Retinal Vascular Growth Research Articles

Mammalian Target of Rapamycin (mTOR) as a Potential Therapeutic Target in Pathological Ocular Angiogenesis.

Pathological ocular angiogenesis is a causative factor of retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. Vascular endothelial growth factor (VEGF) plays an important role in pathological angiogenesis, and anti-VEGF agents have been used to treat the ocular diseases that are driven by pathological angiogenesis. However, adverse effects associated with the blockade of VEGF signaling, including impairments of normal retinal vascular growth and retinal function, were suggested. Therefore, the development of a safe, effective strategy to prevent pathological ocular angiogenesis is needed. Recent studies have demonstrated that inhibitors of the mammalian target of rapamycin (mTOR) target proliferating endothelial cells within the retinal vasculature. Here, we review the potential of targeting the mTOR pathway to treat pathological ocular angiogenesis.

Exposure to high-concentration oxygen in the neonatal period induces abnormal retinal vascular patterning in mice.

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high-concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration-dependent manner. In retinas of hyperoxia-exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short-term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.

Abstract 177: Adenosine Receptor 2A-mediated Endothelial Glycolysis Critically Contributes to Pathological Angiogenesis of Retina

Pathological proliferative retinopathy is the most common cause of blindness in patients of all ages worldwide. Investigation of the molecular mechanisms underlying pathological retina neovascularization is critical to the development of new therapeutic strategies. In the current study, we investigated the effect of macrophage or endothelial cell adenosine receptor 2A (A 2A R) deletion in pathological retinal vascular growth, and further explored the potential mechanism. Our results showed that: (1) A 2A R expression was localized to retinal vessels and was markedly induced in pathological neovascularization tufts in a mouse model of oxygen-induced proliferative retinopathy (OIR) (Figure A); (2) endothelial cell, but not macrophage, -specific A 2A R deletion significantly decreased pathological neovascularization in the mouse OIR model (Figure B);. The levels of glycolytic enzymes, angiogenic factors and lactate secretion were markedly reduced in retina of endothelial cell-specific A 2A R knockout mice; (3) in human retinal microvascular endothelial cells (HRMECs), hypoxia markedly induced the expression of A 2A R, but not A 1 R, A 2B R and A 3 R, by activating HIF-2α. Deletion or blockade of A 2A R by small interfering RNA (siRNA) or antagonist ZM241385 decreased hypoxia or adenosine-induced glycolytic enzyme expression, lactate secretion and cell proliferation via suppression of the cAMP/PKA/CREB/HIF-1α pathway; (4) blockade of glycolysis by 3PO or 2-DG abolished the adenosine-induced HRMEC proliferation under hypoxic conditions. Taken together, we have demonstrated a critical role of endothelial cell A 2A R in promoting pathological vascular development and revealed a novel glycolysis-driven mechanism for endothelial cell A 2A R-mediated pathological angiogenesis in proliferative retinopathy. Thus, A 2A R is a promising therapeutic target in the treatment of proliferative retinopathy.

Short-term treatment with VEGF receptor inhibitors induces retinopathy of prematurity-like abnormal vascular growth in neonatal rats

Retinal arterial tortuosity and venous dilation are hallmarks of plus disease, which is a severe form of retinopathy of prematurity (ROP). In this study, we examined whether short-term interruption of vascular endothelial growth factor (VEGF) signals leads to the formation of severe ROP-like abnormal retinal blood vessels. Neonatal rats were treated subcutaneously with the VEGF receptor (VEGFR) tyrosine kinase inhibitors, KRN633 (1, 5, or 10 mg/kg) or axitinib (10 mg/kg), on postnatal day (P) 7 and P8. The retinal vasculatures were examined on P9, P14, or P21 in retinal whole-mounts stained with an endothelial cell marker. Prevention of vascular growth and regression of some preformed capillaries were observed on P9 in retinas of rats treated with KRN633. However, on P14 and P21, density of capillaries, tortuosity index of arterioles, and diameter of veins significantly increased in KRN633-treated rats, compared to vehicle (0.5% methylcellulose)-treated animals. Similar observations were made with axitinib-treated rats. Expressions of VEGF and VEGFR-2 were enhanced on P14 in KRN633-treated rat retinas. The second round of KRN633 treatment on P11 and P12 completely blocked abnormal retinal vascular growth on P14, but thereafter induced ROP-like abnormal retinal blood vessels by P21. These results suggest that an interruption of normal retinal vascular development in neonatal rats as a result of short-term VEGFR inhibition causes severe ROP-like abnormal retinal vascular growth in a VEGF-dependent manner. Rats treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms underlying the development of plus disease.

