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Background: Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma., Results: Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve., Conclusions: We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

Vascular development in the embryo requires coordinated signaling through several endothelial cell-specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless beta-galactosidase (beta gal) gene behind the ang2 promoter, show constitutive beta gal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for beta gal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: To investigate the relationship between development of the perifoveal blood vessels and formation of the foveal depression., Methods: Retinal sections and flatmounts from monkeys aged between fetal day (Fd)80 and 2 years of age were double labeled using antisera to CD31 or von Willebrand factor to detect vascular endothelial cells and antiserum to glial fibrillary acidic protein to detect astrocytes. Sections were studied by fluorescence or confocal microscopy., Results: From Fd88 to 115, vessels on the horizontal meridian were found only at the level of the ganglion cell layer (GCL)-inner plexiform layer (IPL) border where they form the ganglion cell layer plexus (GCP). Stellate astrocytes accompany GCP vessels and extend closer to the fovea than vessels. The foveal avascular zone was present within the GCP at Fd101, and at Fd105 a shallow foveal depression encircled by the GCP was present. The GCP foveal margin had the same dimensions as the adult foveal pit. Both blood vessels and astrocytes were excluded from the emerging fovea throughout development. After Fd140, capillary plexuses in the outer retina anastomosed with the GCP on the foveal slope to form a perifoveal plexus, but this plexus did not mature until a month or more after birth. After Fd142, astrocytes rapidly disappeared from the GCP and most of central retina., Conclusions: An avascular area is outlined by the GCP before the foveal pit begins to form, suggesting that molecular factors in this region exclude both vessels and astrocytes. These factors may also guide neuronal migration to form the pit. Because the perifoveal plexus is formed during late gestation, both capillary growth and foveal development may be affected adversely by prematurity.

The unique vascular supply of the retina, the ability to visualize the vasculature in vivo, and the ability to selectively express genes in the retina make the retina an ideal model system to study molecular mechanisms of angiogenesis. In addition, this area of investigation has great clinical significance, because retinal and choroidal neovascularization are the most common causes of severe visual loss in developed countries and new treatments are needed. As a result, interest in ocular neovascularization is rapidly growing and there has been considerable recent progress. Use of genetically engineered mice in recently developed murine models provides a means to investigate the role of individual gene products in neovascularization in two distinct vascular beds, the retinal vasculature and the choroidal vasculature. It appears that angiogenesis in different vascular beds has common themes, but also has tissue-specific aspects. This review summarizes recent progress in the field of ocular neovascularization and the prospects that it provides for the development of new treatments., (Copyright 2000 Wiley-Liss, Inc.)

Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.

Combined thromboxane synthase inhibitors and thromboxane receptors antagonists have been shown to have a beneficial effect on different models of thrombosis in vivo. We studied the action of one of these compounds (DT-TX 30) compared with dazoxiben (a thromboxane synthase inhibitor) on retinal vascularity in streptozotocin-diabetic rats. Ten nondiabetic animals were treated with isotonic saline, 30 (10 animals per group) were given 0.4, 4, and 8 mg kg(-1) per day of DT-TX 30 (p.o.) and 30 (10 animals per group) were given 10, 50, and 100 mg kg(-1) per day of dazoxiben (p.o.) over a 90-day study period. DT-TX 30 caused a dose-dependent decrease of platelet aggregation and thromboxane B2 synthesis. There was an increase of 9, 65, and 166% in the synthesis of prostacyclin after treatment with 0.4, 4, and 8 mg kg(-1) per day, respectively. Retinal vascularity increased in 51, 72, and 182% of animals in response to the three doses used. Synthesis of prostacyclin and the degree of retinal vascularity showed a linear correlation (r2=0.6528,p<0.00001). Dazoxiben, at doses that inhibited thromboxane synthesis as much as DT-TX 30, increased prostacyclin production and retinal vascularity with less potency than DT-TX 30. In conclusion, the antagonism of thromboxane receptors may be an important additional effect to the inhibition of thromboxane synthase in the prevention of ischemic retinal lesions in experimental diabetes.

Glial cells in the CNS of vertebrates serve specialized functions in close interaction with surrounding neurons and blood vessels. In the avian eye, the neural tissue (retina) and the supporting vascular structure (pecten oculi) are spatially separated and comprise distinct glial cell types, i.e., the Müller glia and the pecteneal glia, respectively. In the present study we combined morphological and immunocytochemical investigations on the differentiation of the pecteneal glia in comparison to the retinal Müller glia, the retinal pigment epithelium, and the astrocytic cells of the optic nerve head in order to elucidate the nature, origin, and function of the pecteneal glia. Conventional transmission electron microscopy and freeze-fracture imaging revealed striking similarities between the pecteneal glia and retinal pigment epithelial cells at the transition zone to the optic nerve head. Immunofluorescence investigation identified specific labeling for vimentin and glutamine synthetase (GS) but not for glial fibrillary acidic protein (GFAP) in the mature pecteneal glia. Immunogold labeling confirmed the cellular specificity. GS labeling was weak during embryonic development but increasingly strong after hatching. Surprisingly, the intraneuroectodermal endothelial cells were highly immunopositive for GS throughout embryonic development and lost GS expression after hatching. GS expression in the pecteneal glia may participate in pH-regulation of the avian eye. Endothelial GS expression in the developing CNS may detoxify detrimental ammonium concentrations resulting from egg yolk degradation., (Copyright 1999 Wiley-Liss, Inc.)

