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1.  Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration 
Cell metabolism  2013;17(4):549-561.
Pathologic angiogenesis mediated by abnormally polarized macrophages plays a central role in common age-associated diseases such as atherosclerosis, cancer and macular degeneration. Here we demonstrate that abnormal polarization in older macrophages is caused by programmatic changes that lead to reduced expression of ATP binding cassette transporter ABCA1. Downregulation of ABCA1 by microRNA-33 impairs the ability of macrophages to effectively efflux intracellular cholesterol, which in turn leads to higher levels of free cholesterol within senescent macrophages. Elevated intracellular lipid polarizes older macrophages to an abnormal, alternatively activated phenotype that promotes pathologic vascular proliferation. Mice deficient for Abca1, but not Abcg1, demonstrate an accelerated aging phenotype, whereas restoration of cholesterol efflux using LXR agonists or miR-33 inhibitors reverses it. Monocytes from older humans with age-related macular degeneration showed similar changes. These findings provide an avenue for therapeutic modulation of macrophage function in common age-related diseases.
PMCID: PMC3640261  PMID: 23562078
2.  Age-Dependent Changes in FasL (CD95L) Modulate Macrophage Function in a Model of Age-Related Macular Degeneration 
We examined the effect of aging on Fas ligand (FasL) function in a mouse model of choroidal neovascularization (CNV).
Young and aged mice were laser treated to induce CNV. Bone marrow chimeras were performed between young and aged mice. FasL protein expression was examined in the eye and soluble FasL (sFasL) was measured in the blood. Young and aged mice were treated with a matrix metalloprotease (MMP) inhibitor and systemic sFasL was neutralized by antibody treatment. Macrophages from young and aged mice were tested for sFasL-mediated cytokine production and migration.
The elevated CNV response observed with aging was dependent on bone marrow–derived cells. FasL expression in the eye was increased with age, but decreased following laser treatment. Aged mice had higher levels of sFasL in the blood compared to young mice. Systemic treatment with an MMP inhibitor decreased bloodborne sFasL, and reduced CNV in young and aged mice. Systemic neutralization of sFasL reduced CNV only in aged mice. sFasL increased cytokine production in aged macrophages and proangiogenic M2 macrophages. Aged M2 macrophages had elevated Fas (CD95) expression and displayed increased migration in response to sFasL compared to M1 macrophages derived from young animals.
Age modulates FasL function where increased MMP cleavage leads to a loss of function in the eye. The released form of FasL (sFasL) preferentially induces the migration of proangiogenic M2 macrophages into the laser lesions and increases proangiogenic cytokines promoting CNV. FasL may be a viable target for therapeutic intervention in aged-related neovascular disease.
FasL function in the eye decreases with age due to MMP-mediated cleavage, which generates bloodborne sFasL. sFasL preferentially activates proangiogenic M2 macrophages.
PMCID: PMC3738220  PMID: 23821188
macrophages; neovascularization; immune privilege; cytokine; cell migration; age-related macular degeneration
3.  Cost-Effectiveness Analysis of Ranibizumab Plus Prompt or Deferred Laser or Triamcinolone Plus Prompt Laser for Diabetic Macular Edema 
Ophthalmology  2012;119(8):1679-1684.
Perform a cost-effectiveness analysis of the treatment of Diabetic macular edema (DME) with ranibizumab plus prompt or deferred laser versus triamcinolone plus prompt laser. Data for the analysis was drawn from reports of the Diabetic Retinopathy Clinical Research Network (DRCRnet) Protocol I.
Computer simulation based on Protocol I data. Analyses were conducted from the payor perspective.
Simulated participants assigned characteristics reflecting those seen in Protocol I.
Markov models were constructed to replicate Protocol I’s 104 week outcomes using a microsimulation approach to estimation. Baseline characteristics, visual acuity (VA), treatments, and complications were based on Protocol I data. Costs were identified by literature search. One-way sensitivity analysis was performed and the results were validated against Protocol I data.
Main Outcome Measures
Direct cost of care for two years, change in VA from baseline, and incremental cost-effectiveness ratio (ICER) measured as cost per additional letter gained from baseline (ETDRS).
For sham plus laser (S+L), ranibizumab plus prompt laser (R+pL), ranibizumab plus deferred laser (R+dL), and triamcinolone plus laser (T+L), effectiveness through 104 weeks was predicted to be 3.46, 7.07, 8.63, and 2.40 letters correct, respectively. ICER values in terms of dollars per VA letter were $393 (S+L vs. T+L), $5,943 (R+pL vs. S+L), and $20 (R+dL vs. R+pL). For pseudophakics, the ICER value for comparison triamcinolone with laser versus ranibizumab with deferred laser was $14,690 per letter gained. No clinically relevant changes in model variables altered outcomes. Internal validation demonstrated good similarity to Protocol I treatment patterns.
