Role of NLRP3 Inflammasomes in Monocyte and Microglial Recruitments in Choroidal Neovascularization.

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.

Method to Regulate Monocyte Function by Silencing HIF-1α mRNA in a Model of Retinal Neovascularization.

Circulating monocytes migrate into the retina in response to inflammation and neovascularization. Furthermore, under inflammatory conditions such as diabetes, healthy monocytes become activated in the circulation. However, the contribution of activated monocytes to neovascularization is largely unknown. HIF-1α has been shown to contribute to the pathogenesis of neovascularization. We describe here the synthesis of a hybrid nanomaterial for targeted delivery and gene silencing in activated monocytes that are associated with pathological neovascularization. To test the gene silencing ability of AS-shRNA-lipids in vitro, we used the probe to inhibit HIF-1α mRNA induced in mouse monocytes by exposing them to hypoxia. In addition, we tested AS-shRNA-lipids for inhibition of neovascularization in vivo using the mouse model of oxygen-induced retinopathy (OIR). Significant reduction of neovascularization was achieved in mouse OIR by targeting activated monocytes using intraperitoneal injections of AS-shRNA-lipids. Expression of HIF-1α and CD14 mRNA were both inhibited in circulating cells, suggesting normalization of the activated monocytes in P17 OIR animals treated with AS-shRNA-lipids. We hypothesized that inhibition of HIF-1α mRNA in activated monocytes may have a direct impact on VEGF expression in the retinal tissues in vivo. We observed that VEGF mRNA expression was inhibited in P17 retinal tissues after systemic treatment with HIF-1α-targeted AS-shRNA-lipids. These findings may provide a framework for a strategy to inhibit retinal neovascularization by targeting circulating activated monocytes.

Role of NLRP3 inflammasomes in monocyte and microglial recruitments in choroidal neovascularization.

Though the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice to characterize migration of Ccr2RFP positive monocytes and Cx3cr1GFP positive microglial cells into CNV lesions after laser-induced rupture of Bruch's membrane. MCC950 was used as NLRP3 inhibitor. Immunostaining was used to confirm localization of NLRP3 inflammasomes in the LCNV lesions. Confocal microscopy was used to image and quantify LCNV volumes. ELISA and qRT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that RFP positive monocyte-derived macrophages and GFP positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP positive macrophages, Cx3cr1GFP positive microglia, and other cells resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice, showed significantly increased lesion size compared to age-matched controls. Inhibition of NLRP3, resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.

Elucidating glial responses to products of diabetes-associated systemic dyshomeostasis.

Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. DR has non-proliferative stages, characterized in part by retinal neuroinflammation and ischemia, and proliferative stages, characterized by retinal angiogenesis. Several systemic factors, including poor glycemic control, hypertension, and hyperlipidemia, increase the risk of DR progression to vision-threatening stages. Identification of cellular or molecular targets in early DR events could allow more prompt interventions pre-empting DR progression to vision-threatening stages. Glia mediate homeostasis and repair. They contribute to immune surveillance and defense, cytokine and growth factor production and secretion, ion and neurotransmitter balance, neuroprotection, and, potentially, regeneration. Therefore, it is likely that glia orchestrate events throughout the development and progression of retinopathy. Understanding glial responses to products of diabetes-associated systemic dyshomeostasis may reveal novel insights into the pathophysiology of DR and guide the development of novel therapies for this potentially blinding condition. In this article, first, we review normal glial functions and their putative roles in the development of DR. We then describe glial transcriptome alterations in response to systemic circulating factors that are upregulated in patients with diabetes and diabetes-related comorbidities; namely glucose in hyperglycemia, angiotensin II in hypertension, and the free fatty acid palmitic acid in hyperlipidemia. Finally, we discuss potential benefits and challenges associated with studying glia as targets of DR therapeutic interventions. In vitro stimulation of glia with glucose, angiotensin II and palmitic acid suggests that: 1) astrocytes may be more responsive than other glia to these products of systemic dyshomeostasis; 2) the effects of hyperglycemia on glia are likely to be largely osmotic; 3) fatty acid accumulation may compound DR pathophysiology by promoting predominantly proinflammatory and proangiogenic transcriptional alterations of macro and microglia; and 4) cell-targeted therapies may offer safer and more effective avenues for DR treatment as they may circumvent the complication of pleiotropism in retinal cell responses. Although several molecules previously implicated in DR pathophysiology are validated in this review, some less explored molecules emerge as potential therapeutic targets. Whereas much is known regarding glial cell activation, future studies characterizing the role of glia in DR and how their activation is regulated and sustained (independently or as part of retinal cell networks) may help elucidate mechanisms of DR pathogenesis and identify novel drug targets for this blinding disease.

