Research into the Mechanisms of Macular Degeneration

 
The first step towards our goal is to improve our understanding of the disease mechanisms involved in AMD, particularly in the early stages.  Specifically, we want to study the role of the different components of the immune system in AMD.  To accomplish this we are working on developing a pathophysiologically-relevant animal model of the disease.  Our approach exploits the recently discovered genetic association of AMD with a mutation in complement factor H (CFH or Cfh). 
 

 

 

Background:

  1.  Complement factor H (Cfh) is a key regulator of the complement cascade.
  2.  A point mutation (Y402H) increases the risk of AMD up to 7-fold.
  3.  The “at risk” 402H variant of CFH appears to make AMD patients resistant to anti-VEGF agents and t0 anti-oxidant supplements.
  4.  Despite the obvious anatomical differences, mice CAN develop drusen and CNV.
  5.  We need relevant models of AMD that develop drusen and CNV with high enough frequency to allow for research into the early steps in the pathogenesis.

  

 

 

Methods:

In collaboration with Dr. Wojciech Kedzierski, we have generated transgenic mouse lines expressing chimeric (part mouse/part human) Cfh molecules with either the 402-histidine (“at-risk”) variant or the 402-tyrosine (“non-risk”) variant of Cfh.  We aim to use these mice to: (a) identify the molecular basis for both the increased susceptibility to AMD and the resistance to therapy observed in human H-CFH carriers, and to (b) generate a relevant model of AMD that would allow the dissection of the role of the different arms of the immune system in the disease.  We have shown that our mice express the transgenic mRNA both in the liver and in the posterior segment ocular tissues.  They also produce the transgenic protein, which can be found in the serum.

  

 

transgenic mice

  Figure 2: Constructs and design of transgenic animals  

 

graph

 

 

 

 

 

 

 

 

 

  Figure 3: Fold increase in mRNA level by RT-PCR.  The posterior segment of 2 transgenic mouse lines vs. non-transgenic mice were analyzed using primers specific for the transgenic mRNA  

 

western blot

 

 

 

 

 

 

 

 

 

  Figure 4: Western blot and serum concentration of transgenic Cfh in some of our lines.  Lines are 4th generation (313 is 2nd gen).  

 

 

 

We are also using a laser-induced model of AMD as a way to study the acute neovascular phase of the disease.  When applied to our transgenic mice it will help us determine if and how the factor H variants affect the susceptibility to neovascular changes, and the dependence of the neovascular membranes on VEGF and other growth factors.  This laser model is also helping us to examine the potential therapeutic role of some antibodies and small molecules generated here at UTSW in blocking or causing regression of choroidal neovascularization.

 

 

 

 

 

 

 

  Figure 5: Fluorescein angiogram of laser-induced CNV in a B6 mouse.  

 

 

 

Work in Progress:

  1. Does the H402 variant of Cfh predispose to increased risk for choroidal neovascularization (CNV) and/or resistance to anti-VEGF therapy?
        • Frequency and size of CNV induced by laser in H402 vs. Y 402 Cfh transgenic mice
        • Response to anti-VEGF agents

       

  2. Does H402 Cfh change the subretinal microenvironment predisposing to an altered activation of the immune system?
        • Use high-throughput techniques (proteomics and custom-made qPCR array) to screen the
        • subretinal tissues for levels of protein and mRNA of molecules of interest.  Important
        • molecules include: cytokines, chemokines and other chemoattractants, complement
        • components, and enzymes involved in free-radical and lipofuscin methabolism. 

       

  3. Will the Cfh polymorphism lead to a mouse model of AMD with spontaneous CNV?
        • Cross our transgenic mice with human c-reactive protein (hCrp) transgenic mice
        • Look for histopathologic changes, clinically-visible drusen, pigmentary changes and CNV
        • Triggers of inflammation/oxidative stress: light exposure
        • Cross mice to Superoxide Dismutase 1 deficient mice to increase oxidative stress

       

  4. Is the immune system involved in the pathogenesis of drusen or CNV?  Are there subsets of inflammatory cells that are essential?
        • Analyze the deposition of complement, CRP and inflammatory molecules in the subretinal/sub-
        • RPE space in our transgenic mice.
        • Analyze the timing and type of cellular infiltration
        • Deplete subsets of immune cells using antibodies

       

  5. Can our animal model help us identify novel molecules to inhibit the development of drusen or CNV?