In treatment of phakic patients with DME, ranibizumab with deferred laser provided an additional 6 letters correct compared to triamcinolone with laser at an additional cost of $19,216 over two years. That would indicate that if the gain in visual acuity seen at two years is maintained in subsequent years, then the treatment of phakic patients with DME using ranibizumab may meet accepted standards of cost-effectiveness. For pseudophakic patients, first line treatment with triamcinolone appears to be the most cost-effective option.
PMCID: PMC3612959  PMID: 22503301
4.  Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration 
Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.
PMCID: PMC3606733  PMID: 23533946
5.  7-Ketocholesterol Induces Inflammation and Angiogenesis In Vivo: A Novel Rat Model 
PLoS ONE  2013;8(2):e56099.
Accumulation of 7-Ketocholesterol (7KCh) in lipid deposits has been implicated in a variety of chronic diseases including atherosclerosis, Alzheimer's disease and age-related macular degeneration. 7KCh is known to be pro-inflammatory and cytotoxic to various types of cultured cells but little is known about its effects in vivo. In this study we have investigated the effects of 7KCh in vivo by implanting biodegradable wafers into the anterior chamber of the rat eye. The wafers were prepared using a mixture of two biodegradable polymers with different amounts of 7KCh. The 7KCh-containing implants induced massive angiogenesis and inflammation. By contrast, no angiogenesis and very little inflammation were observed with cholesterol-containing implants. The neovessel growth was monitored by fluorescein angiography. Neovessels were observed 4 days post implantation and peaked between 7 to 10 days. The angiography and isolectin IB4 labeling demonstrated that the neovessels originated from the limbus and grew through the cornea. Immunolabeling with anti-CD68 suggested that the 7KCh-containing implants had extensive macrophage infiltration as well as other cell types. A significant increase in VEGF was also observed in 7KCh-containing implants by fluorescent immunolabeling and by immunoblot of the aqueous humor (AH). Direct measurement of VEGF, IL-1β and GRO/KC demonstrated a marked elevation of these factors in the AH of the 7KCh-implants. In summary this study demonstrates two important things: 1) 7KCh is pro-angiogenic and pro–inflammatory in vivo and 2) implants containing 7KCh may be used to create a novel angiogenesis model in rats.
PMCID: PMC3568027  PMID: 23409131
6.  Overexpression of TGF-ß1 in Macrophages Reduces and Stabilizes Atherosclerotic Plaques in ApoE-Deficient Mice 
PLoS ONE  2012;7(7):e40990.
Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.
PMCID: PMC3400574  PMID: 22829904
7.  Hospitalized cardiovascular events in patients with diabetic macular edema 
BMC Ophthalmology  2012;12:11.
Microvascular and macrovascular complications in diabetes stem from chronic hyperglycemia and are thought to have overlapping pathophysiology. The aim of this study was to investigate the incidence rate of hospitalized myocardial infarctions (MI) and cerebrovascular accidents (CVA) in patients with diabetic macular edema (DME) compared with diabetic patients without retinal diseases.
This was a retrospective cohort study of a commercially insured population in an administrative claims database. DME subjects (n = 3519) and diabetes controls without retinal disease (n = 10557) were matched by age and gender. Healthcare claims were analyzed for the study period from 1 January 2002 to 31 December 2005. Incidence and adjusted rate ratios of hospitalized MI and CVA events were then calculated.
The adjusted rate ratio for MI was 2.50 (95% CI: 1.83-3.41, p < 0.001) for DME versus diabetes controls. Predictors of MI events were heart disease, history of acute MI, and prior use of antiplatelet or anticoagulant drugs. The adjusted rate ratio for CVA was 1.98 (95% CI: 1.39-2.83, p < 0.001) for DME versus diabetes controls. Predictors of CVA events were cardiac arrhythmia, Charlson comorbidity scores, history of CVA, hyperlipidemia, and other cerebrovascular diseases.
Event rates of MI or CVA were higher in patients with DME than in diabetes controls. This study is one of few with sufficient sample size to accurately estimate the relationship between DME and cardiovascular outcomes.
PMCID: PMC3395554  PMID: 22646811
8.  Structural and Functional Changes of the Human Macula during Acute Exposure to High Altitude 
PLoS ONE  2012;7(4):e36155.
