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.
| S-EPMC4103428 | biostudies-literature
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: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.
| S-EPMC1809914 | biostudies-literature
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.
| S-EPMC3270960 | biostudies-literature
Project description:Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression.Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription-polymerase chain reaction.In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts.Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.
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Project description:This review compared the clinical results of transepithelial corneal crosslinking (CXL) to epithelium-off (epi-off) CXL in progressive corneal ectasia using a metaanalysis. The Cochrane databases and Medline were searched for randomized controlled trials (RCTs). Seven RCTs involving 505 eyes that met the eligibility criteria were identified. The epi-off CXL group showed significantly better outcomes in postoperative changes in maximum keratometry (K) during 1-year observation periods. Transepithelial CXL resulted in significantly greater post-treatment central corneal thickness and best spectacle-corrected visual acuity (BSCVA). The presence of a postoperative demarcation line was significantly more frequent after epi-off CXL than that after transepithelial CXL. No statistically significant difference was found between other parameters. Although patients in the transepithelial CXL group demonstrated a greater improvement in BSCVA compared with patients in the epi-off CXL group at the 1 year follow-up, transepithelial CXL had less impact on halting progressive corneal ectasia in terms of maximum K than epi-off CXL.
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Project description:ErbB3 harbors weak kinase activity, but strongly activates downstream phosphatidylinositol 3-kinase/Akt signaling through heterodimerization with and activation by other ErbB receptor tyrosine kinases. We report here that ErbB3 loss in the luminal mammary epithelium of mice impaired Akt and MAPK signaling and reduced luminal cell proliferation and survival. ERBB3 mRNA expression levels were highest in luminal mammary populations and lowest in basal cell/stem cell populations. ErbB3 loss in mammary epithelial cells shifted gene expression patterns toward a mammary basal cell/stem cell signature. ErbB3 depletion-induced gene expression changes were rescued upon activation of Akt and MAPK signaling. Interestingly, proliferation and expansion of the mammary basal epithelium (BE) occurred upon ErbB3 targeting in the luminal epithelium, but not upon its targeting in the BE. Multiple cytokines, including interleukin 6, were induced upon ErbB3 depletion in luminal epithelium cells, which increased growth of BE cells. Taken together, these results suggest that ErbB3 regulates the balance of differentiated breast epithelial cell types by regulating their growth and survival through autocrine- and paracrine-signaling mechanisms.
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Project description:Injury to the cornea leads to formation of mediators that initiate and amplify inflammatory responses and neovascularization. Among these are lipid mediators generated by a cytochrome P450 (CYP) enzyme identified as CYP4B1. Increased corneal CYP4B1 expression increases limbal angiogenic activity through the production of 12-hydroxyeicosatrienoic acid (12-HETrE), a potent inflammatory and angiogenic eicosanoid. We used siRNA duplexes targeting CYP4B1 to substantiate the link between CYP4B1 expression, 12-HETrE production and angiogenesis in a model of suture-induced corneal neovascularization. Intrastromal sutures induced a time-dependent neovascular response which was significantly attenuated by CYP4B1-specific siRNAs but not by nonspecific siRNA. CYP4B1 mRNA was reduced by 60% and 12-HETrE's levels were barely detected in corneal homogenates from eyes treated with the CYP4B1-specific siRNA. The decreased neovascular response in CYP4B1 siRNA-treated eyes was associated with a 75% reduction in corneal VEGF mRNA levels. Transfection of rabbit corneal epithelial cells with CYP4B1 cDNA induced VEGF expression. Conversely, treatment with CYP4B1 siRNA or addition of a CYP4B1 inhibitor significantly decreased VEGF mRNA levels; addition of 12-HETrE potently increased them. The results strongly implicate the corneal CYP4B1 as a component of the inflammatory and neovascular cascade initiated by injury and further suggest that CYP4B1-12-HETrE is a proximal regulator of VEGF expression.
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Project description:PurposeTo explore the effect and mechanism of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes on corneal fibrosis.MethodsThe wild-type, NLRP3 knockout (KO), and myeloid cell-specific NLRP3 KO (NLRP3 Lyz-KO) C57 mice were used to establish a corneal scarring model. NLRP3 inhibitor, IL-1β neutralizing antibody, and an IL-1R antagonist were used to investigate the role of NLRP3 and IL-1β in corneal fibrosis. The expression of the NLRP3 signaling pathway related proteins, alpha-smooth muscle actin, TGF-β was determined by quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence staining. Flow cytometry was used to detect the infiltration of macrophages during corneal fibrosis.ResultsThe components of the NLRP3 inflammasomes were elevated and activated during corneal scarring. Additionally, genetic or chemical-mediated blocking of NLRP3 as well as IL-1β significantly alleviated corneal fibrosis. Moreover, neutrophil (CD45+Ly6G+) and macrophage (CD45+ F4/80+) accumulation increased in the cornea during the progression of corneal fibrosis. Intriguingly, the increased concentrations of NLRP3 and IL-1β were prominently colocalized with the infiltrating F4/80+ macrophages. Expectedly, NLRP3 Lyz-KO mice exhibited a marked decrease in their corneal fibrosis symptoms. Mechanistically, the activation of IL-1β or macrophage NLRP3 stimulated the expression of TGF-β1 in the corneal epithelial cells, whereas an NLRP3 deficiency decreased its expression in the corneal epithelium.ConclusionsThese observations revealed that the NLRP3 inflammasome activation in infiltrating macrophages contributes to corneal fibrosis by regulating corneal epithelial TGF-β1 expression. Targeting the NLRP3 inflammasome might be a promising strategy for the treatment of corneal scarring.
| S-EPMC9296889 | biostudies-literature
Project description:PurposeMatrix metalloproteinase 14 (MMP14) has been shown to be required for corneal angiogenesis. We hypothesized that the proangiogenic activity of MMP14 may be based on its selective binding to, and cleaving of, vascular endothelial growth factor receptor 1 (VEGFR1), but not VEGFR2 or VEGFR3.MethodsRecombinant human (rh)VEGFR1, R2, and R3 were incubated with human MMP14, and the reaction mixtures were analyzed by SDS-PAGE and Coomassie blue staining. Surface plasmon resonance was used to determine the equilibrium constants (KD) for binding between MMP14 and VEGFA versus rhVEGFR1, R2, and R3. Extracellular signal-regulated kinase (ERK) phosphorylation was assayed in vascular endothelial cells after incubation with VEGF and various concentrations of MMP14. Ex vivo aortic ring tube formation assays and VEGFA micropocket corneal neovascularization assays were performed using Flk1Cre/Flk1mCherry/MMP14lox and Flk1mCherry/MMP14lox control mice.ResultsMaxtrix metalloproteinase 14 increased VEGFA-induced ERK phosphorylation in a time- and concentration-dependent manner in vascular endothelial cells. Aortic ring assays showed diminished vessel sprouting in vitro in response to VEGFA, but not to basic fibroblast growth factor, in mice with conditional deletion of vascular MMP14 (Flk1creMMP14lox) compared with that in MMP14lox control mice. In addition, diminished VEGFA-induced corneal angiogenesis was seen in flk1creMMP14lox mice compared with MMP14lox mice in vivo.ConclusionsOur findings indicate that VEGFR1 interaction with MMP14 and the enzymatic activity of MMP14 are necessary for VEGFA-induced angiogenesis. Additionally, selective cleavage of VEGFR1 by MMP14 may play an important role in VEGFA-induced corneal angiogenesis.
| S-EPMC5993529 | biostudies-literature