Coats' disease is characterized by abnormal retinal vascular development (so-called 'retinal telangiectasis') which results in massive intraretinal and subretinal lipid accumulation (exudative retinal detachment). The classical form of Coats' disease is almost invariably isolated, unilateral and seen in males. A female with a unilateral variant of Coats' disease gave birth to a son affected by Norrie disease. Both carried a missense mutation within the NDP gene on chromosome Xp11.2. Subsequently analysis of the retinas of nine enucleated eyes from males with Coats' disease demonstrated in one a somatic mutation in the NDP gene which was not present within non-retinal tissue. We suggest that Coats' telangiectasis is secondary to somatic mutation in the NDP gene which results in a deficiency of norrin (the protein product of the NDP gene) within the developing retina. This supports recent observations that the protein is critical for normal retinal vasculogenesis.

In the absence of disease, the vasculature of the mammalian eye is quiescent, in part because of the action of angiogenic inhibitors that prevent vessels from invading the cornea and vitreous. Here, an inhibitor responsible for the avascularity of these ocular compartments is identified as pigment epithelium-derived factor (PEDF), a protein previously shown to have neurotrophic activity. The amount of inhibitory PEDF produced by retinal cells was positively correlated with oxygen concentrations, suggesting that its loss plays a permissive role in ischemia-driven retinal neovascularization. These results suggest that PEDF may be of therapeutic use, especially in retinopathies where pathological neovascularization compromises vision and leads to blindness.

Aims/hypothesis: The growth of retinal vessels is associated with a number of disease conditions, including diabetic retinopathy and proliferative vitreo-retinopathy. In this study we describe a model of human retinal angiogenesis and show how this may be used to explain the mechanisms that are associated with the growth of new retinal vessels., Methods: A 4 mm diameter disc of retinal tissue was placed within a fibrin matrix and the appearance was monitored daily by light microscopy. Immunohistochemical techniques were used for the detection of, glial fibrillary acidic protein, CD68, the Ki-67 antigen, vascular endothelial growth factor, monocarboxylate transporter type 1 and von Willebrand's factor., Results: Vessels were evident extending from the periphery of the explant and the activation of endothelial cells was shown by immuno-peroxidase staining of paraffin embedded sections of the explants for the expression of the Ki-67 antigen, a marker of cell proliferation. The expression of glial fibrillary acidic protein and von Willebrand's factor increased with duration in culture and the presence of activated macrophages or microglia or both was shown by positive immunoreactivity for CD68 and Ki-67 and were identified by day 3. The presence of endogenous vascular endothelial growth factor and the activation of monocarboxylate transporter type 1 by vascular endothelial growth factor, showed the involvement of specific growth factors., Conclusion/interpretation: The explant model provides evidence for the involvement of macrophages and glial fibrillary acidic protein activation in human retinal angiogenesis and for the expression of monocarboxylate transporter type 1, which is likely to be important in the use of lactate in the hypoxic retina.

Purpose: Hypoxia inducible factor-1 (HIF-1) is a transcription factor composed of HIF-1alpha and HIF-1beta subunits. HIF-1 transactivates multiple genes whose products play key roles in oxygen homeostasis, including vascular endothelial growth factor (VEGF). This study was designed to determine whether HIF-1 levels are increased in ischemic retina and whether there is a correlation with increased expression of VEGF., Methods: C57BL/6J mice were killed at time points that span retinal vascular development (PO to adult), or on postnatal day (P) 7 they were placed in a 75% oxygen environment for 5 days and then removed to room air and killed after 0, 2, or 6, or 24 hours and 5 or 14 days. Eyes were frozen, and retinas were isolated and used for immunoblot analysis, or eyes were sectioned for immunohisto chemical staining for HIF-1alpha or HIF-1beta, or for in situ hybridization for VEGF., Results: Immunoblots of retinal lysates showed low levels of HIF-1alpha at PO that were markedly increased at P4, remained high throughout the period of retinal vascular development and then decreased to an intermediate level in adults. HIF-1beta levels were relatively constant at all time points. In mice with oxygen-induced ischemic retinopathy, HIF-1alpha levels were increased in the retina. The peak of increase occurred at 2 hours, and levels returned to baseline by 24 hours. Immunohistochemistry showed increased staining for HIF-1alpha throughout the hypoxic inner retina, but not in the normoxic outer retina. There was no modulation of HIF-1beta levels. There was constitutive expression of VEGF mRNA in the inner nuclear layer that was increased 6 hours after the onset of hypoxia and remained elevated for several days., Conclusions: There are increased levels of HIF-1alpha in ischemic retina that show temporal and spatial correlation with increased expression of VEGF. These findings are consistent with the hypothesis that HIF-1 plays a role in upregulation of VEGF in ischemic retina.

Little is known about how the initial endothelial plexus is remodelled into a mature and functioning vascular network. Studying postnatal remodelling of the retina vasculature, we show that a critical step in vascular maturation, namely pericyte recruitment, proceeds by outmigration of cells positive for (alpha)-smooth muscle actin from arterioles and that coverage of primary and smaller branches lags many days behind formation of the endothelial plexus. The transient existence of a pericyte-free endothelial plexus coincides temporally and spatially with the process of hyperoxia-induced vascular pruning, which is a mechanism for fine tuning of vascular density according to available oxygen. Acquisition of a pericyte coating marks the end of this plasticity window. To substantiate that association with pericytes stabilizes the vasculature, endothelial-pericyte associations were disrupted by intraocular injection of PDGF-BB. Ectopic PDGF-BB caused the detachment of PDGF-beta receptor-positive pericytes from newly coated vessels, presumably through interference with endogenous cues, but had no effect on mature vessels. Disruption of endothelial-pericyte associations resulted in excessive regression of vascular loops and abnormal remodelling. Conversely, intraocular injection of VEGF accelerated pericyte coverage of the preformed endothelial plexus, thereby revealing a novel function of this pleiotropic angiogenic growth factor. These findings also provide a cellular basis for clinical observations that vascular regression in premature neonates subjected to oxygen therapy [i.e. in retinopathy of prematurity] drops precipitously upon maturation of retina vessels and a mechanistic explanation to our previous findings that VEGF can rescue immature vessels from hyperoxia-induced regression.