Characterizing spatial distributions of astrocytes in the mammalian retina.

In addition to being involved in retinal vascular growth, astrocytes play an important role in diseases and injuries, such as glaucomatous neuro-degeneration and retinal detachment. Studying astrocytes, their morphological cell characteristics and their spatial relationships to the surrounding vasculature in the retina may elucidate their role in these conditions. Our results show that in normal healthy retinas, the distribution of observed astrocyte cells does not follow a uniform distribution. The cells are significantly more densely packed around the blood vessels than a uniform distribution would predict. We also show that compared with the distribution of all cells, large cells are more dense in the vicinity of veins and toward the optic nerve head whereas smaller cells are often more dense in the vicinity of arteries. We hypothesize that since veinal astrocytes are known to transport toxic metabolic waste away from neurons they may be more critical than arterial astrocytes and therefore require larger cell bodies to process waste more efficiently. A 1/8th size down-sampled version of the seven retinal image mosaics described in this article can be found on BISQUE (Kvilekval et al., 2010) at http://bisque.ece.ucsb.edu/client_service/view?resource=http://bisque.ece.ucsb.edu/data_service/dataset/6566968.

The effect of blue light on human retinal pigment epithelium cells α1D subunit protein expression and vascular endothelial growth factor and basic fibroblast growth factor secretion in vitro

To investigate the effect of blue light on human retinal pigment epithelium (RPE) α1D subunit protein expression and its relationship with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion in vitro. The fourth generation cultured human RPE cells in vitro were randomly divided into 4 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+nifedipine), group D [exposure to blue light+(-)Bay K8644]. Cells were exposed to blue light (2 000 ± 500) lx for 6 hours, and cells culture completed 24 hours later. VEGF and bFGF concentration were assayed by enzyme linked immunosorbent assay (ELISA). Real-time polymerase chain reaction was used to analysis L-type calcium channel α1D subunit mRNA expression. Western blot was used to examine the protein expression of L-type calcium channel α1D subunit. Analysis of variance was used to compare the difference of α1D subunit mRNA and protein expression, VEGF and bFGF concentration between groups. Correlation analysis was used to show the relationship between α1D subunit protein expression and concentration of VEGF and bFGF. (1) There is significant statistically difference in the population mean of VEGF and bFGF concentration in four groups (F = 99.441, 21.310, P = 0.000,0.000) . VEGF and bFGF concentration in group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) and group D (3 808.01 ± 94.01, 1 485.82 ± 108.97) was higher than it was in group A (2 401.09 ± 228.07, 1 232.42 ± 65.41) , which was statistically different (P = 0.000, 0.000, 0.019, 0.000). And it was higher in group D (3 808.01 ± 94.01, 1 485.82 ± 108.97) compared with group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) (P = 0.000, 0.006). While, it was lower in group C (1 927.28 ± 143.11, 1 149.39 ± 62.99) than it was in group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) (P = 0.000, 0.000) . (2) The mean of mRNA expression of α1D subunit between four groups was statistically significant (F = 50.320, P = 0.000) . It was higher in group B (210 ± 23) , group D (478 ± 80) and group C (232 ± 14) than group A (100 ± 20). It was statistically significant different (P = 0.023, 0.006, 0.010). And it was higher in group D (478 ± 80) than group B (210 ± 23) and C (232 ± 14) (P = 0.032, 0.039). (3) There was statistically significant difference in the expression of L-type calcium channel α1D subunit protein in four groups (F = 1 940.363, P = 0.000) . It was significantly higher (P = 0.000,0.000) in group B (0.974 2 ± 0.014 7) and group D (0.654 9 ± 0.005 0) than group A (0.503 2 ± 0.007 5). And it was higher in group D (0.654 9 ± 0.005 0) than it was in group C (0.413 9 ± 0.008 8) (P = 0.000). (4) There was positive correlation between the L-type calcium channel α1D subunit protein expression and VEGF concentration secreted by retinal pigment epithelium cells (r = 0.674, F = 8.333, P = 0.016), but there was no correlation with bFGF concentration (r = 0.537, F = 4.061, P = 0.072). L-type calcium channel α1D subunit may be involved in blue light induced damage on human retinal pigment epithelial cells. Blue light exposure can induce the mRNA and protein expression of α1D subunit, VEGF and bFGF concentration in retinal pigment epithelium cells Increased. And there was a positive correlation between α1D subunit protein expression and the VEGF concentration.