Effect of cytokine-induced alterations in extracellular matrix composition on diabetic retinopathy-relevant endothelial cell behaviors.

Retinal vascular basement membrane (BM) thickening is an early structural abnormality of diabetic retinopathy (DR). Recent studies suggest that BM thickening contributes to the DR pathological cascade; however, much remains to be elucidated about the exact mechanisms by which BM thickening develops and subsequently drives other pathogenic events in DR. Therefore, we undertook a systematic analysis to understand how human retinal microvascular endothelial cells (hRMEC) and human retinal pericytes (hRP) change their expression of key extracellular matrix (ECM) constituents when treated with diabetes-relevant stimuli designed to model the three major insults of the diabetic environment: hyperglycemia, dyslipidemia, and inflammation. TNFα and IL-1ß caused the most potent and consistent changes in ECM expression in both hRMEC and hRP. We also demonstrate that conditioned media from IL-1ß-treated human Müller cells caused dose-dependent, significant increases in collagen IV and agrin expression in hRMEC. After narrowing our focus to inflammation-induced changes, we sought to understand how ECM deposited by hRMEC and hRP under inflammatory conditions affects the behavior of naïve hRMEC. Our data demonstrated that diabetes-relevant alterations in ECM composition alone cause both increased adhesion molecule expression by and increased peripheral blood mononuclear cell (PBMC) adhesion to naïve hRMEC. Taken together, these data demonstrate novel roles for inflammation and pericytes in driving BM pathology and suggest that inflammation-induced ECM alterations may advance other pathogenic behaviors in DR, including leukostasis.

UGT1A1 genetic variants are associated with increases in bilirubin levels in rheumatoid arthritis patients treated with sarilumab.

Sarilumab is a human monoclonal antibody against interleukin (IL)-6Rα that has been approved for the treatment of adult patients with moderately to severely active rheumatoid arthritis (RA) and an inadequate response or intolerance to one or more disease-modifying antirheumatic drugs (DMARDs). Mild liver function test abnormalities have been observed in patients treated with sarilumab. We describe a genome-wide association study of bilirubin elevations in RA patients treated with sarilumab. Array genotyping and exome sequencing were performed on DNA samples from 1075 patients. Variants in the UGT1A1 gene were strongly associated with maximum bilirubin elevations in sarilumab-treated patients (rs4148325; p = 2.88 × 10-41) but were not associated with aminotransferase elevations. No other independent loci showed evidence of association with bilirubin elevations after sarilumab treatment. These findings suggest that most bilirubin increases during sarilumab treatment are related to genetic variation in UGT1A1 rather than underlying liver injury.

A novel optical imaging probe for targeted visualization of NLRP3 inflammasomes in a mouse model of age-related macular degeneration.

