The vascular geometry of the choriocapillaris is associated with spatially heterogeneous molecular exchange with the outer retina.

Vision relies on the continuous exchange of material between the photoreceptors, retinal pigment epithelium and choriocapillaris, a dense microvascular bed located underneath the outer retina. The anatomy and physiology of the choriocapillaris and their association with retinal homeostasis have proven difficult to characterize, mainly because of the unusual geometry of this vascular bed. By analysing tissue dissected from 81 human eyes, we show that the thickness of the choriocapillaris does not vary significantly over large portions of the macula or with age. Assessments of spatial variations in the anatomy of the choriocapillaris in three additional human eyes indicate that the location of arteriolar and venular vessels connected to the plane of the choriocapillaris is non-random, and that venular insertions cluster around arteriolar ones. Mathematical models built upon these anatomical analyses reveal that the choriocapillaris contains regions where the transport of passive elements is dominated by diffusion, and that these diffusion-limited regions represent areas of reduced exchange with the outer retina. The width of diffusion-limited regions is determined by arterial flow rate and the relative arrangement of arteriolar and venular insertions. These analyses demonstrate that the apparent complexity of the choriocapillaris conceals a fine balance between several anatomical and functional parameters to effectively support homeostasis of the outer retina. KEY POINTS: The choriocapillaris is the capillary bed supporting the metabolism of photoreceptors and retinal pigment epithelium, two critical components of the visual system located in the outer part of the retina. The choriocapillaris has evolved a planar multipolar vascular geometry that differs markedly from the branched topology of most vasculatures in the human body. Here, we report that this planar multipolar vascular geometry is associated with spatially heterogenous molecular exchange between choriocapillaris and outer retina. Our data and analyses highlight a necessary balance between choriocapillaris anatomical and functional parameters to effectively support homeostasis of the outer retina.

Clinical, histological and genetic findings in a donor with a clinical history of type 1 Autoimmune Polyendocrinopathy Syndrome.

PURPOSE: Autoimmune Polyendocrinopathy Syndrome (APS) is a rare condition caused by an autoimmune failure of two or more endocrine glands. In this case, we report the ocular findings and correlated histopathology from a human eye donor with a prior clinical history of Type 1 APS. OBSERVATIONS: The 23 year-old patient originally presented with blurred vision at the 20/125 level caused by papilledema of the right eye. Bilateral pigmentary changes in the peripheral retinal were also noted. The patient passed away due to electrolyte abnormalities related to autoimmune illness. Histopathology of the posterior segments documents that these pigmentary changes were caused by pigment deposition around inner retinal vessels with corresponding outer retina atrophy. Postmortem genetic sequence analyses revealed a homozygous R257X (C to T substitution) mutation within exon 6 of the AIRE gene. CONCLUSIONS AND IMPORTANCE: The retinal findings in Type 1 Autoimmune Polyendocrinopathy Syndrome resemble those observed in individuals with retinitis pigmentosa, suggesting that similar pathological processes occur in both.

Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium.

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151-171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389-407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227-3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE-Bruch's membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE-BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

Assessment of Proteins Associated With Complement Activation and Inflammation in Maculae of Human Donors Homozygous Risk at Chromosome 1 CFH-to-F13B.

