Reducing Treatment Burden in AMD.

VEGF-A antagonists have revolutionized wet AMD treatment. Several challenges remain including high treatment burden requiring repeated intraocular injections for persistent disease. Brolucizumab directly inhibits VEGF-A function, providing visual outcomes comparable to aflibercept (an FDA-approved VEGF-A antagonist). Anatomic retinal outcomes including retinal fluid, a marker of disease activity, favored brolucizumab. To view this Bench to Bedisde, open or download the PDF.

VEGF in Signaling and Disease: Beyond Discovery and Development.

The discovery of vascular endothelial-derived growth factor (VEGF) has revolutionized our understanding of vasculogenesis and angiogenesis during development and physiological homeostasis. Over a short span of two decades, our understanding of the molecular mechanisms by which VEGF coordinates neurovascular homeostasis has become more sophisticated. The central role of VEGF in the pathogenesis of diverse cancers and blinding eye diseases has also become evident. Elucidation of the molecular regulation of VEGF and the transformative development of multiple therapeutic pathways targeting VEGF directly or indirectly is a powerful case study of how fundamental research can guide innovation and translation. It is also an elegant example of how agnostic discovery and can transform our understanding of human disease. This review will highlight critical nodal points in VEGF biology, including recent developments in immunotherapy for cancer and multitarget approaches in neovascular eye disease.

Gene Therapy for Retinal Degeneration.

Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.

Visualizing the Heterogeneity of Retinal Microglia.

In this issue of Immunity, O'Koren et al. (2019) report that murine retinal microglia are long lived and are divided into two spatially and functionally distinct niches in the retina. In models of retinal neurodegeneration, retinal microglia migrate to the subretinal space, an inducible disease-associated niche, where they are neuroprotective.

Dry eye disease in mice activates adaptive corneal epithelial regeneration distinct from constitutive renewal in homeostasis.

Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.

Dysregulated CD200-CD200R signaling in early diabetes modulates microglia-mediated retinopathy.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

Association of Systemic Lupus Erythematosus Disease Activity With Choroidal Thickness.

OBJECTIVE: To determine the association between disease activity and choroidal thickness in patients with systemic lupus erythematosus (SLE). METHODS: We conducted a cohort study of 24 SLE patients and 13 healthy controls recruited at Washington University School of Medicine between June 2019 and November 2021. SLE disease activity was assessed using the SLE Disease Activity Index-2000 Responder Index-50 (S2K RI-50). Patients were divided into four groups: high disease activity/no lupus nephritis (HDA/no LN; S2K RI-50 > 4), HDA/active LN (HDA/active LN; S2K RI-50 > 4), low disease activity/inactive LN (LDA/inactive LN; S2K RI-50 ≤ 4), and LDA/no LN (LDA/no LN; S2K RI-50 ≤ 4). LDA/no LN patients were age-, sex-, and race-matched to healthy controls and patients in other SLE groups. Choroidal thickness of the right eye was measured blinded to disease activity on a horizontal section through the fovea on optical coherence tomography images taken within a week of disease assessment. RESULTS: Patients with HDA had choroidal thickening compared with matched patients with LDA. After controlling for multiplicity, choroidal thinning remained statistically significant at 1000 µm nasal to the fovea (308 ± 68 vs 228 ± 64 µm, p = 0.001). Choroidal thickness was not different between LDA/no LN and LDA/inactive LN or healthy controls. CONCLUSION: HDA in patient with SLE is associated with increased choroidal thickness whereas comorbid inactive LN did not affect choroidal thickness. Additional studies in a larger longitudinal cohort are needed to study whether choroidal thickness may be used as a noninvasive, adjunctive measure for disease activity in SLE.

Macrophage Plasticity and Function in the Eye and Heart.

Macrophages are important mediators of inflammation and tissue remodeling. Recent insights into the heterogeneity of macrophage subpopulations have renewed interest in their functional diversity in homeostasis and disease. In addition, their plasticity enables them to perform a variety of functions in response to changing tissue contexts, such as those imposed by aging. These qualities make macrophages particularly intriguing cells given their dichotomous role in protecting against, or accelerating, diseases of the cardiovascular system and the eye, two tissues that are particularly susceptible to the effects of aging. We review novel perspectives on macrophage biology, as informed by recent studies detailing the diversity of macrophage identity and function, as well as mechanisms influencing macrophage behavior that might offer opportunities for new therapeutic strategies.

Dexamethasone implant improves anatomic response to anti-VEGF therapy in treatment-resistant polypoidal choroidal vasculopathy.