This study aimed to quantify structural and functional changes at the macula during acute exposure to high altitude and to assess their structure/function relationship. This work is related to the Tuebingen High Altitude Ophthalmology (THAO) study.
Methodology/Principal Findings
Spectral domain optical coherence tomography and microperimetry were used to quantify changes of central retinal structure and function in 14 healthy subjects during acute exposure to high altitude (4559 m). High-resolution volume scans and fundus-controlled microperimetry of the posterior pole were performed in addition to best-corrected visual acuity (BCVA) measurements and assessment of acute mountain sickness. Analysis of measurements at altitude vs. baseline revealed increased total retinal thickness (TRT) in all four outer ETDRS grid subfields during acute altitude exposure (TRTouter = 2.80±1.00 μm; mean change±95%CI). This change was inverted towards the inner four subfields (TRTinner = −1.89±0.97 μm) with significant reduction of TRT in the fovea (TRTfoveal = −6.62±0.90 μm) at altitude. BCVA revealed no significant difference compared to baseline (0.06±0.08 logMAR). Microperimetry showed stable mean sensitivity in all but the foveal subfield (MSfoveal = −1.12±0.68 dB). At baseline recordings before and >2 weeks after high altitude exposure, all subjects showed equal levels with no sign of persisting structural or functional sequels.
During acute exposure to high altitude central retinal thickness is subject to minor, yet statistically significant changes. These alterations describe a function of eccentricity with an increase in regions with relatively higher retinal nerve fiber content and vascular arcades. However, these changes did not correlate with measures of central retinal function or acute mountain sickness. For the first time a quantitative approach has been used to assess these changes during acute, non-acclimatized high altitude exposure.
PMCID: PMC3340355  PMID: 22558365
9.  Dietary Lactoferrin Alleviates Age-Related Lacrimal Gland Dysfunction in Mice 
PLoS ONE  2012;7(3):e33148.
Decrease in lacrimal gland secretory function is related to age-induced dry eye disease. Lactoferrin, the main glycoprotein component of tears, has multiple functions, including anti-inflammatory effects and the promotion of cell growth. We investigated how oral administration of lactoferrin affects age-related lacrimal dysfunction.
Methods and Findings
Twelve-month-old male C57BL/6Cr Slc mice were randomly divided into a control fed group and an oral lactoferrin treatment group. Tear function was measured at a 6-month time-point. After euthanasia, the lacrimal glands were subjected to histological examination with 8-hydroxy-2′-deoxyguanosine (8-OHdG) antibodies, and serum concentrations of 8-OHdG and hexanoyl-lysine adduct (HEL) were evaluated. Additionally, monocyte chemotactic protein-1(MCP-1) and tumor necrosis factor-α (TNF-α) gene expression levels were determined by real-time PCR. The volume of tear secretion was significantly larger in the treated group than in the control. Lactoferrin administration reduced inflammatory cell infiltration and the MCP-1 and TNF-α expression levels. Serum concentrations of 8-OHdG and HEL in the lactoferrin group were lower than those in the control group and were associated with attenuated 8-OHdG immunostaining of the lacrimal glands.
Oral lactoferrin administration preserves lacrimal gland function in aged mice by attenuating oxidative damage and suppressing subsequent gland inflammation.
PMCID: PMC3314001  PMID: 22479365
10.  Mitochondrial Haplogroups and Control Region Polymorphisms in Age-Related Macular Degeneration: A Case-Control Study 
PLoS ONE  2012;7(2):e30874.
Onset and development of the multifactorial disease age-related macular degeneration (AMD) are highly interrelated with mitochondrial functions such as energy production and free radical turnover. Mitochondrial dysfunction and overproduction of reactive oxygen species may contribute to destruction of the retinal pigment epithelium, retinal atrophy and choroidal neovascularization, leading to AMD. Consequently, polymorphisms of the mitochondrial genome (mtDNA) are postulated to be susceptibility factors for this disease. Previous studies from Australia and the United States detected associations of mitochondrial haplogroups with AMD. The aim of the present study was to test these associations in Middle European Caucasians.
Methodology/Principal Findings
Mitochondrial haplogroups (combinations of mtDNA polymorphisms) and mitochondrial CR polymorphisms were analyzed in 200 patients with wet AMD (choroidal neovascularization, CNV), in 66 patients with dry AMD, and in 385 controls from Austria by means of multiplex primer extension analysis and sequencing, respectively. In patients with CNV, haplogroup H was found to be significantly less frequent compared to controls, and haplogroup J showed a trend toward a higher frequency compared to controls. Five CR polymorphisms were found to differ significantly in the two study populations compared to controls, and all, except one (T152C), are linked to those haplogroups.
