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1.  A2E accumulation influences retinal microglial activation and complement regulation 
Neurobiology of aging  2012;34(3):943-960.
Age-related macular degeneration (AMD) is an outer retinal disease that involves aging and immune dysfunction. In the aging retina, microglia aggregate in the outer retina and acquire intracellular autofluorescent lipofuscin deposits. In this study, we investigated whether accumulation of A2E, a key bisretinoid constituent of ocular lipofuscin, alters the physiology of retinal microglia in pathologically relevant ways. Our findings show that sublethal accumulations of intracellular A2E in cultured retinal microglia increased microglial activation and decreased microglial neuroprotection of photoreceptors. Increased A2E accumulation also lowered microglial expression of chemokine receptors and suppressed microglial chemotaxis, suggesting that lipofuscin accumulation may potentiate subretinal microglial accumulation. Significantly, A2E accumulation altered microglial complement regulation by increasing CFB and decreasing CFH expression, favoring increased complement activation and deposition in the outer retina. Taken together, our findings highlight the role of microglia in the local control of complement activation in the retina and present the age-related accumulation of ocular lipofuscin in subretinal microglia as a cellular mechanism capable of driving outer retinal immune dysregulation in AMD pathogenesis.
doi:10.1016/j.neurobiolaging.2012.06.010
PMCID: PMC3480997  PMID: 22819137
microglia; retina; lipofuscin; A2E; aging; age-related macular degeneration; complement; activation; neuroprotection; photoreceptors; chemokine
2.  Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease 
Nature genetics  2012;44(8):890-894.
We performed a meta-analysis of 2 genome-wide association studies of coronary artery disease comprising 1,515 cases with coronary artery disease and 5,019 controls, followed by de novo replication studies in 15,460 cases and 11,472 controls, all of Chinese Han descent. We successfully identified four new loci for coronary artery disease reaching genome-wide significance (P < 5 × 10−8), which mapped in or near TTC32-WDR35, GUCY1A3, C6orf10-BTNL2 and ATP2B1. We also replicated four loci previously identified in European populations (PHACTR1, TCF21, CDKN2A/B and C12orf51). These findings provide new insights into biological pathways for the susceptibility of coronary artery disease in Chinese Han population.
doi:10.1038/ng.2337
PMCID: PMC3927410  PMID: 22751097
3.  HIF-1α 1772 C/T and 1790 G/A Polymorphisms Are Significantly Associated with Higher Cancer Risk: An Updated Meta-Analysis from 34 Case-Control Studies 
PLoS ONE  2013;8(11):e80396.
Background
HIF-1 activates various genes in cancer progression and metastasis. HIF-1α 1772 C/T and 1790 G/A polymorphisms are reportedly associated with cancer risk; however, the results are inconclusive.
Methodology/Principal Findings
A meta-analysis of 34 studies that involved 7522 cases and 9847 controls for 1772 C/T and 24 studies that involved 4884 cases and 8154 controls for 1790 G/A was conducted to identify the association of C/T and G/A polymorphisms with cancer risk. Odds ratio (OR) and 95% confidence intervals (95% CI) were used to assess the strength of association.
HIF-1α 1772 C/T and 1790 G/A polymorphisms were associated with higher cancer risk in homozygote comparison (1772C/T: TT vs. CC: OR = 2.45, 95% CI: 1.52, 3.96; Pheterogeneity = 0.028; 1790G/A: AA vs. GG: OR=4.74, 95% CI: 1.78, 12.6; Pheterogeneity < 0.01), dominant model (1772C/T: TT/CT vs. CC: OR = 1.27, 95% CI: 1.04, 1.55; Pheterogeneity < 0.01, 1790G/A: AA/GA vs. GG: OR = 1.65, 95% CI: 1.05, 2.60; Pheterogeneity < 0.01), T allele versus C allele (T vs. C: OR = 1.42, 95% CI: 1.18, 1.70; Pheterogeneity < 0.01), and A allele versus G allele (A vs. G: OR = 1.83, 95% CI: 1.13, 2.96; Pheterogeneity < 0.01). On a subgroup analysis, the 1772 C/T polymorphism was significantly linked to higher risks for breast cancer, lung cancer, prostate cancer, and cervical cancer, whereas the 1790 G/A polymorphism was significantly linked to higher risks for lung cancer and prostate cancer. A significantly increased cancer risk was found in both Asians and Caucasians for 1772C/T polymorphism, whereas a significantly increased cancer risk was found in Caucasians in the heterozygote comparison and recessive model for 1790G/A polymorphism.
