Targeting Lipid Metabolism for the Treatment of Age-Related Macular Degeneration: Insights from Preclinical Mouse Models

Differences between mouse and human retinas

Notable distinctions exist between the mouse and human retina. For the most part, mouse and human eyes undergo similar early developmental programs, but these programs diverge to allow for the complex structure of the human retina. ²⁰⁴ Importantly, mice do not possess a cone-dominant macular region but rather a rod-rich retina that is similar to the rod-rich parafovea in humans. ²⁰⁵ This similarity is an advantage of using the mouse retina for modeling early to intermediate AMD since this rod-rich region of the human macula is where macular degeneration is first detected. ²⁰⁶ The cone-dominant macular region in the human retina allows for high visual acuity, but mice lacking this type of retinal structure have extremely low visual acuity that is equivalent to 20/2,000. ²⁰⁷ Rods and cones have different energy demands ²⁰⁸ and transcriptional profiles, ²⁰⁹ thus, there could be difficulty in translating findings of ophthalmic drug studies in mice to humans.

A potent physiological difference between a mouse and human retina is the interaction between photoreceptors and RPE cells. RPE cells are tasked with delivering nutrients to photoreceptors and ingesting photoreceptor outer segment wastes. ²¹⁰ The central part of the mouse retina contains a higher density of photoreceptors than the central part of the human retina, equating to a higher proportion of photoreceptors to RPE cells in the central murine retina. ²⁰⁵ In addition, there are more smaller and denser RPE cells in the center of the mouse retina than the central human retina. ²⁰⁵ These differences suggest that murine RPE may have increased phagocytosis of photoreceptor outer segments and may explain transcriptional differences between mouse and human RPE, including increased expression of oxidative stress and outer retinal barrier genes. ²¹¹ It has been shown that mice have higher basal metabolic rates than humans ²¹² and could suggest higher metabolic rates in mouse RPE cells than human RPE cells. Furthermore, since RPE cells have coordinated metabolic relationships with both rod and cone photoreceptors, ²¹³ it is plausible that there are variations in the metabolic ecosystem within the murine retina versus human retina and may confound therapeutic studies, especially those focusing on metabolism.

Differences between mouse and human lipid metabolism

Mice and humans have similar expression of genes involved in lipid metabolism within the retina,^214,215 but it is unknown if there are differences between lipid metabolic functions between mouse and human RPE. One important expression difference between these species is the absence of CETP in the mouse. ²¹⁶ CETP facilitates the transfer of cholesterol between HDL to very-low-density lipoprotein (VLDL) and LDL. ²¹⁷ Because of the CETP deficiency in mice, most plasma cholesterol is confined to HDL particles. ²¹⁸ This is in stark contrast to humans where most plasma cholesterol is found in LDL particles. ²¹⁸ Recapitulating the plasma lipid profiles of humans in mice can be achieved by diet intervention but often these diets do not represent typical diets consumed by humans. Strikingly, regardless of the absence of CETP and diet intervention, both mice and humans have comparable lipoprotein proteomes that may indicate similar functions of lipoproteins between mice and humans. ²¹⁸ Lastly, differences in transcription factors involved in lipid metabolism between mice and humans may influence lipid-targeting drug studies in AMD mouse models. ²¹⁹ Thus, caution is warranted when extrapolating conclusions from lipid metabolism, notably with regard to cholesterol metabolism, studies in mice to humans.

Translatability of mouse studies to the clinic

The anatomical differences between the mouse and human retina have called into question the usefulness of mice in evaluating therapies for AMD since these studies may not translate well to the clinic. However, it is important to remember that AMD mouse models are only 1 step in the bench-to-bedside pipeline for translating basic research to clinical treatments (Fig. 3). We strongly advocate for the validation of preclinical AMD mouse model studies with other models of AMD. In parallel with the mouse models presented in this review, advances have been made in cell-culture systems such as fetal human RPE, ²²⁰ induced-pluripotent stem cell-derived RPE, ²²¹ primary porcine RPE, ²²² and retinal organoids ²²³ that allow for confirmation of mouse study findings in human cells (Fig. 3). Ultimately, any therapeutic showing promising effects should be tested in higher order animal models of AMD, including rats, rabbits, pigs, or nonhuman primates (Fig. 3). No model of AMD has represented the full complex spectrum of AMD but combining multiple models together is essential for the translating results from mouse studies to humans.