It can be concluded that haplogroup J is a risk factor for AMD, whereas haplogroup H seems to be protective for AMD.
PMCID: PMC3278404  PMID: 22348027
11.  Optical Scattering Measurements of Laser Induced Damage in the Intraocular Lens 
PLoS ONE  2012;7(2):e31764.
This study optically determines whether the amount of light scatter due to laser-induced damage to the intraocular lens (IOL) is significant in relation to normal straylight values in the human eye. Two IOLs with laser-induced damage were extracted from two donor eyes. Each IOL had 15 pits and/or cracks. The surface area of each pit was measured using a microscope. For 6 pits per intraocular lens the point spread function (PSF) in terms of straylight was measured and the total straylight for all 15 pits was estimated. The damage in the IOLs was scored as mild/moderate. The total damaged surface areas, for a 3.5 mm pupil, in the two IOLs were 0.13% (0.0127 mm2) and 0.66% (0.064 mm2), respectively. The angular dependence of the straylight caused by the damage was similar to that of the normal PSF. The total average contribution to straylight was log(s) = −0.82 and −0.42, much less than the straylight value of the normal eye.
The straylight due to normal levels of laser induced damage of the IOL is much lower than normal straylight values found clinically for the normal eye and may therefore be considered not significant.
PMCID: PMC3277504  PMID: 22348128
12.  Vision-Related Quality of Life and Self-Rated Satisfaction Outcomes of Rhegmatogenous Retinal Detachment Surgery: Three-Year Prospective Study 
PLoS ONE  2011;6(12):e28597.
Subjective functional outcomes measurements, such as vision health-related quality of life (VRQoL) and self-rated satisfaction measures can provide helpful multidimensional vision health information that is more comprehensive than traditional objective measures, such as best corrected visual acuity (BCVA). The purpose of this study is to demonstrate 3-year longitudinal postoperative VRQoL and self-rated satisfaction changes after rhegmatogenous retinal detachment (RRD) surgery.
Methodology/Principal Findings
A prospective case series report was conducted in 92 RRD patients who underwent surgery during January 2004 through December 2006. Preoperative, 3-month, 1-year and 3-year postoperative patient VRQoL and self-rated satisfaction were assessed by face-to-face interviews. The importance of objective variables for predicting three dependent variables: CLVQOL composite scores change, 3-year postoperative CVLQOL composite score and self-rated satisfaction degree scores were calculated by stepwise multivariate linear or logistic regression analysis methods.
The total CLVQOL composite scores change ranged between -48 and 90 (mean±standard deviation: 19.48±31.34), including positive changes in 62 patients. The self-rated satisfaction degree scores ultimately improved in 86 patients as compared with preoperative degrees. Statistically significant increases occurred only in the composite scores of subscale mobility and self-rated satisfaction degrees in the first 3 months, while the composite scores of the remaining subscales, and the total CLVQOL, BCVA in the RRD eye and weighted average BCVA, increased steadily throughout the first postoperative year. A better 3-year postoperative weighted average BCVA was associated with all of the 3 dependent outcome variables.
VRQoL of RRD patients improved substantially after surgery and they were satisfied with their postoperative vision. The BCVA, VRQoL and self-rated satisfactory degree scores recovered in different patterns, and supplemented each other in the RRD surgery outcomes evaluated. Surgeons are advised to pay closer attention to binocular vision in RRD patients, and make efforts to explain the results of surgery.
PMCID: PMC3230609  PMID: 22162779
13.  An ERP Assessment of Hemispheric Projections in Foveal and Extrafoveal Word Recognition 
PLoS ONE  2011;6(9):e23957.
The existence and function of unilateral hemispheric projections within foveal vision may substantially affect foveal word recognition. The purpose of this research was to reveal these projections and determine their functionality.
Single words (and pseudowords) were presented to the left or right of fixation, entirely within either foveal or extrafoveal vision. To maximize the likelihood of unilateral projections for foveal displays, stimuli in foveal vision were presented away from the midline. The processing of stimuli in each location was assessed by combining behavioural measures (reaction times, accuracy) with on-line monitoring of hemispheric activity using event-related potentials recorded over each hemisphere, and carefully-controlled presentation procedures using an eye-tracker linked to a fixation-contingent display.