Risk factors and rate of progression for zone I versus zone II type 1 retinopathy of prematurity

To compare the risk factors and rate of progression of zone I versus zone II type 1 retinopathy of prematurity (ROP). The medical records of consecutive preterm infants with bilateral type 1 ROP in zone I and age-matched control infants with type 1 ROP in zone II were retrospectively analyzed. Fundus findings at each screening examination and systemic parameters were compared between groups. Univariate and conditional multivariate regression analyses were employed to identify variables significantly associated with zone I ROP. A total of 30 cases and 30 controls were included. The mean gestational age of included infants was 24.6 weeks in both groups, and the mean birth weights were 685 g in the zone I group and 667 g in the zone II group. The postmenstrual age (PMA) at the time of initial ROP detection did not differ between groups, but the PMA at the time of type 1 ROP detection was significantly earlier in the zone I group (mean, 34.9 vs 37.6 weeks). Conditional multiple logistic regression revealed that mechanical ventilation for 30 days or more was significantly associated with the type 1 ROP in zone I compared with zone II (OR, 3.5; 95% CI, 1.2-10.0). Zone I ROP exhibited rapid progression, necessitating close monitoring and prompt treatment. Compromised pulmonary function with associated mechanical ventilation in early life may restrict retinal vascular growth and increase the likelihood of zone I type 1 ROP.

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.

Anti-angiogenic effects of mammalian target of rapamycin inhibitors in a mouse model of oxygen-induced retinopathy.

Ocular pathologic angiogenesis is a causative factor for retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. In the present study, we examined the effects of rapamycin and everolimus, inhibitors of mammalian target of rapamycin (mTOR), on retinal pathologic angiogenesis in mice with oxygen-induced retinopathy (OIR), an animal model of proliferative ischemic retinopathy. Mice were exposed to 80% oxygen from postnatal day (P) 7 to P10, and were then brought into room air and subcutaneously injected with rapamycin and everolimus. The neovascular tufts, the size of the central avascular zone, and the immunoreactivity for phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTOR activity, were evaluated in flat-mounted retinas. Retinal neovascular tufts and vascular growth in the avascular zone were observed in P15 mice with OIR. In addition, intense immunoreactivity for pS6 was detected in the neovascular tufts and in endothelial cells located at the vascular-avascular border. Both rapamycin and everolimus reduced the extent of retinal neovascular tufts and pS6 immunoreactivity, but they also increased the size of the avascular zone. Thus, activation of the mTOR pathway in endothelial cells contributes to retinal pathologic angiogenesis, and mTOR inhibitors that target proliferating endothelial cells are promising candidates as anti-angiogenic agents for the treatment of vasoproliferative retinal diseases.

Treatment of newborn mice with inhibitors of vascular endothelial growth factor receptor tyrosine kinase induces abnormal retinal vascular patterning.

We have previously reported that treatment of newborn mice with KRN633, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, delayed retinal vascularization leading to abnormal retinal vascular growth and patterns. To determine whether similar abnormalities are observed in newborn mice treated with other VEGF receptor tyrosine kinase inhibitors, we administered axitinib to mice on the day of birth and on the following day. When compared with control pups, a significant delay in retinal vascularization was observed in pups treated with axitinib (5 mg/kg). Axitinib-treated pups had a very dense capillary network on postnatal day (P) 6 and fewer central arteries and veins on P8 and P12. Central veins, but not arteries, were significantly enlarged on P8. These abnormalities were similar to those observed in KRN633-treated pups and probably represent a common phenotype induced by short-term treatment with VEGF receptor inhibitors in newborn mice. Therefore, mice treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms of retinal vascular formation and patterning.