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FIG. 3.

Bench-to-bedside pipeline of AMD therapeutics. Preclinical AMD mouse models can be used to evaluate therapeutics for AMD but these therapeutics need to be tested in cell cultures modeling AMD and/or higher order animal models of AMD. Then, promising therapies can advance to clinical trials to determine if they are viable treatments for AMD. Multiple models should be utilized to translate preclinical AMD mouse model studies to clinically relevant applications.

Clearance of pathogenic lipid or protein components in Sub-RPE deposits

Testing therapies directed at preventing the formation and/or accumulation of BLamDs in AMD mouse models is a good use of these models with the caveat that these deposits are not completely analogous to pathogenic drusen in AMD,^{21,77,80,102,104,105,109,110,117–119,128,132,224} such that therapies that work in these preclinical models may not necessarily translate well to human clinical trials. In support of this approach, it is important to note that many drusen constituents are found in BLamDs, including complement components,^{21,81,83,105,106,108–110,118,148,224} apolipoproteins, ⁸¹ amyloid beta, ⁸¹ extracellular membranous debris,^104,109,224 and long-spaced collagen.^{77,102,107,109} Targeting specific components within BLamDs has led to amelioration of AMD-like pathologies in AMD mouse models. For example, aged transgenic APOE mice with targeted replacement of mouse ApoE with human APOE4 develop an AMD-like ocular phenotype, including decreased visual function, CNV, RPE damage, and BLamD accumulation after an 8-week high-fat, cholesterol-enriched (HFC) diet. ⁸⁰ APOE4 is associated with decreased risk for AMD in humans but the pathogenicity caused by the E4 allele in aged mice fed a HFC diet is unknown.^59–62

The BLamDs of APOE4∼HFC mice contain complement activated products and amyloid-beta. ⁸¹ Targeting amyloid beta with a systemic anti-amyloid-beta immunotherapy prevented amyloid-beta accumulation, sub-RPE complement activation, and AMD-like pathologies, although there was no change in BLamD load. ⁸¹ Preventing the accumulation of toxic inflammatory components within drusen-like amyloid beta may be a viable therapeutic approach for AMD. However, a humanized monoclonal antibody against amyloid beta was tested in a clinical trial for the treatment of geographic atrophy but it failed to slow the geography atrophy enlargement in patients, ²²⁵ indicating not all therapies assessed in mice are translatable to human diseases. Still, it should be noted that the anti-amyloid antibody, RN6G, which showed efficacy in the mouse and targets the c-terminus of both Ab40 and Ab42 was not the one tested in clinical trials. ⁸¹

Similar to amyloid beta, oxidized lipids can be pathogenic to RPE cells. ²⁶ A therapeutic strategy for targeting inflammatory lipids in AMD has emerged from studies of AMD mouse models with varying complement factor H (CFH) activity.^20,21,138 CFH is a major AMD susceptibility gene^226–229 that functions as the main soluble regulator of the alternative complement pathway by serving as a cofactor for factor I-mediated proteolytic inactivation of C3b ²³⁰ and accelerating the decay of the C3 convertase that is responsible for the intial activation and propagation of the complement cascade. ²³¹ The importance of CFH in regulating the formation of the C3 convertase is exemplified by the absence of intact plasma C3 in Cfh knockout (Cfh^−/−) mice due to uncontrolled C3 cleavage.^21,232

In addition to its canonical functions, we have found that CFH can bind and decrease adherence of ApoE- and ApoB-containing lipoproteins to BrM. ²¹ To examine the significance of impaired CFH binding to lipoproteins in vivo, C57BL/6J, Cfh heterozygous (Cfh^+/−), and Cfh^−/− mice were aged to ninety weeks and then fed a HFC diet for 8 weeks to exacerbate the subtle AMD-like phenotype seen in aged Cfh^−/− mice.^21,108,109 Consumption of a HFC diet leads to increased circulating lipoproteins in mice. ²³³ Both aged Cfh^+/− and Cfh^−/− mice accumulate sub-RPE basal deposits in response to the HFC diet. ²¹ Notably, only the aged Cfh^+/− and not the Cfh^−/− null mice fed a HFC diet, developed sustained vision loss and RPE damage. ²¹ Aged Cfh^−/− mice may be protected against HFC-induced ocular damage from pathogenic sub-RPE basal deposits because they lack a reservoir of complement components and they display increased expression of membrane-bound complement regulators in the posterior eye, whereas Cfh^+/− mice possess an intact complement system, but express only half the levels of Cfh as wild-type mice.^21,234,235

