AMD is an significant disease affecting millions of persons worldwide. Although the neovascular (wet) form of AMD represents only 10% of reported cases, more than 90% of the blindness from this disorder is caused by this form. Consequently, treatments that target neovascularization have important implications for the quality of life of many patients. Current anti-VEGF therapies have been effective,
31 but there is no therapy that successfully targets other proinflammatory components of this disorder. Because the eye is an immune-privileged site and contains constitutive mechanisms to prohibit inflammation and angiogenesis,
2,30 we tested the idea that modulating immune privilege might have important therapeutic implications. Our results show that we can prevent blood vessel growth by injection of cytotoxic sFasL into the vitreous cavity. In addition, we can inhibit neovascularization by increasing RPE cell expression of FasL by systemic treatment with doxycycline. We conclude that targeting the Fas/FasL pathway may be a viable treatment for pathogenic neovascularization in the eye.
FasL is an important component of the immune privilege, and loss of this protein can have significant consequences for inflammation,
3 corneal transplantation,
32,33 and neovascularization.
8–10 FasL is expressed on parenchymal cells throughout the eye and forms a barrier to inflammatory cells and sprouting endothelial cells.
10,11,34 In the retina, FasL is expressed in the RPE cells.
3,6,34,35 Studies have shown that FasL expression is tightly regulated in both lymphoid and nonlymphoid cell populations, thereby preventing unwanted damage to Fas
+ cells and tissues throughout the body.
1,36 Thus, cells that use FasL as a barrier to cellular invasion or as an effector molecule typically do not display the protein on their surfaces. It is either stored in secretory vesicles for rapid mobilization to the cell surface
37,38 or produced de novo after cellular activation, stress, or injury.
1,36 Our data support this idea because we have shown that FasL is not expressed on the surfaces of RPE cells; rather, surface expression is rapidly increased on laser injury. Interestingly, even FasL upregulation in response to laser injury does not provide an absolute barrier to penetration of the eye by endothelial cells because laser-treated mice with increased surface FasL expression develop neovascularization. One possible explanation for this is the sensitivity of cell surface FasL to the activity of MMPs, which can cleave the protein from the cell surface, thereby rapidly downregulating its activity.
27,37 As a result, increased surface expression is offset by enzymatic activity resulting in less efficient FasL function. Our results show that the use of a potent MMP inhibitor can increase FasL expression and effectively reduce FasL cleavage, thereby eliminating the neovascular response.
Doxycycline is an effective antimicrobial agent that has potent anti-inflammatory effects in a number of experimental models.
25,28,39 Besides being a potent MMP inhibitor, doxycycline can inhibit IL-1β production,
29 decrease nitric oxide production,
40 inhibit stress kinase activation,
28 and prevent cellular proliferation.
10 These properties can easily explain the potent effect of doxycycline on CNV in the laser model because angiogenesis can depend on an initial inflammatory response.
16–18 However, our data suggest that though these functions of doxycycline may be important to its antiangiogenic properties, the ultimate effect of doxycycline on the CNV response is mediated by its effect on FasL expression. That doxycycline treatment is ineffective in mice without functional expression of this proapoptotic protein (i.e., in B6-
gld mice) supports this idea.
Doxycycline has also been shown to have effects on several VEGF-related functions.
25,28 In the current studies we observed a decrease in VEGF after doxycycline treatment, suggesting that this may be a potential mechanism. However, VEGF production was unaffected in FasL-deficient
gld mice, suggesting that the reduction in VEGF production may be related to the decreased angiogenesis in doxycycline-treated mice, not the result of a direct effect on VEGF production.
Recently, we demonstrated that FasL was also expressed on macrophages and that these cells were capable of inhibiting laser-induced CNV. Consequently, we examined FasL expression on the macrophage infiltrates in doxycycline-treated mice. We observed little FasL expression on CD11b
+ cells, further supporting our previous study demonstrating that RPE cell-derived FasL was the major effector in the antiangiogenic properties of FasL in the eye. In addition, doxycycline treatment did not alter the infiltration of CD11b
+ cells into the laser lesions. We conclude that the major effect of FasL in this model is likely the inhibition of endothelial cell function through apoptosis, as has been demonstrated previously.
8–10
As a final point, we used doses of doxycycline that are much higher than the appropriate therapeutic doses of the compound.
26 We found that these levels were well tolerated in short-term experiments, but we do not have data on any long-term effects of doxycycline treatment. However, we are not specifically proposing doxycycline as a long-term treatment for CNV but are suggesting that targeting an important molecule involved in immune privilege with the use of a potent MMP inhibitor has therapeutic efficacy. Thus, targeting the Fas-FasL pathway alone, or in combination with other treatments, may be viable clinical approach to the treatment of neovascular eye disease.
Supported by National Institutes of Health Grants EY06765, EY015570, and EY02687 (Department of Ophthalmology and Visual Science Core Grant); a Department of Ophthalmology and Visual Science grant from Research to Prevent Blindness; and the Macular Vision Research Foundation.