Growth factors involved in angiogenesis are critical to both the normal and pathological vascular development in the retina and choroid. In the present experiment, the relationship between the vascular endothelial growth factor (VEGF) expression and the retinochoroidal vasculogenesis in Sprague-Dawley rats was investigated using in situ hybridization and immunohistochemistry. It was found that VEGF was produced mainly by astrocytes and Muller cells in the neural retina, and this was correlated temporally and spatially with the retinal vasculogenesis. In addition, it was observed that, although the VEGF expression in the retinal pigment epithelium (RPE) decreased with increasing age, it persisted from the embryonic stage to adulthood. These findings indicate that the VEGF expression in RPE may play a role in the development of the choroidal vessels as well as in the maintenance of the normal structure and permeability of the choriocapillaris in adults.

Patterns of vascularisation were examined in whole-mounted retinae from tadpole stages to adulthood in the tree frog Litoria moorei using perfusion with Indian ink. Changing cell densities in the underlying ganglion cell layer were studied in a parallel Cresyl-stained series. Throughout development, the vasculature was pan-retinal and the hyaloid vessel was prominent. In early tadpole stages, capillaries were arranged as a honeycomb, and their number increased at a rate sufficient to maintain high densities in the face of increasing retinal area; major arteries and veins condensed within the capillary network. By early post-metamorphic life, the retinal vasculature was remodelled by the loss of four-fifths of the capillaries; the reduction in their density was far greater than could be accounted for by continuing retinal growth. This loss resulted in a change from the honeycomb appearance to one with largely parallel vessels linked by fewer connecting ones, an arrangement that became increasingly pronounced. In post-metamorphic life, the number of branch points increased such that their density decreased only slightly in the face of considerable increases in retinal area. The density of branch points varied across the retina and changed with age. Initially, the vasculature was most dense centrally, but by mid-larval life densities were highest in two patches located in the mid-temporal and mid-nasal retina. Thereafter, the vasculature increasingly assumed gradients resembling an area centralis and visual streak, a profile that survived the vascular remodelling. The development of density gradients in the vasculature preceded that of cells in the ganglion cell layer, the latter appearing only following metamorphosis. However, in post-metamorphic life, the topographies of the retinal vasculature and cells in the ganglion cell layer were closely related.

Purpose: To assess the role of astrocytes in controlling the growth of developing retinal vessels., Methods: Growth of retinal vessels in the neonatal rat retina was examined in three conditions: normal development, cyclic hyperoxia, and normoxia (1 day 70% to 75% oxygen, 1 day room air for up to seven cycles from birth, and room air for up to 16 days), and direct hypoxia (10% oxygen from postnatal day 3 [P3]). Retinas were examined as wholemounts labeled for astrocytes, microglia, and blood vessels and in some experiments for the fragmentation of DNA characteristic of apoptosis., Results: In normoxia, superficial retinal vessels formed to the processes of astrocytes. In cyclic hyperoxia, the depletion of superficial retinal vessels and subsequent neovascularization described by others were confirmed. The neovascularization was preceded by the depletion by apoptotic death of the astrocyte population, first between vessels but eventually breaching the glia limitans along vessels. The earliest forms of neovascularization resembled microaneurysms, each protruding through a defect in the glia limitans of a capillary. Neurons of the ganglion cell layer survived. Direct hypoxia from P3 caused hypertrophy of superficial vessels. Between P3 and P6, some vessels accelerated past the still-spreading astrocytes, often growing out of the retina into the vitreous humor. Direct hypoxia also caused astrocyte degeneration, but capillaries retained astrocyte investment and were not the site of vascular damage. By P8, breaches in the astrocytic glia limitans became prominent but were restricted to large veins. At such breaches, bleeding into the vitreous humor was common., Conclusions: Retinal vessels normally develop in close association with astrocytes. Where that association is broken, preretinal vessels may grow or bleed into the vitreous humor. Astrocytes play important roles in constraining retinal vessels to the retina and in maintaining their integrity.

Age-related morphological, ultrastructural and morphometric changes in the capillaries of the superficial and deep plexuses of the rat retina were studied in animals aged from 3 to 15 months. Our results suggest that age-related morphological alterations start occurring in the retina of rats at about 12 months of age. Increased glycogen deposits, pinocytotic vesicles, residual bodies and cell debris were observed in both the endothelial and pericytic cells of 12- and 15-month-old animals. In addition, heterogeneous osmiophilic accumulations, electron-transparent spaces were observed in the basement membrane as well as projections of the basement membrane towards the neighboring cells. Morphometric examination of the two vascular plexuses studied did not show differences in the area of the endothelial or pericytic cells, basement membrane or vascular lumen between rats of different ages.

Purpose: Raised arterial carbon dioxide levels have been suggested as a risk factor for retinopathy of prematurity (ROP). We investigated the effect of raised inspired CO2 on normal postnatal vasculogenesis in the neonatal rat retina., Methods: One hundred fifty newborn rat pups were divided among 15 mothers (n = 10 for each litter). Five litters were exposed to low CO2 (0.2%), 5 litters exposed to 6%, and 5 litters to 10% CO2. On day 7 of life the rats were sacrificed and the total retinal and vascularized retinal areas analyzed., Results: The vascularized retinal area and ratio of vascularized to total retinal area were reduced in rats exposed to 6% and 10% CO2., Conclusions: Raised inspired CO2 was associated with retardation of normal retinal vascular development and increased peripheral avascular area in neonatal rats. Raised CO2 may be a risk factor for the development of abnormal neovascularization such as in ROP.

