Inflammasome activation aggravates choroidal neovascularization.

Inflammasome activation is implicated in diseases of aberrant angiogenesis such as age-related macular degeneration (AMD), though its precise role in choroidal neovascularization (CNV), a characteristic pathology of advanced AMD, is ill-defined. Reports on inhibition of inflammasome constituents on CNV are variable and the precise role of inflammasome in mediating pathological angiogenesis is unclear. Historically, subretinal injection of inflammasome agonists alone has been used to investigate retinal pigmented epithelium (RPE) degeneration, while the laser photocoagulation model has been used to study pathological angiogenesis in a model of CNV. Here, we report that the simultaneous introduction of any of several disease-relevant inflammasome agonists (Alu or B2 RNA, Alu cDNA, or oligomerized amyloid ß (1-40)) exacerbates laser-induced CNV. These activities were diminished or abrogated by genetic or pharmacological targeting of inflammasome signaling constituents including P2rx7, Nlrp3, caspase-1, caspase-11, and Myd88, as well as in myeloid-specific caspase-1 knockout mice. Alu RNA treatment induced inflammasome activation in macrophages within the CNV lesion, and increased accumulation of macrophages in an inflammasome-dependent manner. Finally, IL-1ß neutralization prevented inflammasome agonist-induced chemotaxis, macrophage trafficking, and angiogenesis. Collectively, these observations support a model wherein inflammasome stimulation promotes and exacerbates CNV and may be a therapeutic target for diseases of angiogenesis such as neovascular AMD.

Kamuvudine-9 Protects Retinal Structure and Function in a Novel Model of Experimental Rhegmatogenous Retinal Detachment.

Purpose: Rhegmatogenous retinal detachment (RRD) is a vision-threatening event that benefits from surgical intervention. While awaiting surgical reattachment, irreversible hypoxic and inflammatory damage to the retina often occurs. An interim therapy protecting photoreceptors could improve functional outcomes. We sought to determine whether Kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors (NRTIs) that inhibits inflammasome activation, and the NRTIs lamivudine (3TC) and azidothymidine (AZT) could protect the retina following RRD. Methods: RRD was induced in mice via subretinal injection (SRI) of 1% carboxymethylcellulose (CMC). To simulate outcomes following the clinical management of RRD, we determined the optimal conditions by which SRI of CMC induced spontaneous retinal reattachment (SRR) occurs over 10 days (RRD/SRR). K-9, 3TC, or AZT was administered via intraperitoneal injection. Inflammasome activation pathways were monitored by abundance of cleaved caspase-1, IL-18, and cleaved caspase-8, and photoreceptor death was assessed by TUNEL staining. Retinal function was assessed by full-field scotopic electroretinography. Results: RRD induced retinal inflammasome activation and photoreceptor death in mice. Systemic administration of K-9, 3TC, or AZT inhibited retinal inflammasome activation and photoreceptor death. In the RRD/SRR model, K-9 protected retinal electrical function during the time of RRD and induced an improvement following retinal reattachment. Conclusions: K-9 and NRTIs exhibit anti-inflammatory and neuroprotective activities in experimental RRD. Given its capacity to protect photoreceptor function during the period of RRD and enhance retinal function following reattachment, K-9 shows promise as a retinal neuroprotectant and warrants study in RRD. Further, this novel RRD/SRR model may facilitate experimental evaluation of functional outcomes relevant to RRD.

Subretinal injection in mice to study retinal physiology and disease.

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a 'successful' SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with 'procedure success' defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7-14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. Conclusions: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain­containing protein 3, and apoptosis-associated speck-like protein­containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA­driven diseases.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-ß induced retinal pigmented epithelium degeneration.

Nonfibrillar amyloid-ß oligomers (AßOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AßOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AßOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AßO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors-nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines-both inhibit AßOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

Assessment of a New Nanostructured Microemulsion System for Ocular Delivery of Sorafenib to Posterior Segment of the Eye.

Eye drop formulations allowing topical treatment of retinal pathologies have long been sought as alternatives to intravitreal administration. This study aimed to assess whether a novel nanostructured microemulsions system (NaMESys) could be usefully employed to deliver sorafenib to the retina following topical instillation. NaMESys carrying 0.3% sorafenib (NaMESys-SOR) proved to be cytocompatible in vitro on rabbit corneal cells, and well-tolerated following b.i.d. ocular administration to rabbits during a 3-month study. In rats subject to retinal ischemia-reperfusion, NaMESys-SOR significantly inhibited retinal expression of tumor necrosis factor-alpha (TNFα, 20.7%) and inducible nitric oxide synthase (iNos, 87.3%) mRNAs in comparison to controls. Similarly, in streptozotocin-induced diabetic rats, NaMESys-SOR inhibited retinal expression of nuclear factor kappa B (NFκB), TNFα, insulin like growth factor 1 (IGF1), IGF1 receptor (IGF1R), vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2) mRNAs by three-fold on average compared to controls. Furthermore, a reduction in TNFα, VEGFR1 and VEGFR2 protein expression was observed by western blot. Moreover, in mice subject to laser-induced choroidal neovascularization, NaMESys-SOR significantly inhibited neovascular lesions by 54%. In conclusion, NaMESys-SOR was shown to be a well-tolerated ophthalmic formulation able to deliver effective amounts of sorafenib to the retina, reducing proinflammatory and pro-angiogenic mediators in reliable models of proliferative retinopathies. These findings warrant further investigations on the full therapeutic potential of NaMESys-SOR eye drops, aiming to address unmet needs in the pharmacotherapy of retinal neovascular diseases.

Prolyl 3-Hydroxylase 2 Is a Molecular Player of Angiogenesis.

Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3H2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.