Conclusions
HIF-1α 1772 C/T and 1790 G/A polymorphisms are significantly associated with higher cancer risk.
doi:10.1371/journal.pone.0080396
PMCID: PMC3832403  PMID: 24260383
4.  Validation of Reliable Reference Genes for Real-Time PCR in Human Umbilical Vein Endothelial Cells on Substrates with Different Stiffness 
PLoS ONE  2013;8(6):e67360.
Background
The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness.
Methodology/Principal Findings
Four algorithms, GeNormPLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes.
Conclusion/Significance:
Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study.
doi:10.1371/journal.pone.0067360
PMCID: PMC3696109  PMID: 23840676
5.  Preservation of cone photoreceptors after a rapid yet transient degeneration and remodeling in cone-only Nrl−/− mouse retina 
Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor NRL. The loss of Nrl (Nrl−/−) in mice results in a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. Here we report that Nrl−/− retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by four months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl−/− mouse illustrates the long-term viability of cones in the absence of rods and RPE defects in a rodless retina. We propose that Nrl−/− retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.
doi:10.1523/JNEUROSCI.3591-11.2012
PMCID: PMC3567450  PMID: 22238088
6.  Perivascular Mural Cells of the Mouse Choroid Demonstrate Morphological Diversity That Is Correlated to Vasoregulatory Function 
PLoS ONE  2013;8(1):e53386.
Objective
Perivascular mural cells of the choroid have been implicated in physiological functioning as well as in retinal disease pathogenesis. However details regarding their form and function are not well understood. We aim to characterize choroidal mural cells in the adult mouse choroid in terms of their distribution and morphology, and correlate these to their contractile behavior.
Methods
Sclerochoroidal flat-mounted explants were prepared from albino transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives the expression of green fluorescent protein (GFP). α-SMA-expressing smooth muscle cells and pericytes in the living choroid were thereby rendered fluorescent and imaged with confocal microscopy and live-cell imaging in situ.
Results
Choroidal perivascular mural cells demonstrate significant diversity in terms of their distribution and morphology at different levels of the vasculature. They range from densely-packed circumferentially-oriented cells that provide complete vascular coverage in primary arteries to widely-spaced stellate-shaped cells that are distributed sparsely over terminal arterioles. Mural cells at each level are immunopositive for contractile proteins α-SMA and desmin and demonstrate vasoconstrictory contractile movements in response to endothelin-1 and the calcium ionophore, A23187, and vasodilation in response to the calcium chelator, BAPTA. The prominence of vasoregulatory contractile responses varies with mural cell morphology and density, and is greater in vessels with dense coverage of mural cells with circumferential cellular morphologies. In the choriocapillaris, pericytes demonstrate a sparse, horizontal distribution and are selectively distributed only to the scleral surface of the choriocapillaris.
Conclusions
Diversity and regional specialization of perivascular mural cells may subserve varying requirements for vasoregulation in the choroid. The model of the α-SMA-GFP transgenic albino mouse provides a useful and intact system for the morphological and functional study of choroidal mural cells.
doi:10.1371/journal.pone.0053386
PMCID: PMC3537675  PMID: 23308209
7.  Intersubband absorption properties of high Al content AlxGa1−xN/GaN multiple quantum wells grown with different interlayers by metal organic chemical vapor deposition 
Nanoscale Research Letters  2012;7(1):649.
High Al content AlxGa1−xN/GaN multiple quantum well (MQW) films with different interlayers were grown by metal organic chemical vapor deposition. These MQWs were designed to achieve intersubband (ISB) absorption in the mid-infrared spectral range. We have considered two growth conditions, with AlGaN interlayer and GaN/AlN superlattice (SL) interlayer, both deposited on GaN-on-sapphire templates. Atomic force microscopy images show a relatively rough surface with atomic-step terraces and surface depression, mainly dominated by dislocations. High-resolution X-ray diffraction and transmission electron microscopy analyses indicate that good crystalline quality of the AlGaN/GaN MQW layer could be achieved when the AlGaN interlayer is inserted. The ISB absorption with a peak at 3.7 μm was demonstrated in MQW films with AlGaN interlayer. However, we have not observed the infrared absorption in MQW films with GaN/AlN SL interlayer. It is believed that the high dislocation density and weaker polarization that resulted from the rough interface are determinant factors of vanished ISB absorption for MQW films with the GaN/AlN SL interlayer.