BACKGROUND: A significant proportion of eyes with polypoidal choroidal vasculopathy (PCV) can be resistant to anti-vascular endothelial growth factor (VEGF) injections. We evaluated the efficacy of a combination of dexamethasone intravitreal implant (DXI) and anti-VEGF therapy in eyes resistant to anti-VEGF monotherapy. METHODS: In this retrospective study, patients with PCV resistant to anti-VEGF injections were additionally injected with a DXI along with an anti-VEGF agent. Best-corrected visual acuity (BCVA), slit-lamp examination, fundus evaluation, and optical coherence tomography (OCT) data were analyzed. Anatomical response on OCT was the primary outcome measure. Change in visual acuity and injection-free interval after DXI were evaluated as secondary outcome measures. RESULTS: Twelve eyes of 11 patients were included in the study. Mean age of patients at presentation was 64.7 ± 9.5 years (range, 49-78.8 years), and there were seven females (63.6%). Median number of anti-VEGF injections prior to DXI was 4 (interquartile range IQR, 3-7). Median follow-up duration after DXI was 32.2 months (IQR, 6.6-41.6 months). Median logMAR BCVA immediately prior to DXI was 0.41 (IQR, 0.30-0.88) and after injection was 0.40 (IQR, 0.30-1.05), which was not significantly different (p = 0.85). Median Central Retinal Thickness (CRT) after DXI was 305.5 µm (IQR, 249-409 µm), which was significantly (p = 0.003) lesser than pre-injection thickness of 547 µm (IQR, 431-771 µm). Median injection-free interval in these eyes after DXI was 5 months (IQR, 2.8-6.4 months). Kaplan-Meier estimates of first injection after DXI were 27.3% at 3 months, 67.3% at 6 months, and 89.1% at 12 months. CONCLUSIONS: Dexamethasone implant combined with anti-VEGF treatment can prolong the treatment-free interval in eyes with PCV resistant to anti-VEGF injection while maintaining visual acuity.

FGF2-induced STAT3 activation regulates pathologic neovascularization.

Cell-autonomous endothelial cell (EC) fibroblast growth factor receptor (FGFR) signaling through FGFR1/2 is essential for injury-induced wound vascularization and pathologic neovascularization as in blinding eye diseases such as age-related macular degeneration. Which FGF ligand(s) is critical in regulating angiogenesis is unknown. Utilizing ex vivo models of choroidal endothelial sprouting and in vivo models of choroidal neovascularization (CNV), we demonstrate here that only FGF2 is the essential ligand. Though FGF-FGFR signaling can activate multiple intracellular signaling pathways, we show that FGF2 regulates pathogenic angiogenesis via STAT3 activation. The identification of FGF2 as a critical mediator in aberrant neovascularization provides a new opportunity for developing multi-target therapies in blinding eye diseases especially given the limitations of anti-VEGF monotherapy.

SURGICAL OUTCOMES AFTER INVERTED INTERNAL LIMITING MEMBRANE FLAP VERSUS CONVENTIONAL PEELING FOR VERY LARGE MACULAR HOLES.

PURPOSE: To evaluate the anatomical and visual outcomes of inverted flap technique of peeling of internal limiting membrane (ILM) versus standard peeling of ILM for macular holes of basal diameter more than 800 µm. METHODS: Patients with very large idiopathic macular holes more than 800 µm in basal diameter (ranging from 243 µm to 840 µm in minimum diameter) were retrospectively included in the study. In Group A, 18 eyes of 18 patients underwent ILM peeling using the inverted flap technique. In Group B, 18 eyes of 18 patients underwent conventional ILM peeling. The primary endpoint was the rate of hole closure at 6 months after surgery. The secondary outcome measure was the change in best-corrected visual acuity at 6 months after surgery. RESULTS: There were no significant differences in ocular characteristics of the study groups at baseline except for the age distribution. Mean macular hole diameter was 1,162.8 ± 206.0 µm and 1,229.6 ± 228.1 µm in Group A and Group B, respectively. The hole closure rate was 88.9% (16/18) in Group A and 77.8% (14/18) in Group B (P = 0.66). The mean gain in best-corrected visual acuity was higher in Group A than in Group B (P = 0.12) at 6 months, but this was not statistically significant. There were no severe ocular adverse events in either group. CONCLUSION: In this multicenter series, inverted ILM flap technique did not lead to significantly higher anatomical closure rates than conventional ILM peeling in large macular holes more than 800 µm in diameter.

Disrupted cholesterol metabolism promotes age-related photoreceptor neurodegeneration.

Photoreceptors have high intrinsic metabolic demand and are exquisitely sensitive to metabolic perturbation. In addition, they shed a large portion of their outer segment lipid membranes in a circadian manner, increasing the metabolic burden on the outer retina associated with the resynthesis of cell membranes and disposal of the cellular cargo. Here, we demonstrate that deletion of both ABCA1 and ABCG1 in rod photoreceptors leads to age-related accumulation of cholesterol metabolites in the outer retina, photoreceptor dysfunction, degeneration of rod outer segments, and ultimately blindness. A high-fat diet significantly accelerates rod neurodegeneration and vision loss, further highlighting the role of lipid homeostasis in regulating photoreceptor neurodegeneration and vision.