Purpose: To determine the development of astrocytes and their vascular relations in Macaca monkey retina., Methods: Sections and wholemounts of retinas from fetal day (Fd) 65 to adult animals were analyzed immunohistochemically to detect glial fibrillary acidic protein (GFAP) and vimentin., Results: Astrocytes appeared first near the optic disc, then subsequently further peripherally, but avoided the fovea. In the nerve fiber layer, round and ovoid cells extended processes parallel to ganglion cell axons. In the ganglion cell layer, ovoid and stellate cells exhibited anisotropic processes or a honeycomb network. The inner lamina of astrocytes developed ahead of the outer lamina, and both reached their final positions before birth. Astrocytes lay more peripherally than did developing blood vessels, and the growing edge of nerve fiber layer vessels lay between the two astrocytic layers. Spindle cells, which may be vascular precursor cells, often aligned along linear astrocytic processes. Occasional spindle-shaped cells containing GFAP or vimentin were identified as immature glia. Astrocytes and blood vessels coincided regionally during development, but astrocyte processes were typically not in register with the meshwork of growing blood vessels. Astrocyte-vessel associations increased during fetal life and postnatally., Conclusions: During development, astrocytes display the same bilaminar pattern and morphologies present in adult retina. Astrocytes and blood vessels exhibit a similar regional distribution, but develop in distinct spatial patterns. Vessel investment by astrocytic processes increases during fetal life but is variable at all ages.

Angiogenesis is a complex biologic process that occurs normally in development and in turnover and remodeling of mature vascular networks. Pathological angiogenesis and neovascularization occur in association with retinal and ocular ischemic diseases, in retinopathy of prematurity and other developmental disorders, and in tumor growth and metastasis. We describe current understanding of cellular and molecular mechanisms of retinal vascular development, highlighting aspects that relate to eye diseases, that provide sites of therapeutic intervention in ophthalmology and that are potential avenues for research.

Purpose: To characterize developing retinal blood vessels with vascular markers and to relate the histochemical profile of maturing vessels to morphologic stages in retinal vascular development., Methods: Vessels were examined in frozen and paraffin-embedded retinas and in wholemounts of Macaca monkeys ranging in age from fetal day 75 (F75) to adulthood. Endothelial cells were visualized immunohistochemically using antisera to von Willebrand's factor and CD31 with lectins Ulex europaeus, Bandeiraea simplicifolia, peanut agglutinin, Ricinis communis, and wheat germ agglutinin, and by ATPase and ADPase enzymatic histochemistry. Antibodies to vascular basement membrane and matrix markers laminin, fibronectin, and collagen types I and VIII, and antisera recognizing cell cycle-specific nuclear proteins (cyclin, Ki-67, Mib-1) also were used., Results: Newly formed and mature vessels were reactive with reagents specific for CD31, von Willebrand's factor, types I and VIII collagens, laminin, fibronectin, U. europaeus, R. communis, and peanut agglutinin. Wheat germ agglutinin labeled vessels only after pretreatment with neuraminidase. All vascular markers appeared simultaneously, but some were distributed differentially between capillaries and larger vessels, along the central-peripheral extent of a vascular plexus, and among different vascular laminae. Markers of vessels failed to label spindle-shaped presumed vascular precursor cells lying peripheral to the advancing vessels during development. Spindle cells exhibited cyclin, Ki-67, and Mib-1 immunoreactivity., Conclusions: Immature and mature vitread and sclerad vessels displayed histochemical profiles that were qualitatively similar but that had subtle quantitative differences. Results do not support identification of spindle-shaped cells as vascular precursors in the developing monkey retina and are discussed in relation to mechanisms of retinal vascularization.

Vascular endothelial growth factor (VEGF) has been identified as an endothelial cell-specific mitogen with potent angiogenic properties. VEGF is overexpressed in pathologic angiogenesis observed in tumor growth, rheumatoid arthritis, and retinal angiogenic diseases such as diabetic retinopathy and retinopathy of prematurity. VEGF expression in physiologic angiogenesis, i.e., vasculogenesis, has also been reported in the embryonic organs such as brain, kidney, spleen, and lung. However, the details of VEGF expression in vasculogenesis remain largely unclear. To determine if VEGF contributes to vasculogenesis in the developing tissues, VEGF expression was studied by both immunohistochemistry and in situ hybridization in newborn rat retinas on postnatal days 3, 7, 14, and 30. Vasculogenesis was assessed by both the ink perfusion method and a histologic examination. To identify the cell types of VEGF-expressing cells, immunohistochemistry for cell markers such as glial fibrillary acidic protein and von Willebrand factor was performed. On postnatal days 3 and 7, when retinal vasculogenesis was active, VEGF mRNA and protein(s) were prominently expressed in the ganglion cell and the inner nuclear layers. In rats, as well as humans, these two layers are where the retinal vessels develop, and these two layers depend solely on the retinal vessels. In addition to the ganglion and the inner nuclear layers, VEGF protein(s) were located in the endothelial cells of the developing vessels and the angioblasts, i.e., endothelial precursors. On postnatal day 14, when vasculogenesis became inactive, VEGF mRNA expression markedly decreased. These results indicated that VEGF expression in the developing retinas is temporally and spatially correlated with retinal vasculogenesis.