doi:10.1186/1556-276X-7-649
PMCID: PMC3526456  PMID: 23181766
Quantum wells; Interface; Intersubband; TEM; PACS; 61.72.Lk; 61.05.cp; 68.37.-d; 61.72.uj
8.  Epstein-Barr Virus Downregulates MicroRNA 203 through the Oncoprotein Latent Membrane Protein 1: a Contribution to Increased Tumor Incidence in Epithelial Cells 
Journal of Virology  2012;86(6):3088-3099.
The Epstein-Barr virus (EBV) is highly associated with nasopharyngeal carcinoma (NPC), and it regulates some microRNAs (miRNAs) that are involved in the development of cancer. The role of EBV in the deregulation of cellular miRNAs and how this affects the progression of NPC remain to be investigated. An analysis of the miRNA profile in an EBV-infected cell line revealed that miRNA 203 (miR-203) was downregulated. miR-203 is expressed specifically in epithelial cells. This downregulation of miR-203 was further verified and functionally analyzed. miR-203 was downregulated substantially in epithelial cells and NPC tissues that were latently infected with EBV. Downregulation of miR-203 also occurred during the early stage of EBV infection. Furthermore, the viral oncoprotein, latent membrane protein 1 (LMP1), was responsible for downregulation of miR-203. Removal of the latent EBV genome or suppression of LMP1 resulted in restoration of miR-203 expression. EBV-LMP1 mediated the downregulation of miR-203 at the primary transcript level. E2F3 and CCNG1 were identified as target genes of miR-203. Ectopic expression of miR-203 inhibited EBV-induced S-phase entry and transformation in vivo. Overexpression of the targets overcame the effects of miR-203 mimics on the cell cycle, and the expression of target genes in tumor models was inhibited by miR-203. Inhibitors of Jun N-terminal protein kinase (JNK) and NF-κB blocked miR-203 downregulation. These results imply that EBV promotes malignancy by downregulating cellular miR-203, which contributes to the etiology of NPC.
doi:10.1128/JVI.05901-11
PMCID: PMC3302296  PMID: 22205737
9.  Minocycline Inhibits Alkali Burn-Induced Corneal Neovascularization in Mice 
PLoS ONE  2012;7(7):e41858.
The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV). A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS) or minocycline twice a day (60 mg/kg or 30 mg/kg) for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs). The mRNA expression levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs), interleukin-1α, 1β, 6 (IL-1α, IL-1β, IL-6) were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1β and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1β, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1β, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d.) can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.
doi:10.1371/journal.pone.0041858
PMCID: PMC3405025  PMID: 22848638
10.  Age-related Alterations in the Dynamic Behavior of Microglia 
Aging cell  2010;10(2):263-276.
Summary
Microglia, the primary resident immune cells of the CNS, exhibit dynamic behavior involving rapid process motility and cellular migration that is thought to underlie key functions of immune surveillance and tissue repair. Although age-related changes in microglial activation have been implicated in the pathogenesis of neurodegenerative diseases of aging, how dynamic behavior in microglia is influenced by aging is not fully understood. In this study, we employed live imaging of retinal microglia in situ to compare microglial morphology and behavioral dynamics in young and aged animals. We found that aged microglia in the resting state have significantly smaller and less branched dendritic arbors, and also slower process motilities, which likely compromise their ability to continuously survey and interact with their environment. We also found that dynamic microglial responses to injury were age-dependent. While young microglia responded to extracellular ATP, an injury-associated signal, by increasing their motility and becoming more ramified, aged microglia exhibited a contrary response, becoming less dynamic and ramified. In response to laser-induced focal tissue injury, aged microglia demonstrated slower acute responses with lower rates of process motility and cellular migration compared to young microglia. Interestingly, the longer term response of disaggregation from the injury site was retarded in aged microglia, indicating that senescent microglial responses, while slower to initiate, are more sustained. Together, these altered features of microglial behavior at rest and following injury reveal an age-dependent dysregulation of immune response in the CNS that may illuminate microglial contributions to age-related neuroinflammatory degeneration.
doi:10.1111/j.1474-9726.2010.00660.x
PMCID: PMC3056927  PMID: 21108733
microglia; aging; retina; age-related macular degeneration; imaging; laser
11.  Adaptive Müller cell responses to microglial activation mediate neuroprotection and coordinate inflammation in the retina 
Purpose
Microglia and Müller cells are prominent participants in retinal responses to injury and disease that shape eventual tissue adaptation or damage. This investigation examined how microglia and Müller cells interact with each other following initial microglial activation.
