Most patients with neovascular age-related macular degeneration (nvAMD), the leading cause of severe vision loss in elderly Americans, respond inadequately to current therapies targeting a single angiogenic mediator, vascular endothelial growth factor (VEGF). Here we report that aqueous levels of a second vasoactive mediator, angiopoietin-like 4 (ANGPTL4), can help predict the response of nvAMD patients to anti-VEGF therapies. ANGPTL4 expression was higher in patients who required monthly treatment with anti-VEGF therapies compared to patients who could be effectively treated with less frequent injections. We further demonstrate that ANGPTL4 acts synergistically with VEGF to promote the growth and leakage of choroidal neovascular (CNV) lesions in mice. Targeting ANGPTL4 expression was as effective as targeting VEGF expression for treating CNV in mice, while simultaneously targeting both was more effective than targeting either factor alone. To help translate these findings to patients, we used a soluble receptor that binds to both VEGF and ANGPTL4 and effectively inhibited the development of CNV lesions in mice. Our findings provide an assay that can help predict the response of nvAMD patients to anti-VEGF monotherapy and suggest that therapies targeting both ANGPTL4 and VEGF will be a more effective approach for the treatment of this blinding disease.
Yu Qin, Aumreetam Dinabandhu, Xuan Cao, Jaron C. Sanchez, Kathleen Jee, Murilo Rodrigues, Chuanyu Guo, Jing Zhang, Jordan Vancel, Deepak Menon, Noore-Sabah Khan, Tao Ma, Stephany Y. Tzeng, Yassine J. Daoud, Jordan J. Green, Gregg L. Semenza, Silvia Montaner, Akrit Sodhi
Disruption of the neurovascular unit (NVU) underlies the pathophysiology of various CNS diseases.(1-3) One strategy to repair NVU dysfunction would use stem/progenitor cells to provide trophic support to the NVU’s functionally coupled and interdependent vasculature and surrounding CNS parenchyma.(4) A subset of endothelial progenitor cells, endothelial colony forming cells (ECFCs) with high expression of the CD44 hyaluronan receptor (CD44hi), provides such neurovasculotrophic support via a paracrine mechanism.(5) Here, we report that bioactive extracellular vesicles from CD44hi ECFCs (EVshi) are paracrine mediators, recapitulating the effects of intact cell therapy in murine models of ischemic/neurodegenerative retinopathy; vesicles from ECFCs with low expression levels of CD44 (EVslo) were ineffective. Small RNA sequencing comparing the microRNA (miR) cargo from EVshi and EVslo identified candidate miRs that contribute to these effects. EVshi may be used to repair NVU dysfunction through multiple mechanisms to stabilize hypoxic vasculature, promote vascular growth, and support neural cells.
Kyle V. Marra, Edith Aguilar, Wei Guoqin, Ayumi Usui-Ouchi, Yochiro Ideguchi, Susumu Sakimoto, Martin Friedlander
Rhodopsin (RHO)-associated retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified non-retinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules towards the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Further, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL) indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that non-retinoid chaperones are promising drug candidates in treating RHO-associated RP.
Abhishek Vats, Yibo Xi, Bing Feng, Owen D. Clinger, Anthony J. St. Leger, Xujie Liu, Archisha Ghosh, Chase D. Dermond, Kira L. Lathrop, Gregory P. Tochtrop, Serge Picaud, Yuanyuan Chen
Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). Very low-density lipoprotein receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids (FA). Since FA uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We find that excess circulating lipids restrain retinal autophagy, which contributes to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived FA sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased Sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.
