Normal Retinal Vascular Development Research Articles

Lama1 mutations lead to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice

BackgroundValuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants.ResultsAlthough astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy.ConclusionsLama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.

Épidémiologie et physiopathologie de la rétinopathie du prématuré

Retinopathy of prematurity (ROP) is a major cause of visual impairment in premature infants. It is characterized by an arrest in normal retinal vascular development associated with microvascular degeneration, followed by an abnormal hypoxiainduced neovascularization. Recent studies point out that ROP is a multifactorial disease, implicating both oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent factors leading to microvascular degeneration include generation of reactive oxygen species and suppression of specific oxygen-regulated vascular survival factors, such as vascular endothelial growth factor (VEGF) and erythropoietin. The other major mechanism for the initial capillary loss is oxygen-independent and implicates a deficit in growth factor IGF-1/IGFBP3. The proliferative, second phase of ROP is triggered by increases in vascular growth factors concentrations, in an attempt to compensate for the hypoxic retina. Novel signaling pathways for vascular repair, implicating both metabolite signaling and inflammatory lipids signaling, represent new therapeutic avenues for ROP.

Effect of VEGF Trap on Normal Retinal Vascular Development and Oxygen-Induced Retinopathy in the Dog

Purpose. To evaluate the effects of a vascular endothelial growth factor trap (VEGF Trap) on retinal vascular development and pathologic neovascularization (NV) in the canine model of oxygen-induced retinopathy (OIR). Methods. Newborn dogs (postnatal day [P]1) were exposed to 100% O(2) and then returned to room air on P5. VEGF Trap (5, 25, or 250 μg) was injected intravitreally in one eye and human FC (hFc) injected in the fellow eye of air control and oxygen-treated dogs on P8. The retinal vasculature and NV were evaluated on P21. Other oxygen-exposed animals received 5 μg of VEGF Trap or hFc on P22 after confirmation of retinopathy of prematurity (ROP)-like pathology and were evaluated at P45. Results. In air controls, both the vascularized area of the retina and the density of superficial capillaries were reduced in 250 or 25 μg VEGF Trap-injected eyes, and deep capillaries were absent. Eyes that received the 5 μg dose were indistinguishable from controls. In oxygen-treated animals, all eyes injected with VEGF Trap exhibited markedly less intravitreal NV than that of hFc-injected fellow eyes, irrespective of dose. Retinal vascular area in OIR animals was significantly reduced in eyes injected with 250 or 25 μg of VEGF Trap, but the 5 μg dose did not inhibit retinal revascularization. Eyes with existing NV that received 5 μg VEGF Trap at P22 exhibited substantial resolution of OIR pathology at P45. Conclusions. The VEGF Trap inhibited the formation of NV, but higher doses also inhibited revascularization of retina when injected at P8. In contrast, the lowest dose tested effectively blocked NV and caused regression of existing NV, without appreciably affecting vasculogenesis or retinal revascularization. These findings suggest that dose selection is an important variable when considering the use of VEGF-targeting agents for the treatment of ROP.

Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life

Retinopathy of prematurity (ROP) is a major complication of preterm birth. It encompasses a spectrum of pathologies that affect vision, from mild disease that resolves spontaneously to severe disease that causes retinal detachment and subsequent blindness. The pathologies are characterized by an arrest in normal retinal vascular development associated with microvascular degeneration. The resulting ischemia and retinal hypoxia lead to excessive abnormal compensatory blood vessel growth. However, this neovascularization can lead to fibrous scar formation and culminate in retinal detachment. Present therapeutic modalities to limit the adverse consequences of aberrant neovascularization are invasive and/or tissue-destructive. In this Review, we discuss current concepts on retinal microvascular degeneration, neovascularization, and available treatments, as well as present future perspectives toward more profound elucidation of the pathogenesis of ROP.