Principal Findings
Event-related potentials 100–150 ms and 150–200 ms after stimulus onset indicated that stimuli in extrafoveal and foveal locations were projected unilaterally to the hemisphere contralateral to the presentation hemifield with no concurrent projection to the ipsilateral hemisphere. These effects were similar for words and pseudowords, suggesting this early division occurred before word recognition. Indeed, event-related potentials revealed differences between words and pseudowords 300–350 ms after stimulus onset, for foveal and extrafoveal locations, indicating that word recognition had now occurred. However, these later event-related potentials also revealed that the hemispheric division observed previously was no longer present for foveal locations but remained for extrafoveal locations. These findings closely matched the behavioural finding that foveal locations produced similar performance each side of fixation but extrafoveal locations produced left-right asymmetries.
These findings indicate that an initial division in unilateral hemispheric projections occurs in foveal vision away from the midline but is not apparent, or functional, when foveal word recognition actually occurs. In contrast, the division in unilateral hemispheric projections that occurs in extrafoveal locations is still apparent, and is functional, when extrafoveal word recognition takes place.
PMCID: PMC3174137  PMID: 21935368
14.  Visual Acuity and Associated Factors. The Central India Eye and Medical Study 
PLoS ONE  2011;6(7):e22756.
Visual acuity is a major parameter for quality of vision and quality of life. Information on visual acuity and its associated factors in rural societies almost untouched by any industrialization is mostly non-available. It was, therefore, the purpose of our study to determine the distribution of visual acuity and its associated factors in a rural population not marked influenced by modern lifestyle. The population-based Central India Eye and Medical Study included 4711 subjects (aged 30+ years), who underwent a detailed ophthalmologic examination including visual acuity measurement. Visual acuity measurements were available for 4706 subjects with a mean age of 49.5±13.4 years (range: 30–100 years). BCVA decreased significantly (P<0.001) from the moderately hyperopic group (0.08±0.15 logMAR) to the emmetropic group (0.16±0.52 logMAR), the moderately myopic group (0.28±0.33 logMAR), the highly hyperopic group (0.66±0.62 logMAR) and finally the highly myopic group (1.32±0.92 logMAR). In multivariate analysis, BCVA was significantly associated with the systemic parameters of lower age (P<0.001), higher level of education (P<0.001), higher body stature (P<0.001) and higher body mass index (P<0.001), and with the ophthalmic parameters of more hyperopic refractive error (spherical equivalent) (P<0.001), shorter axial length (P<0.001), lower degree of nuclear cataract (P<0.001), and lower intraocular pressure (P = 0.006). The results suggest that in the rural population of Central India, major determinants of visual acuity were socioeconomic background, body stature and body mass index, age, refractive error, cataract and intraocular pressure.
PMCID: PMC3150376  PMID: 21829503
15.  Blockade of VEGFR1 and 2 Suppresses Pathological Angiogenesis and Vascular Leakage in the Eye 
PLoS ONE  2011;6(6):e21411.
VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.
Materials and Methods
Neutralizing antibodies specific for mouse VEGFR1 (MF1) and VEGFR2 (DC101) were administrated systemically. CNV was induced by laser photocoagulation and assessed 14d after laser treatment. Retinal NV was generated in oxygen-induced ischemic retinopathy (OIR) and assessed at p17. NV quantification was determined by measuring NV tufts and vascular leakage was quantified by measuring [3H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.
VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.
Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents.
PMCID: PMC3120882  PMID: 21731737
16.  Visual Advantage in Deaf Adults Linked to Retinal Changes 
PLoS ONE  2011;6(6):e20417.
The altered sensory experience of profound early onset deafness provokes sometimes large scale neural reorganisations. In particular, auditory-visual cross-modal plasticity occurs, wherein redundant auditory cortex becomes recruited to vision. However, the effect of human deafness on neural structures involved in visual processing prior to the visual cortex has never been investigated, either in humans or animals. We investigated neural changes at the retina and optic nerve head in profoundly deaf (N = 14) and hearing (N = 15) adults using Optical Coherence Tomography (OCT), an in-vivo light interference method of quantifying retinal micro-structure. We compared retinal changes with behavioural results from the same deaf and hearing adults, measuring sensitivity in the peripheral visual field using Goldmann perimetry. Deaf adults had significantly larger neural rim areas, within the optic nerve head in comparison to hearing controls suggesting greater retinal ganglion cell number. Deaf adults also demonstrated significantly larger visual field areas (indicating greater peripheral sensitivity) than controls. Furthermore, neural rim area was significantly correlated with visual field area in both deaf and hearing adults. Deaf adults also showed a significantly different pattern of retinal nerve fibre layer (RNFL) distribution compared to controls. Significant correlations between the depth of the RNFL at the inferior-nasal peripapillary retina and the corresponding far temporal and superior temporal visual field areas (sensitivity) were found. Our results show that cross-modal plasticity after early onset deafness may not be limited to the sensory cortices, noting specific retinal adaptations in early onset deaf adults which are significantly correlated with peripheral vision sensitivity.