PURPOSE: To determine the effects of chromosome 1 genotype and cigarette smoking on levels of complement activation and inflammation in the human macula. METHODS: Donor macular tissue was stratified into three groups by diplotype at the AMD-associated CFH-to-F13B locus: homozygous "risk" (n = 9, 56-78 years), homozygous neutral (n = 2, 64-79 years), and homozygous "protective" (n = 6, 61-78 years) diplotype. Importantly, all donors were homozygous nonrisk at the ARMS2/HTRA1 locus, so that purely chromosome 1-directed pathways were examined. Immunohistochemistry was performed by using 14 antibodies, mostly against markers of complement and inflammation, followed by confocal microscopy and immunofluorescence quantification (all masked to donor status). RESULTS: Donors homozygous risk at CFH-to-F13B exhibited significantly higher levels of terminal complement complex (TCC) in macular Bruch's membrane (BM; P = 0.03), choriocapillaris (CC; P = 0.04), and choriocapillaris intercapillary septa (CC IS; P = 0.03), compared to homozygous protected donors. Smoking was associated with increased TCC in BM (P = 0.05), CC IS (P = 0.03), and choroidal stroma (CS; P = 0.01), and with substantially elevated C-reactive protein (CRP) levels in RPE (P = 0.04), BM (P = 0.01), CC (P = 0.05), and CS (P = 0.05). Smoking was associated with higher levels of oxidative stress in macular RPE (P = 0.04) and CS (P = 0.01). CONCLUSIONS: Genetic risk at the CFH-to-F13B locus was associated with higher levels of complement activation at the human macular RPE-choroid interface, as was cigarette smoking. Levels of CRP were substantially elevated in risk donors with smoking history. Examination of human macular tissue from donors with "pure" diplotypes allows assessment of AMD-associated pathways driven solely by CFH-to-F13B. These findings have important implications for identifying chromosome 1-directed pathways and therapeutic targets.

Ultrastructure of the Gregarina niphandrodes nucleus through stages from unassociated trophozoites to gamonts in syzygy and the syzygy junction.

In Gregarina niphandrodes, an apicomplexan parasite, the sexual stage of its life cycle begins with the association of 2 gamonts. Here, we describe the ultrastructure of the syzygy junction and the nucleus during the transition from unassociated trophozoites to paired gamonts to gamonts in syzygy. Throughout this process, the folds within the syzygy junction undergo changes that correspond to changes of the epicytic folds. The nucleus goes through dramatic changes from multiple spheres of condensed chromatin in unassociated trophozoites, to mostly uncondensed chromatin in paired gamonts, to a large single sphere of condensed chromatin encasing many smaller spheres in gamonts in syzygy. These differing nuclear ultrastructures reflect the dramatic cellular and transcriptional changes associated with life cycle transitions and are indicative of the numerous cell divisions that follow.

Gregarina niphandrodes may lack both a plastid genome and organelle.

Gregarines are early diverging apicomplexans that appear to be closely related to Cryptosporidium. Most apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess both plastids and corresponding plastid genomes. Cryptosporidium lacks both the organelle and the genome. To investigate the evolutionary history of plastids in the Apicomplexa, we tried to determine whether gregarines possess a plastid and/or its genome. We used PCR and dot-blot hybridization to determine whether the gregarine Gregarina niphandrodes possesses a plastid genome. We used an inhibitor of plastid function for any reduction in gregarine infection, and transmission electron microscopy to search for plastid ultrastructure. Despite an extensive search, an organelle of the appropriate ultrastructure in transmission electron microscopy, was not observed. Triclosan, an inhibitor of the plastid-specific enoyl-acyl carrier reductase enzyme, did not reduce host infection by G. niphandrodes. Plastid-specific primers produced amplicons with the DNA of Babesia equi, Plasmodium falciparum, and Toxoplasma gondii as templates, but not with G. niphandrodes DNA. Plastid-specific DNA probes, which hybridized to Babesia equi, failed to hybridize to G. niphandrodes DNA. This evidence indicates that G. niphandrodes is not likely to possess either a plastid organelle or its genome. This raises the possibility that the plastid was lost in the Apicomplexan following the divergence of gregarines and Cryptosporidium.

Expressed sequence tag (EST) analysis of Gregarine gametocyst development.

Gregarines are protozoan parasites of invertebrates in the phylum Apicomplexa. We employed an expressed sequence tag strategy in order to dissect the molecular processes of sexual or gametocyst development of gregarines. Expressed sequence tags provide a rapid way to identify genes, particularly in organisms for which we have very little molecular information. Analysis of approximately 1800 expressed sequence tags from the gametocyst stage revealed highly expressed genes related to cell division and differentiation. Evidence was found for the role of degradation and recycling in gametocyst development. Numerous additional genes uncovered by expressed sequence tag sequencing should provide valuable tools to investigate gametocyst development as well as for molecular phylogenetics, and comparative genomics in this important group of parasites.