Person:

Saint-Geniez, Magali

During embryogenesis, the development and differentiation of the eye requires the concomitant formation of the neural/glial elements along with a dense vascular network. The adult neural retina is supported by two distinct vascular systems, the proper retinal vessels and the choroidal vessels. The two beds differ not only in their pattern of embryonic differentiation, but also in their function in the adult organism. The retinal vasculature has barrier properties similar to those observed in the brain, whereas the choroidal vessels display a highly fenestrated phenotype. The hyaloid vasculature is a transient embryonic vascular bed which is complete at birth in mammals and regresses contemporaneously with the formation of the retinal vasculature. The dependence of the retina on its blood supply makes it highly vulnerable to any vascular changes and indeed ocular diseases, such as proliferative retinopathy, age-related macular degeneration and the hyperplastic primary vitreous, which are associated with abnormalities of the different vascular beds of the eye. A number of factors have been implicated in developmental and pathological changes in vessel formation and regression, including fibroblast growth factors, platelet-derived endothelial growth factor and vascular endothelial growth factor, among others. The purpose of this review is to describe and discuss new insights into the mechanisms and molecular cues involved in the development of the normal and pathological vascular systems of the eye. The characterization of the molecules and cell-cell interactions involved in the formation, stabilization and regression of new vessels has led to the identification of potential control points for therapeutic intervention.

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-β signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-β signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases.

purpose. To determine the role of vascular endothelial growth factor (VEGF) in embryonic eye development and lens differentiation.

methods. Expression of components of the VEGF signaling pathway during lens development and in adults was characterized by β-galactosidase staining of VEGF-LacZ mice, immunohistochemistry, and real-time (q) PCR. Embryonic eyes from wild-type mice and VEGF120/120 mice were analyzed by light microscopy and immunohistochemistry. VEGF function during lens development was analyzed using eye explants treated with VEGF-neutralizing antibody. Direct function of VEGF was demonstrated on the human lens epithelial cell line, HLE-B3.

results. Embryonic lens epithelium and posterior lens fibers expressed VEGF and VEGFR2. qPCR revealed VEGF164 as the major isoform in embryonic lens. Transgenic mice expressing only VEGF120 (VEGF120/120 mice) showed major defects in eye development, including microphthalmia, failed lens differentiation, and hyperplastic hyaloid vessels. The lens displayed abnormal cell patterning and differentiation associated with altered c-Maf, Prox1, and p57 expression pattern in the anterior epithelium. The number of proliferating epithelial cells was drastically reduced in VEGF120/120 lenses. Altered MIP26 cellular localization and reduced E-cadherin expression in the lens epithelium were observed. VEGF-neutralization led to reduced fiber elongation of eye explants. Exogenous VEGF increased survival and proliferation of HLE-B3 cell in a dose-dependent manner.

conclusions. Abnormalities in ocular development in VEGF120/120 mice suggest a role for VEGF not only in the formation of ocular vascular beds but also in the differentiation of the lens itself.

Background

Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.

Methods and Findings

Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.

Conclusions

These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

Although the role of vascular endothelial growth factor (VEGF) in developmental and pathological angiogenesis is well established, its function in the adult is less clear. Similarly, although transforming growth factor (TGF) β is involved in angiogenesis, presumably by mediating capillary (endothelial cell [EC]) stability, its involvement in quiescent vasculature is virtually uninvestigated. Given the neurological findings in patients treated with VEGF-neutralizing therapy (bevacizumab) and in patients with severe preeclampsia, which is mediated by soluble VEGF receptor 1/soluble Fms-like tyrosine kinase receptor 1 and soluble endoglin, a TGF-β signaling inhibitor, we investigated the roles of VEGF and TGF-β in choroid plexus (CP) integrity and function in adult mice. Receptors for VEGF and TGF-β were detected in adult CP, as well as on ependymal cells. Inhibition of VEGF led to decreased CP vascular perfusion, which was associated with fibrin deposition. Simultaneous blockade of VEGF and TGF-β resulted in the loss of fenestrae on CP vasculature and thickening of the otherwise attenuated capillary endothelium, as well as the disappearance of ependymal cell microvilli and the development of periventricular edema. These results provide compelling evidence that both VEGF and TGF-β are involved in the regulation of EC stability, ependymal cell function, and periventricular permeability.