Reducing equivalents for the electron transport chain are generated within the mitochondria by the Krebs cycle and in cytoplasm by processes like lipid metabolism. Two mitochondrial enzymes, succinate dehydrogenase (SDH), a prominent enzyme in the Krebs cycle, and alpha-glycerophosphate dehydrogenase (alpha-GPDH), half of the glycerophosphate shuttle system for bringing reducing equivalents from cytoplasm to mitochondria, were examined enzyme histochemically to assess the contribution of each to metabolism of the developing canine retina. SDH activity, a common marker for oxidative metabolism, was insignificant at birth. By 4 days of age, activity was observed only in developing photoreceptor inner segments. By 21 days of age SDH activity was present throughout the retina, especially in photoreceptor inner segments and plexiform layers, and approached the level observed in the adult dog. Menadione-linked alpha-GPDH (M-alpha-GPDH) activity, however, was prominent in developing vasculature and outermost portion of the neuroblastic layer of the 1 day-old retina. Most notable was localization in vascular precursors, angioblasts, found distant from formed vessels in the peripheral nerve fiber layer. Retinal dependence on an oxidative metabolism in neuronal elements, as represented by SDH activity, occurs only when the vasculature is well established.

Many animal models of retinal disease use the neonatal rat. Raising rat pups in large litters has been shown to result in postnatal growth retardation. We investigated the effect of litter size on the normal postnatal vascularization of the neonatal rat retina. Sixty-six newborn rat pups were divided among 5 nursing mothers into 3 small litters (n = 10) and 2 large litters (n = 18). On day 6 of life the rats were sacrificed and total retinal and vascularized retinal areas analyzed. The total retinal area was reduced in the rats raised in larger litters (28.6 mm2 vs. 25.9 mm2 p < 0.001) but there was a more pronounced reduction in vascularized retinal area (67% vascularized vs. 54% vascularized, p < 0.001). Postnatal vascularization of the normal rat retina may be influenced by litter size.

We have studied the role of the hypoxia-inducible angiogenic growth factor vascular endothelial growth factor (VEGF) in the induction and control of vessel growth in the developing retina of rats and cats, using in situ hybridization techniques. VEGF is expressed successively in two layers of neural retina, the innermost (axon) layer and the inner nuclear layer (INL). In the axon layer, VEGF is expressed transiently by astrocytes as they spread across the layer, closely preceding the formation of superficial vessels. In the INL, VEGF is expressed transiently by somas at the middle of the layer (presumably Müller cells), closely preceding the formation of the deep layer of retinal vessels. We propose that hypoxia caused by the onset of neuronal activity is detected by strategically located populations of neuroglia, first astrocytes, then Müller cells. In response they secrete VEGF, inducing formation of the superficial and deep layers of retinal vessels, respectively. As the vessels become patent, they relieve the hypoxic stimulus, so vessel formation is matched to oxygen demand. This hypothesis was tested experimentally in three ways. Expression of the high affinity flk-1 receptor for VEGF was demonstrated in newly formed retinal vessels, confirming that the secreted VEGF acts on the vessels, in a paracrine fashion. Direct hypoxic regulation of VEGF expression by macroglia was demonstrated in primary cultures of astrocytes and in cells of a glioma line. Hypoxic regulation of VEGF expression in the intact developing retina was demonstrated by showing that oxygen-enriched atmospheres that inhibit vessel formation also suppress endogenous VEGF production.

Purpose: To assess the role of oxygen in normal retinal vasculogenesis., Methods: A new preparation for studying cytogenesis in retinal wholemounts was developed. Nuclei of dividing cells were labeled with a monoclonal antibody to bromodeoxyuridine (BrdU), and vascular cells were covisualized with Griffonia simplicifolia lectin. The topography and time course of vasculogenic cell division and vessel formation were determined in the kitten retina during normal development and under experimental hyperoxia and hypoxia., Results: During normal development, vasculogenic cell division was maximal at the leading edge of the forming vessels. Normal vessel formation was initially proliferative, and cell division was high. However, after vessel formation occurred, which presumably relieved tissue hypoxia, the mitogenic process was markedly reduced, and many excess capillary segments underwent retraction. The rate of vasculogenic cell division and vessel formation increased when the inner layers of the retina were made avascular after exposure to hyperoxia, and it decreased when there was an increase in inspired oxygen., Conclusions: The authors have shown that between 17% and 45% oxygen, the extent of vasculogenic cell division is inversely proportional to the level of oxygen in the inspired gas mixture. They have further shown that dividing vascular cells have a peak density in a region proximal to the edge of the forming vasculature. The density is maximal between P7 and P8, a time when formation of photoreceptor outer segment begins, only a few days before the onset of retinal function. These results led the authors to conclude that the stimulus for normal vasculogenesis is a transient but physiological level of hypoxia induced by the increasing activity of retinal neurons.

Vasculogenesis is the formation of blood-vessels by differentiation of vascular precursor cells. Experiments using retinal models were designed to test the hypothesis that astrocytes influence this process by effects on the composition of the extracellular matrix. Retinal vasculogenesis was studied in relation to the migration of astrocytes and expression of the extracellular matrix proteins laminin and fibronectin by in vivo experiments in neonatal rats. The results show that astrocytes spread into the retina just ahead of the newly formed vessels, where they probably initiate vasculogenesis. They also establish that fibronectin, but not laminin, is expressed in the zone of vasculogenesis immediately prior to vessel formation. Increased amounts of fibronectin mRNA indicate that fibronectin is synthesized by cells within this same region during this same time period. Later, as the new vessels form, differentiation of endothelial cells is correlated with the appearance of pericytes in the vessel wall and laminin in the vascular basement membrane. In vitro experiments using conditioned medium approaches showed that astrocytes stimulate endothelial cell fibronectin expression. Taken together with the in vivo observations these in vitro results suggest that fibronectin expression is an essential component in the initiation of retinal vasculogenesis. This study is the first indication that astrocytes influence the fibronectin component of the extracellular matrix during retinal vasculogenesis and that expression of fibronectin precedes that of laminin in this process.

