The Essential Role of the Choriocapillaris in Vision: Novel Insights from Imaging and Molecular Biology.

The choriocapillaris, a dense capillary network located at the posterior pole of the eye, is essential for supporting normal vision, supplying nutrients, and removing waste products from photoreceptor cells and the retinal pigment epithelium. The anatomical location, heterogeneity, and homeostatic interactions with surrounding cell types make the choroid complex to study both in vivo and in vitro. Recent advances in single-cell RNA sequencing, in vivo imaging, and in vitro cell modeling are vastly improving our knowledge of the choroid and its role in normal health and in age-related macular degeneration (AMD). Histologically, loss of endothelial cells (ECs) of the choriocapillaris occurs early in AMD concomitant with elevated formation of the membrane attack complex of complement. Advanced imaging has allowed us to visualize early choroidal blood flow changes in AMD in living patients, supporting histological findings of loss of choroidal ECs. Single-cell RNA sequencing is being used to characterize choroidal cell types transcriptionally and discover their altered patterns of gene expression in aging and disease. Advances in induced pluripotent stem cell protocols and 3D cultures will allow us to closely mimic the in vivo microenvironment of the choroid in vitro to better understand the mechanism leading to choriocapillaris loss in AMD.

Choroidal endothelial and macrophage gene expression in atrophic and neovascular macular degeneration.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Local factor H production by human choroidal endothelial cells mitigates complement deposition: implications for macular degeneration.

Activation of the alternative complement pathway is an initiating event in the pathology of age-related macular degeneration (AMD). Unchecked complement activation leads to the formation of a pro-lytic pore, the membrane attack complex (MAC). MAC deposition is observed on the choriocapillaris of AMD patients and likely causes lysis of choroidal endothelial cells (CECs). Complement factor H (FH, encoded by the gene CFH) is an inhibitor of complement. Both loss of function of FH and reduced choroidal levels of FH have been reported in AMD. It is plausible that reduced local FH availability promotes MAC deposition on CECs. FH is produced primarily in the liver; however, cells including the retinal pigment epithelium can produce FH locally. We hypothesized that CECs produce FH locally to protect against MAC deposition. We aimed to investigate the effect of reduced FH levels in the choroid to determine whether increasing local FH could protect CECs from MAC deposition. We demonstrated that siRNA knockdown of FH (CFH) in human immortalized CECs results in increased MAC deposition. We generated AMD iPSC-derived CECs and found that overexpression of FH protects against MAC deposition. These results suggest that local CEC-produced FH protects against MAC deposition, and that increasing local FH protein may be beneficial in limiting MAC deposition in AMD. © 2022 The Pathological Society of Great Britain and Ireland.

In vitro Measurement of Membrane Attack Complex in RPE Cells.

Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the outer blood retinal barrier. RPE cells are highly polarized with apical microvilli and basolateral contact with Bruch's membrane. In order to obtain biologically relevant polarized RPE cultures in vitro, RPE cells are seeded onto the apical side of a transwell filter and cultured for 4 weeks in low serum media. MAC formation on RPE cells has been reported to be sub-lytic. MAC formation can be achieved in vitro by introduction of normal human serum (NHS) to media following serum starvation for 24 h. NHS contains all serum complement proteins required to initiate complement activation and MAC formation. We combined in vitro RPE polarization and complement activation to visualize MAC formation in vitro utilizing confocal microscopy allowing for high resolution MAC imaging.

Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells.

BACKGROUND: Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically. METHODS: We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function. RESULTS: We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors. CONCLUSION: ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age.

Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration.

Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration.

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Interleukin-36 cytokines alter the intestinal microbiome and can protect against obesity and metabolic dysfunction.

Members of the interleukin-1 (IL-1) family are important mediators of obesity and metabolic disease and have been described to often play opposing roles. Here we report that the interleukin-36 (IL-36) subfamily can play a protective role against the development of disease. Elevated IL-36 cytokine expression is found in the serum of obese patients and negatively correlates with blood glucose levels among those presenting with type 2 diabetes. Mice lacking IL-36Ra, an IL-36 family signalling antagonist, develop less diet-induced weight gain, hyperglycemia and insulin resistance. These protective effects correlate with increased abundance of the metabolically protective bacteria Akkermansia muciniphila in the intestinal microbiome. IL-36 cytokines promote its outgrowth as well as increased colonic mucus secretion. These findings identify a protective role for IL-36 cytokines in obesity and metabolic disease, adding to the current understanding of the role the broader IL-1 family plays in regulating disease pathogenesis.

Type 1 IFN Induction by Cytosolic Nucleic Acid Is Intact in Neonatal Mononuclear Cells, Contrasting Starkly with Neonatal Hyporesponsiveness to TLR Ligation Due to Independence from Endosome-Mediated IRF3 Activation.

