ß-secretase 1 overexpression by AAV-mediated gene delivery prevents retina degeneration in a mouse model of age-related macular degeneration.

We reported previously that ß-site amyloid precursor protein cleaving enzyme (BACE1) is strongly expressed in the normal retina and that BACE1-/- mice develop pathological phenotypes associated with age-related macular degeneration (AMD). BACE1 expression is increased within the neural retina and retinal pigment epithelium (RPE) in AMD donor eyes suggesting that increased BACE1 is compensatory. We observed that AAV-mediated BACE1 overexpression in the RPE was maintained up to 6 months after AAV1-BACE1 administration. No significant changes in normal mouse visual function or retinal morphology were observed with low-dose vector while the high-dose vector demonstrated some early pathology which regressed with time. No increase in ß-amyloid was observed. BACE1 overexpression in the RPE of the superoxide dismutase 2 knockdown (SOD2 KD) mouse, which exhibits an AMD-like phenotype, prevented loss of retinal function and retinal pathology, and this was sustained out to 6 months. Furthermore, BACE1 overexpression was able to inhibit oxidative stress, microglial changes, and loss of RPE tight junction integrity (all features of AMD) in SOD2 KD mice. In conclusion, BACE1 plays a key role in retina/RPE homeostasis, and BACE1 overexpression offers a novel therapeutic target in the treatment of AMD.

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage- c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

BACE1 Inhibition Increases Susceptibility to Oxidative Stress by Promoting Mitochondrial Damage.

BACE1 is a key enzyme facilitating the generation of neurotoxic ß-amyloid (Aß) peptide. However, given that BACE1 has multiple substrates we explored the importance of BACE1 in the maintenance of retinal pigment epithelial (RPE) cell homeostasis under oxidative stress. Inhibition of BACE1 reduced mitochondrial membrane potential, increased mitochondrial fragmentation, and increased cleaved caspase-3 expression in cells under oxidative stress. BACE1 inhibition also resulted in significantly lower levels of mitochondrial fusion proteins OPA1 and MFN1 suggesting a higher rate of mitochondrial fission while increasing the levels of mitophagic proteins Parkin and PINK1 and autophagosome numbers. In contrast, BACE2 had minimal effect on cellular response to oxidative stress. In summary, our results emphasize the importance of BACE1 in augmenting cellular defense against oxidative stress by protecting mitochondrial dynamics.

Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model.

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow-derived CD34+ cells and vascular wall-derived endothelial colony-forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

Improving the Transduction of Bone Marrow-Derived Cells with an Integrase-Defective Lentiviral Vector.

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow-derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells.

Enteral Arg-Gln Dipeptide Administration Increases Retinal Docosahexaenoic Acid and Neuroprotectin D1 in a Murine Model of Retinopathy of Prematurity.

Purpose: Low levels of the long chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) have been implicated in retinopathy of prematurity (ROP). However, oral DHA suffers from poor palatability and is associated with increased bleeding in premature infants. We asked whether oral administration of the neutraceutical arginine-glutamine (Arg-Glu) could increase retinal DHA and improve outcomes in a mouse model of oxygen-induced retinopathy (OIR). Methods: Postnatal day 7 (P7) pups were maintained at 75% oxygen for 5 days and then returned to room air on P12. Pups were gavaged twice daily with Arg-Gln or vehicle from P12 to P17 and eyes were harvested for analysis on P17. Vaso-obliteration and vascular density were assessed on retinal flat mounts and preretinal neovascularization was assessed on retinal cross sections. Retinas were used for measurement of DHA and 10,17S-docosatriene (neuroprotectin D1, NPD1), a key DHA-derived lipid, and for analysis by reverse-phase protein array (RPPA). Results: With Arg-Gln treatment, retinal DHA and NPD1 levels were increased in OIR pups. Arg-Gln reduced preretinal neovascularization by 39 ± 6% (P < 0.05) relative to vehicle control. This was accompanied by a restoration of vascular density of the retina in the pups treated with Arg-Gln (73.0 ± 3.0%) compared to vehicle (53.1 ± 3.4%; P < 0.05). Arg-Gln dipeptide restored OIR-induced signaling changes toward normoxia and was associated with normalization of insulin-like growth factor receptor 1 signaling and reduction of apoptosis and an increase in anti-apoptosis proteins. Conclusions: Arg-Gln may serve as a safer and easily tolerated nutraceutical agent for prevention or treatment of ROP.

Repurposing antimalarial aminoquinolines and related compounds for treatment of retinal neovascularization.

