluster, canonical, stepwise) were applied on 20 metrical variables to discriminate among different genetic groups. Results identified four genetic groups: Damascus, Mountain, Dhaiwi and Desert in addition to a population of crossbred goats. Distribution of Mountain and Damascus breeds and the crossbred population was over the five clusters with varying frequencies. Dhaiwi and Desert breeds showed similar distribution of their populations over the clusters. All pair-wise Mahalanobis distances were significant (P < 0.001). Mountain goat breed tends to have the closest distance to all other native goats in Jordan. The largest distance was that between Dhaiwi and Damascus breeds (22.23). The dendrogram showed two large clusters: cluster one included Damascus breed as a large group and two sub clusters of Mountain breed and crossbred goats. Cluster two included Desert and Dhaiwi breeds. The canonical discriminant analysis and the stepwise discriminant analysis revealed that nose shape was the most discriminating variable among different pair-wise breeds’ comparisons, followed by withers height (WH) then body weight, ear type, color and teat placement. Chest width (CW), withers depth (WD), rump width showed small discriminatory power. Crossbred goats have been found to be derived mainly from Damascus and Mountain breeds, with more relation to the latter. [Copyright &y& Elsevier]

Retinopathy of prematurity (ROP) is a growing cause of lifelong blindness and visual defects as improved neonatal care worldwide increases survival in very-low-birthweight preterm newborns. Advancing ROP is managed by laser surgery or a single intravitreal injection of anti-VEGF, typically at 33–36 weeks gestational age. While newer methods of scanning and telemedicine improve monitoring ROP, the above interventions are more difficult to deliver in developing countries. There is also concern as to laser-induced detachment and adverse developmental effects in newborns of anti-VEGF treatment, spurring a search for alternative means of mitigating ROP. Pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor appears late in gestation, is undetected in 25–28 week vitreous, but present at full term. Its absence may contribute to ROP upon transition from high-to-ambient oxygen environment or with intermittent hypoxia. We recently described antiangiogenic PEDF-derived small peptides which inhibit choroidal neovascularization, and suggested that their target may be laminin receptor, 67LR. The latter has been implicated in oxygen-induced ischemic retinopathy (OIR). Here we examined the effect of a nonapeptide, PEDF 336, in a newborn mouse OIR model. Neovascularization was significantly decreased in a dose-responsive manner by single intravitreal (IVT) injections of 1.25–7.5 μg/eye (1.0–6.0 nmol/eye). By contrast, anti-mouse VEGFA 164 was only effective at 25 ng/eye, with limited dose-response. Combination of anti-VEGFA 164 with PEDF 336 gave only the poorer anti-VEGF response while abrogating the robust inhibition seen with peptide-alone, suggesting a need for VEGF in sensitizing the endothelium to the peptide. VEGF stimulated 67LR presentation on endothelial cells, which was decreased in the presence of PEDF 336. Mouse and rabbit eyes showed no histopathology or inflammation after IVT peptide injection. Thus, PEDF 336 is a potential ROP therapeutic, but is not expected to be beneficial in combination with anti-VEGF. • PEDF-derived 9-mer peptide, PEDF 336, previously shown to inhibit laser-induced CNV in mice was tested in OIR. • IVT injection of 1 μL PEDF 336 (1.25–7.5 μg) in neonatal mouse eyes at transition from 75% O2 to room air (P12) reduced NV and VO at P17. • While PEDF 336 displayed dose-responsive efficacy, anti-mouse VEGFA164 was active only at 25 ng/eye, but not at half or twice this dose. • Combination of both peptide and anti-VEGF showed only antibody efficacy but not robust peptide activity, thus the latter may require VEGF. • PEDF 336 contains a 5-mer epitope, Y- - - R, that binds laminin receptor, 67LR,which increased on choroidal EC after exposure to VEGF. [ABSTRACT FROM AUTHOR]

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-2Flox/Flox) and Pdgfrb-cre (Bcl-2PC mice) or Gfap-cre (Bcl-2AC mice). Bcl-2PC and Bcl-2AC mice demonstrated increased retinal vascular cell apoptosis, reduced numbers of pericytes and endothelial cells and fewer arteries and veins in the retina. Bcl-2PC mice also demonstrated delayed advancement of the superficial retinal vascular layer and aberrant vascularization of the deep vascular plexus and central retina. Although pathologic neovascularization in oxygen-induced ischemic retinopathy (OIR) was not affected by lack of expression of Bcl-2 in either pericytes or astrocytes, laser-induced choroidal neovascularization (CNV) was significantly reduced in Bcl-2PC mice compared to littermate controls. Together these studies begin to reveal how cell autonomous modulation of apoptosis in vascular cells impacts development and homeostasis. [ABSTRACT FROM AUTHOR]

Purpose: 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. [ABSTRACT FROM AUTHOR]