The cornea stays clear by expressing a soluble form of a receptor that traps factors enabling growth of vision-obstructing blood vessels, researchers say.
When sflt-1, a free-floating receptor for vascular endothelial growth factor A, is eliminated, vision-obstructing blood vessels start growing, teams of researchers led by the Medical College of Georgia and University of Kentucky report in Nature. The paper was published online Oct. 18 and will be in the Oct. 26 print issue.
"Sflt-1 is a handcuff essentially," says Dr. Balamurali K. Ambati, corneal specialist at MCG and the Veterans Affairs Medical Center in Augusta and the study's first author. Using multiple approaches to unlock those cuffs, from neutralizing antibodies to gene ablation, mice corneas consistently developed blood vessels.
"The standard paradigm has been the cornea is avascular because it has lots of anti-angiogenic molecules. And it does," he says. "But knockdown of the others does not cause blood vessels to enter the cornea."
Flt-1's role as VEGF receptor has been known; it is abundant on cell membranes of blood vessel walls where it helps initiate blood vessel growth. In fact, its soluble form has been studied for anti-tumor potential.
However its newfound role in corneal clarity opens the door for exploring its use to eliminate unwanted blood vessels in the cornea that can follow injury, including contact lens use or a chemical burn, as well as blinding proliferation occurring in the retina with macular degeneration and diabetic retinopathy.
"If we understand what keeps the cornea avascular in the first place, that will hopefully help us restore it when that is breached," Dr. Ambati says of the cornea, which lets light into the eye and focuses two-thirds of it.
"The molecule responsible for corneal avascularity is much like the holy grail of vascular biology and our identification of VEGF receptor-1 as that candidate has far-reaching implications for a variety of neovascular diseases such as macular degeneration, diabetic retinopathy, cancer and atherosclerosis," said Dr. Jayakrishna Ambati, ophthalmologist and vice chair of the University of Kentucky Department of Ophthalmology & Visual Sciences.
In two animal models known to have blood vessels in their corneas – corn1 and Pax6 mice – they found no corneal expression of sflt-1. When they gave recombinant sflt-1, the animals' corneas cleared. Pax6 mice have a mutant version of Pax6 protein, which is involved in eye development; humans with a rare disease called aniridia, in which the irises are missing, also have this mutation.
They found the corneas of manatees, which have unusual, naturally vascularized corneas, also do not express sflt-1. Interestingly, most marine life, including whales, have clear corneas, as do shallow-water dwelling dugongs or sea cows – which are the same order of mammals as manatees – and elephants, the closest known terrestrial evolutionary relative of manatees, the researchers say.
"The correlation between sflt-1 expression and corneal avascularity in diverse mammals supports an evolutionarily conserved role for sflt-1 conferring the cloak of corneal avascularity," they write.
The finding of sflt-1's critical role in corneal clarity also opens a Pandora's box, because the avascular tissue is typically used to study drugs that stop dangerous new blood vessel growth that can occur with cancer, diabetes and macular degeneration.
"The cornea is a logical place to study these drugs because you don't have to wonder which blood vessels are abnormal: they all are," says Dr. Balamurali Ambati. "But the finding that sflt-1 is responsible for corneal avascularity has implications for the relevance of these tests because we would want to know if a candidate drug is really working or working through sflt-1 in preventing angiogenesis."
No doubt sflt-1 is vigilant, keeping blood vessels at bay when the cornea's oxygen is compromised by contact lenses or even just sleeping. Since the cornea is avascular, it counts on air for oxygen, so any barrier, even an eyelid, could cause problems. Instead researchers found levels of the VEGF-binder increase dramatically when oxygen availability drops, such as during sleep.
As they pursue its clinical potential, researchers want to study the regulators that switch the gene from producing membrane-bound flt-1 to making the roaming soluble form. "They have the same parent gene," says Dr. Balamurali Ambati. "Why sometimes does it make one and sometimes it makes the other? What controls the switch is of great interest."
Other contributing institutions include Department of Ophthalmology, Nagoya City University Medical School, Nagoya, Japan; Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville; Molecular and Cell Biology Laboratory, Rome; University of Florida College of Veterinary Medicine, Gainesville, Fla.; Department of Pathology, Microbiology and Immunology, University of California, Davis; Department of Pathology, Sea World, San Diego; The Eye Pathology Laboratory, Wilmer Institute and Department of Pathology, Johns Hopkins Medical Institutions, Baltimore; Division of Human Gene Therapy, The Gene Therapy Center, University of Alabama at Birmingham; School of Tropical Environment Studies and Geography; James Cook University, Australia; Department of Medical Genetics, University of Wisconsin, Madison; School of Medical Sciences, University of Aberdeen, United Kingdom; Department of Physiology & Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia; Institute of Medical Science, University of Tokyo, Japan; Department of Molecular Oncology, Genentech, Inc., South San Francisco; and the Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Naples.