Emilie Heckel, Gael Cagnone, Tapan Agnihotri, Bertan Cakir, Ashim Das, Jin Sung Kim, Nicholas Kim, Geneviève Lavoie, Anu Situ, Sheetal Pundir, Ye Sun, Florian Wünnemann, Kerry A. Pierce, Courtney Dennis, Grant A. Mitchell, Sylvain Chemtob, Flavio A. Rezende, Gregor Andelfinger, Clary B. Clish, Philippe P. Roux, Przemyslaw Sapieha, Lois E.H. Smith, Jean-Sébastien Joyal
BACKGROUND Outcome measures sensitive to disease progression are needed for ATP-binding cassette, sub-family A, member 4–associated (ABCA4-associated) retinopathy. We aimed to quantify ellipsoid zone (EZ) loss and photoreceptor degeneration beyond EZ-loss in ABCA4-associated retinopathy and investigate associations between photoreceptor degeneration, genotype, and age.METHODS We analyzed 132 eyes from 66 patients (of 67 enrolled) with molecularly confirmed ABCA4-associated retinopathy from a prospective natural history study with a median [IQR] follow-up of 4.2 years [3.1, 5.1]. Longitudinal spectral-domain optical coherence tomography volume scans (37 B-scans, 30° × 15°) were segmented using a deep learning (DL) approach. For genotype-phenotype analysis, a model of ABCA4 variants was applied with the age of criterion EZ-loss (6.25 mm2) as the dependent variable.RESULTS Patients exhibited an average (square-root-transformed) EZ-loss progression rate of [95% CI] 0.09 mm/y [0.06, 0.11]. Outer nuclear layer (ONL) thinning extended beyond the area of EZ-loss. The average distance from the EZ-loss boundary to normalization of ONL thickness (to ±2 z score units) was 3.20° [2.53, 3.87]. Inner segment (IS) and outer segment (OS) thinning was less pronounced, with an average distance from the EZ-loss boundary to layer thickness normalization of 1.20° [0.91, 1.48] for the IS and 0.60° [0.49, 0.72] for the OS. An additive model of allele severity explained 52.7% of variability in the age of criterion EZ-loss.CONCLUSION Patients with ABCA4-associated retinopathy exhibited significant alterations of photoreceptors outside of EZ-loss. DL-based analysis of photoreceptor laminae may help monitor disease progression and estimate the severity of ABCA4 variants.TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT01736293.FUNDING National Eye Institute Intramural Research Program and German Research Foundation grant PF950/1-1.
Maximilian Pfau, Catherine A. Cukras, Laryssa A. Huryn, Wadih M. Zein, Ehsan Ullah, Marisa P. Boyle, Amy Turriff, Michelle A. Chen, Aarti S. Hinduja, Hermann E.A. Siebel, Robert B. Hufnagel, Brett G. Jeffrey, Brian P. Brooks
Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with non-syndromic and syndromic retinitis pigmentosa. Here we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicated that ADIPOR1 encodes the most abundantly expressed ceramidase in mice and one of the two most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1-/- retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine and L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1-/- mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC-treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina, and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.
Dominik Lewandowski, Andrzej T. Foik, Roman Smidak, Elliot H. Choi, Jianye Zhang, Thanh Hoang, Aleksander Tworak, Susie Suh, Henri Leinonen, Zhiqian Dong, Antonio F.M. Pinto, Emily Tom, Jennings C. Luu, Joan Y. Lee, Xiuli Ma, Erhard Bieberich, Seth Blackshaw, Alan Saghatelian, David C. Lyon, Dorota Skowronska-Krawczyk, Marcin Tabaka, Krzysztof Palczewski
BACKGROUND. >1,500 variants in the ABCA4 locus underlie a heterogeneous spectrum of retinal disorders ranging from aggressive childhood-onset chorioretinopathy to milder, late-onset macular disease. Genotype-phenotype correlation studies have been limited in clinical applicability as patient cohorts are typically small and seldom capture the full natural history of individual genotypes. To overcome these limitations, we constructed a genotype-phenotype correlation matrix that provides quantifiable probabilities of long-term disease outcomes associated with specific ABCA4 genotypes from a large, age-restricted patient cohort. METHODS. The study included 112 unrelated patients ≥50 years of age in whom 2 pathogenic variants were identified after sequencing of the ABCA4 locus. Clinical characterization was performed using the results of best-corrected visual acuity, retinal imaging and full-field electroretinogram testing. RESULTS. Four distinct prognostic groups were defined according to the spatial severity of disease features across the fundus. Recurring genotypes were observed in milder prognoses including those associated with a newly defined class of rare hypomorphic alleles. PVS1 (predicted null) variants were enriched in the most severe prognoses; however, missense variants comprised a larger than expected fraction of these patients. Analysis of allele combinations and their respective prognostic severity, showed that certain variants such as p.(Gly1961Glu), and both rare and frequent hypomorphic alleles, are “clinically dominant” with respect to patient phenotypes irrespective of the allele in trans. CONCLUSION. These results provide much needed structure to the complex genetic and clinical landscape of ABCA4 disease and adds a tool to the clinical repertoire to quantitatively assess individual genotype-specific prognoses in patients.