Understanding ischemic retinopathies: emerging concepts from oxygen-induced retinopathy

Ischemic retinopathies, such as retinopathy of prematurity and diabetic retinopathy are characterized by an initial microvascular degeneration, followed by an abnormal hypoxia-induced neovascularization. Oxygen-induced retinopathy (OIR) is a well-established in vivo model of ischemic retinopathies, which, although the triggering insult varies, all share a common end result of capillary loss. Understanding the mechanisms of normal retinal vascular development as well as the pathophysiological processes leading to the primary vascular loss is the key to develop treatments to prevent the sight-threatening neovascularization associated with human ischemic retinopathies. The importance of oxygen-dependent vascular endothelial growth factor in the pathophysiology of both phases of OIR has long been recognized. However, recent studies point out that OIR is a multifactorial disease, resulting from additive effects of an unbalanced expression of pro- and anti-angiogenic factors, interrelated with protective effects of nutritional factors and cytotoxic effects of oxidative and nitro-oxidative stress-dependent mediators. This review summarizes the most recent aspects of the research on OIR conducted in our laboratory and others, with a particular focus on the role of new mediators of nitro-oxidative stress, the trans-arachidonic acids, in microvascular degeneration, and on a novel pathway of metabolic signaling where hypoxia-driven succinate, via receptor GPR91, governs normal and pathological retinal angiogenesis.

MCP-1 Deficiency Delays Regression of Pathologic Retinal Neovascularization in a Model of Ischemic Retinopathy

The present study investigates whether retinal neovascularization (NV) and apoptosis are altered in MCP-1-deficient ((-/-)) mice in the OIR model. Postnatal day (P) 7 MCP-1(-/-) and C57BL/6 (B6) mice were exposed to 75% oxygen for 5 days and then recovered in room air. Immunostaining was performed to localize macrophages/microglia within retinal whole mounts and cross-sections. Retinopathy was qualitatively assessed in FITC-dextran-perfused retinas, and preretinal NV was quantified on P17, P21, and P24. TUNEL analysis was used to compare apoptosis between B6 and MCP-1(-/-) mice. MCP-1(-/-) and B6 mice revealed normal vascular development in room air controls and similar vaso-obliteration in oxygen-exposed mice on P12. MCP-1(-/-) mice exhibited significantly reduced vascular tuft-associated F4/80(+) cells compared with B6 mice. FITC-dextran-perfused retinas exhibited prominent neovascular tufts on P17, and quantification of preretinal nuclei revealed no significant differences between MCP-1(-/-) and B6 mice. In contrast, on P21 and P24, MCP-1(-/-) mice exhibited significant increases in preretinal neovascular nuclei compared with B6 controls. These increases in NV in the MCP-1(-/-) mice were associated with a significant reduction in vascular tuft apoptosis. The results demonstrate that the absence of MCP-1 does not alter normal retinal vascular development. Furthermore, MCP-1(-/-) mice exhibit a similar neovascular response on P17. However, the reduction in tuft-associated macrophages/microglia in the MCP-1(-/-) mice correlates with reduced vascular tuft apoptosis and delayed regression of retinal NV. These findings suggest that macrophages/microglia may contribute to tuft regression through their proapoptotic properties.

Hyperoxia inhibits several critical aspects of vascular development

Normal human retinal vascular development uses angiogenesis and vasculogenesis, both of which are interrupted in the vaso-obliteration phase of retinopathy of prematurity (ROP). Canine oxygen-induced retinopathy (OIR) closely resembles human ROP. Canine retinal endothelial cells (ECs) and angioblasts were used to model OIR and characterize the effects of hyperoxia on angiogenesis and vasculogenesis. Cell cycle analysis showed that hyperoxia reduced the number of G1 phase cells and showed increased arrest in S phase for both cell types. Migration of ECs was significantly inhibited in hyperoxia (P < 0.01). Hyperoxia disrupted the cytoskeleton of angioblasts but not ECs after 2 days. Differentiation of angioblasts into ECs (determined by acetylated low-density lipoprotein uptake) was evaluated after basic fibroblast growth factor treatment. Differentiation of angioblasts into pericytes was determined by smooth muscle actin expression after treatment with platelet-derived growth factor. Differentiation into ECs was significantly inhibited by hyperoxia (P < 0.0001). The percentage of CXCR4(+) cells (a marker for retinal vascular precursors) increased in both treatment groups after hyperoxia. These data show novel mechanisms of hyperoxia-induced disruption of vascular development.