Methods
Mouse Müller cells were cultured alone, or co-cultured with activated or unactivated retinal microglia, and their morphological, molecular, and functional responses were evaluated. Müller cell-feedback signaling to microglia was studied using Müller cell-conditioned media. Corroborative in vivo analyses of retinal microglia-Müller cell interactions in the mouse retina were also performed.
Results
Our results demonstrate that Müller cells exposed to activated microglia, relative to those cultured alone or with unactivated microglia, exhibit marked alterations in cell morphology and gene expression that differed from those seen in chronic gliosis. These Müller cells demonstrated in vitro (1) an upregulation of growth factors such as GDNF and LIF, and provide neuroprotection to photoreceptor cells, (2) increased pro-inflammatory factor production, which in turn increased microglial activation in a positive feedback loop, and (3) upregulated chemokine and adhesion protein expression, which allowed Müller cells to attract and adhere to microglia. In vivo activation of microglia by intravitreal injection of lipopolysaccharide (LPS) also induced increased Müller cell-microglia adhesion, indicating that activated microglia may translocate intraretinally in a radial direction using Müller cell processes as an adhesive scaffold.
Conclusion
Our findings demonstrate that activated microglia are able to influence Müller cells directly, and initiate a program of bidirectional microglia-Müller cell signaling that can mediate adaptive responses within the retina following injury. In the acute aftermath following initial microglia activation, Müller cell responses may serve to augment initial inflammatory responses across retinal lamina and to guide the intraretinal mobilization of migratory microglia using chemotactic cues and adhesive cell contacts. Understanding adaptive microglia-Müller cell interactions in injury responses can help discover therapeutic cellular targets for intervention in retinal disease.
doi:10.1186/1742-2094-8-173
PMCID: PMC3251543  PMID: 22152278
Müller cell; microglia; retina; cytokine; cellular interaction; gliosis; migration; adhesion; inflammation; neuroprotection
12.  catena-Poly[[[(acetato-κ2 O,O′)cadmium]-μ-acetato-κ3 O,O′:O′-μ-{1,2-bis­[4-(pyridin-3-yl)pyrimidin-2-ylsulfan­yl]ethane}-κ2 N 4,N 4′] trihydrate] 
The title compound, {[Cd(CH3COO)2(C20H16N6S2)]·3H2O}n, exists as a one-dimensional zigzag polymer in which the CdII ion shows a seven-coordinate [CdO5N2] distorted penta­gonal–bipyramidal geometry with the N atoms in axial positions and an N—Cd—N angle of 176.94 (13)°. The metal atoms are bridged by 1,2-bis­[4-(pyridin-3-yl)pyrimidin-2-ylsulfan­yl]ethane ligands, giving a polymeric chain extending along the b axis. Adjacent chains related by an inversion center are further bridged by Cd—O bonds formed between the O atom of one of the acetate ligands and the metal atom. The five Cd—O bond lengths are in the range 2.329 (3)–2.485 (3) Å. There are π–π stacking inter­actions between the aromatic rings of adjacent polymeric chains, the centroid–centroid distances being 3.556 (3) and 3.698 (3) Å, organizing the chains into a three-dimensional framework. This framework is additionally stabilized by extensive O—H⋯O and O—H⋯N hydrogen bonding between water mol­ecules and the ligands.
doi:10.1107/S1600536811046794
PMCID: PMC3238649  PMID: 22199540
13.  Naloxone Ameliorates Retinal Lesions in Ccl2/Cx3cr1 Double-Deficient Mice via Modulation of Microglia 
Naloxone significantly reduces the progress of focal retinal degeneration via modulation of microglia and inflammatory molecules in a murine AMD model.
Purpose.