Purpose: Wet form of age-related macular degeneration (wet AMD) is a progressive vascular disease that mainly affects older adults and causes severe and irreversible vision loss. A key complication of wet AMD is choroidal neovascularization (CNV), which may be driven in part by NLRP3 inflammasomes that are associated with macrophages migration to CNV lesions. Since activated NLRP3 is correlated with CNV, visualizing NLRP3 inflammasomes and their associated macrophages is of great interest to monitor wet AMD progression and develop effective therapies against it. However, to the best of our knowledge, current ophthalmic imaging systems do not permit such targeted imaging. Therefore, in this study, we developed InflammaProbe-1, an optical imaging probe for targeted visualization of NLRP3 inflammasomes in CNV lesions. Methods: InflammaProbe-1 was synthesized by conjugating a clinically relevant fluorophore, Oregon Green® 488, to the selective NLRP3 inhibitor, CY-09. The ability of InflammaProbe-1 to target NLRP3 was assessed with an enzyme-linked immunosorbent assay by comparing its ability to inhibit NLRP3-mediated secretion of IL-1ß to that of CY-09 in LPS-primed and nigericin-stimulated BMDMs. In vitro confocal imaging of NLRP3 was performed on InflammaProbe-1-stained BMDMs that had been induced to express NLRP3 with LPS. In vivo imaging of NLRP3 was conducted on mouse laser induced choroidal neovascularization (LCNV), a model of AMD, 6 h after an intraperitoneal injection of InflammaProbe-1 at 10 mg/kg on day 4 post-LCNV. Results: InflammaProbe-1 was just as effective as CY-09 at inhibiting IL-1ß secretion (p < 0.01 at 10 µM for both the InflammaProbe-1 and CY-09 groups relative to the control). InflammaProbe-1-stained BMDMs that had been induced to express NLRP3 showed significantly brighter fluorescence than untreated cells (p < 0.0001 for LPS treatment group and p < 0.001 for LPS and nigericin treatment group). Furthermore, in vivo molecular imaging of NLRP3 was achieved in mouse LCNV. Conclusion: We propose that InflammaProbe-1 may be a useful molecular imaging probe to monitor the onset, progression, and therapeutic response of AMD and other NLRP3-mediated diseases.

Nuclear factor of activated T-cells (NFAT) regulation of IL-1ß-induced retinal vascular inflammation.

Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1ß has the reputation of being a major driver of cytokine-induced inflammation. IL-1ß and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1ß-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1ß-induced expression of IL-1ß and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1ß-induced increases of IL-1ß, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1ß-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1ß-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.

Cytochrome P450-epoxygenated fatty acids inhibit Müller glial inflammation.

Free fatty acid dysregulation in diabetics may elicit the release of inflammatory cytokines from Müller cells (MC), promoting the onset and progression of diabetic retinopathy (DR). Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including IL-1ß, which induces the production of itself and other inflammatory cytokines in the retina as well. In this study we propose that experimental elevation of cytochrome P450 epoxygenase (CYP)-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), will reduce PA- and IL-1ß-induced MC inflammation. Broad-spectrum CYP inhibition by SKF-525a increased MC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP significantly decreased PA- and IL-1ß-induced MC expression of IL-1ß and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1ß-induced MC and TNFα in PA-induced MC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1ß-treated MC. GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor, significantly reduced PA- and IL-1ß-stimulated MC cytokine expression. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1ß-induced NFκB-dependent transcriptional activity. These data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation in part by blocking NFκB-dependent transcription and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR.

A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization.

Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA-lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA-lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA-lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

Exome sequencing and characterization of 49,960 individuals in the UK Biobank.

The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world1. Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, including PIEZO1 on varicose veins, COL6A1 on corneal resistance, MEPE on bone density, and IQGAP2 and GMPR on blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenic BRCA1 and BRCA2 variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.

Visualizing HIF-1α mRNA in a Subpopulation of Bone Marrow-Derived Cells to Predict Retinal Neovascularization.

Bone marrow-derived progenitor cells and macrophages are known to migrate into the retina in response to inflammation and neovascularization. These migratory cells might play important regulatory roles in the pathogenesis of neovascularization, a common complication observed in diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion. Hypoxia-inducible factor 1α (HIF-1α) has been shown to contribute to the pathogenesis of retinal inflammation and neovascularization. However, contributions of monocyte-derived macrophages to neovascularization are largely unknown. We hypothesized that selective visualization of these microglia/macrophages could be a powerful method for predicting the onset of neovascularization and its progression at the molecular level. In this report, we describe the synthesis of a new hybrid nanoparticle to visualize HIF-1α mRNA selectively in microglia/macrophages in a mouse model of neovascularization. HIF-1α expression was confirmed in MRC-1 positive monocytes/macrophages as well as in CD4 positive T-cells and CD19 positive B-cells using single-cell RNA sequencing data analysis. The imaging probes (AS- or NS-shRNA-lipid) were synthesized by conjugating diacyl-lipids to short hairpin RNA with an antisense sequence complementary to HIF-1α mRNA and a fluorophore that is quenched by a black hole quencher. We believe that imaging mRNA selectively in tissue specific microglia/macrophages could be a powerful method for predicting the onset of neovascularization, its progression, and its response to therapy.

