Corneal avascularity is due to soluble VEGF receptor-1 - PubMed (original) (raw)

. 2006 Oct 26;443(7114):993-7.

doi: 10.1038/nature05249. Epub 2006 Oct 18.

Miho Nozaki, Nirbhai Singh, Atsunobu Takeda, Pooja D Jani, Tushar Suthar, Romulo J C Albuquerque, Elizabeth Richter, Eiji Sakurai, Michael T Newcomb, Mark E Kleinman, Ruth B Caldwell, Qing Lin, Yuichiro Ogura, Angela Orecchia, Don A Samuelson, Dalen W Agnew, Judy St Leger, W Richard Green, Parameshwar J Mahasreshti, David T Curiel, Donna Kwan, Helene Marsh, Sakae Ikeda, Lucy J Leiper, J Martin Collinson, Sasha Bogdanovich, Tejvir S Khurana, Masabumi Shibuya, Megan E Baldwin, Napoleone Ferrara, Hans-Peter Gerber, Sandro De Falco, Jassir Witta, Judit Z Baffi, Brian J Raisler, Jayakrishna Ambati

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Corneal avascularity is due to soluble VEGF receptor-1

Balamurali K Ambati et al. Nature. 2006.

Abstract

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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Figures

Figure 1

Figure 1. Avascular cornea contains sflt-1 bound to VEGF-A

a, Photo of human eye demonstrates abrupt termination of blood vessels in the conjunctiva (CJ) at its border with the cornea (C), the limbus (asterisks). b, Representative non-reducing western blot of mouse cornea reveals immunoreactive bands of VEGF-A at 100–130 kDa corresponding to bound forms, and negligible immunoreactivity at 45–50 kDa corresponding to the free form. n = 5. c, sflt-1 (lane 1) and VEGF-A (lane 3) transcripts in mouse cornea identified by representative polymerase chain reaction with reverse transcription (RT–PCR). Lane 2 is water (template-negative) control. n = 5. bp, base pairs; aa, amino acids. d, e, sflt-1 mRNA detected by in situ hybridization in mouse corneal epithelium (epi) and stroma (str). Antisense RNA probes show purple-brown reactivity. Sense RNA probes show negligible reactivity. f, Immunolocalization (brown) of sflt-1 protein in mouse cornea. g, Representative reducing western blots (WB) with an antibody against the amino (N) terminus of flt-1 that recognizes both mbflt-1 and sflt-1 and an antibody against the unique C terminus of sflt-1 show that mouse cornea (1) contains primarily sflt-1 (60 kDa) whereas the conjunctiva (2) contains mainly mbflt-1 (190 kDa). h, Representative western blot of two independent mouse cornea samples immunoprecipitated (IP) with anti-VEGF-A antibody and immunoblotted (WB) with a biotinylated antibody against the N terminus of flt-1 that recognizes both mbflt-1 and sflt-1 shows that VEGF-A interacts with sflt-1 (60 kDa). Subsequent immunoblot with a biotinylated anti-VEGF-A antibody confirms the pull-down of VEGF-A by the immunoprecipitating antibody. n = 6.

Figure 2

Figure 2. Topical enzymatically active Cre recombinase abolishes corneal avascularity in flt-1loxP/loxP mice

NLS–Cre but not NLS–β-galactosidase induces Cre expression (brown) in cornea within 1 h of eye drop application as shown by immunolocalization in cell nuclei stained red (a, b), and by representative reducing western blot (WB; c). d, X-gal staining of corneal flat mount of ROSA26R lacZ reporter mouse confirms expression of β-galactosidase (blue) 2 days after Cre expression. e, f, Representative corneal flat mounts show CD31+ (green)/LYVE-1− blood vessels in flt-1loxP/loxP mouse corneas 14 days after treatment with NLS–Cre eye drops (e) but not with NLS–β-galactosidase (f). No corneal vascularization occurs in wild-type mice after topical NLS–Cre (g) or NLS–β-galactosidase (h). n = 4 (a, b, d), n = 6 (c), n = 8–11 (e–h).

Figure 3

Figure 3. Knockdown of sflt-1 mRNA abolishes corneal avascularity

a–c, Real-time RT–PCR reveals reduced sflt-1 mRNA (a) and enzyme-linked immunosorbent assay (ELISA) reveals reduced sflt-1 protein (b) and increased free VEGF-A protein (c) in wild-type mouse corneas 3 days after injection of pshRNA–sflt-1 but not pshRNA–mbflt-1. Asterisk denotes P < 0.05, Bonferroni corrected Mann–Whitney _U_-test. n = 8–12. Error bars depict s.e.m. d–f, pshRNA–sflt-1 but not pshRNA–mbflt-1 induces corneal vascularization in wild-type mice. n = 36. Photo of eye (d) and corneal flat mounts showing CD31+ (green, vascular endothelial cells) LYVE-1− (lymphatic vessel endothelial hyaluronan receptor) blood vessels 14 days after injection (e, f). Scale bars, 500 μm.

Figure 4

Figure 4. Spontaneously vascularized corneas lacking sflt-1 are rescued by sflt-1 administration

a, Representative reducing western blots (WB) reveal deficiency of sflt-1 in corneas of corn1 and Pax6+/− mice compared with background strain A/J and Pax6+/+ mice. n = 10–12. b, c, Administration of sflt-1-Fc inhibits corneal vascularization in corn1 and Pax6+/− mice compared with administration of IgG1-Fc (by 87 ± 2% in corn1; n = 12; P = 0.01; by 85 ± 3% in Pax6+/−; n = 10; P = 0.03) and with control untreated mice (by 87 ± 2% in corn1; n = 12; P = 0.01; by 84 ± 3% in Pax6+/−; n = 10; P = 0.03). Significance by Bonferroni corrected Mann–Whitney _U_-test. Error bars depict s.e.m. (b). Representative flat mounts show CD31+ (green)/LYVE-1− corneal blood vessels (c). d, Immunostaining reveals deficiency of sflt-1 (brown) in the cornea of a 32-yr-old woman (top) with aniridia-associated vascularization, revealed by vascular cell adhesion molecule-1 (VCAM-1) staining (red) compared to the avascular cornea (lack of VCAM-1 staining) of a 38-yr-old man without aniridia (bottom). e–i, Marked deficiency of sflt-1 (reddish brown) staining in cornea of Antillean manatee (e) compared with dugong (f), African elephant (g) and beaked whale (h). Nuclear counterstain is blue (d–h). i, Representative reducing western blots with an antibody against the N terminus of flt-1 reveal presence of sflt-1 (60 kDa) and absence of mbflt-1 (190 kDa) in corneas of bottlenose dolphin (1) and Asian elephant (2); n = 4. Scale bars, 200 μm.

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