Bilateral idiopathic juxtafoveolar retinal telangiectasis

Bilateral idiopathic juxtafoveolar retinal telangiectasis (JRT) is a condition of unknown etiology characterized by telangiectatic retinal vessels located temporal to the fovea. Most patients with bilateral JRT have associated telangiectasis, dilated capillaries, minimal exudation, retinal crystals, right angle venules, and retinal pigment hyperplasia. An unusual case of bilateral retinal telangiectasis with associated asymmetric cystoid macular edema in a middle-aged female is presented. This case is unusual due to the presence of cystoid macular edema. Classification, differential diagnosis and management of idiopathic juxtafoveolar retinal telangiectasis are reviewed.     

LRP5 Is Required for Vascular Development in Deeper Layers of the Retina

Background

The low-density lipoprotein receptor-related protein 5 (LRP5) plays an important role in the development of retinal vasculature. LRP5 loss-of-function mutations cause incomplete development of retinal vessel network in humans as well as in mice. To understand the underlying mechanism for how LRP5 mutations lead to retinal vascular abnormalities, we have determined the retinal cell types that express LRP5 and investigated specific molecular and cellular functions that may be regulated by LRP5 signaling in the retina.

Methods and Findings

We characterized the development of retinal vasculature in LRP5 mutant mice using specific retinal cell makers and a GFP transgene expressed in retinal endothelial cells. Our data revealed that retinal vascular endothelial cells predominantly formed cell clusters in the inner-plexiform layer of LRP5 mutant retina rather than sprouting out or migrating into deeper layers to form normal vascular network in the retina. The IRES-β-galactosidase (LacZ) report gene under the control of the endogenous LRP5 promoter was highly expressed in Müller cells and was also weakly detected in endothelial cells of the retinal surface vasculature. Moreover, the LRP5 mutant mice had a reduction of a Müller cell-specific glutamine transporter, Slc38a5, and showed a decrease in b-wave amplitude of electroretinogram.

Conclusions

LRP5 is not only essential for vascular endothelial cells to sprout, migrate and/or anastomose in the deeper plexus during retinal vasculature development but is also important for the functions of Müller cells and retinal interneurons. Müller cells may utilize LRP5-mediated signaling pathway to regulate vascular development in deeper layers and to maintain the function of retinal interneurons.     

The Notch signaling pathway in retinal dysplasia and retina vascular homeostasis

The retina is one of the most essential elements of vision pathway in vertebrate. The dysplasia of retina cause congenital blindness or vision disability in individuals, and the misbalance in adult retinal vascular homeostasis leads to neovaseularization-associated diseases in adults, such as diabetic retinopathy or age-related macular degeneration. Many developmental signaling pathways are involved in the process of retinal development and vascular homeostasis. Among them, Notch signaling pathway has long been studied, and Notch signaling-interfered mouse models show both neural retina dysplasia and vascular abnormality. In this review, we discuss the roles of Notch signaling in the maintenance of retinal progenitor cells, specification of retinal neurons and glial cells, and the sustaining of retina vascular homeostasis, especially from the aspects of conditional knockout mouse models. The potential of Notch signal mampulation may provide a powerful cell fate- and neovascularization-controlling tool that could have important applications in la'eatment of retinal diseases.     

Hypoxia-induced insulin-like growth factor II contributes to retinal vascularization in ocular development

In ocular development, retinal physiological hypoxia in response to the retinal metabolic activity controls retinal vascular development, which is regulated by variable angiogenic factors. Herein, we demonstrated that hypoxia-induced IGF-II could contribute to retinal vascularization in ocular development. In the developing retina, IGF-II expression appears to be predominant on retinal vessels, which was chronologically increased and peaked during active retinal angiogenesis similar to VEGF expression. Under hypoxic condition, IGF-II as well as VEGF was significantly up-regulated in retinal vascular endothelial cells. In addition, IGF-II treatment could also increase VEGF expression in retinal vascular endothelial cells. The VEGF expression induced by IGF-II was mediated by ERK-1/2 activation. Moreover, IGF-II strongly promoted angiogenic processes of migration and tube formation of retinal microvascular endothelial cells. In conclusion, our results provided that hypoxia-induced IGF-II may regulate retinal vascular development not only directly by IGF-II-mediated angiogenic activity, but also indirectly by IGF-II-induced VEGF expression. Therefore, the potential contribution of IGF-II to pathological retinal angiogenesis should be furthermore explored for the development of novel treatments to vaso-proliferative retinopathies.     

Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.     

Agents that bind annexin A2 suppress ocular neovascularization

TM601 is a synthetic polypeptide with sequence derived from the venom of the scorpion Leiurus quinquestriatus that has anti‐neoplastic activity. It has recently been demonstrated to bind annexin A2 on cultured tumor and vascular endothelial cells and to suppress blood vessel growth on chick chorioallantoic membrane. In this study, we investigated the effects of TM601 in models of ocular neovascularization (NV). When administered by intraocular injection, intravenous injections, or periocular injections, TM601 significantly suppressed the development of choroidal NV at rupture sites in Bruch's membrane. Treatment of established choroidal NV with TM601 caused apoptosis of endothelial cells and regression of the NV. TM601 suppressed ischemia‐induced and vascular endothelial growth factor‐induced retinal NV and reduced excess vascular permeability induced by vascular endothelial growth factor. Immunostaining with an antibody directed against TM601 showed that after intraocular or periocular injection, TM601 selectively bound to choroidal or retinal NV and co‐localized with annexin A2, which is undetectable in normal retinal and choroidal vessels, but is upregulated in endothelial cells participating in choroidal or retinal NV. Intraocular injection of plasminogen or tissue plasminogen activator, which like TM601 bind to annexin A2, also suppressed retinal NV. This study supports the hypothesis that annexin A2 is an important target for treatment of neovascular diseases and suggests that TM601, through its interaction with annexin A2, causes suppression and regression of ocular NV and reduces vascular leakage and thus may provide a new treatment for blinding diseases such as neovascular age‐related macular degeneration and diabetic retinopathy. J. Cell. Physiol. 225: 855–864, 2010. © 2010 Wiley‐Liss, Inc.     

Angiopoietin 1 prevents retinal detachment in an aggressive model of proliferative retinopathy, but has no effect on established neovascularization

Vascular endothelial growth factor (VEGF) plays a central role in vasoproliferative diseases in the retina, however, other gene products modulate its effects. The angiopoietins are particularly important in this regard. Angiopoietin 2 (Ang2) collaborates with VEGF to stimulate neovascularization (NV) in some situations, but in other situations causes regression of NV. Ang2 also causes a transient increase in vascular density during retinal vascular development. In this study, we sought to determine if Ang1 has similar activities. The effects of Ang1 were tested in double transgenic mice with inducible expression of Ang1. Increased expression of Ang1 in the retina during retinal vascular development did not cause a detectable alteration in vascular density. Also, unlike Ang2, increased expression of Ang1 had no effect on established retinal or choroidal NV. However, when Ang1 expression was initiated simultaneously with that of VEGF, it strongly suppressed VEGF-induced NV and prevented retinal detachment. These data indicate that the timing of Ang1 expression is a critical determinate of its effects on VEGF-induced NV in the retina; it effectively blocks the initiation and progression of NV, but cannot reverse established NV or reduce leakage from NV. These data suggest that increased expression of Ang1 may be a good strategy for prophylaxis of retinal NV, but is unlikely to be effective as monotherapy of established NV.     

Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina

Mice or humans with photoreceptor degenerations experience permeability and dropout of retinal capillaries. Loss of photoreceptors results in decreased oxygen usage and thinning of the retina with increased oxygen delivery to the inner retina. To investigate the possibility that increased tissue oxygen plays a role in the vascular damage, we exposed adult mice to hyperoxia, which also increases oxygen in the retina. After 1, 2, or 3 weeks of hyperoxia, there was a statistically significant decrease in retinal vascular density that was not reversible, and endothelial cell apoptosis was demonstrated by TUNEL staining. Mice exposed to hyperoxia and mice with photoreceptor degeneration both showed decreased expression of VEGF in the retina. After complete or near-complete degeneration of photoreceptors, there was increased expression of VEGF in RPE cells, which may explain the association of photoreceptor degeneration and neovascularization in or around the RPE. Increased expression of VEGF in photoreceptors of transgenic mice failed to prevent hyperoxia-induced retinal capillary dropout. These data suggest that increased oxygen in the retina, either by increased inspired oxygen or by photoreceptor degeneration, results in endothelial cell death and dropout of capillaries. Decreased expression of VEGF may be a contributing factor, but the situation may be more complicated for mature retinal vessels than it is for immature vessels, because VEGF replacement does not rescue mature retinal vessels, suggesting that other factors may also be involved.     

视网膜微血管内皮细胞在糖尿病视网膜病变中的研究现状

糖尿病视网膜疾病是导致成年人失明的主要因素,是糖尿病的一种令人恐惧的并发症,高血糖被认为是促进其发展的主要原因。高血糖不断地破坏视网膜的微血管系统最终导致视网膜的许多代谢,结构和功能的紊乱。视网膜微血管内皮细胞在微脉管系统中形成树枝状供应视网膜神经,这些内皮细胞的解剖和生理符合重要视觉保护的营养需求[1]。一方面,内皮组织务必确保氧的供应和代谢活跃的视网膜营养供应;另一方面,内皮细胞有助于血-视网膜屏障将循环产生的毒素分子,白细胞促炎性物质排出体外来保护视网膜,这种特性也可能会引起疾病,比如:视网膜血管的渗漏和新生血管,炎性物质转移,因此,视网膜内皮细胞在视网膜缺血性病变,血管炎中起到重要作用,包括糖尿病视网膜病变和视网膜炎症或感染尤其是后葡萄膜炎。使用基因表达和蛋白质组学分析等研究方法,有助于了解这些疾病的发病机制。为了进一步开展对糖尿病视网膜疾病的研究,有必要就目前有关糖尿病视网膜病变患者微血管内皮细胞的研究进展予以综述,旨在为糖尿病视网膜病变的深入研究提供参考依据。     

Increased Oxidative and Nitrative Stress Accelerates Aging of the Retinal Vasculature in the Diabetic Retina

Hyperglycemia-induced retinal oxidative and nitrative stress can accelerate vascular cell aging, which may lead to vascular dysfunction as seen in diabetes. There is no information on whether this may contribute to the progression of diabetic retinopathy (DR). In this study, we have assessed the occurrence of senescence-associated markers in retinas of streptozotocin-induced diabetic rats at 8 and 12 weeks of hyperglycemia as compared to normoglycemic aging (12 and 14 months) and adult (4.5 months) rat retinas. We have found that in the diabetic retinas there was an up-regulation of senescence-associated markers SA-β-Gal, p16^INK4a and miR34a, which correlated with decreased expression of SIRT1, a target of miR34a. Expression of senescence-associated factors primarily found in retinal microvasculature of diabetic rats exceeded levels measured in adult and aging rat retinas. In aging rats, retinal expression of senescence associated-factors was mainly localized at the level of the retinal pigmented epithelium and only minimally in the retinal microvasculature. The expression of oxidative/nitrative stress markers such as 4-hydroxynonenal and nitrotyrosine was more pronounced in the retinal vasculature of diabetic rats as compared to normoglycemic aging and adult rat retinas. Treatments of STZ-rats with the anti-nitrating drug FeTPPS (10mg/Kg/day) significantly reduced the appearance of senescence markers in the retinal microvasculature. Our results demonstrate that hyperglycemia accelerates retinal microvascular cell aging whereas physiological aging affects primarily cells of the retinal pigmented epithelium. In conclusion, hyperglycemia-induced retinal vessel dysfunction and DR progression involve vascular cell senescence due to increased oxidative/nitrative stress.     