Neovascularization is the pathological driver of blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The loss of vision resulting from these diseases significantly impacts the productivity and quality of life of patients, and represents a substantial burden on the health care system. Current standard of care includes biologics that target vascular endothelial growth factor (VEGF), a key mediator of neovascularization. While anti-VGEF therapies have been successful, up to 30% of patients are non-responsive. Therefore, there is a need for new therapeutic targets, and small molecule inhibitors of angiogenesis to complement existing treatments. Apelin and its receptor have recently been shown to play a key role in both developmental and pathological angiogenesis in the eye. Through a cell-based high-throughput screen, we identified 4-aminoquinoline antimalarial drugs as potent selective antagonists of APJ. The prototypical 4-aminoquinoline, amodiaquine was found to be a selective, non-competitive APJ antagonist that inhibited apelin signaling in a concentration-dependent manner. Additionally, amodiaquine suppressed both apelin-and VGEF-induced endothelial tube formation. Intravitreal amodaiquine significantly reduced choroidal neovascularization (CNV) lesion volume in the laser-induced CNV mouse model, and showed no signs of ocular toxicity at the highest doses tested. This work firmly establishes APJ as a novel, chemically tractable therapeutic target for the treatment of ocular neovascularization, and that amodiaquine is a potential candidate for repurposing and further toxicological, and pharmacokinetic evaluation in the clinic.

Ferrochelatase is a therapeutic target for ocular neovascularization.

Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.

A novel small molecule ameliorates ocular neovascularisation and synergises with anti-VEGF therapy.

Ocular neovascularisation underlies blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. These diseases cause irreversible vision loss, and provide a significant health and economic burden. Biologics targeting vascular endothelial growth factor (VEGF) are the major approach for treatment. However, up to 30% of patients are non-responsive to these drugs and they are associated with ocular and systemic side effects. Therefore, there is a need for small molecule ocular angiogenesis inhibitors to complement existing therapies. We examined the safety and therapeutic potential of SH-11037, a synthetic derivative of the antiangiogenic homoisoflavonoid cremastranone, in models of ocular neovascularisation. SH-11037 dose-dependently suppressed angiogenesis in the choroidal sprouting assay ex vivo and inhibited ocular developmental angiogenesis in zebrafish larvae. Additionally, intravitreal SH-11037 (1 µM) significantly reduced choroidal neovascularisation (CNV) lesion volume in the laser-induced CNV mouse model, comparable to an anti-VEGF antibody. Moreover, SH-11037 synergised with anti-VEGF treatments in vitro and in vivo. Up to 100 µM SH-11037 was not associated with signs of ocular toxicity and did not interfere with retinal function or pre-existing retinal vasculature. SH-11037 is thus a safe and effective treatment for murine ocular neovascularisation, worthy of further mechanistic and pharmacokinetic evaluation.

A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.

PURPOSE: Therapeutic efficacy is routinely assessed by measurement of lesion size using flatmounted choroids and confocal microscopy in the laser-induced choroidal neovascularization (L-CNV) rodent model. We investigated whether optical coherence tomography (OCT) quantification, using an ellipsoid volume measurement, was comparable to standard ex vivo evaluation methods for this model and whether this approach could be used to monitor treatment-related lesion changes. METHODS: Bruch's membrane was ruptured by argon laser in the dilated eyes of C57BL/6J mice, followed by intravitreal injections of anti-VEGF164 or vehicle, or no injection. In vivo OCT images were acquired using Micron III or InVivoVue systems at 7, 10, and/or 14 days post-laser and neovascular lesion volume was calculated as an ellipsoid. Subsequently, lesion volume was compared to that calculated from confocal Z-stack images of agglutinin-stained choroidal flatmounts. RESULTS: Ellipsoid volume measurement of orthogonal 2-dimensional OCT images obtained from different imaging systems correlated with ex vivo lesion volumes for L-CNV (Spearman's ρ=0.82, 0.75, and 0.82 at days 7, 10, and 14, respectively). Ellipsoid volume calculation allowed temporal monitoring and evaluation of CNV lesions in response to antivascular endothelial growth factor treatment. CONCLUSIONS: Ellipsoid volume measurements allow rapid, quantitative use of OCT for the assessment of CNV lesions in vivo. This novel method can be used with different OCT imaging systems with sensitivity to distinguish between treatment conditions. It may serve as a useful adjunct to the standard ex vivo confocal quantification, to assess therapeutic efficacy in preclinical models of CNV, and in models of other ocular diseases.