Ocular Neovascularization: Basic Mechanisms and Therapeutic Advances

The vast majority of diseases that cause catastrophic loss of vision do so as a result of ocular neovascularization. During normal retinal vascular development, vascular endothelial cells proliferate and migrate through the extracellular matrix in response to a variety of cytokines, leading to the formation of new blood vessels in a highly ordered fashion. During abnormal neovascularization of the iris, retina, or choroid, angiogenesis is unregulated and usually results in the formation of dysfunctional blood vessels. When these newly formed vessels leak fluid, hemorrhage, or are associated with fibrous proliferation, retinal edema, retinal/vitreous hemorrhage, or traction retinal detachments may occur resulting in potentially catastrophic loss of vision. In this review, we will briefly discuss the scope of the clinical problem and the general underlying principles of angiogenesis. We will focus on recent laboratory advances that have led to the development of therapeutics useful in the treatment of neovascular eye diseases. We will describe compounds currently in pre-clinical development stages as well as the results of clinical trials involving the use of these drugs as treatments for ocular neovascularization.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

Desmin Ensheathment Ratio as an Indicator of Vessel Stability: Evidence in Normal Development and in Retinopathy of Prematurity

We developed a measure of pericyte/endothelial interaction, the desmin ensheathment ratio (DER), using the intermediate filament desmin as an indicator of pericyte ensheathment and have examined the DER in normal retinal vascular development and in the kitten retinopathy of prematurity (ROP) model. We also examined the role of mural cells in the pathogenesis of ROP. Postnatal day 1 to 45 kitten retinae were labeled for desmin, alpha-smooth muscle actin (SMA), and isolectin-B4. Newborn kittens exposed to hyperoxia and then returned to room air for 0 to 40 days (dRA) were similarly labeled. The ratio of desmin to lectin labeling on confocal images yielded the DER. Ultrastructural studies showed that mural cells were present on even the most primitive vessels. During normal development, immature vascular beds had DERs of 0.3 to 0.6 whereas mature beds, which predominated by postnatal day 28, had DERs greater than 0.9. Immature pericytes and smooth muscle cells did not prevent hyperoxia-induced vessel regression. During the vasoproliferative stage of ROP, the DERs of intra- and preretinal vessels ranged between 0.2 and 0.5. In the recovery stage, the DER increased in parallel with regression of pathology, reaching 0.9 at 34 dRA. Stabilization of the DER by the fifth postnatal week was temporally coincident with the development of resistance to hyperoxia-induced vessel regression previously reported in the kitten. These observations lead us to suggest that a DER of 0.9 represents a vascular stability threshold and that a low DER observed during ROP raises the possibility that mural cell abnormalities play a key role in the pathogenesis of ROP.

238 The Roles of Oxygen and Vascular Growth Factors in Pathogenesis of Retinopathy of Prematurity in a Murine Model

Purpose: To investigate the changes of VEGF°¢FGF-2°¢IGF-1 and ER mRNA and protein levels in mice retinae of normal retinal vascular development and retinopathy of prematurity to elucidate risk factors and pathogenesis of retinopathy of prematurity (ROP). Methods: Four hundred and seventy-four 7-day-old (P7) C57BL/6J mice, half female and half male, were assigned to four groups according to oxygen therapy and the gender. Total RNA was extracted from 2 mice for one sample. VEGF, FGF-2 and ER mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of VEGF, FGF-2, IGF-1 and ER were determined by immunohistochemistry. Results: Gender and oxygen therapy couldn't affect the expression of VEGF, bFGF, IGF-1 and ER (P0.05). However, the age was the independent factor which could affect their expression (p<0.05). While hyperoxia and age were integrated, they obviously affected these factors expression (p<0.0001). The level of VEGF mRNA increased since P7, peaked at P9, and declined since P11 to a low level and maintained to P17 in nomoxic groups. In hyperoxic groups, it declined since P8 and remained declining during oxygen exposure; while it increased slowly since mice were taken back to room air and increased rapidly since P15 which is significant compared to the controls, indicating a close relationship with changes of O2 concentration. The level of FGF-2 mRNA maintained low in normoxia; while in hyperoxic groups it had no change during the hyperoxia exposure and increased since 3 days after back to room air and maintained to P21. The level of ER mRNA increased since P7, peaked at P9, declined since P11 and maintained to P17 in nomoxic group. In hyperoxic groups, it had no change during the hyperoxic period and rised since 5 days after back to room air and maintained to P21. The changes of protein levels of these three factors were later than that of their mRNA, but had the same trend. The protein of IGF-1 declined since P7 and maintained low since P11, while in hyperoxic group, it had no change during the hyperoxic exposure, increased since mice were back to room air and declined since P16 to a low level. Conclusion: Our study suggested that hyperoxia followed by hypoxia was very important in the pathogenesis of ROP. VEGF, FGF-2, IGF-1 and ER played important roles in the development of normal retina vascularization and the pathogenesis of ROP. VEGF may be the most important factor.