Expression and significance of thrombonspondin-1 in oxygen-induced retinopathy in mice

To examine the expression and function of thrombonspondin-1 (TSP-1) in oxygeninduced retinopathy in new-born mice, and to investigate its role in retinal neovascularization. A total of 40 C57BL / 6J newborn mice were divided equally into a model group (n=20) and a normal control group (n=20). Mice in the model group were exposed to 75% oxygen to establish the oxygen-induced retinopathy (OIR) model. On the 7th, 9th, and 11th day after the birth of mice, 5 mice were randomly selected each time from the 2 groups to examine the expression of TSP-1 mRNA with reverse transcription polymerase chain reaction (RT-PCR). After that, 5 mice were selected on the 11th day to observe the retinal neovascularization by fluorescein angiography retinal flatmount. On the 11th day, fluorescein angiography retinal flatmount showed that the retinal blood vessels presented mean network distribution in the normal control group, while in the model group, a lot of dilatated areas in the retinal main vessels surrounded the optic disc. Meanwhile lots of new blood vessels were found surrounding the optic disc with irregular distribution but well distributed peripheral retinal small vessels, which was typical of early stage OIR. There was no significant difference in the retinal TSP-1 mRNA level between the model group and the normal control group in the postnatal 7-day mice (P>0.05). Compared with the normal control group, the expression of TSP-1 mRNA in the model group was significantly lower in postnatal 9-day and 11-day mice (P<0.05, P<0.01) , and the expression of TSP-1 mRNA in postnatal 9-day mice was lower than that in the postnatal 11-day mice(P<0.05). In the early stage of OIR model (retinal vascular growth and development stage), the expression of TSP-1 mRNA in the retinal tissue is gradually decreased, implying that TSP-1 (as a negative regulatory factor) may be involved in the formation of retinal neovascularization in the early stage.

Kindliche Ablationes: ROP und Myopie

This article reviews the cause and treatment options for retinopathy of prematurity (ROP)-related tractional detachment and detachments in ROP residuals during childhood and early adulthood. Retinal vascularisation is incomplete after premature birth. Phase I of ROP consists of a delayed retinal vascular growth and vessel loss after premature birth resulting in hypoxia, phase II results in hypoxia-induced vascular proliferation and as a consequence to vitreoretinal traction. The anatomic and functional outcome of tractional detachment in ROP is determined by the previous treatment (e.g., laser to the avascular periphery or anti-VEGF). While the literature reports re-attachment rates > 70 % in ROP IV a, functional and anatomic outcome in the later stages is limited. ROP residuals may cause rhegmatogenous rather than tractional detachments in childhood or early adulthood. Myopia is associated with ROP and may further complicate the retinal situation and the risk for rhegmatogenous detachment. The retinal changes due to ROP warrant lifelong controls.

The effect of Delta-like ligand 4 monoclonal antibody on retinal neovascularization and vascular endothelial growth factor expression

Background Studieshowed thaDelta-like ligand 4 (Dll4) participatein the deveopmenof retinal celland angiogenesis.The Dll4-Notch pathway and vasculaendothelial growth facto(VEGF) are thoughto be critical mediatorof neovascularization undehypoxiconditions.The relationship between Dll4 and VEGF inovery cleaand furtheresearch ineeded.Objective Thistudy wato observe the inhibition of Dll4 on experimental retinal neovascularization and VEGF expression.MethodThe retinal neovascularization animal model wainduced by oxygen-induced retinopathy (OIR) in 5-day-old SPF SD ratby rearing the new postnatal ratwith the motherattogethein closed box with oxygen level a(80±2) ％ till 12-day-old.The ratwere then raised in normal aifo5 days.Aftethat,2.5μl (0.5 μg) of Dll4 monoclonal antibody wainjected into the mid-vitreoucavity in the righeye(Dll4 injected group) and PBwaused in the same way in the fellow eye(PBcontrol group) in the 12-day-old rats.Retinawere isolated in the 17-day-old rats,and retinal vasculamorphology waexamined by adenosine diphosphatease (ADPase) staining of retinal flatmounts,and the endotheliocyte nuclei above the internal limiting membrane were counted in the retinal tissue-slices.Reverse transcription PC(RT-PCR) waused to detecthe mRNexpression level of Dll4,VEGF,VEGF receptor-1 (VEGFR-1),VEGFR-2 and neuropilin-1 mRNin the retinas.Statistical analysiwaperformed by the paired t-test.The care and use of the animalcomplied with the Guidance Suggestion issued by the Ministry of Science and Technology of Chinin 2006.ResultThe Dll4 mRNexpression in the retin(Dll4 mRNA/β-actin mRNA) wa0.22± 0.06 and 0.98 ± 0.13 in the Dll4 injected group and the PBcontrol group,respectively,with statistically significandifference (=21.839,P =0.000).No significandifferencewere found in the expression of the VEGF mRNA,VEGFR-1 mRNand VEGFR-2 mRNin the retinabetween the two group(t=0.463,P=0.649;=1.687,P=0.109;=-1.674,P=0.111).Compared with the PBcontrol group,the expression of neuropilin-1 mRNwasignificantly elevated in the Dll4-injected group (0.73±0.08 vs.0.64±0.07) (t=-2.677,P=0.015).ADPase staining showed thathere were much more new blood vesselin the Dll4 injected group than those of the PBcontrol group.The numbeof nuclei structurally adjacento the vitreal side of the internal limiting membrane wa(63.6± 11.6)/slide in the Dll4 injected group,which wamore than thaof the PBcontrol group a(35.1±5.2)/slide (=-7.879,P =0.000).ConclusionDll4 playan essential role in the procesof pathological angiogenesiin the retina.Dll4 ithoughto be feedback regulatoof VEGFR,which participatein the procesof restraining pathological vasculogenesis. Key words: Delta-like ligand; Vascular endothelial growth factor; Retina; Oxygen-induced retinopathy; Neovascularization