Purpose: The temporal and spatial sequence of development of laminar vascular plexuses was determined qualitatively and quantitatively in monkey and human retina., Methods: Histologic and cytochemical methods were used to study Macaca monkey eyes from fetal day 55 (F55d; birth = F168d) to 17 years, and human retina from fetal 21 weeks to adult., Results: In monkey retina, spindle-shaped, presumed vascular precursor, cells appear at F55d in the nerve fiber layer (NFL) adjacent to the optic nerve. The vascular plexuses in the NFL-ganglion cell layer appear first and form in the presence of spindle cells. Nerve fiber layer vessels extended radially to reach the temporal ora at F95d and nasal ora at F110d. The capillary plexus at the inner border of the inner nuclear layer (INL) appears at F120d near the optic disc, whereas the plexus at the outer INL border appears at F130d. Both reach their final position before birth. The INL plexuses form by endothelial budding from more vitread vessels in the absence of spindle cells. In the NFL, vessel growth to match retinal growth at the ora also involves endothelial budding. The growth rate of all plexuses was approximately 225 microns/day. The central fovea and the most peripheral retina adjacent to the ora serrata remained avascular throughout development. Differences between humans and monkeys include: Human vessels complete maturation after birth; human vessels reach the nasal ora earlier than the temporal ora; and spindle cells are more abundant and dispersed over a greater area within human NFL. Growth rates of human plexuses were comparable to those in monkeys., Conclusion: In both primates, deeper capillary plexuses form only by extension from existing vessels (angiogenesis). In the NFL, early vessel formation involves spindle precursor cells (vasculogenesis). The main difference between monkey and human in these processes is that the mature monkey vascular pattern is established well before birth.

In this study, regression of the hyaloid vessels has been followed in the tunica vasculosa lentis (TVL) of the Wistar rat using light, transmission and scanning electron microscopy. The investigation extended from the 1st to the 32nd postnatal day. On day one, the posterior tunica vasculosa lentis is made up of radiating capillaries connected by side-arm branches, the vascular walls of which possess a continuous endothelium, a basement membrane and an incomplete pericyte covering. Endothelial cell specialization is apparent in the form of extreme thinning and fenestration in capillary regions lying opposite the lenticular capsule. The earliest detectable regressive changes become apparent on approximately day 3 and initially involve the short connecting capillaries surrounding the posterior pole of the lens and proceed from there. Regression takes place in the presence of rarefaction of vessel wall cells and the accumulation of endothelial cells in the adjacent capillaries. This leads to the formation of acellular channels which are made up of only basement membrane tubes. After the complete disappearance of these transitional acellular channels, the capillary meshwork coarsens. Remnants of these capillaries are detectable until the 30th postnatal day.

Plasminogen activators (PAs) regulate a variety of processes involved in tissue morphogenesis and differentiation. We used a coculture system in which microvascular endothelial cells are induced by glial cells to form capillary-like structures in order to examine the role of urokinase-type PA (uPA) during microvessel morphogenesis within the central nervous system (CNS). Endothelia-derived uPA activity decreased sevenfold within glial-endothelial cocultures when capillary-like structures were formed. Incubation of cocultures with concentrations of phorbol 12-myristate 13-acetate (0.1 and 1.0 nM) that induced endothelial uPA activity (45-210%) inhibited endothelial differentiation (25-70%). Furthermore, incubation of cocultures with proteolytically active low molecular weight uPA (5-500 IU/ml) inhibited endothelial differentiation (37-75%), whereas the amino terminal cell-binding fragment of uPA had minimal effect. Inhibition of plasminogen activation in cocultures with the serine protease/plasmin inhibitors aprotinin and soybean trypsin inhibitor increased glia-induced capillary-like structure formation (96-98%). These findings establish a paracrine/autocrine function for urokinase and its inhibitors in regulating endothelial responses to perivascular glia and provide insight into mechanisms of microvascular reactions to CNS pathology.

The effect of the alpha-glucosidase inhibitor acarbose on retinal capillary basement membrane thickening was examined in the spontaneously diabetic BB/W-rat. Four months of diabetes resulted in significant thickening of the basement membranes of both the superficial and deep capillary nets of the retina. This characteristic change of the retinal microvasculature in diabetes was completely prevented by acarbose treatment that substantially reduced postprandial hyperglycemia. A similar but less pronounced effect was seen on the age-related increase in basement membrane thickening in acarbose-treated non-diabetic control rats who demonstrated decreased glycated hemoglobin levels compared to non-treated control rats. Significant positive correlations between basement membrane thickness and glycated hemoglobin area suggest that diabetic retinal microangiopathy may be prevented by lowering the cumulative glucose exposure to the microvasculature, and that age-related basement membrane thickening is mediated by long-term exposure to normal glucose levels.