Winston Lee, Jana Zernant, Pei-Yin Su, Takayuki Nagasaki, Stephen H. Tsang, Rando Allikmets
Ischemic retinopathies including diabetic retinopathy are major causes of blindness. While neurons and Müller glia are recognized as important regulators of reparative and pathologic angiogenesis, the role of mononuclear phagocytes (MPs), such as microglia/macrophages, is unclear, particularly microglia, the resident retinal immune cells. Here we found microglial/macrophage activation in human diabetic retinopathy, especially in neovessels from human neovascular membranes in proliferative retinopathy, including TNF-α expression. There was similar activation in the mouse oxygen-induced retinopathy (OIR) model of ischemia-induced neovascularization. Glucagon-like peptide-1 receptor (GLP-1R) agonists are in clinical use for glycemic control in diabetes and are also known to modulate microglia. We investigated the effect of a long-acting GLP-1R agonist, NLY01. Following intravitreal administration, NLY01 selectively localized to MPs in OIR retina. NLY01 modulated MP but not retinal endothelial cell viability, apoptosis, and tube formation in vitro. In OIR, NLY01 treatment inhibited MP infiltration and activation, including microglia/macrophage expression of cytokines in vivo. NLY01 significantly suppressed global induction of retinal inflammatory cytokines, promoted reparative angiogenesis, and suppressed pathologic retinal neovascularization. Collectively, these findings indicate the important role of microglia/macrophages in regulation of retinal vascularization in ischemia and suggest modulation of MPs as a new treatment strategy for ischemic retinopathies.
Lingli Zhou, Zhenhua Xu, Yumin Oh, Rico Gamuyao, Grace Lee, Yangyiran Xie, Hongkwan Cho, Seulki Lee, Elia J. Duh
Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor β (TGFβ). Previously, αV integrins were found to be critical for the onset of TGFβ-mediated PCO in vivo, however, the functional heterodimer was unknown. Here, β8 integrin conditional knockout (β8ITGcKO) lens cells (LCs) were observed to attenuate their fibrotic responses, while both β5 and β6 integrin null LCs underwent fibrotic changes similar to WT at 5 days PCS. RNAseq revealed that β8ITGcKO LCs attenuated their upregulation of integrins and their ligands, as well as known targets of TGFβ induced signaling at 24 hours PCS. Treatment of β8ITGcKO eyes with active TGFβ1 at the time of surgery rescued the fibrotic response. Treatment of wild type mice with an anti- αVβ8 integrin function blocking antibody at the time of surgery ameliorated both canonical TGFβ signaling and LC fibrotic response PCS, and treatment at 5 days PCS, after surgically induced fibrotic responses are established, largely reversed this fibrotic response. These data suggest that αVβ8 integrin is a major regulator of TGFβ activation by LCs PCS and that therapeutics targeting αVβ8 integrin could be effective for fibrotic PCO prevention and treatment.
Mahbubul H. Shihan, Samuel G. Novo, Yan Wang, Dean Sheppard, Amha Atakilit, Thomas D. Arnold, Nicole M. Rossi, Adam P. Faranda, Melinda K. Duncan
Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multi-organ ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used superresolution fluorescence microscopy and electron microscopy (TEM) to localize CEP290 in the CC and in primary cilia of cultured cells with sub-diffraction resolution, and to determine effects of CEP290 deficiency in three mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in hRPE-1 cells. We found Y-shaped links, ciliary sub-structures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. There were fewer cilia and no normal outer segments in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.
Valencia L. Potter, Abigail R. Moye, Michael A. Robichaux, Theodore G. Wensel
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