CFH also acts to regulate RPE-derived lipoprotein accumulations in BrM. Evidence for a unique RPE-derived lipoprotein comes from biochemical assessments of human donor eyes.^23,236 RPE cells possess the machinery required to generate lipoproteins ²³⁷ and have been validated by detecting secreted lipoproteins in the media of RPE cell cultures.^236–238 Our recent study of transgenic mice expressing equal amounts of the full-length normal human CFH Y402 versus the AMD-risk-associated CFH H402 variant on a Cfh null background (CFH-Y/0 and CFH-H/H, respectively) revealed a correlation between RPE-derived lipoproteins and pathologies. ²⁰ The CFH Y402H polymorphism is one of the most replicated genetic variants associated with AMD risk,^226–229 so these mice were developed to test the in vivo effect of the Y402H CFH risk variant on AMD pathobiology.^20,109 CFH-Y/0 and CFH-H/H mice were aged to 90 weeks on a normal mouse chow diet (ND) and then switched to a HFC diet for 8 weeks to test for an AMD phenotype as aging and the consumption of a HFC diet are sufficient to elicit AMD-like pathology development in Cfh heterozygous mice and APOE4-targeted replacement mice.^21,80 Only the old CFH-H/H mice fed HFC (CFH-H/H∼HFC) developed AMD-like pathologies. ²⁰

Quantitation of plasma lipoproteins in these mice revealed decreases in plasma LDLs and its markers, apolipoprotein B100 (ApoB100), and ApoE in aged CFH-H/H∼HFC mice compared with aged CFH-Y/0 mice fed HFC (CFH-Y/0∼HFC), but no change in any other lipoprotein class. ²⁰ Strikingly, however, biochemical analyses revealed that changes in eyecup apolipoproteins correlated with the AMD-like phenotype seen in the CFH-H/H∼HFC, where apolipoproteins B48 (ApoB48) and A1 (ApoA-1) are elevated in the RPE/choroid of the aged CFH-H/H∼HFC mice compared with age-matched control CFH-Y/0 after an 8-week HFC diet. ²⁰ Thus, we are the first to establish a functional consequence of the Y402H polymorphism in vivo, promoting AMD-like pathology and affecting lipoprotein levels in aged mice. ²⁰

The identity of molecular pathways involved in RPE lipoprotein synthesis and secretion are largely unknown, but insight may be gleaned from a dietary intervention study using aged CFH-H/H mice performed in our laboratory. Diets with similar macronutrient composition as the HFC diet previously used, which either had the dietary cholesterol removed (HF) or the fat removed [high cholesterol (HC)], were used to test the role of dietary cholesterol and fat on circulating lipoprotein and visual function in aged CFH-H/H mice. Aged CFH-H/H mice consuming a HFC or HC diet have increased chylomicron (CM)-, VLDL-, and LDL-containing cholesterol compared with aged CFH-H/H mice after a ND or HF diet (Fig. 5A). The area under each lipoprotein curve was calculated and the confirmed levels of these lipoproteins were statistically different between these groups (Fig. 5B). Visual function of aged CFH-H/H mice was assessed by scotopic electroretinography (ERG) after the consumption of an 8-week HF or HC diet. ⁸¹ Using another cohort of aged CFH-H/H mice, these mice developed a similar decrease of scotopic ERG b-wave responses after the consumption of an 8-week HFC diet (Fig. 5C), as previously described. ²⁰ No change was observed in aged CFH-H/H∼HF mice relative to aged CFH-H/H∼ND mice (Fig. 5D). Notably, aged CFH-H/H∼HC mice developed significantly attenuated ERG b-wave responses compared with aged CFH-H/H∼ND mice (Fig. 5E).

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FIG. 5.