A biologically active fluorescent derivative of recombinant human basic fibroblast growth factor (bFGF) was prepared by immobilization on heparin-Sepharose 4B (HS) and derivatization with the fluorophore, Texas Red (TR). TR-bFGF was separated from free dye and carrier protein by elution from HS using 1.5 M NaCl. TR-bFGF contained an average of two dye molecules bound per bFGF, retained its mitogenic activity and was visible using a fluorescence microscope equipped with silicon intensified target camera (SIT). TR-bFGF stimulated the growth of bovine aortic endothelial cells (BAEC), microvessel endothelial cells (MVEC) and BHK-21 cells grown in culture. BAEC, MVEC and BHK-21 cells treated with 20 ng ml-1 (1 nM) TR-bFGF for 72 hr were stimulated over serum controls by 87, 26 and 6%, respectively. TR-bFGF stimulated EC growth was inhibited in a dose-dependent fashion when cells were coincubated with microM chloroquine. When EC were treated with TR-bFGF at 4 degrees C and then monitored at 37 degrees C, bright, focal, cytoplasmic spots were observed, which accumulated as punctate, perinuclear fluorescence. EC internalization of TR-bFGF was inhibited 80% by the addition of 100-fold molar excess unlabeled bFGF or by maintaining cultures at 4 degrees C. TR-bFGF colocalized with an EC lysosomal marker, but TR-bFGF was not detected in the nucleus. Results of these localization studies suggest that TR-bFGF stimulates EC proliferation without entering the nucleus.

Rodent photoreceptor dystrophies are characterized by late stage ingrowth of retinal blood vessels into the retinal pigment epithelium (RPE) where they proliferate. Some of these vessels develop the fenestrated phenotype of the choriocapillaris (CC). To determine if development of fenestrae in these endothelial cells is a function of the duration of time the endothelial cell had been encapsulated by the RPE, we did an ultrastructural morphometric study of these vessels in urethane induced photoreceptor degeneration in Long-Evans rats. Retinas of animals aged 20, 24, 40 and 56 weeks were studied. The fraction of vessel profiles within the RPE that had fenestrated endothelial cells increased from 10% to 90% between 20 to 56 weeks. The average number of fenestrae per vessel increased approximately 25 fold between 20 and 24 weeks but stabilized after that, despite a decrease in the number of vessels present at 56 weeks. A large number of degenerated retinal vessel profiles were seen in the RPE at 40 weeks. These facts support the idea that the presence of the RPE induces endothelial cell fenestrae, and also show that a complex process of remodelling including proliferation and degeneration is occurring in these vessels. Analogies between the basic cell biology of neovascularization occurring in these rodent models and that of proliferative diabetic retinopathy and age-related macular degeneration are discussed.

Steroid hormones alter several aspects of microvascular function within the CNS. Both microvessel formation and blood-brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte-endothelial interactions, we studied their effects on astroglial-induced microvessel morphogenesis in vitro. C6 astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary-like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6-induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80-100% at 1 microM. Tetrahydrocortisone and 17 alpha-hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone-mediated inhibition. Astroglial cells were further implicated as the effectors of steroid-mediated inhibition because none of the steroids inhibited astroglial-independent capillary-like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor-mediated mechanism.

In the Multicenter Trial of Cryotherapy for Retinopathy of Prematurity (ROP), 4099 infants weighing less than 1251 g at birth underwent sequential ophthalmic examinations, beginning at age 4 to 6 weeks, to monitor the incidence and course of ROP. Overall, 65.8% of the infants developed ROP to some degree; 81.6% for infants of less than 1000 g birth weight. As expected, ROP incidence and severity were higher in lower birth weight and gestational age categories. Black infants appeared less susceptible to ROP, of all severity categories, than nonblack infants. The timing of retinal vascular events correlated more closely with postconceptional age than with postnatal age, implicating the level of maturity more than postnatal environmental influences in governing the timing of these vascular events. These results include the current incidence of various severity stages of ROP found in the United States and provide new insight into the development of ROP.

Presented is the personal material comprising cases treated surgically for primary retinal detachment; analysed is the causal connection between postoperatively occurring proliferative vitreoretinopathy and the risk factors. The comparison of personal results with results of the other authors shows that in case of diagnosis of risk factors the reduction of the surgical trauma reduces the threat of proliferative vitreoretinopathy after operation of retinal detachment.

Examined were 91 eyes with non-proliferative and proliferative retinopathy with coexisting maculopathy in 51 patients in whom segmental or retinal pan-photocoagulation were performed. The period of observation--3 to 4.5 years. Stabilization of the pathological process was observed in 37.3 p.c. of cases, 54.9 p.c. of patients showed further deterioration and 7.6 p.c. loss of vision. The most frequent cause of deterioration was the progression of the macular lesions (72 p.c.), a smaller number of cases (28 p.c.) was due to other changes.

Analysed were the results of surgical treatment, causes of the failure and early recurrence in 108 patients with retinal detachment in whom was performed an indentation of the sclera by means of a balloon (1st group--50) or by an episcleral implant (2d group--58). In the first group the apposition was achieved in 92 p.c., in the 2d group in 90 p.c. An early recurrence (2 weeks to 2 months) was observed in 9 eyes which were operated again achieving an apposition of the retina in 6 eyes. There were following causes of the failure: the existence of a second hole, rupture of the balloon, PVR before surgery and traction of the retina in the region of the hole. An early recurrence occurred in the consequence of intensification of the PVR together with inaccurate localization of the implant, existence of the PVR before the operation and traction of the retina in the vicinity of the hole, formation of a new hole.

Astrocytes migrate into the cat retina from the optic nerve, beginning from embryonic day (E) 52. Once they have entered the retina they concentrate along major axon bundles and fail to enter regions of the retina with high densities of neurones, in particular the area centralis region of the ganglion cell layer. These nonuniformities appear as the astrocytes spread over the retina during development, and in this study we have examined factors that might control their spread. First we examined astrocytes in a retina in which the axon bundles had degenerated following an optic nerve lesion at birth. The area over which astrocytes had spread was normal, suggesting that their spread does not depend on the presence of intact axons. Second, we noted that, despite the degeneration of all ganglion cells following the nerve lesion, astrocytes still did not spread over the area centralis. Their spread is apparently not inhibited by concentrations of neurones. Third, we examined astrocytes in retinas of animals raised in an atmosphere containing 70-80% oxygen, which prevents the formation of retinal vessels. Again, the area over which the astrocytes had spread was normal, suggesting that their spread does not depend on the presence of patent blood vessels. These negative findings led us to compare the distribution of spindle cells (precursors of retinal vasculature) and astrocytes in the cat during development. The close correspondence in their topographical distribution and the earlier spread of the spindle cells lead us to suggest that spindle cells provide a basal lamina component that may guide the migration of astrocytes.