Angiogenesis is largely controlled by hypoxia-driven transcriptional up-regulation and secretion of vascular endothelial growth factor (VEGF) and its binding to the endothelial cell tyrosine receptor kinases, VEGFR1 and VEGFR2. Recent expression analysis suggests that VEGF is expressed in a cell-specific manner in normoxic adult tissue; however, the transcriptional regulation and role of VEGF in these tissues remains fundamentally unknown. In this report we demonstrate that VEGF is coordinately up-regulated during terminal skeletal muscle differentiation. We reveal that this regulation is mediated in part by MyoD homo- and hetero-dimeric transcriptional mechanisms. Serial deletions of the VEGF promoter elucidated a region containing three tandem CANNTG consensus MyoD sites serving as essential sites of direct interaction for MyoD-mediated up-regulation of VEGF transcription. VEGF-null embryonic stem (ES) cells exhibited reduced myogenic differentiation compared with wild-type ES cells, suggesting that VEGF may serve a role in skeletal muscle differentiation. We demonstrate that VEGFR1 and VEGFR2 are expressed at low levels in myogenic precursor cells and are robustly activated upon VEGF stimulation and that their expression is coordinately regulated during skeletal muscle differentiation. VEGF stimulation of differentiating C2C12 cells promoted myotube hypertrophy and increased myogenic differentiation, whereas addition of sFlt1, a VEGF inhibitor, resulted in myotube hypotrophy and inhibited myogenic differentiation. We further provide evidence indicating VEGF-mediated myogenic marker expression, mitogenic activity, migration, and prosurvival functions may contribute to increased myogenesis. These data suggest a novel mechanism whereby VEGF is coordinately regulated as part of the myogenic differentiation program and serves an autocrine function regulating skeletal myogenesis.

purpose. Previous studies have demonstrated a role for the retinal pigment epithelium (RPE) in the development and maintenance of the choroidal vasculature, suggesting that RPE serves a trophic role for the choroidal vessels. The goal of this study was to determine the expression pattern of vascular endothelial growth factor (VEGF) and its receptors and their activation status in embryonic and adult choroid, with the purpose of providing cues regarding the role of VEGF in development and stabilization of the choroidal vasculature.

methods. Transgenic VEGF-LacZ mice were used to examine VEGF expression in embryonic and adult eyes. Expression of VEGF isoforms and receptors in the RPE-choroid complex was assessed by RT-PCR and real-time PCR. VEGF receptor 2 expression was assessed by immunohistochemistry and its activation state was examined by immunoprecipitation followed by phosphotyrosine blot.

results. VEGF is expressed by RPE throughout the choroidal vascular development and in the adult. The major VEGF isoforms detected in adult RPE were VEGF120 and VEGF164, with almost no detectable VEGF188. RT-PCR analysis showed expression of VEGF receptors and coreceptors in the RPE–choroid complex. VEGFR2 was detected in the choriocapillaris underlying the RPE. Immunoprecipitation and phosphotyrosine blot of this receptor revealed that VEGFR2 is activated in adult mouse and bovine choroids.

conclusions. The observations suggest that VEGF signaling is involved, not only in choroidal vessel formation, but perhaps also in the maintenance of the choriocapillaris.

Transparency of the cornea, the window of the eye, is a prerequisite for vision. Angiogenesis into the normally avascular cornea is incompatible with good vision and, therefore, the cornea is one of the few tissues in the human body where avascularity is actively maintained. Here, we provide evidence for a critical mechanism contributing to corneal avascularity. VEGF receptor 3, normally present on lymphatic and proliferating blood vascular endothelium, is strongly constitutively expressed by corneal epithelium and is mechanistically responsible for suppressing inflammatory corneal angiogenesis.