Dietary intervention effects on plasma lipoprotein levels and visual function of aged CFH-H/H mice. Male CFH-H/H mice over 90 weeks of age, housed conventionally and maintained on ND (Isopurina 5001; Prolab) were either continued on ND or switched to a HFC (Envigo #88051), HF (Envigo #98232) or HC (Envigo #91342) diet for 8 weeks. All mice were negative for the rd8 mutation. Protocols for FPLC fractionation, cholesterol quantification, ERG, and statistical analysis are described in Landowski et al. ²⁰ (A) FPLC fractions of male aged CFH-H/H mice after an 8-week ND, HFC, HF, and HC diet. (B) Averages of the area under the FPLC curve for aged male CFH-H/H mice after an 8-week ND, HFC, HF, and HC diet. Consumption of dietary cholesterol increases CM/VLDL and LDL cholesterol fractions in aged male CFH-H/H mice relative to the ND- and HF-fed groups. (C–E) Visual function in aged male CFH-H/H mice after an 8-week ND, HFC, HF, and HC diet. Analysis of scotopic ERG b-wave responses reveals statistically lower b-wave amplitudes in aged CFH-H/H mice fed a HFC and HC diet compared with ND-fed controls. Data are presented as fitted lines of the average. Mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. CFH, complement factor H; CM, chylomicron; ERG, electroretinography; FPLC, fast protein liquid chromatography; HC, high cholesterol with no added cocoa butter fat; HDL, high-density lipoprotein; HF, high fat with no added cholesterol; HFC, high-fat, cholesterol-enriched; LDL, low-density lipoprotein; ND, normal mouse chow diet; nd, not detected; VLDL, very-low-density lipoprotein.

The correlation of decreased visual function with consumption of dietary cholesterol in aged CFH-H/H mice fed a HC diet but not when fed a HF diet, supports the notion that therapeutic strategies targeting cholesterol intake through the small intestine (such as ezetimibe) may prevent visual loss in aged CFH-H/H∼HFC mice. Ezetimibe interacts with the Neimann Pick C1-like intracellular cholesterol transporter 1 (NPC1) to prevent cholesterol uptake by intestinal enterocytes and thereby lowering the level of plasma LDLs containing cholesterol. ²³⁹ Ongoing studies are examining whether ezetimibe could mimic the effects of dietary modulation in aged CFH-H/H mice and if HFC diet-induced increases in eyecup ApoB48 and ApoA-1 in aged CFH-H/H mice is influenced by dietary cholesterol.

ApoA-1 is a major protein constituent of HDL, a lipoprotein associated with AMD risk.^48–51 In the posterior eye, the major site of interaction between CFH and HDL occurs at heparan sulfation within BrM.^22,240 We postulate that augmenting CFH concentrations or soluble heparan sulfate in the posterior eye may prevent toxic accumulations of lipoproteins such as HDL in BrM and protect against RPE damage and death. In support of this, treatment of BrM explants from human donor eyes with short heparan sulfate oligosaccharides or an ApoA-1 mimetic was sufficient to remove lipoproteins from BrM. ²³ In addition, aged nonhuman primates that got an intravitreal injection of an ApoA-1 mimetic had less neutral lipid, esterified cholesterol, and activated complement components in BrM than placebo-treated controls. ²⁴¹ Pharmaceutical interventions aimed at lipoprotein binding in BrM may be effective in the treatment of AMD and should be considered in future studies involving AMD mouse models.

Restoration of lipid processing in the RPE and BrM

Restoration of pathways that the RPE cells use to process lipids could be a means to therapeutically target lipid metabolism in AMD. For example, multiple transcription factors, including sterol regulatory element-binding proteins (SREBPs), ²⁴² liver X receptors (LXRs), ²⁴³ retinoid X receptors (RXRs), ²⁴⁴ and peroxisome proliferator-activated receptors (PPARs) ²⁴⁵ participate in intracellular lipid homeostasis. The SREBP, LXR, RXR, and PPAR pathways are present in RPE cells^100,123,215 and their importance in lipid homeostasis of the RPE is supported by reports of visual loss and lipid deposition in mice lacking LXR and PPAR β/δ signaling.^100,123

In a study aimed at determining if activating LXRs can prevent AMD-like pathology development, 3-month-old mice with a mutation in apolipoprotein B100 (apob100) that prevents the formation of its alternative splice variant apolipoprotein B48 (apob48) were fed a low-fat diet for 5 months and given GW3965, an LXR agonist. ¹⁰⁰ Limiting expression to only ApoB100 in mice increased LDL triglyceride and cholesterol levels, resembling lipoprotein profiles in humans. ²⁴⁶ As a consequence of this metabolic change, age-related progression of lipid deposition occurs in the BrM of apob100 mice but no other pathologies were noted in these animals. ¹⁰¹ GW3965 treatment improved hypopigmented regions in fundus images, dampened neuroinflammation, and decreased lipid deposition in the apob100 mice after the 5-month low-fat diet. ¹⁰⁰ Differences in the ocular phenotypes of the apob100 mice may be explained by the standard mouse chow and low-fat diet consumed by mice in these 2 studies.^100,101 More investigations of LXR agonists and other agents modulating transcription factors in other AMD mouse models are needed to confirm the promising results after treating apob100 mice with GW3965.