Two million infants die each year from infectious diseases before they reach 12 mo; many of these diseases are vaccine preventable in older populations. Pattern recognition receptors represent the critical front-line defense against pathogens. Evidence suggests that the innate immune system does not fully develop until puberty, contributing to impaired response to infection and impaired vaccine responses in neonates, infants, and children. The activity of the pattern recognition receptor family of cytosolic nucleic acid (CNA) sensors in this pediatric population has not been reported. We show that in direct contrast to weak TLR-induced type I IFN in human cord blood mononuclear cells, cord blood mononuclear cells are capable of initiating a potent response to CNA, inducing both antiviral type I IFN and, unexpectedly, proinflammatory TNF-α. A deficiency in Rab11-GTPase endosome formation and consequent lack of IRF3 activation in neonatal monocytes is at least in part responsible for the marked disparity in TLR-induced IFN production between neonatal and adult monocytes. CNA receptors do not rely on endosome formation, and therefore, these responses remain intact in neonates. Heightened neonatal responses to CNA challenge are maintained in children up to 2 y of age and, in marked contrast to TLR4/9 agonists, result in IL-12p70 and IFN-γ generation. CNA sensors induce robust antiviral and proinflammatory pathways in neonates and children and possess great potential for use as immunostimulants or vaccine adjuvants for targeted neonatal and pediatric populations to promote cell-mediated immunity against invasive infectious disease.

Toll-Like Receptors and Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of central vision loss in the over 50s worldwide. Activation of the immune system has been implicated in disease progression, but while polymorphisms in genes associated with the immune system have been identified as risk factors for disease, the underlying pathways and mechanisms involved in disease progression remain incompletely characterised. Typically inflammatory responses are mediated by microbial infection; however, in chronic conditions, a form of 'sterile' inflammation exists whereby immune responses occur in areas of the body, in the absence of microbes; 'sterile' inflammation is likely to be central to AMD. In this case the innate immune response is triggered when alarm signals released by stressed cells or damaged tissue are identified by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are a family of membrane-spanning PRRs for which host-derived ligands have been identified; these include heat shock proteins, extracellular matrix breakdown products, mRNA from necrotic cells and modified lipids. Here we review the evidence for TLR involvement in the pathogenesis of AMD.

IL-18 Immunotherapy for Neovascular AMD: Tolerability and Efficacy in Nonhuman Primates.

PURPOSE: Age-related macular degeneration is the most common form of central retinal blindness in the elderly. Of the two end stages of disease, neovascular AMD-although the minority form-is the most severe. Current therapies are highly successful at controlling progression of neovascular lesions; however, a significant number of patients remain refractory to treatment and the development of alternative and additive therapies to anti-VEGFs is essential. METHODS: In order to address the translational potential of interleukin (IL)-18 for use in neovascular AMD, we initiated a nonhuman primate tolerability and efficacy study for the use of intravitreally (IVT) administered clinical grade human IL-18 (SB-485232). Cynomolgus monkeys were injected IVT with increasing doses of human IL-18 (two each at 1000, 3000, and 10,000 ng per eye). In tandem, 21 monkeys were administered nine laser burns in each eye prior to receiving IL-18 as an IVT injection at a range of doses. Fundus fluorescein angiography (FFA) was performed on days 8, 15, and 22 post injection and the development of neovascular lesions was assessed. RESULTS: We show intravitreal, mature, recombinant human IL-18 is safe and can reduce choroidal neovascular lesion development in cynomolgus monkeys. CONCLUSIONS: Based on our data comparing human IL-18 to current anti-VEGF-based therapy, clinical deployment of IL-18 for neovascular AMD has the potential to lead to a new adjuvant immunotherapy-based treatment for this severe form of central blindness.

IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common form of central retinal blindness globally. Distinct processes of the innate immune system, specifically activation of the NLRP3 inflammasome, have been shown to play a central role in the development of both "dry" and neovascular ("wet") forms of the disease. We show that the inflammatory cytokine interleukin-18 (IL-18) can regulate choroidal neovascularization formation in mice. We observed that exogenous administration of mature recombinant IL-18 has no effect on retinal pigment epithelial (RPE) cell viability, but that overexpression of pro-IL-18 or pro-IL-1ß alone can cause RPE cell swelling and subsequent atrophy, a process that can be inhibited by the promotion of autophagy. A direct comparison of local and systemic administration of mature recombinant IL-18 with current anti-VEGF (vascular endothelial growth factor)-based therapeutic strategies shows that IL-18 treatment works effectively alone and more effectively in combination with anti-VEGF therapy and represents a novel therapeutic strategy for the treatment of wet AMD.

Plasmonics-enhanced and optically modulated delivery of gold nanostars into brain tumor.

Plasmonics-active gold nanostars exhibiting strong imaging contrast and efficient photothermal transduction were synthesized for a novel pulsed laser-modulated plasmonics-enhanced brain tumor microvascular permeabilization. We demonstrate a selective, optically modulated delivery of nanoprobes into the tumor parenchyma with minimal off-target distribution.