The role of naloxone, an opioid receptor antagonist, on microglial inhibition and neuroprotective effects has been reported in lipopolysaccharide (LPS)-induced neurodegeneration and light-induced photoreceptor degeneration. The authors evaluated the effects of naloxone on Ccl2−/−/Cx3cr1−/− (DKO) mice, a murine model of age-related macular degeneration (AMD).
Methods.
Two-month-old DKO and wild-type controls were given daily intraperitoneal injections of naloxone or PBS for 2 months. Animals were examined monthly by funduscopy. Ocular tissue was analyzed histologically and in retinal flat mount preparations. Ocular A2E was measured using HPLC. Quantitative RT-PCR analyzed TNF-α, IL-1β, IL-10 and TLR4 transcripts in the DKO eyes and LPS activated culture microglial cells. Serum nitrite was measured using Griess colorimetric reaction.
Results.
Naloxone ameliorated the clinical progression and severity of retinal lesions in the DKO mice compared with those of untreated controls. Histopathology also showed less focal retinal degeneration in the treated DKO mice than in controls. The aggregation of microglia in the outer retina in DKO mice was significantly reduced in naloxone-treated animals compared with control untreated DKO. Ocular TNF-α, IL-1β, and TLR4 transcripts and A2E were significantly lower in naloxone-treated DKO animals and cultured microglial cells than in controls, as were serum nitrite levels.
Conclusions.
Naloxone significantly reduces the progress of retinal lesions in DKO mice. Naloxone modulates microglia accumulation and activation at the site of retinal degeneration, which may be mediated by inhibition of the proinflammatory molecules of NO, TNF-α, and IL-β. The potential therapeutic effects of naloxone on retinal degeneration, including AMD, warrants further investigation.
doi:10.1167/iovs.10-6114
PMCID: PMC3109007  PMID: 21245403
14.  Subclinical atherosclerosis in northern and southern China: the Chinese paradox 
Background
The incidence of coronary heart disease (CHD) is higher in Northern than that in Southern China, however differences in traditional CHD risk factors do not fully explain this. No study has examined the differences in subclinical atherosclerosis that may help explain the differences in incidence. This study examined these differences in subclinical atherosclerosis using coronary computed tomography (CT) for calcification between the Northern and Southern China.
Methods
We selected a random sample of participants in a large multi-center ongoing epidemiologic study for coronary calcium scanning in one northern city (North) (Beijing, n = 49) and in two southern cities (South) (Shanghai, n = 50, and Guangzhou, n = 50). Participants from the three field centers (mean age 67 years) underwent coronary risk factor evaluation and cardiac CT scanning for coronary calcium measurement using the Multi-Ethnic Study of Atherosclerosis scanning protocol.
Results
Adjusted log-transformed coronary artery calcium score in North China (Beijing) was 3.1 ± 0.4 and in South China (Shanghai and Guangzhou) was 2.2 ± 0.3 (P = 0.04). Mean calcium score for the northern city of Beijing was three times higher than that of the southern city of Guangzhou (P = 0.01) and 2.5 times higher than for the southern city of Shanghai (P = 0.03).
Conclusions
The extent of subclinical atherosclerosis is significantly higher in the northern city of Beijing than that in the two southern cities of Guangzhou and Shanghai, even after adjusting for standard cardiac risk factors. This finding suggests that standard risk factors do not fully explain north south differences in clinical CHD incidence.
doi:10.3724/SP.J.1263.2011.00072
PMCID: PMC3390074  PMID: 22783288
coronary calcium; CT scanning; atherosclerosis; epidemiology; China
15.  A Subretinal Matrigel Rat Choroidal Neovascularization (CNV) Model and Inhibition of CNV and Associated Inflammation and Fibrosis by VEGF Trap 
A novel subretinal Matrigel model of choroidal neovascularization (CNV) was devised, with several unique features that mimic those in human exudative (wet) AMD. With this model and VEGF Trap, a potent receptor-based inhibitor of VEGF-A and PlGF, the data show that inhibition of VEGF-A, and perhaps PlGF as well, not only stops the growth and induces regression of experimental CNV, but also inhibits the associated inflammation and fibrotic responses.
Purpose.
The exudative, or the wet form of age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). A subretinal Matrigel (BD Biosciences, Bedford MA) model of CNV is described here, along with the effects of vascular endothelial growth factor (VEGF) neutralization on the development of CNV and associated inflammation and fibrosis.
Methods.