PMCID: PMC3105994  PMID: 21673805
17.  Targeting Immune Privilege to Prevent Pathogenic Neovascularization 
The authors demonstrate that augmenting the immune privilege of the eye can successfully inhibit pathogenic neovascularization.
Current studies suggest that the immune system plays a critical role in blinding eye disorders. The eye is an immune-privileged site, and FasL expression is a major part of that mechanism because Fas/FasL interactions regulate inflammation and neovascularization, preventing damage to delicate ocular structures. These studies were undertaken to test the idea that modulating immune privilege might be an effective therapeutic approach to pathogenic angiogenesis in the eye.
C57BL/6 mice or FasL-defective B6-gld mice were laser treated to induce choroidal neovascularization (CNV). Mice were injected with cytotoxic FasL in the vitreous cavity or were treated with oral doxycycline in the drinking water. They were evaluated for CNV 7 days later. In some experiments eye tissue was harvested and evaluated for FasL expression, macrophage influx by immunohistochemistry, and release of sFasL.
Injection of cytotoxic FasL successfully prevented neovascularization in a mouse model of CNV. Oral doxycycline increased functional FasL in the eye and substantially inhibited neovascularization. Doxycycline treatment increased FasL expression on the RPE cells and reduced circulating and tissue-associated sFasL. Treatment was ineffective in B6-gld mice, demonstrating that CNV inhibition was mediated by FasL.
Targeting immune privilege using cytotoxic molecules or by increasing expression of the proapoptotic protein FasL may be a viable approach to treating neovascular eye disease.
PMCID: PMC2904009  PMID: 20164456
19.  Interleukin-10 Overexpression Promotes Fas-Ligand-Dependent Chronic Macrophage-Mediated Demyelinating Polyneuropathy 
PLoS ONE  2009;4(9):e7121.
Demyelinating polyneuropathy is a debilitating, poorly understood disease that can exist in acute (Guillain-Barré syndrome) or chronic forms. Interleukin-10 (IL-10), although traditionally considered an anti-inflammatory cytokine, has also been implicated in promoting abnormal angiogenesis in the eye and in the pathobiology of autoimmune diseases such as lupus and encephalomyelitis.
Principal Findings
Overexpression of IL-10 in a transgenic mouse model leads to macrophage-mediated demyelinating polyneuropathy. IL-10 upregulates ICAM-1 within neural tissues, promoting massive macrophage influx, inflammation-induced demyelination, and subsequent loss of neural tissue resulting in muscle weakness and paralysis. The primary insult is to perineural myelin followed by secondary axonal loss. Infiltrating macrophages within the peripheral nerves demonstrate a highly pro-inflammatory signature. Macrophages are central players in the pathophysiology, as in vivo depletion of macrophages using clodronate liposomes reverses the phenotype, including progressive nerve loss and paralysis. Macrophage-mediate demyelination is dependent on Fas-ligand (FasL)-mediated Schwann cell death.
These findings mimic the human disease chronic idiopathic demyelinating polyneuropathy (CIDP) and may also promote further understanding of the pathobiology of related conditions such as acute idiopathic demyelinating polyneuropathy (AIDP) or Guillain-Barré syndrome.
PMCID: PMC2743195  PMID: 19771172
20.  An assay for macrophage mediated regulation of endothelial cell proliferation 
Immunobiology  2008;213(9-10):695-699.
We have developed an assay that quantifies the potential of macrophages to regulate proliferation of endothelial cells. We show that young mice macrophages can be distinguished from old mice macrophages by their ability to inhibit vascular endothelial cell proliferation. While young mice macrophages robustly inhibit proliferation, old mice macrophages fail to do so and actually promote the proliferation of endothelial cells. In this report, we outline a technique that directly assesses the effect of macrophages on modulation of endothelial cell proliferation. This assay will help us in understanding the mechanisms of macrophage function in several disease states characterized by abnormal angiogenesis including cancers, angiogenic eye disease and atherosclerotic heart disease.
PMCID: PMC2572032  PMID: 18926285
angiogenesis; endothelial; immunity; innate; macrophage; vascular
21.  Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development 
PLoS ONE  2008;3(10):e3381.