Sirtuin1 Over-Expression Does Not Impact Retinal Vascular and Neuronal Degeneration in a Mouse Model of Oxygen-Induced Retinopathy

Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy.     

Idiopathic uveal effusion syndrome

Idiopathic uveal effusion syndrome (IUES) is a rare condition characterized by a spontaneous detachment of the choroid, often with a secondary non-rhegmatogenous retinal detachment, in healthy middle-aged men. It is hypothesized that the primary cause of the condition is a congenital scleral anomaly causing abnormal uveoscleral protein transport. The following case report reviews a typical presentation of IUES. The patient was treated with lamellar sclerectomy and sclerostomy with resolution of the choroidal detachment over the next several months. Three months later, the patient developed unilateral disswelling and a non-ischemic central retinal vein occlusion which eventually resolved without sequelae.     

Cysteine-rich protein 61 (CCN1) and connective tissue growth factor (CCN2) at the crosshairs of ocular neovascular and fibrovascular disease therapy

Abstract

The vasculature forms a highly branched network investing every organ of vertebrate organisms. The retinal circulation, in particular, is supported by a central retinal artery branching into superficial arteries, which dive into the retina to form a dense network of capillaries in the deeper retinal layers. The function of the retina is highly dependent on the integrity and proper functioning of its vascular network and numerous ocular diseases including diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity are caused by vascular abnormalities culminating in total and sometimes irreversible loss of vision. CCN1 and CCN2 are inducible extracellular matrix (ECM) proteins which play a major role in normal and aberrant formation of blood vessels as their expression is associated with developmental and pathological angiogenesis. Both CCN1 and CCN2 achieve disparate cell-type and context-dependent activities through modulation of the angiogenic and synthetic phenotype of vascular and mesenchymal cells respectively. At the molecular level, CCN1 and CCN2 may control capillary growth and vascular cell differentiation by altering the composition or function of the constitutive ECM proteins, potentiating or interfering with the activity of various ligands and/or their receptors, physically interfering with the ECM-cell surface interconnections, and/or reprogramming gene expression driving cells toward new phenotypes. As such, these proteins emerged as important prognostic markers and potential therapeutic targets in neovascular and fibrovascular diseases of the eye. The purpose of this review is to highlight our current knowledge and understanding of the most recent data linking CCN1 and CCN2 signaling to ocular neovascularization bolstering the potential value of targeting these proteins in a therapeutic context.     

Attenuation of retinal vascular development and neovascularization during oxygen-induced ischemic retinopathy in Bcl-2-/- mice

Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, we investigated the physiological role of bcl-2 in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibited a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2-/- mice. We observed, in bcl-2-/- mice, delayed development of retinal vasculature and remodeling, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2-/- mice exhibited a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization was significantly reduced in bcl-2-/- mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR.     

Retinal vascular endothelium expresses fibronectin and class II histocompatibility complex antigens in experimental autoimmune uveitis

To analyze the role of the retinal vascular endothelial cells in the development of experimental autoimmune uveitis (EAU), we studied the presence of Ia antigen and FN in retinal vessels of Lewis rats immunized with retinal S antigen. Immunopathologic studies were performed on frozen tissues obtained during various stages of the disease. Our results show that Ia antigen was not present in the normal rat retina, and there was very little FN present in a few retinal vessels. One to two days prior to the histologic and clinical onset of EAU, FN was found to be increased in the retinal vessels. Ia antigen was found to be present in the retinal vessels coincident with the first signs of cellular infiltration. During the stage of maximal cellular infiltration, FN was present diffusely throughout the retina, as well as in the subretinal space, and Ia antigen was found diffusely in the cellular infiltrate. Therefore, FN and Ia antigen reflect the immunomodulation of vascular endothelial cells in EAU, which may be very important in the pathogenesis of retinal S antigen-induced uveitis. Two possible mechanisms for the role of the activation of the retinal vascular endothelium in the development of retinal inflammation in uveitis are discussed.     