CNV was induced in adult Sprague-Dawley rats by subretinal injection of Matrigel. CNV growth and associated leukocyte infiltration and collagen deposition were examined. VEGF Trap (Regeneron Pharmaceuticals, Tarrytown, NY), a recombinant protein that comprises portions of the extracellular domains of VEGF receptors 1 and 2 and that binds all isoforms of VEGF-A as well as placental growth factor with high affinity, was administered subcutaneously.
Results.
Initiation of CNV was detected 4 days after Matrigel injection and then increased progressively in size. Systemic administration of VEGF Trap beginning on day 2 and 6 completely prevented development of CNV. When CNV was allowed to develop for 10 days before treatment was initiated, VEGF Trap not only prevented its further progression, but also induced substantial regression of existing lesions. In addition, VEGF Trap treatment reduced the total lesion volume and largely prevented the progressive leukocyte infiltration and fibrosis associated with CNV.
Conclusions.
The subretinal Matrigel CNV model provides a convenient tool for the study of the diverse components of complex CNV lesions. The data not only confirm the critical roles of VEGF in the development and maintenance of CNV, but further demonstrate that VEGF and other VEGF receptor 1 ligands promote CNV-associated inflammation and fibrosis.
doi:10.1167/iovs.09-4956
PMCID: PMC3061520  PMID: 20538989
16.  Oncostatin M Protects Rod and Cone Photoreceptors and Promotes Regeneration of Cone Outer Segment in a Rat Model of Retinal Degeneration 
PLoS ONE  2011;6(3):e18282.
Retinitis pigmentosa (RP) is a group of photoreceptor degenerative disorders that lead to loss of vision. Typically, rod photoreceptors degenerate first, resulting in loss of night and peripheral vision. Secondary cone degeneration eventually affects central vision, leading to total blindness. Previous studies have shown that photoreceptors could be protected from degeneration by exogenous neurotrophic factors, including ciliary neurotrophic factor (CNTF), a member of the IL-6 family of cytokines. Using a transgenic rat model of retinal degeneration (the S334-ter rat), we investigated the effects of Oncostatin M (OSM), another member of the IL-6 family of cytokines, on photoreceptor protection. We found that exogenous OSM protects both rod and cone photoreceptors. In addition, OSM promotes regeneration of cone outer segments in early stages of cone degeneration. Further investigation showed that OSM treatment induces STAT3 phosphorylation in Müller cells but not in photoreceptors, suggesting that OSM not directly acts on photoreceptors and that the protective effects of OSM on photoreceptors are mediated by Müller cells. These findings support the therapeutic strategy using members of IL-6 family of cytokines for retinal degenerative disorders. They also provide evidence that activation of the STAT3 pathway in Müller cells promotes photoreceptor survival. Our work highlights the importance of Müller cell-photoreceptor interaction in the retina, which may serve as a model of glia-neuron interaction in general.
doi:10.1371/journal.pone.0018282
PMCID: PMC3068173  PMID: 21479182
17.  Bis[μ-2,2′-dimethyl-1,1′-(3-oxapentane-1,5-di­yl)di-1H-benzimidazole-κ2 N 3:N 3′]bis­[bis­(4-meth­oxy­benzoato)-κO;κ2 O,O′-cobalt(II)] 
The complete mol­ecule of the title complex, [Co2(C8H7O3)4(C20H22N4O)2], is a dimer of the paddle-wheel-type generated by crystallographic inversion symmetry. The CoII ion is penta­coordinated by three O atoms from two 4-meth­oxy­benzoate anions (one bidentate and one monodentate) and two N atoms from two 2,2′-bis­(2-methyl-1H-benzimidazole)­ether ligands. This results in a very distorted trigonal–bipyramidal geometry for the metal ion, with both N atoms in equatorial sites. The dihedral angle between the benzimidazole ring systems in the ligand is 60.04 (8)°. The configuration of the mol­ecule is supported by intra­molecular C—H⋯O hydrogen bonds.
doi:10.1107/S160053681004568X
PMCID: PMC3011569  PMID: 21589242
18.  Benzene-1,3,5-tricarb­oxy­lic acid–1,10-bis­(1,2,4-triazol-1-yl)deca­ne–water (1/1/2) 
In the title 1:1:2 association, C14H24N6·C9H6O6·2H2O, the alkyl chain in the 1,10-bis­(1,2,4-triazol-1-yl)decane mol­ecule adopts an extended conformation and the dihedral angle between the aromatic rings is 10.28 (13)°. The benzene-1,3,5-tricarb­oxy­lic acid mol­ecule is close to being planar (r.m.s. deviation = 0.052 Å). In the crystal, the components are linked by O—H⋯O and O—H⋯N hydrogen bonds, generating a layered network.
doi:10.1107/S1600536810044673
PMCID: PMC3011396  PMID: 21589390
19.  Retinal Vascular Repair and Neovascularization are not dependent on CX3CR1 Signaling in a Model of Ischemic Retinopathy 
Experimental eye research  2009;88(6):1004-1013.
