Mar. 3, 1999 St. Louis, March 1, 1999 -- Investigators at Washington University School of Medicine in St. Louis have found that a mechanism key to immune-privilege in the eye also plays a role in age-related macular degeneration, the leading cause of blindness in people over the age of 60.
Reporting today in the March issue of Nature Medicine, the investigators link a form of macular degeneration to potential problems involving the interaction between two proteins called Fas and Fas ligand (FasL). Contact between these two molecules prevents immune system activity in the front of the eye. At the back of the eye in the retina, the interaction between Fas and FasL is involved in preventing growth of blood vessels that interfere with vision in the "wet" form of age-related macular degeneration. When one of these is deficient, vision loss can occur.
"There are two forms of the disease," said investigator Henry J. Kaplan, M.D. professor of ophthalmology and visual sciences at the School of Medicine. "The dry form is the more common type, but the wet form is associated with severe loss of vision. In the wet form of the disease, abnormal vessels grow beneath the retina and interfere with central vision. In the dry form, while there is vision loss, there are no unwanted vessels growing beneath the retina."
The vessels grow from the choroid, a structure beneath the retina. For 10 years, Kaplan and colleagues have attempted to surgically remove abnormal vessels and scar tissue. The limitation of this approach is that while removing the vessels, the surgeon also is likely to remove a thin layer of cells at the back of the retina called the retinal pigment epithelium (RPE). These cells nourish photoreceptor cells that convert light impulses into vision. If a surgeon removes abnormal vessels along with the RPE layer, the surgery probably will not restore vision.
The search for a different treatment for the disease led Kaplan, a retina surgeon, to Thomas A. Ferguson, Ph.D., associate professor of ophthalmology and pathology at the School of Medicine. A basic scientist, Ferguson was the first to discover how Fas and FasL function in the front of the eye.
In 1995, Ferguson reported that FasL found on the surface of corneal cells protects the eye by intercepting invading immune system cells. When those cells, such as T cells, are activated during an immune response to a viral infection or some other problem, they make a receptor protein called Fas. When these Fas molecules encounter FasL on the surface of cells in the eye, the activated immune cells are destroyed through programmed cell death.
Following that initial discovery, Ferguson showed that the presence of FasL helps explain why the vast majority of corneal transplants succeed even without anti-rejection drugs. In the lab, he also has found FasL in other parts of the eye.
"It's very interesting," he explained. "Fas ligand is located in all of the areas where there's the potential for interaction between the eye and the outside environment, especially where there are blood vessels -- in the iris, all over the cornea and in the retinal pigment epithelium, which forms the outermost layer of the retina."
The observation that FasL was present in the retinal pigment epithelium led Ferguson and Kaplan to look at the cells that migrate from the choroid when blood vessels invade the retina in the wet form of macular degeneration. They looked under the microscope at specimens taken from human subjects and found that the RPE cells were positive for FasL and that the cells on the surface of the growing blood vessels were positive for Fas.
To learn more about the importance of Fas and FasL in the retina, Ferguson and Kaplan took their experiments into a strain of mice used to study interactions between the two proteins. In a mouse model of macular degeneration, they found that if the mice were deficient in Fas or FasL, there was a large increase in the growth of new retinal vessels.
To create conditions similar to macular degeneration, the investigators used a laser to make small scars in the mouse retina. In normal mice that expressed both Fas and FasL, less than a quarter of the mice developed new blood vessels at the scar sites. In addition, where new vessels did grow, the investigators found that those vessels were surrounded by FasL-positive RPE cells, suggesting that the retina was working to prevent the growth of those blood vessels.
But in the mouse strain deficient in Fas, the lpr mouse, new vessels grew about 44 percent of the time, and in those deficient in FasL, the gld mouse, the investigators observed new vessel growth 55 percent of the time.
"When Fas ligand was mutated in this mouse strain, the blood vessels just went crazy," Ferguson said. "They grew vigorously beneath the laser spot. They grew inside the laser spot. They grew around the laser spot. The amount of new blood vessel growth was incredibly large."
In addition to the observations in humans and mice, the investigators also looked at choroidal vascular cells in culture and found that those cells could form connections and grow only in the absence of FasL.
The investigators believe their experiments, taken together, demonstrate that FasL on RPE cells protects the retina against the growth of new blood vessels that can result in macular degeneration. The findings could mean that the wet form of macular degeneration develops only in people with abnormal FasL that cannot destroy Fas-positive vascular cells invading the retina from the choroid.
"Clearly, we need to try to determine whether RPE cells express less Fas ligand or Fas ligand that's non-functional as patients get older and age-related macular degeneration develops," Kaplan said. "If that is the case, there may be drugs that could be used to increase the expression of Fas ligand in the retina and inhibit the growth of these invading blood vessels."
He also hopes to measure the amount of FasL expressed on RPE cells in the retina and to follow the expression of FasL over time to learn whether problems develop that could contribute to the new vessel growth seen in macular degeneration.
Beyond macular degeneration, Kaplan and Ferguson believe the interaction between Fas and FasL may be important for patients with diabetes. A complication of diabetes is the formation of new blood vessels that results in diabetic retinopathy, leading to vision loss. The researchers have a grant from the Kilo Foundation to look at a potential role for FasL to inhibit new vessel growth in diabetic retinopathy.
Kaplan and Ferguson also believe the interaction between Fas and FasL may have implications beyond the eye, however. Already understood as key to immune system responses, the interaction of Fas and FasL in new vessel formation may be important in understanding other diseases where blood vessel formation is a problem.
"You don't have to go far in the literature now to find tumors that are Fas ligand-positive," Ferguson said. "There are melanomas, adenocarcinomas, colon carcinomas, all kinds of cancerous tumors that are positive for Fas ligand. Whether that regulates blood vessel growth in those tumors is something no one has looked at yet. Most scientists are looking at angiostatin and those sorts of molecules, but I think that this discovery may change that."
This research was supported by grants from the National Eye Institute, the Foundation Fighting Blindness and Research to Prevent Blindness, Inc.
Kaplan HJ, Leibole MA, Tezel T, Ferguson, TA. Fas Ligand (CD95 ligand) Controls Angiogenesis Beneath the Retina. Nature Medicine, vol. 5 (3), March 1999.
The full-time and volunteer faculty of Washington University School of Medicine are the physicians and surgeons of Barnes-Jewish and St. Louis Children's hospitals. The School of Medicine is one of the leading medical research, teaching and patient care institutions in the nation. Through its affiliations with Barnes-Jewish and St. Louis Children's hospitals, the School of Medicine is linked to BJC Health System.
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