Another pathway critical for lipid metabolism within the RPE is autophagy, a conserved catabolic pathway induced under cellular stresses. ²⁴⁷ RPE cells depend on autophagy for the daily breakdown of photoreceptor outer segments. ¹⁴⁴ Autophagy is increased in RPE cells of aged non-AMD eyes, but decreased in RPE cells of human donor eyes diagnosed with AMD. ²⁴⁸ Mice with decreased autophagy such as microtubule-associated protein 1 light chain 3 B (LC3B) knockout, lysosomal-associated membrane protein 2 (Lamp2) knockout, ¹¹⁴ and RPE-specific RB1-inducible coiled-coil 1 (Rb1cc1) knockout mice have lipid accumulations within the RPE.^142,144 As a consequence, these mouse lines develop visual loss, migration of immune cells into the subretinal space, and subRPE deposits.^114,142,144 Studies utilizing RPE cell cultures identified a synthetic lignan secoisolariciresinol diglucoside, LGM2605, ²⁴⁹ and flubendazole ²⁵⁰ as inducers of autophagy that were able to reduce intracellular RPE lipid levels. Testing these therapies in mice with increased RPE lipid accumulation like RPE-specific ABCA1 and ABCG1 knockout mice ⁶⁶ could be an effective means testing if targeting autophagy may be a therapy for AMD.

Preservation of lipid oxidative pathways

Another possible intervention is to sustain the lipid oxidative function of RPE cells through preservation of mitochondrial health. Mitochondria are critical organelles required for the breakdown of fatty acids for ATP production through beta-oxidation ²⁵¹ and regulation of reactive oxygen species that, when in excess, leads to lipid peroxidation. ²⁵² With age, mitochondrial function declines and impacts retinal homeostasis. ³³ Multiple AMD mouse models are based on mitochondrial dysfunction, including the Tmem135 transgenic (Tmem135 TG) ¹²¹ and superoxide dismutase 2 knockout (Sod2^−/−).^117,118,253 It is possible that other mouse models may have mitochondrial impairment such as the DICER1-deficient mice.^120,152,153 Not only is there a consequence of an accumulation of Alu elements in DICER1-deficient mice, they also have decreased mitochondrial genome-encoded small RNAs and consequent reduced mitochondrial gene expression. ²⁵⁴ RPE degeneration is a common feature in these AMD mouse models.^{117,118,120,121,153,253,254} The RPE degeneration in these models phenocopies mice with RPE-specific ablation of mitochondrial transcription factor A (Tfam) ²⁵⁵ and global knockout of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), ²⁵⁶ proteins required for mitochondrial biogenesis.

The RPE-specific Tfam knockout mice have increased activation of the mammalian target of rapamycin (mTOR) pathway, ²⁵⁵ a pathway crucial for nutrient sensing and homeostasis. ²⁵⁷ Abnormal mTOR activation can cause RPE degeneration ²⁵⁸ but inhibiting mTOR in mice with RPE-specific ablation of TFAM or in mice treated with sodium iodate, alleviates RPE pathologies. ²⁵⁵ Furthermore, activation of mTOR signaling through ablation of tuberous sclerosis complex 1 (Tsc1) in rod photoreceptors leads to RPE abnormalities. ¹²² Conversely, activating mTOR in mouse models of retinitis pigmentosa is sufficient to reduce cone photoreceptor cell death,^259–262 whereas inhibiting mTOR through rapamycin can decrease retinal vascularization and lead to hypoxia. ²⁶³

One important aspect of the mTOR pathway is its ability to coordinate protein synthesis. ²⁶⁴ One study mimicked inhibition of the mTOR pathway by administering anisomycin, which curbs eukaryotic protein synthesis, to rd16 and wild-type C57BL/6J mice. ²⁶⁵ The rd16 mice had accelerated retinal degeneration, whereas wild-type mice developed retinal pathologies after anisomycin treatment. ²⁶⁵ Restoring mitochondrial function, rather than targeting the mTOR pathway, may be a safer avenue for an AMD therapy, as it is less likely to damage other nearby ocular tissues.