Wnt Signaling Mediates Pathological Vascular Growth in Proliferative Retinopathy

Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor-related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5(-/-) vessels. Blocking claudin5 significantly suppresses Wnt pathway-driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.

The Roles of Vitreal Macrophages and Circulating Leukocytes in Retinal Neovascularization

To analyze the roles of vitreal macrophages and circulating leukocytes in retinal vascular growth. Bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P)1 mice after irradiation. The mice were exposed to 76% to 78% oxygen (P7-P12), to initiate oxygen-induced retinopathy (OIR). The eyes were collected at P8, P17, and P30, to analyze the engraftment of GFP-positive cells in the retina. GFP-positive peritoneal macrophages, clodronate liposomes, or control liposomes were injected into the eyes at P5 or P12 to examine the effects at P8 or P17. The number of Iba1-positive vitreal macrophages was quantified from histologic sections at P12 and P17. Few transplanted GFP-positive cells were found in the retina at P8 in both wild-type and OIR mice. However, their number increased at P17 during retinal neovascularization in OIR. Most GFP-positive cells were Iba1-positive microglia, which comprised a minority of the total retinal microglia. Intravitreal injection of peritoneal macrophages showed only incidental migration of these cells into the wild-type retinas (P8), whereas the engraftment was more robust, typically around the neovascularization, in OIR mice (P17). Furthermore, native macrophages in the vitreous cavity became fewer (37.7% reduction) during neovascularization in OIR at P17. The selective depletion of vitreal macrophages by clodronate liposomes at P12 reduced retinal neovascularization in OIR mice by 59.0% at P17. Vitreal macrophages are attracted to the site of pathologic angiogenesis triggered by retinal ischemia, where they actively participate in vascular development.

Regulation of Pathologic Retinal Angiogenesis in Mice and Inhibition of VEGF-VEGFR2 Binding by Soluble Heparan Sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.

Effect of Ethylene Diurea on Oxygen-induced Ischemic Retinopathy in the Neonatal Rat

This study investigated the effect of N[2(2-oxo-1-imidazolidinyl) ethyl]-N-phenylurea (ethylene diurea, EDU) on oxygen-induced ischemic retinopathy (OIR) in a neonatal rat model. OIR was induced by maintaining daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and a progressive return to 80% oxygen (3 h) for 12 days (postnatal day : P12). The rats were treated intraperitoneally with EDU (30 mg / kg body weight) or distilled water (DW) from P6 to P17. At P18, the percentage of avascular areas in the total retinal area (%AVA) was measured, and retinal neovascularization (NV) was scored in ADPase-stained retinas. Retinal superoxide dismutase (SOD) activity in the retina was also determined by a chemiluminescence method. The mean %AVA in the EDU-treated group (9.3±1.7%, n= 16) was lower than in the DW group (18.2 ± 4.7%, n = 17). EDU did not signi cantly affect NV, but significantly increased SOD activity (1.36±0.13 units / mg protein, n = 4) compared to DW treatment (1.04±0.01 units / mg protein, n = 4, P= 0.032) at P18. These results suggest that EDU treatment decreased the %AVA, accompanied by an increase in normal retinal vascular growth and / or a decrease in vessel proliferation. The increased SOD activity observed in the present study is likely to involve the EDU-mediated effects.