Proliferative retinal neovascularization occurring in response to ischemia is a common mechanism underlying many retinal diseases. In recent studies, retinal microglia have been shown to influence pathological neovascularization, likely through an exchange of cellular signals with associated vascular elements. CX3CR1 is a chemokine receptor located specifically on microglia; its ligand, CX3CL1 (also known as fractalkine or neurotactin) displays pro-angiogenic activity both in in vivo and in vitro. Discovering the regulatory role, if any, that CX3CR1 signaling may have in ischemic retinopathy will shed light on the molecular nature of microglial-vascular interactions and clarify potential targets for future therapy. In this study, we examined this question by inducing and comparing ischemic vascular changes in transgenic mice in which CX3CR1 signaling is either preserved or ablated. Using a well-known oxygen-induced retinopathy (OIR) model, we induced ischemic retinopathy in transgenic mice in which the gene for CX3CR1 has been replaced by green fluorescent protein (GFP) and their wild type controls. CX3CR1+/+, CX3CR1+/GFP, and CX3CR1GFP/GFP transgenic mice were exposed to 75% oxygen for 5 days starting from postnatal day (P) 7, and then transferred back to room air. At P12 and P17, the extents of vascular repair and neovascularization, and associated changes in retinal microglia distribution, were quantified and compared between mice of different genotypes. Neuronal loss in the retina following ischemia was also evaluated in paraffin sections. Our results show that: (1) CX3CR1 signaling is not required for normal vascular, microglial, and neuronal development in the retina in the first postnatal week, (2) the processes of retinal vascular repair and neovascularization following ischemia occur similarly with and without CX3CR1 signaling, (3) microglia redistribution in the retina and their association with vascular elements occurring concurrently is independent of CX3CR1, and (4) CX3CR1 does not influence the extent of neuronal cell loss in the retina following ischemia. Taken together, our findings indicate that the regulatory signals exchanged between microglia and vascular elements in the ischemic retinopathy animal model are unlikely to involve CX3CR1. These results have implications on therapeutic approaches to, pathological neovascularization involving the modulation of chemokine signaling in general, and the regulation of CX3CR1 signaling specifically.
doi:10.1016/j.exer.2008.12.013
PMCID: PMC2683176  PMID: 19176215
retina; microglia; ischemia; neovascularization; CX3CR1; vascular repair
20.  Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI 
Nature protocols  2008;3(11):1703-1708.
We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.
doi:10.1038/nprot.2008.172
PMCID: PMC2811090  PMID: 18846097
21.  Inhibition of mouse alkali burn induced-corneal neovascularization by recombinant adenovirus encoding human vasohibin-1 
Molecular Vision  2010;16:1389-1398.
Purpose
To evaluate the activity of recombinant adenovirus encoding human vasohibin-1 (Ad-Vasohibin-1) on mouse corneal neovasularization induced by alkali burn.
Methods
For the treatment group, 50 mice each received subconjunctival injection (5 μl) of 109 plaque forming units of replication-defective Ad-Vasohibin-1. Control group mice received the same dosage of blank adenoviral vector (AdNull). Five days after injection, corneal neovascularization (CNV) was induced by placing 2.5 μl of 0.1 M NaOH on the right cornea for 30 s. Subsequently, CNV was observed and photographed every 3 days for a total duration of 9 days after the alkali burn. The percentage of neovascularized area was measured and compared with the AdNull control. The expression of human vasohibin-1 protein was detected by immunohistochemistry and western blotting at 5, 8, and 14 days after injection. The mRNA expression levels of murine vascular endothelial growth factor (Vegf), VEGF receptor 1 and 2 (Vegfr1, Vegfr2), and vasohibin-1 (Vash1) were analyzed and compared by real time quantitative reverse-transcription polymerase chain reaction.