Aberrant angiogenesis in the eye is the most common cause of blindness. The current study examined the role of interleukin-10 (IL-10) in ischemia-induced pathological angiogenesis called neovascularization during postnatal development. IL-10 deficiency resulted in significantly reduced pathological retinal angiogenesis. In contrast to the choroicapillaris where IL-10 interferes with macrophage influx, IL-10 did not prevent anti-angiogenic macrophages from migrating to the retina in response to hypoxia. Instead, IL-10 promoted retinal angiogenesis by altering macrophage angiogenic function, as macrophages from wild-type mice demonstrated increased vascular endothelial growth factor (VEGF) and nitric oxide (NO) compared to IL-10 deficient macrophages. IL-10 appears to directly affect macrophage responsiveness to hypoxia, as macrophages responded to hypoxia with increased levels of IL-10 and STAT3 phosphorylation as opposed to IL-10 deficient macrophages. Also, IL-10 deficient macrophages inhibited the proliferation of vascular endothelial cells in response to hypoxia while wild-type macrophages failed to do so. These findings suggest that hypoxia guides macrophage behavior to a pro-angiogenic phenotype via IL-10 activated pathways.
PMCID: PMC2557127  PMID: 18852882
22.  Clinical phenotypes associated with the Complement Factor H Y402H variant in age-related macular degeneration 
American journal of ophthalmology  2007;144(3):404-408.
To determine whether the complement factor H (CFH) Y402H variant is associated with specific age-related macular degeneration (AMD) clinical phenotypes.
Retrospective, case-control study.
188 Caucasian subjects with AMD and 189 control subjects were genotyped for the T-to-C polymorphism in exon-9 of the CFH gene by restriction-fragment length analysis and DNA sequencing using genomic DNA from mouthwash samples. AMD phenotypes were characterized by clinical examination, fundus photography, and fluorescein angiography.
Heterozygosity for the at-risk genotype (TC) increased the likelihood for AMD 2.1-fold (95% CI 1.3–3.3) while homozygosity for the genotype (CC) increased the likelihood for AMD 6.5-fold (95% CI 3.4–12.5) in our population. The C allele was significantly associated with predominantly classic choroidal neovascularization (OR 2.01, 95% CI 1.34–3.30). Neovascular lesion size was similar among the three genotypes (p=0.67).
The Y402H CFH variant carried a significantly increased risk for developing AMD in our population. Genotype/phenotype correlations regarding choroidal neovascular lesion type were observed
PMCID: PMC2140051  PMID: 17631852
23.  Senescence regulates macrophage activation and angiogenic fate at sites of tissue injury in mice 
The Journal of Clinical Investigation  2007;117(11):3421-3426.
Abnormal angiogenesis plays a key role in diseases of aging such as heart disease, certain cancers, and eye diseases including age-related macular degeneration. Macrophages have been shown previously to be both anti- and proangiogenic, and their role in regulating angiogenesis at sites of tissue injury is critical and complex. In this study, we analyzed cytokine gene expression patterns of mouse macrophages by real-time quantitative PCR and tested the functional effects of senescence on gene expression and macrophage polarization. Following laser injury to the retina, IL-10 was upregulated and Fas ligand (FasL), IL-12, and TNF-α were downregulated in ocular macrophages of old mice (>18 months of age). Downregulation of FasL on macrophages led to a loss of the antiangiogenic phenotype, as evidenced by the inability of these macrophages to inhibit vascular endothelial cells. Our results demonstrate that senescence, FasL, and IL-10 are key determinants of macrophage function that alter the growth of abnormal postdevelopmental blood vessels in disease processes including blinding eye disease.
PMCID: PMC2045608  PMID: 17975672
24.  Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration 
PLoS Medicine  2006;3(8):e310.
Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 y of age in at least three continents. Choroidal neovascularization (CNV) is the process by which abnormal blood vessels develop underneath the retina. CNV develops in 10% of patients with AMD but accounts for up to 90% of the blindness from AMD.
Although the precise etiology of CNV in AMD remains unknown, the macrophage component of the inflammatory response, which has been shown to promote tumor growth and support atherosclerotic plaque formation, is thought to stimulate aberrant angiogenesis in blinding eye diseases. The current theory is that macrophage infiltration promotes the development of neovascularization in CNV.
Methods and Findings
We examined the role of macrophages in a mouse model of CNV. IL-10−/− mice, which have increased inflammation in response to diverse stimuli, have significantly reduced CNV with increased macrophage infiltrates compared to wild type. Prevention of macrophage entry into the eye promoted neovascularization while direct injection of macrophages significantly inhibited CNV. Inhibition by macrophages was mediated by the TNF family death molecule Fas ligand (CD95-ligand).
Immune vascular interactions can be highly complex. Normal macrophage function is critical in controlling pathologic neovascularization in the eye. IL-10 regulates macrophage activity in the eye and is an attractive therapeutic target in order to suppress or inhibit CNV in AMD that can otherwise lead to blindness.