NAD+-dependent deacetylase SIRT3 in adipocytes is dispensable for maintaining normal adipose tissue mitochondrial function and whole body metabolism.

Mitochondrial dysfunction in adipose tissue is involved in the pathophysiology of obesity-induced systemic metabolic complications, such as type 2 diabetes, insulin resistance, and dyslipidemia. However, the mechanisms responsible for obesity-induced adipose tissue mitochondrial dysfunction are not clear. The aim of present study was to test the hypothesis that nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin-3 (SIRT3) in adipocytes plays a critical role in adipose tissue mitochondrial biology and obesity. We first measured adipose tissue SIRT3 expression in obese and lean mice. Next, adipocyte-specific mitochondrial Sirt3 knockout (AMiSKO) mice were generated and metabolically characterized. We evaluated glucose and lipid metabolism in adult mice fed either a regular-chow diet or high-fat diet (HFD) and in aged mice. We also determined the effects of Sirt3 deletion on adipose tissue metabolism and mitochondrial biology. Supporting our hypothesis, obese mice had decreased SIRT3 gene and protein expression in adipose tissue. However, despite successful knockout of SIRT3, AMiSKO mice had normal glucose and lipid metabolism and did not change metabolic responses to HFD-feeding and aging. In addition, loss of SIRT3 had no major impact on putative SIRT3 targets, key metabolic pathways, and mitochondrial function in white and brown adipose tissue. Collectively, these findings suggest that adipocyte SIRT3 is dispensable for maintaining normal adipose tissue mitochondrial function and whole body metabolism. Contrary to our hypothesis, loss of SIRT3 function in adipocytes is unlikely to contribute to the pathophysiology of obesity-induced metabolic complications.

WNT7A/B promote choroidal neovascularization.

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

Inflammation-Induced Photoreceptor Cell Death.

Neuroinflammation is an important aspect of many diseases of the eye, and experimental animal models have been widely used to determine its impact on retinal homeostasis and neuron survival. Physical separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE) results in activation and infiltration of macrophages. Numerous studies have shown the critical role of macrophages in retinal disease processes. In retinal detachment, accumulation of macrophages in the subretinal space is associated with changes in cytokine and chemokine profile which lead to photoreceptor cell death. Targeted disruption of macrophage chemotaxis significantly reduces retinal detachment-induced photoreceptor degeneration. Apoptosis is the predominant mechanism of cell death; however regulated necrosis is also a contributor of photoreceptor loss. Therefore, effective neuroprotective approaches could integrate combined inhibition of both apoptotic and regulated necrosis pathways.

Role of Sirtuins in Retinal Function Under Basal Conditions.

Sirtuins are NAD+-dependent enzymes that govern cellular homeostasis by regulating the acylation status of their diverse target proteins. We recently demonstrated that both rod and cone photoreceptors rely on NAMPT-mediated NAD+ biosynthesis to meet their energetic requirements. Moreover, we found that this NAD+-dependent retinal homeostasis relies, in part, on maintenance of optimal activity of the mitochondrial sirtuins and of SIRT3 in particular. Nonetheless, it is unknown whether other sirtuin family members also play important roles in retinal homeostasis. Our results suggest that SIRT1, SIRT2, SIRT4, and SIRT6 are dispensable for retinal survival at baseline, as individual deletion of each of these sirtuins does not cause retinal degeneration by fundus biomicroscopy or retinal dysfunction by ERG. These findings have significant implications and inform future studies investigating the mechanisms underlying the central role of NAD+ biosynthesis in retinal survival and function.

Plasma lipoprotein subfraction concentrations are associated with lipid metabolism and age-related macular degeneration.

Disturbance in lipid metabolism has been suggested as a major pathogenic factor for age-related macular degeneration (AMD). Conventional lipid measures have been inconsistently associated with AMD. Other factors that can alter lipid metabolism include lipoprotein phenotype and genetic mutations. We performed a case-control study to examine the association between lipoprotein profile and neovascular AMD (nAMD) and whether the cholesterylester transfer protein (CETP) D442G mutation modulates these associations. Patients with nAMD had significantly higher concentrations of HDL and IDL compared with controls. The increase in HDL particles in nAMD patients was driven by an excess of medium-sized particles. Concurrently, patients with nAMD also had lower Apo A-1, lower VLDL and chylomicron lipoprotein. Many of these associations showed a dose-dependent association between controls, early AMD cases, and nAMD cases. Adjustment for the presence of the D442G mutation at the CETP locus did not significantly alter the increased AMD risk associated with HDL particle concentration. AMD is associated with variation in many lipoprotein subclasses, including increased HDL and IDL particles and decreased Apo A-1, VLDL, and chylomicron particles. These data suggest widespread systemic disturbance in lipid metabolism in the pathogenesis of AMD, including possible alterations in lipoprotein carrier capacity.