Results
The percentage of neovascularized area within the cornea was significantly reduced in mice treated with Ad-Vasohibin-1 compared to mice treated with AdNull at every time point after alkali-induced injury (7.11%±3.91% and 15.48%±1.79% of corneal area in the treatment and control groups, respectively, on day 3; 31.64%±4.71% and 43.93%±6.15% on day 6, and 45.02%±9.98% and 66.24%±7.17% on day 9, all p<0.001). Human vasohibin-1 protein was detected at the injection sites on day 3 after corneal burn and was highly expressed in the central subepithelial stroma and co-localized with neovascularized vessels within the alkali-treated cornea on day 6. On day 9, the peripheral cornea exhibited a similar staining pattern as the central cornea, but a more intense vasohibin-1 immunostaining signal was detected in the deep stroma. Some of the vasohibin-1 stain signal diffused into the frontal and deep stroma of the central cornea and was not co-localized with new vessels. By contrast, in mice injected with AdNull or normal corneas, no vasohibin-1 stain signal was detected within the corneas. Vasohibin-1 protein expression within treated corneas was also further confirmed by western blotting on day 5. Expression appeared to peak by day 8 and was maintained at high levels until day 14. However, Vasohibin-1 protein was not detected in the corneas of normal mice or mice treated with AdNull. Real-time quantitative reverse-transcription polymerase chain reaction analysis showed that expression of Vegfr2 and endogenous Vash1 mRNA were significantly decreased in the treatment versus control group (t1=–2.161, p1=0.047; t2=–2.236, p2=0.041). In contrast, there were no significant differences in Vegf and Vegfr1 mRNA expression levels between the treatment and control groups (p>0.05 for both).
Conclusions
Subconjunctival injection of Ad-Vasohibin-1 significantly reduces corneal neovascularization in alkali-treated mouse corneas. This effect of anti-neovascularization may be related to the downregulation of Vegfr2 expression.
PMCID: PMC2913137  PMID: 20680097
22.  Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD 
PLoS ONE  2009;4(11):e7945.
Background
Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.
Methodology/Principal Findings
In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.
Conclusions/Significance
These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.
doi:10.1371/journal.pone.0007945
PMCID: PMC2775955  PMID: 19936204
23.  Clinical significance of 4 patients with chronic hepatitis B achieving HBsAg clearance by treated with pegylated interferon alpha-2a for less than 1 year: a short report 
Virology Journal  2009;6:97.
We report 4 chinese patients with hepatitis B e antigen-positive chronic hepatitis B achieving clearance of HBsAg by using pegylated interferon alpha-2a for less than 1 year, to provide one clinical clue for the treatment of chronic hepatitis B.
doi:10.1186/1743-422X-6-97
PMCID: PMC2714489  PMID: 19583877
24.  Triaqua­chlorido[3-dimethyl­amino-1-(2-pyrid­yl)prop-2-en-1-one-κN 1]manganese(II) chloride 
In the title compound, [MnCl(C10H12N2O)(H2O)3]Cl, the MnII ion has a distorted octa­hedral coordination environment formed by one N and one O atom from the chelating 3-dimethyl­amino-1-(2-pyrid­yl)prop-2-en-1-one ligand, one chloride anion and three coordinated water mol­ecules. Inter­molecular O—H⋯O and O—H⋯Cl hydrogen bonds link the cations and anions into layers parallel to the ac plane.
doi:10.1107/S1600536809024192
PMCID: PMC2977233  PMID: 21583324
25.  Diaqua­dibromidobis[3-dimethyl­amino-1-(4-pyridyl-κN)prop-2-en-1-one]cadmium(II) 
In the title compound, [CdBr2(C10H12N2O)2(H2O)2], the CdII ion is located on an inversion center and is six-coordinated by two N atoms [Cd—N = 2.377 (3) Å] from two different 3-dimethyl­amino-1-(4-pyrid­yl)prop-2-en-1-one ligands, two O atoms [Cd—O = 2.355 (2) Å] from two coordinated water mol­ecules and two bromide anions [Cd—Br = 2.6855 (5) Å]. Inter­molecular O—H⋯O hydrogen bonds link the mol­ecules into layers parallel to the bc plane.
doi:10.1107/S1600536809007028
PMCID: PMC2968920  PMID: 21582316

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