Retinopathy of prematurity

Retinopathy of prematurity (ROP) is a common blinding disease in children in the developed world despite current treatment, and is becoming increasingly prevalent in the developing world. ROP progresses in two phases. The first phase begins with delayed retinal vascular growth after birth and partial regression of existing vessels, followed by a second phase of hypoxia-induced pathological vessel growth. Two major risk factors of ROP are the use of oxygen and a decreased gestation period. Excessive oxygen contributes to ROP through regulation of vascular endothelial growth factor (VEGF). Suppression of VEGF by oxygen in phase I of ROP inhibits normal vessel growth, whereas elevated levels of VEGF induced by hypoxia in phase II of ROP precipitate pathological vessel proliferation. Insulin-like growth factor 1 (IGF-1) is a critical non-oxygen-regulated factor in ROP. We have found that serum levels of IGF-1 in premature babies directly correlate with the severity of clinical ROP. IGF-1 acts indirectly as a permissive factor by allowing maximal VEGF stimulation of vessel growth. Lack of IGF-1 in preterm infants prevents normal retinal vascular growth in phase I of ROP, despite the presence of VEGF. As infants mature, rising levels of IGF-1 in phase II of ROP allows VEGF stimulated pathological neovascularization. These findings suggest that restoration of IGF-1 to normal levels might be useful in preventing ROP in preterm infants.

Postnatal Head Growth Deficit Among Premature Infants Parallels Retinopathy of Prematurity and Insulin-like Growth Factor-1 Deficit

We hypothesized that in premature infants, retinal vascular growth retardation between birth and postmenstrual age of approximately 30 to 32 weeks that initiates retinopathy of prematurity is paralleled by brain growth retardation. In a prospective longitudinal study, we measured postnatal head growth, retinopathy of prematurity stage, protein and energy intake, severity of illness and serum insulin-like growth factor-1 levels in 58 preterm infants (mean gestational age at birth: 27.6 weeks) from birth until postmenstrual age of approximately 40 weeks. Premature infant head growth decelerates dramatically after birth until postmenstrual age of approximately 30 weeks. Head growth retardation coincides with retinal vascular growth suppression. Accelerated growth follows between post menstrual ages of approximately 30 to 32 weeks and approximately 40 weeks. The degree of head growth retardation up to postmenstrual age of 31 weeks corresponds to the degree of retinopathy of prematurity and to the degree of suppression of serum levels of insulin-like growth factor-1. At postmenstrual age of 31 weeks, if a child's head circumference SD is below -2.5, then the probability of also developing at least stage 3 retinopathy of prematurity increases fivefold compared with head circumference above -2.5 SD (32% vs 6%) suggesting parallel processes in brain and retina. Serum insulin-like growth factor-1 levels correlate positively with head circumference SD score and with the degree of retinopathy of prematurity. The correlation between head and retinal growth is consistent with insulin growth factor-1 being one of the postnatal growth factors involved in this multifactorial process and also suggests that factors that contribute to retinopathy of prematurity during this critical period may also affect neurological dysfunction. Additional studies are required to establish this connection.

Recommandations pour la pratique clinique : Standards, Options et Recommandations 2003 pour le traitement médical de première ligne des patientes atteintes de tumeurs épitheliales malignes de l'ovaire, mise à jour (rapport abrégé)

Retinopathy of prematurity (ROP) is a major cause of blindness in children in developed countries. ROP is a two-phase disease, beginning with delayed retinal vascular growth after premature birth (Phase I). Phase II follows when Phase I-induced hypoxia releases factors to stimulate new blood vessel growth. Both oxygen-regulated and non-oxygen-regulated factors contribute to normal vascular development and retinal neovascularization. Vascular endothelial growth factor (VEGF) is an important oxygen-regulated factor. A critical non-oxygen-regulated growth factor is insulin-like growth factor-I (IGF-I). In knockout mice, lack of IGF-I prevents normal retinal vascular growth, despite the presence of VEGF, important to vessel development. In vitro, low IGF-I levels prevent VEGF-induced activation of Akt, a kinase critical for vascular endothelial cell survival. We found that premature infants who develop ROP have low levels of serum IGF-I compared to age-matched infants without disease. IGF-I is critical to normal vascular development. Low IGF-I predicts ROP in premature infants, and restoration of IGF-I to normal levels might prevent ROP.