Despiramine and TO901316

ABCA4 is a member of the ABC transporter family found on rod and cone photoreceptor outer segments.^266–268 It is responsible for the clearance of all-trans-retinal from the disc membranes after phototransduction through the transport of N-retinylidene-phosphatidylethanolamine (PE), a product of the reaction of all-trans-retinal with PE. ²⁶⁹ If N-retinylidene-PE is not removed from the disc membranes, N-retinylidene-PE can react with all-trans-retinal to form toxic accumulations of N-retinylidene-N-retinylethanolamine (A2E) and other bisretinoids in the RPE. ²⁶⁹ Ablation of the murine Abca4 gene recapitulates STGD1-like phenotypes in mice, including age-dependent vision loss, delayed dark adaptation, increased retinal PE, deposition of A2E in the RPE, and additional toxic phototransduction byproducts.^166,270,271

The spatial distribution of lipids in Abca4^−/− RPE detected through a combination of matrix-assisted laser desorption ionization and Fourier transform ion cyclotron resonance imaging mass spectrometry reveals increased bis(monoacylglycerol)phosphate (BMP) lipid species. ²⁷² BMP lipid species are commonly observed in endosomal/lysosomal storage diseases and regulate cholesterol levels in endosomes. ²⁷³ Kaur et al. reported enlarged early endosomes in the RPE of Abca4^−/− mice that allow more extracellular complement component 3 (C3) intake and activation of the C3-proteolytic cleavage product, C3a. ²⁷⁴ Inhibiting acid sphingomyelinase with desipramine can decrease the size of early endosomes and prevent C3a activation in Abca4^−/− RPE. ²⁷⁴ Promoting cholesterol efflux with a LXR agonist, TO901316, can mimic the effects of desipramine on early endosome size in the Abca4^−/− RPE. ²⁷⁴ Decreasing the size of early endosomes in the RPE may be a viable therapeutic strategy for AMD, although more phenotypic work is needed to determine the functional effects of desipramine and TO901316 on the RPE in Abca4^−/− mice and other models with RPE pathologies.

DHA treatment

Mutations in ELOVL4 are known to cause mislocalization and aggregation of ELOVL4 from the endoplasmic reticulum to other organelles in photoreceptors leading to STGD3.^275,276 ELOVL4 is a fatty acid elongase that preferentially uses eicosapentaenoic acid (EPA) as a substrate to generate very-long-chain polyunsaturated fatty acids (VLC-PUFAs). ²⁷⁷ To model STGD3, researchers identified a 5 base pair deletion in ELOVL4 of a STGD3 patient ¹⁹⁵ and generated transgenic mice expressing ELOVL4 with either the same 5 base pair deletion in human ELOVL4 ¹⁷⁵ or mouse Elovl4. ¹⁷⁶ Regarding the efficacy of omega-3 VLC-PUFAs as a treatment for STGD3, a recent long-term clinical study testing diets supplemented with omega-3 VLC-PUFAs, such as EPA and DHA, in STGD3 patients found no changes in the disease progression. ²⁷⁸ Dietary DHA supplementation was also tested in mice with a mutant ELOVL4 allele but these studies yielded conflicting results.^279,280 This could be due to the differential effect of the mutant ELOVL4 and Elovl4 allele on VLC-PUFA synthesis in the mouse retina. Only the mice with a mutant Elovl4 allele displayed a 50% reduction in retinal VLC-PUFAs. ¹⁷⁴ Further studies have confirmed the essential role of ELOVL4 in retinal VLC-PUFA synthesis by ablating Elovl4 in cone and rod photoreceptors. ²⁸¹ Therefore, it would be important to test if DHA supplementation could be a viable therapeutic strategy in other mouse models of disrupted ELOVL4 function. This is especially important since DHA was added to the AREDS2 formulation and could explain why there was no reduction in AMD risk with the AREDS2 formulation. ²⁸²