The urokinase/urokinase receptor system in retinal neovascularization: inhibition by A6 suggests a new therapeutic target.

The objective of the study was to determine the role of urokinase (uPA) and the urokinase receptor (uPAR) in retinal angiogenesis, and whether loss of uPAR or the inhibition of uPA/uPAR interactions could suppress the extent of retinal neovascularization in an animal model of ischemic retinopathy. Retinal neovascularization was induced by exposing newborn mice to 75% oxygen on postnatal day 7 for 5 days, followed by exposure to room air on days 12 to 17. The expression of uPAR in the retina was investigated by RT-PCR and immunohistochemistry. The role of uPAR in ischemic retinopathy was investigated by quantitating the extent of retinal neovascularization in the uPAR(-/-) mouse. The effects of inhibiting the uPA/uPAR interaction on the development of retinal neovascularization were studied in this animal model with a uPA-derived peptide, A6. Animals were treated with an intraperitoneal injection of A6 at a dose of 5, 10, or 100 mg/kg once a day on days 12 to 16. Control animals included oxygen-exposed mice treated with similar amounts of PBS only on days 12 to 16. The effect of A6 on the expression of uPAR in the retina was examined by real-time RT-PCR. The expression of uPAR mRNA was upregulated in experimental animals during the period of angiogenesis and was localized to endothelial cells in the superficial layers of the retina. The uPAR(-/-) mouse demonstrated normal retinal vascular development; however, the absence of functional uPAR resulted in a significant reduction in the extent of retinal neovascularization. Histologic analysis of mice treated with A6 peptide showed significant inhibition of retinal neovascularization, and the response was dose dependent. The RT-PCR analysis of the retinas of the A6-treated animals showed a greater than twofold decrease in uPAR expression. Expression of the urokinase receptor uPAR is essential to the development of retinal neovascularization. Inhibition of the activity of uPAR suppresses retinal neovascularization, possibly through a reduction in cell-associated proteolytic activity, cell signaling, or cell-matrix adhesion necessary for cell migration during angiogenesis. The uPA/uPAR interaction may be an important therapeutic target in the management of proliferative retinopathies.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression.

Vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PlGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PlGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PlGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PlGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PlGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Dexamethasone reduces oxygen induced retinopathy in a mouse model.

Dexamethasone is widely used in the postnatal period. Its impact on retinopathy of prematurity (ROP) is extremely controversial; published studies have found a detrimental, protective, or no effect on ROP. The goal of this study was to test the hypothesis that use of dexamethasone during the injury phase (oxygen exposure) reduces the severity of oxygen-induced retinopathy (OIR) in a mouse model. C57BL6 mice pups were exposed to either room air or hyperoxia (75% FiO2) from postnatal d 7 through 12 (PN7-12) with or without dexamethasone (0.5 mg/kg/d s.c.) and killed on PN17-21. Retinopathy was assessed by a scoring system of retinal flat mount preparations and periodic acid-Schiff (PAS) staining of retinal sections. Pups exposed to dexamethasone and oxygen had a lower median retinopathy score of 5 (4, 6) [median (25th, 75th quartile)] compared with animals exposed to oxygen alone with median score of 9 (6, 10) with p < 0.001. PAS staining for extra retinal neovascularization in the dexamethasone and oxygen treated animals showed a significant reduction in number of nuclei extending beyond the inner limiting membrane when compared with oxygen exposed alone (p = 0.04). Animals treated with dexamethasone had decreased weight gain compared with control animals. Dexamethasone did not appear to affect the normal development of retinal vasculature as assessed by the scoring system when compared with control animals. Thus, dexamethasone decreases severity of OIR without having an adverse effect on normal retinal vascular development in the mouse model. We speculate that dexamethasone decreases the injury response that occurs during the hyperoxic phase, thus protecting the developing vasculature and improving the subsequent retinopathy.

SHORT COMMUNICATION: The effect of raised inspired carbon dioxide on normal retinal vascular development in the neonatal rat

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 CO 2 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 CO 2 (0.2%), 5 litters exposed to 6%, and 5 litters to 10% CO 2. 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% CO 2. CONCLUSIONS. Raised inspired CO 2 was associated with retardation of normal retinal vascular development and increased peripheral avascular area in neonatal rats. Raised CO 2 may be a risk factor for the development of abnormal neovascularization such as in ROP.

The effect of litter size on normal retinal vascular development in the neonatal rat.

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.