Selective permeability of mouse blood-aqueous barrier as determined by¹⁵N-heavy isotope tracing and mass spectrometry

The blood-aqueous barrier plays a key role in regulating aqueous humor homeostasis by selectively restricting passage of proteins into the eye. The kinetics of aqueous flow are traditionally measured using artificial markers; however, these marker molecules do not address the barrier’s selective permeability to plasma proteins. Here we applied stable isotope labeling of all serum proteins with nitrogen-15 (¹⁵N) atoms. Following systemic injection of this “heavy” serum in mice, the ¹⁵N-to-endogenous nitrogen-14 (¹⁴N) ratio of each protein in aqueous was measured by mass spectrometry. By monitoring the kinetic changes in these ratios, we determined the permeability profiles of hundreds of serum proteins. Meanwhile, we subjected one of the eyes to neoangiogenic wound healing by inflicting injury to the corneal limbus and compared the ¹⁵N proteomes between the normal eyes and the recovering eyes at 2 weeks after injury. In the injured eye, we detected markedly enhanced permeability to inhibitory complement regulator proteins, such as Cfh, Cfhr, Cfb, Cfi, Cfd, and Vtn. Many of the proteins in this group are implicated in age-related macular degeneration associated with leakage of the blood-retinal barrier due to inflammation. To rule out the possibility that the observed leakage was due simply to physical damage of the blood vessels, we separately created a neovascularization model using an alkali burn of the avascular cornea. In this latter model, elevated levels of Cfh and Cfb were evident. These findings suggest that ocular neovascularization is associated with enhanced permeability to serum complement regulators.