By David N. Leff
The customary trajectory of hands-on biotechnology experimentation goes from in vitro (culture) to in vivo (animal model) to in Homo (clinical trials).
Research ophthalmologist Henry Kaplan and vision scientist Thomas Ferguson, at Washington University, in St. Louis, took the opposite route. Their experiments began with human specimens, went on to mouse models and finally to cell culture.
The target of their inquiry was, and is, age-related macular degeneration (AMD), leading cause of blindness in the elderly populations of Western countries. ¿As we age, in each decade, starting with age 55,¿ Kaplan told BioWorld Today, ¿the percentage of individuals with AMD increases. So that by age 75, for instance, approximately 20 percent of the population has macular degeneration.
¿The strongest association is with senescence,¿ he said. ¿We know that smoking is an aggravating factor. Hypotheses suggest that perhaps exposure to sun, perhaps antioxidants in the diet, are related to development of AMD. It¿s quite clear that aging is a major factor, and there is a strong genetic predisposition as well. AMD is a familial disease.¿
Eyes, brains and testes are the only mammalian organ systems that raise a protective barrier against their body¿s own immune systems. ¿This little-understood mechanism of immune privilege,¿ Kaplan suggested, ¿is probably multi-factorial, but one component of it is very important. It¿s the expression of Fas ligand [FasL] within the eye on resident ocular cells, such as in the ciliary body and the corneal endothelium. Those cells express FasL. As lymphocytes enter the eye and undergo proliferation, they start to express Fas, the receptor for FasL. Then, they come in contact with those resident cells that are expressing FasL, and undergo apoptosis. This programmed cell death is a way the eye has of naturally protecting itself from an immune response, which, if too vigorous, could result in damage to the fine ocular structures for visual function.¿
That deadly duo, FasL and Fas, can trigger cell death in a wide variety of tissues, including malignant tumors. (See BioWorld Today, June 4, 1997, p. 1.)
¿In the normal eye,¿ Kaplan explained, ¿the retinal epithelium, which is the layer under the retinal surface, expresses FasL very abundantly. The hypothesis we have presented, which the data supports, is that under normal circumstances, FasL acts as a barrier to angiogenesis ¿ new blood vessels growing beneath the retina. In other words, macular degeneration. As we age, we assume, but do not know, that the expression of FasL is probably altered. We know that the retinal pigment endothelial cells die, and therefore there may be a total reduction in the amount of FasL produced.
¿We hypothesized that with aging, because there¿s some abnormality of FasL, the blood vessels beneath the retina start to grow, are no longer stopped; can no longer bind Fas on the endothelial cells, and so abort the apoptotic cascade,¿ Kaplan said.
Kaplan is first author, and Ferguson senior author, of a paper in the current Nature Medicine, dated March 1999. Its title is ¿Fas ligand (CD95) controls angiogenesis beneath the retina.¿
He related how he and his co-authors advanced experimentally from human specimens to mouse models to cell culture.
¿Ten years ago,¿ Kaplan said, ¿in patients with AMD, we pioneered an operation to remove the abnormal blood vessels and scar tissue from beneath the retina. And, recently, when we removed these cells from six patients, we examined them histologically to see if the cells were compatible with our hypothesis ¿ namely, that FasL is expressed on the retinal pigment epithelial cells [RPE] and that Fas is present on the vascular endothelial cells. If this indeed was true in our specimens, which contain both cell types, we should have found FasL expressed on the RPE cells in an attempt to control the new blood-vessel formation. Also, we should have found Fas on the vascular endothelial cells, as a consequence of their proliferation and division. And indeed that is what we did find.
Three Rodent Strains Tested
¿That observation suggested that our hypothesis might be true,¿ Kaplan said. ¿Therefore, we decided to look at it in an experimental animal model, where we could more precisely control the expression of Fas and FasL. That brought us to the mouse.¿
Those experiments featured three rodent strains: normal wild type; a variant mutant that did not express Fas; and one that did not express FasL. ¿What we did,¿ Kaplan said, ¿was use a model of angiogenesis that mimics the new vessel formation in AMD by means of laser treatment. When the laser punctured the retina, new blood vessels frequently formed beneath it. In the normal wild-type animals, approximately one out of four laser spots resulted in such de novo blood-vessel formation. But in both the Fas-deficient and FasL-deficient variants, the incidence went from one out of four to two out of four. Also, in the FasL-deficient mice, those abnormal vessels ¿ rather than growing as small, skinny little strands characteristic of the wild type angiogenesis ¿ spread as a dramatically abundant bush of vessels.
¿That indeed lent support to our hypothesis that both Fas and FasL were very important in the incidence and extent of new vessel formation,¿ Kaplan continued. ¿We then extended those studies in vitro, using vascular endothelial cells as a model of angiogenesis. This culture assay showed that, if we bind the Fas that¿s on the vascular endothelial cells, we can inhibit new vessel formation. All of which led us to the conclusion that Fas and FasL are in the pathway of apoptosis, which regulates neovascularization.¿
Kaplan added, ¿We¿re clearly not close to clinical applications for this. The theoretical direction is that if this Fas/FasL interaction is important in controlling angiogenesis in macular degeneration, diabetic retinopathy and tumor metastasis, then one might be able to pharmacologically up-rate the expression of FasL in resident cells where such disease has occurred.
¿So,¿ he concluded, ¿it opens up the potential for regulating the expression of FasL to treat, and perhaps even prevent, some of the complications that occur.¿ n