Notch signaling in ocular vasculature development and diseases

Ocular angiogenesis, characterized by the formation of new blood vessels in the avascular area in eyes, is a highly coordinated process involved in retinal vasculature formation and several ocular diseases such as age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. This process is orchestrated by complicated cellular interactions and vascular growth factors, during which endothelial cells acquire heterogeneous phenotypes and distinct cellular destinations. To date, while the vascular endothelial growth factor has been identified as the most critical angiogenic agent with a remarkable therapeutic value, the Notch signaling pathway appears to be a similarly important regulator in several angiogenic steps. Recent progress has highlighted the involvement, mechanisms and therapeutic potential of Notch signaling in retinal vasculature development and pathological angiogenesis-related eye disorders, which may cause irreversible blindness.     

Retinal Vascular Caliber Is Associated with Cardiovascular Biomarkers of Oxidative Stress and Inflammation: The POLA Study

Purpose

Retinal vascular caliber has been linked with increased cardiovascular risk and is predictive of cardiovascular pathology, including stroke and coronary heart disease. Oxidative stress, as well as inflammatory mechanisms, plays a major role in the pathogenesis and progression of atherosclerosis, plaque rupture and vascular thrombotic propensity. The purpose of this study is to explore the relationship between retinal vascular calibers and biomarkers of oxidative stress and inflammation, in subjects free of cardiovascular pathology.

Patients and Methods

Cross-sectional analysis from a community-dwelling cohort comprising 1224 individuals aged 60 years and over, without a history of coronary or peripheral artery disease or stroke. Retinal vascular caliber was measured from fundus photographs using semi-automated standardized imaging software. Oxidative stress was evaluated using plasma superoxide dismutase 2 and glutathione peroxidase (GPx-3) activities, and inflammatory state was assessed using plasma high sensitivity C-reactive protein (hsCRP) and orosomucoid.

Results

In a multivariate model controlling for cardiovascular risk factors, larger retinal arteriolar caliber was independently related to higher level of GPx-3 activity (p = 0.003) whereas larger venular caliber was associated with higher levels of hsCRP (p = 0.0001) and orosomucoid (p = 0.01).

Conclusion

In the present study, biomarkers of oxidative stress regulation and inflammation were independently associated with retinal vascular calibers. This suggests that an assessment of retinal vessels may offer early and non-invasive detection of subclinical vascular pathology.     

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.     

Angiopoietin-2 plays an important role in retinal angiogenesis

Angiopoietin 2 (Ang2) expression in the retina is increased during physiologic and pathologic neovascularization suggesting that it may be involved. In this study, we used Ang2-deficient mice to test that hypothesis. Mice deficient in Ang2 showed delayed and incomplete development of the superficial vascular bed of the retina, which develops primarily by vasculogenesis, and complete absence of the intermediate and deep vascular beds which develop by angiogenesis. In addition to incomplete retinal vascular development, Ang2-deficient mice showed lack of regression of the hyaloid vasculature, resulting in a phenotype that mimics infants with persistent fetal vasculature (PFV), a relatively common congenital abnormality. Exposure to high levels of oxygen resulted in partial regression of the retinal vessels, indicating that oxygen-induced regression of retinal vessels does not require Ang2. When these oxygen-exposed mice with few retinal vessels were moved to room air, there was no ischemia-induced retinal neovascularization. These data support the hypothesis that Ang2 plays a critical role in physiologic and pathologic angiogenesis, and physiologic, but not oxygen-induced vascular regression. The data also suggest that infants with PFV should be examined for genetic modifications that would be expected to cause perturbations in Tie2 signaling.     

Perturbation of Mouse Retinal Vascular Morphogenesis by Anthrax Lethal Toxin

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.