The peroxisome proliferator-activated receptor-ß/δ antagonist GSK0660 mitigates retinal cell inflammation and leukostasis.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARß/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARß/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARß/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARß/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARß/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARß/δ inhibition is a potential therapeutic strategy against early DR pathology.

Human Umbilical Tissue-Derived Cells Secrete Soluble VEGFR1 and Inhibit Choroidal Neovascularization.

Exudative age-related macular degeneration (AMD), characterized by choroidal neovascularization (CNV), is the leading cause of irreversible blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) drugs are the standard treatment for AMD, but they have limitations. Cell therapy is a promising approach for ocular diseases, and it is being developed in the clinic for the treatment of retinal degeneration, including AMD. We previously showed that subretinal injection of human umbilical tissue-derived cells (hUTCs) in a rodent model of retinal degeneration preserved photoreceptors and visual function through rescue of retinal pigment epithelial (RPE) cell phagocytosis. Here we investigated the effect of hUTCs on a rat model of laser-induced CNV and on a human RPE cell line, ARPE-19, for VEGF production. We demonstrate that subretinal injection of hUTCs significantly inhibited CNV and lowered choroidal VEGF in vivo. VEGF release from ARPE-19 decreased when co-cultured with hUTCs. Soluble VEGF receptor 1 (sVEGFR1) is identified as the only factor in hUTC conditioned medium (CM) that binds to VEGF. The level of exogenous recombinant VEGF in hUTC CM was dramatically reduced and could be recovered with sVEGFR1-neutralizing antibody. This suggests that hUTC inhibits angiogenesis through the secretion of sVEGFR1 and could serve as a novel treatment for angiogenic ocular diseases, including AMD.

Photothermal Optical Coherence Tomography of Anti-Angiogenic Treatment in the Mouse Retina Using Gold Nanorods as Contrast Agents.

PURPOSE: Optical coherence tomography (OCT) is widely used for ocular imaging in clinical and research settings. OCT natively provides structural information based on the reflectivity of the tissues it images. We demonstrate the utility of photothermal OCT (PTOCT) imaging of gold nanorods (GNR) in the mouse retina in vivo in the laser-induced choroidal neovascularization (LCNV) model to provide additional image contrast within the lesion. METHODS: Wild-type C57BL/6 mice were imaged following the intravenous injection of ICAM2-targeted or untargeted GNR. Mice were also imaged following the injection of ICAM2-targeted GNR with or without the additional ocular delivery of a neutralizing monoclonal anti-vascular endothelial growth factor (anti-VEGF) antibody. RESULTS: Mice cohorts injected with untargeted or ICAM2-targeted GNR demonstrated increased lesion-associated photothermal signal during subsequent imaging relative to phosphate-buffered saline (PBS)-injected controls. Additionally, intravitreal injection of anti-VEGF antibody caused a detectable reduction in the extent of anatomic laser damage and lesion-associated photothermal signal density in mice treated in the LCNV model and injected with ICAM2-targeted GNR. CONCLUSIONS: These experiments demonstrate the ability of PTOCT imaging of GNR to detect anti-VEGF-induced changes in the mouse retina using the LCNV model. TRANSLATIONAL RELEVANCE: This study shows that PTOCT imaging of GNR in the LCNV model can be used to detect clinically relevant, anti-VEGF-induced changes that are not visible using standard OCT systems. In the future this technology could be used to aid in early detection of disease, monitoring disease progress, and assessing its response to therapies.

