Browsing by Author "Bowes Rickman, Catherine"

Age-related macular degeneration (AMD), a major cause of irreversible vision loss in elderly populations, has been associated genetically with the complement system, and especially with complement factor H (CFH). However, it remains unknown how CFH affects risk of developing AMD. Humans additionally express a truncated splice variant of CFH, Factor H-like 1 (FHL-1). The retinal pigmented epithelium (RPE) is separated from circulation by Bruch’s membrane (BrM), and it has been reported that CFH is unable to diffuse through BrM, while FHL-1 can. Therefore, it is uncertain whether FHL-1 is responsible for most complement regulation in BrM and on RPE cells, or if local production of CFH is more important. We examined this question in Cfh-/- animals through the use of AAVs, allowing expression of both FHL-1 or truncated versions of CFH retaining the C-terminal end of protein, 18tCFH and 12tCFH, from either local sources following subretinal injection or from systemic sources following tail vein injection. While all constructs showed regulation of the complement system using Western blots in circulation, only 18tCFH, the most complete of the constructs, showed definitive regulation of the complement system following subretinal injection. Additionally, there was more evidence of complement regulation following expression locally in the eye, as opposed to circulation. These results suggest that the C-terminus of the protein is essential for complement regulation in the eye, and that local delivery of the protein is the better delivery route. These findings are important for the design of future gene therapies for complement regulation in AMD patients.

Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/- and Cfh-/- mice fed a high fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (RPE) deposit formation, specifically basal laminar deposits, following high fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/- and Cfh-/- mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/- mice. We demonstrate that such pathology is a function of excess complement activation and C5a production, associated with monocyte recruitment, in Cfh+/- mice versus complement deficiency in Cfh-/- animals. Due to the CFH dependent increase in sub-RPE deposit height we interrogated the potential of CFH as a novel regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Interestingly, although the CFH H402 variant shows altered binding to BrM, this does not affect its ability to remove endogenous lipoproteins. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly in developed countries. Oxidative stress is implicated in the pathobiology of AMD and initially targets the retinal pigmented epithelium (RPE). A major function of the RPE is to process photoreceptor outer segments and its polarity is responsible for the directional secretion of proteins, lipoproteins, and extracellular vesicles (including exosomes). Dysfunction of the RPE leads to the development of AMD. There are no non-imaging biomarkers currently available to detect AMD. A potential approach to detect AMD is through analysis of exosomes, which are nanovesicles that can be purified from biofluids (e.g., plasma and urine). Exosomes are known to be secreted during homeostasis and diseased states. Exosomes transfer their cargo containing protein and genetic material (mostly microRNAs) to other cells, resulting in activation of signaling pathways. The role of RPE-derived exosomes in AMD has been largely understudied. Our lab has established and published a protocol for isolating exosomes and identifying their cargo from primary porcine RPE grown on Transwells. To test the role RPE-derived exosomes at early stages of AMD, we used polarized primary RPE cells and exposed them to subtoxic chronic oxidative stress. Unbiased proteomic analyses of the content of basolateral exosomes isolated from RPE cultures under oxidative stress revealed changes to a number of desmosome and hemidesmosome proteins, in which we identified a stress profile. We also identified significant changes in proteins accumulating in the basal sub-RPE extracellular matrix during oxidative stress. To further interrogate the role of exosomes in RPE function, we analyzed the miRNA cargo from exosomes secreted apically and basally from control and chronic oxidative stressed primary RPE cells. We identified a set of miRNAs associated with RPE homeostasis. Unbiased miRNA analysis of secreted exosomes revealed differences based on polarity, exosome isolation method, and oxidative stress. Our data supports that proteins and miRNAs in exosomes contribute to RPE homeostasis and function in a polarized manner. Our findings suggest that exosomes are early indicators of RPE dysfunction prior to overt morphological changes, supporting further studies of RPE exosomal cargo as biomarkers for AMD.

Age-related macular degeneration (AMD) is a complex retinal degeneration present in elderly populations of first world countries with limited available therapeutic interventions. Risk for AMD is strongly conferred by advanced aging but is also modulated by genetic variants and environmental stresses. One of the most replicated genetic variants associated with AMD is the Complement Factor H (CFH) Y402H polymorphism. CFH is a critical regulator of the complement cascade but how the H402 variant impairs its function and contributes to AMD development is unclear. Herein, the role of the H402 variant in the development of AMD-like pathologies was interrogated using two different mouse models based on advanced aging, CFH perturbation and environmental stress.

First, aged CFH hemizygous knockout (Cfh+/-) mice were fed a high fat, cholesterol-enriched (HFC) diet for eight weeks to induce AMD-like pathologies such as vision loss, increased retinal pigmented epithelium (RPE) damage, increased sub-RPE deposit formation and immune cell recruitment to the RPE/choroid interface. To determine if the recruitment of immune cells drives the formation of the AMD-like pathologies in aged Cfh+/-~HFC mice, aged Cfh+/- mice were concurrently treated with a systemic anti-C5a therapy during the eight week HFC diet treatment to block the complement-mediated recruitment of immune cells to the eye. However, the ocular phenotype was unchanged in aged Cfh+/-~HFC mice treated with the anti-C5a therapy despite the decrease of recruited immune cells to the posterior eye. This data suggests the risk associated with the H402 CFH variant is not solely attributable to complement-mediated immune cell recruitment to the posterior eye.

Second, aged transgenic mice expressing equal concentrations of the normal human CFH Y402 (CFH-Y:Cfh-/-) or risk-associated CFH H402 (CFH-HH:Cfh-/-) protein were fed an eight week HFC diet. Remarkably, vision loss, increased RPE damage and increased sub-RPE deposit formation was only observed in aged CFH-HH:Cfh-/- mice following diet treatment. Biochemical analysis of aged CFH:Cfh-/- mice revealed differences in plasma and ocular lipoproteins, but not complement, between aged CFH-Y:Cfh-/-~HFC and CFH-HH:Cfh-/-~HFC mice. Thus, we targeted plasma lipoprotein levels through dietary intervention in aged CFH-HH:Cfh-/- mice and observed visual loss in these mice that coincided with dietary cholesterol-induced increases of plasma LDL. Based on our findings we hypothesize that the risk-associated with the H402 CFH variant and AMD is due to the interaction of CFH with lipoproteins and not its complement regulatory roles. These new insights may help explain why current therapies targeting complement inhibition for AMD have failed and, importantly, support targeting lipoprotein metabolism, as a treatment for AMD.