During the recovery period after a high oxygen injury in the kitten, chronic hypoxia adversely affects the resulting retinopathy, but increasing oxygen breathing to 28% improves it. To test the effects of chronic hypoxia without an antecedent hyperoxic injury in the kitten, the animals were raised in 13% or 21% (room air) oxygen and their retinas examined at 3, 7, 14, or 21 days. They were also studied after 14 days of exposure to 30% or 40% oxygen to compare the graded effects of elevated oxygen to that of hypoxia on the development of the retinal capillary bed. Chronic hypoxia alone did not affect somatic growth or cause retinopathy. An inverse relationship was found between the rate of retinal vascularization and ambient oxygen. As inspired oxygen rose from 13%-40%, the proportion of the retina vascularized at 14 days fell from 76 +/- 12%-18 +/- 8% (mean +/- standard deviation, P less than 0.01). All 14-day animals had similar capillary density at the advancing edge of their retinal vasculature (mean diameter of the capillary meshwork = 71 +/- 12 microns) despite the impairment of forward progress observed in elevated oxygen. However, the width of the periarteriolar capillary-free zone increased from 65 +/- 10 microns in 13% oxygen-breathing animals to 104 +/- 5 microns in 40% oxygen-breathing animals (P less than 0.001). The animals raised in hypoxic conditions had more mature-appearing retinal vasculature at 21 days than did controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of the pecten oculi, a structure peculiar to the avian eye, was studied by scanning electron microscopy (SEM) correlated with light microscopy (LM) in embryonic and adult chickens. The development of the chick pecten was divided into 4 phases: (1) formation of the primordial pecten (Hamburger-Hamilton's stages 27 to 29), (2) formation of the plate-like pecten (stages 30 to 34), (3) pleat formation and pigmentation (stages 35 to 37), and (4) bridge formation and high-vascularization (stage 38 to adult). The primordial pecten is formed entirely from the ectoderm by fusion of the inwardly-projecting edges of the optic fissure. The primordial pecten grows into a tall, thin plate rising from a broad base. The pecten begins to fold slightly at stage 35. The number of pleats increases rapidly, from 7 at stage 35, to 16 at stage 36, 18 at stage 37, and 19 to 20 at stage 40. The bridge begins to form at stage 38 by a swelling of the apical edge of the pecten and completes its development by the twentieth post-hatch day. Blood vessels appear first in the broad base of the plate-like pecten, then become more numerous and gradually extend into the pleats. The pecten becomes more vascular than cellular at stage 43, and it is highly vascularized in the adult. The pleat surface becomes conspicuously irregular with increased vascularization. The peripectinate cells, located on the pecten, are already present at stage 27.

Two principal processes can be distinguished in the development of the retinal circulation in the cat. One process, which forms most of the inner layer of vasculature, involves three stages. First, beginning prior to E (embryonic day) 26, spindle cells of mesenchymal origin spread over the inner surface of the retina. Second, beginning at approximately E48, a network of coarse capillaries forms, apparently derived from spindle cells. Third, major vessels differentiate from the capillary plexus, and the capillaries become thinner and more widely spaced. All three stages begin at the optic disc and spread towards the margin of the retina. The other process involves budding of capillary sized vessels from existing vasculature. This process forms the inner layer of vasculature at the area centralis, the outer layer of vasculature, and the radial peripapillary capillaries. It begins between P (postnatal day) 7 and P10 at the area centralis and spreads to the margins of the retina. The radial peripapillary capillaries form at a later stage (P20). The different topographies of the two processes suggest that they are controlled by distinct mechanisms. In the first process, the formation of vessels follows a pattern set by the early migration of spindle cells. In the second process, the vessels form in a pattern determined by the metabolic needs of the developing retina.

27 eyes of 20 premature and full-term infants were subjected to trypsin digestion, and a study was made of the extension of the temporal retinal vessels and of the formation of the radial peripapillary capillaries (RPCs) and the capillary-free zone in the macula. The advancing borders of the temporal retinal vessels exhibited a V-shaped appearance in cases of 23-29 weeks in gestational age. The RPCs were observed in cases after the 27th week of gestation. The capillary-free zone in the macula was observed in cases after the 25th week of gestation. There were two eyes having extremely small and incomplete capillary-free zones: in one eye a vascular network was present in the region corresponding to the center of the macula. It was thought that the vascular network in the temporal horizontal aspect of the macula is formed by anastomosis of the superior and inferior temporal vessels, and that the capillary-free zone in the macula develops secondarily as the result of retraction of the capillaries.

Scanning electron microscopy of vascular casts was used to investigate three experimental models of neovascularization. In each experimental situation, the casts provided a valuable three dimensional representation of the newly formed blood vessels and permitted subclassification of the vessels within normal and proliferating vascular networks. They defined also the predominant origin of new vessels from venules and capillaries, and enabled the evolution of proliferating vessels into arterioles and venules to be documented. Although vascular casts must be interpreted with caution in light of the possibility of incomplete filling and other artifacts, they are a valuable tool in the study of ocular vasoproliferation.