Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.
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ABSTRACT:
SUBMITTER: Cursiefen C
PROVIDER: S-EPMC1544098 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1(Sema-) mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.
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Project description:Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.
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Project description:The cornea fends off chemicals, dirt, and infectious particles and provides most of the eye's focusing power. Corneal transparency is of paramount importance to normal vision, yet how it is established and maintained remains unclear. Here, we ablated Notch1 in keratocytes using Twist2-Cre mice and found that Twist2-Cre; Notch1f/f mice developed stroma expansion and neovascularization, followed by hyperproliferation and metaplasia of corneal epithelial progenitor cells and plaque formation at central cornea, leading to loss of transparency. Development of these phenotypes does not involve bacteria-caused inflammation; instead, Notch1 deletion upregulates Vegfa and Vegfc via Hif1α in keratocytes. Vascular endothelial growth factor (VEGF) receptor inhibitor axitinib prevented development of these anomalies in Twist2-Cre; Notch1f/f mice, suggesting that VEGFs secreted by keratocytes promote not only neovascularization but also proliferation and metaplasia of epithelial progenitor cells at central cornea. This study uncovers a Notch1-Hif1α-VEGF pathway in keratocytes that maintains corneal transparency and represents a potential target for treatment of related corneal disorders.
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Project description:PURPOSE: To determine the molecular basis of corneal avascularity during wound healing and determine the role of angiogenic and antiangiogenic factors in corneal vasculogenesis. METHODS: The expression of proangiogenic factors (vascular endothelial growth factor [VEGF]; basic fibroblast growth factor [bFGF]; matrix metalloproteinase-2 [MMP-2]; and membrane-type 1-MMP [MT1-MMP]) and antiangiogenic factors (pigment epithelium-derived factor [PEDF]; angiostatin; restin; and endostatin) was analyzed in avascular corneas and in models of corneal neovascularization (bFGF pellet implantation, intrastromal injection of MT1-MMP cDNA, and surgically induced partial limbal deficiency). RESULTS: Immunohistochemistry demonstrated the presence of antiangiogenic factors (PEDF, angiostatin, restin, and endostatin) and proangiogenic molecules (VEGF, bFGF, MMP-2, and MT1-MMP) in the cornea after wounding. Proangiogenic MMPs were upregulated in stromal fibroblasts in the vicinity of invading vessels following bFGF pellet implantation. Corneal neovascularization (NV) was also induced by intrastromal injection of MT1-MMP naked cDNA in conjunction with de-epithelialization. Partial limbal deficiency (HLD-) resulted in corneal NV in MMP-7 and MMP-3 knockout mice but not in wild type controls. CONCLUSIONS: Corneal angiogenic privilege is an active process involving the production of antiangiogenic factors to counterbalance the proangiogenic factors (which are upregulated after wound healing even in the absence of new vessels). Our finding that the potent antiangiogenic factors, angiostatin and endostatin, are colocalized with several MMPs during wound healing suggests that MMPs may be involved in the elaboration of these antiangiogenic molecules by proteolytic processing of substrates within the cornea.
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Project description:PurposeIntraflagellar transport 46 (IFT46) is an integral subunit of the IFT-B complex, playing a key role in the assembly and maintenance of primary cilia responsible for transducing signaling pathways. Despite its predominant expression in the basal body of cilia, the precise role of Ift46 in ocular development remains undetermined. This study aimed to elucidate the impact of neural crest (NC)-specific deletion of Ift46 on ocular development.MethodsNC-specific conditional knockout mice for Ift46 (NC-Ift46F/F) were generated by crossing Ift46F mice with Wnt1-Cre2 mice, enabling the specific deletion of Ift46 in NC-derived cells (NCCs). Sonic Hedgehog (Shh) and Notch signaling activities in NC-Ift46F/F mice were evaluated using Gli1lacZ and CBF:H2B-Venus reporter mice, respectively. Cell fate mapping was conducted using ROSAmTmG reporter mice.ResultsThe deletion of Ift46 in NCCs resulted in a spectrum of ocular abnormalities, including thickened corneal stroma, hypoplasia of the anterior chamber, irregular iris morphology, and corneal neovascularization. Notably, this deletion led to reduced Shh signal activity in the periocular mesenchyme, sustained expression of key transcription factors Foxc1, Foxc2 and Pitx2, along with persistent cell proliferation. Additionally, it induced increased Notch signaling activity and the development of ectopic neovascularization within the corneal stroma.ConclusionsThe absence of primary cilia due to Ift46 deficiency in NCCs is associated with anterior segment dysgenesis (ASD) and corneal neovascularization, suggesting a potential link to Axenfeld-Rieger syndrome, a disorder characterized by ASD. This underscores the pivotal role of primary cilia in ensuring proper anterior segment development and maintaining an avascular cornea.
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Project description:Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis.
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