Targeted Imaging of VCAM-1 mRNA in a Mouse Model of Laser-Induced Choroidal Neovascularization Using Antisense Hairpin-DNA-Functionalized Gold-Nanoparticles.

Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.

Genetic inactivation of ANGPTL4 improves glucose homeostasis and is associated with reduced risk of diabetes.

Angiopoietin-like 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk, and nutrient partitioning. We hypothesize that loss of ANGPTL4 function might improve glucose homeostasis and decrease risk of type 2 diabetes (T2D). We investigate protein-altering variants in ANGPTL4 among 58,124 participants in the DiscovEHR human genetics study, with follow-up studies in 82,766 T2D cases and 498,761 controls. Carriers of p.E40K, a variant that abolishes ANGPTL4 ability to inhibit lipoprotein lipase, have lower odds of T2D (odds ratio 0.89, 95% confidence interval 0.85-0.92, p = 6.3 × 10-10), lower fasting glucose, and greater insulin sensitivity. Predicted loss-of-function variants are associated with lower odds of T2D among 32,015 cases and 84,006 controls (odds ratio 0.71, 95% confidence interval 0.49-0.99, p = 0.041). Functional studies in Angptl4-deficient mice confirm improved insulin sensitivity and glucose homeostasis. In conclusion, genetic inactivation of ANGPTL4 is associated with improved glucose homeostasis and reduced risk of T2D.

Profiling and Leveraging Relatedness in a Precision Medicine Cohort of 92,455 Exomes.

Large-scale human genetics studies are ascertaining increasing proportions of populations as they continue growing in both number and scale. As a result, the amount of cryptic relatedness within these study cohorts is growing rapidly and has significant implications on downstream analyses. We demonstrate this growth empirically among the first 92,455 exomes from the DiscovEHR cohort and, via a custom simulation framework we developed called SimProgeny, show that these measures are in line with expectations given the underlying population and ascertainment approach. For example, within DiscovEHR we identified ∼66,000 close (first- and second-degree) relationships, involving 55.6% of study participants. Our simulation results project that >70% of the cohort will be involved in these close relationships, given that DiscovEHR scales to 250,000 recruited individuals. We reconstructed 12,574 pedigrees by using these relationships (including 2,192 nuclear families) and leveraged them for multiple applications. The pedigrees substantially improved the phasing accuracy of 20,947 rare, deleterious compound heterozygous mutations. Reconstructed nuclear families were critical for identifying 3,415 de novo mutations in ∼1,783 genes. Finally, we demonstrate the segregation of known and suspected disease-causing mutations, including a tandem duplication that occurs in LDLR and causes familial hypercholesterolemia, through reconstructed pedigrees. In summary, this work highlights the prevalence of cryptic relatedness expected among large healthcare population-genomic studies and demonstrates several analyses that are uniquely enabled by large amounts of cryptic relatedness.

Palmitic Acid Induces Müller Cell Inflammation that is Potentiated by Co-treatment with Glucose.

Chronic hyperglycemia is thought to be the major stimulator of retinal dysfunction in diabetic retinopathy (DR). Thus, many diabetes-related systemic factors have been overlooked as inducers of DR pathology. Cell culture models of retinal cell types are frequently used to mechanistically study DR, but appropriate stimulators of DR-like factors are difficult to identify. Furthermore, elevated glucose, a gold standard for cell culture treatments, yields little to no response from many primary human retinal cells. Thus, the goal of this project was to demonstrate the effectiveness of the free fatty acid, palmitic acid and compare its use alone and in combination with elevated glucose as a stimulus for human Müller cells, a retinal glial cell type that is activated early in DR pathogenesis and uniquely responsive to fatty acids. Using RNA sequencing, we identified a variety of DR-relevant pathways, including NFκB signaling and inflammation, intracellular lipid signaling, angiogenesis, and MAPK signaling, that were stimulated by palmitic acid, while elevated glucose alone did not significantly alter any diabetes-relevant pathways. Co-treatment of high glucose with palmitic acid potentiated the expression of several DR-relevant angiogenic and inflammatory targets, including PTGS2 (COX-2) and CXCL8 (IL-8).