Apolipoprotein mimetics

Repurposing lipid-lowering pharmacologic agents that have been effective in other diseases may be beneficial to treat AMD. For example, a major risk factor for cardiovascular disease is increased LDL and decreased HDL. ²⁸³ The opposite has been shown for AMD where increased HDL has emerged as a major risk factor.^48–51 One possible strategy to treat cardiovascular disease is to promote HDL function through ApoA1 mimetics. HDL particles that harbor ApoA1 are critical for reverse cholesterol transport, which removes lipids from cells and provides anti-inflammatory protection. ²⁸⁴ ApoA1 mimetics vary in peptide length and promote reverse cholesterol transport and are anti-inflammatory. ²⁸⁵ However, the effectiveness of ApoA1 mimetics as a cardiovascular disease treatment has been variable with some reports indicating no beneficial changes in patients.^286–288 and others describing increased HDL function in patients. ²⁸⁹

To determine if ApoA1 mimetics could be effective against AMD, 10-month-old Apoe^−/− mice were treated with a single intravitreal injection of the ApoA1 mimetic, 4F, and assessed 30 days postinjection for changes in BrM. ²⁹⁰ 4F mimetic treatment sufficiently lowered esterified cholesterol and prevented ultrastructural changes in Apoe^−/− mice. ²⁹⁰ In addition, based on our findings of increased ApoA1 in CFH-H/H∼HFC mice, we tested the ApoA1 mimetic 5A for 8 weeks in CFH-H/H∼HFC and were able to block HFC diet-induced changes in their plasma HDL proteome. ²³ We are currently testing whether the ApoA1 5A mimetic treatment correlate with changes in visual function that occur in both Apoe^−/− and CFH-H/H after a HF, cholesterol-enriched diet in these genotypes.^20,78

Other apolipoprotein mimetics have been tested in preclinical AMD mouse models such as the hybrid ApoE and apolipoprotein J (ApoJ) mimetic, HM-10/10 in the intravenous sodium iodate treatment model of geographic atrophy. ²⁹¹ Treatment of C57BL/6J mice with a diet chow containing HM-10/10 after induction of sodium iodate-induced RPE injury was sufficient to partially protect the retina and prevent caspase-3 cleavage in RPE cells. ²⁹¹ Since recruitment of immune cells is a major consequence of sodium iodate injury in the retina, ²⁹² it would be interesting to investigate whether HM-10/10 can reduce sodium iodate-induced immune cell recruitment to the retina as this frequently occurs in AMD mouse models and AMD-afflicted eyes. ²⁹³

Statins

Pharmaceuticals that could work for AMD and revolutionized the treatment of cardiovascular disease are statins, a class of lipid-lowering agents inhibiting the activity of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase to increase the uptake of LDLs and decrease plasma cholesterol. ²⁹⁴ A recent pilot study, which was randomized and placebo controlled, showed that taking simvastatin (40 mg/day) slowed progression of nonadvanced AMD especially for those with the CFH Y402H genotype. ²⁹⁵ Additionally, an open-label prospective pilot multicenter clinical trial found that a high dose of statins given to 26 patients resolved drusenoid pigment epithelial detachments and improved visual acuity without any progression to RPE atrophy or CNV formation. ²⁹⁶ However, the potency of statins as a treatment for AMD is controversial as evident by varying conclusions of case/control cross-sectional studies where statin usage had a beneficial,^297–299 worsened,^300,301 or no effect in AMD.^302–314

To determine efficacy in an animal model, female C57BL/6 mice were orally treated with simvastatin, atorvastatin, rosuvastatin, and pravastatin at similar concentrations where simvastatin had the highest accumulation in the retina. ³¹⁵ Oral gavage of simvastatin led to a 24% reduction in retinal cholesterol content, ³¹⁵ suggesting simvastatin can inhibit cholesterol synthesis in the retina. C57BL/6 mice fed a HF diet for 30 weeks were treated concurrently with simvastatin to evaluate the effect of simvastatin on retinal pathology development. ⁸² Simvastatin treatment decreased fundus abnormalities and BrM thickness, but did not significantly ameliorate visual function induced by the 30-week HF diet regime. ⁸² The absence of a visual function change could be due to an upregulation of cluster of differentiation 36 (CD36), an oxidized LDL receptor critical for ingestion of photoreceptor outer segments, ³¹⁶ in the mouse retina. ³¹⁵ Intake of oxidized LDL in the posterior eye can lead to increased expression of genes regulating oxidative stress, inflammation, and angiogenesis,^317–320 as well as an increase in the number of apoptotic RPE cells.^317,321,322 Further analysis of the C57BL/6∼HF mouse retina may answer whether statins could work as a treatment for AMD.