LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.

Quick-freeze/deep-etch electron microscopy visualization of the mouse posterior pole.

The mouse is one of the most commonly used mammalian systems to study human diseases. In particular it has been an invaluable tool to model a multitude of ocular pathologies affecting the posterior pole. The aim of this study was to create a comprehensive map of the ultrastructure of the mouse posterior pole using the quick-freeze/deep-etch method (QFDE). QFDE can produce detailed three-dimensional images of tissue structure and macromolecular moieties, without many of the artifacts introduced by structure-altering post-processing methods necessary to perform conventional transmission electron microscopy (cTEM). A total of 18 eyes from aged C57BL6/J mice were enucleated and the posterior poles were processed, either intact or with the retinal pigment epithelium (RPE) cell layer removed, for imaging by either QFDE or cTEM. QFDE images were correlated with cTEM cross-sections and en face images through the outer retina. Nicely preserved outer retinal architecture was observed with both methods, however, QFDE provided excellent high magnification imaging, with greater detail, of the apical, central, and basal planes of the RPE. Furthermore, key landmarks within Bruch's membrane, choriocapillaris, choroid and sclera were characterized and identified. In this study we developed methods for preparing the outer retina of the mouse for evaluation with QFDE and provide a map of the ultrastructure and cellular composition of the outer posterior pole. This technique should be applicable for morphological evaluation of mouse models, in which detailed visualization of subtle ocular structural changes is needed or in cases where post-processing methods introduce unacceptable artifacts.

Flow-Induced Crystallization of Collagen: A Potentially Critical Mechanism in Early Tissue Formation.

The type I collagen monomer is one of nature's most exquisite and prevalent structural tools. Its 300 nm triple-helical motifs assemble into tough extracellular fibers that transition seamlessly across tissue boundaries and exceed cell dimensions by up to 4 orders of magnitude. In spite of extensive investigation, no existing model satisfactorily explains how such continuous structures are generated and grown precisely where they are needed (aligned in the path of force) by discrete, microscale cells using materials with nanoscale dimensions. We present a simple fiber drawing experiment, which demonstrates that slightly concentrated type I collagen monomers can be "flow-crystallized" to form highly oriented, continuous, hierarchical fibers at cell-achievable strain rates (<1 s(-1)) and physiologically relevant concentrations (∼50 µM). We also show that application of tension following the drawing process maintains the structural integrity of the fibers. While mechanical tension has been shown to be a critical factor driving collagen fibril formation during tissue morphogenesis in developing animals, the precise role of force in the process of building tissue is not well understood. Our data directly couple mechanical tension, specifically the extensional strain rate, to collagen fibril assembly. We further derive a "growth equation" which predicts that application of extensional strains, either globally by developing muscles or locally by fibroblasts, can rapidly drive the fusion of already formed short fibrils to produce long-range, continuous fibers. The results provide a pathway to scalable connective tissue manufacturing and support a mechano-biological model of collagen fibril deposition and growth in vivo.