Apte and colleagues examined the role of macrophages in a mouse model of choroidal neovascularization, and showed that normal macrophage function is critical in controlling pathologic neovascularization in the eye.
Editors' Summary
The most common cause of poor eyesight in later life in the developed world is known as age-related macular degeneration (AMD). The macula is the central part of the retina (the film-like membrane at the back of the eye), which is the most sensitive and important for sharp central vision. There are two types of advanced AMD: so-called wet, or neovascular, AMD (neovascular means “new vessel”) and dry, or geographic atrophy, AMD (atrophy means “to waste away”). Wet AMD occurs when abnormal, fragile new blood vessels grow under the macula behind the retina. These blood vessels often leak blood and fluid, which lift the macula. Dry AMD occurs as the light-sensitive cells in the macula (the rods and cones) break down. To study this disease further, researchers use animal models. One such animal model is made by using a laser to damage the back of the eye in a mouse, which causes the formation of new vessels. Various treatments can then be tested to see if they have any effect on the damage.
Why Was This Study Done?
One theory about AMD is that the immune system may be involved in determining how severe the damage at the back of the eye is, and how much new vessel formation occurs. The researchers wanted to look at the effect of the immune system on AMD, in particular, the effect of one type of cell called a macrophage, and a substance, IL-10, that is secreted from bone marrow cells and that affects how these macrophages work.
What Did the Researchers Do and Find?
The researchers used a mouse strain in which IL-10 was absent, induced damage in the eyes that mimicked AMD, and then looked at what role macrophages had in the eye abnormalities. They found that in the eyes of mice that lacked IL-10, there was reduced new vessel formation and increased numbers of macrophages compared to mice that had normal amounts of IL-10. Also, preventing macrophages from getting into the eyes of such mice by injecting IL-10 into the eyes made the new vessel formation worse, while direct injection of macrophages made it better.
What Do These Findings Mean?
Although animal models cannot completely replicate disease in humans, they can give us an idea of how diseases might come about and suggest possible treatment strategies. It is possible that inhibiting the effect of IL-10, or other strategies that make macrophages more efficient in the eye, may be a useful treatment for AMD. In a related Perspective (DOI: 10.1371/journal.pmed.0030364), Susan Lightman and Virginia Calder discuss the findings further, including suggesting new experiments that will need to be done as a next step.
Additional Information.
Please access these Web sites via the online version of this summary at
MedlinePlus encyclopedia entry on macular degeneration
National Institutes of Health Senior Health page of information on AMD
National Eye Institute AMD fact page
PMCID: PMC1539093  PMID: 16903779
To determine penetration of moxifloxacin 0.5% into human aqueous and vitreous via topical and collagen shield routes of administration.
Moxifloxacin 0.5% was administered prior to vitrectomy surgery through one of three routes: topical drops every 2 hours for 3 days, versus topical drops every 6 hours for 3 days, versus delivery using a 24-hour dissolvable cross-linked corneal collagen shield. Aqueous and vitreous moxifloxacin concentrations were assayed using high-performance liquid chromatography
Mean moxifloxacin concentrations in the every-2-hour group for aqueous (n = 9) and vitreous (n = 10) were 2.28 ± 1.23 μg/mL and 0.11 ± 0.05 μg/mL, respectively. Mean moxifloxacin concentrations in the every-6-hour group for aqueous (n = 10) and vitreous (n = 9) were 0.88 ± 0.88 μg/mL and 0.06 ± 0.06 μg/mL, respectively. Levels of minimum inhibitory concentration at which 90% of isolates are inhibited (MIC90) were far exceeded in the aqueous for a wide spectrum of pathogens that most commonly cause postoperative endophthalmitis. Moxifloxacin concentration in the vitreous did not exceed the MIC90 for several key organisms. Delivery of moxifloxacin via a collagen shield revealed a mean aqueous concentration of 0.30 ± 0.17 μg/mL 4 hours after placement (n = 5). Vitreous levels at 4 hours, as well as aqueous and vitreous levels at 24 hours, were negligible using this route of administration.
The findings of this investigation reveal that topically administered moxifloxacin 0.5% can achieve relatively high aqueous concentrations. Although aqueous moxifloxacin levels achieved through the use of a collagen shield delivery device are lower, there are several advantages to this route of delivery that make it appealing in the immediate postoperative period. Future studies will be needed to precisely define the role of fourth-generation fluoroquinolones and presoaked collagen shields in the prophylaxis or management of intraocular infections.
PMCID: PMC1280095  PMID: 15747753

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