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 disorders1-4. But the molecular underpinnings of the avascular phenotype have until now remained obscure5-10 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 trap11 by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/− mice12,13 and Pax6+/− patients with aniridia14 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 corneas15, 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.

beta-Lactoglobulin and whey protein isolate (WPI) were heated in aqueous solutions at pH 2 and 7 at 80 degrees C, spread onto freshly cleaved mica surfaces, and visualized under butanol using atomic force microscopy. Fine-stranded aggregates were formed at pH 2, the diameter of strands being ca. 4 nm for beta-lactoglobulin and 10 nm for WPI. At pH 7, aggregates were composed of ellipsoidal particles, regardless of the concentration of added NaCl. This observation supports the previously proposed two-step aggregation model at neutral pH (Aymard, P.; Gimel, J. C.; Nicolai, T.; Durand, D. J. Chim. Phys. 1996, 93, 987-997), consisting of the formation of primary globular particles and the subsequent aggregation of those primary particles. The AFM provides the first direct evidence for the anisotropic shape of these primary particles. The heights of primary particles increased from ca. 11 to 27 nm with increasing concentrations of added NaCl from 0 to 0.3 M in the case of WPI. The rate of aggregation was also accelerated with increasing NaCl concentrations, which appeared to induce transitions in gel networks from fine-stranded toward particulate networks. The present study provides structural information essential for understanding the diverse physical properties of heat-induced whey protein gels.

The effects of ionic strength on fractal structures in heat-induced gels prepared from globular proteins were investigated in the framework of a fractal aggregation of colloidal particles. All gels formed at 90 °C exhibited power law relationships between the storage shear modulus (G′) and protein concentration. At 25 mM NaCl, the fractal dimension, d (∼2.2), calculated based on the value of the power law exponent agreed with those for reaction-limited cluster-cluster aggregation. Further addition of NaCl (50, 80, 500, 1000 mM) decreased the values of d (∼1.8), which agreed with d for diffusion-limited cluster-cluster aggregation. These results suggest that the predominant effect of an increase in ionic strength on globular protein gelation is ascribed to shielding charges on the surface of the proteins, thereby increasing the reaction probability of protein aggregation. The effective structure-determining rheological properties of heat-induced protein gels are characterized by fractal dimensions deduced from the rheological model, which also suggests that the size of primary flocs building fractal structures is one of the important factors that determines the linear viscoelastic properties of the gels.