Gene loss, adaptive evolution and the co-evolution of plumage coloration genes with opsins in birds.

BACKGROUND: The wide range of complex photic systems observed in birds exemplifies one of their key evolutionary adaptions, a well-developed visual system. However, genomic approaches have yet to be used to disentangle the evolutionary mechanisms that govern evolution of avian visual systems. RESULTS: We performed comparative genomic analyses across 48 avian genomes that span extant bird phylogenetic diversity to assess evolutionary changes in the 17 representatives of the opsin gene family and five plumage coloration genes. Our analyses suggest modern birds have maintained a repertoire of up to 15 opsins. Synteny analyses indicate that PARA and PARIE pineal opsins were lost, probably in conjunction with the degeneration of the parietal organ. Eleven of the 15 avian opsins evolved in a non-neutral pattern, confirming the adaptive importance of vision in birds. Visual conopsins sw1, sw2 and lw evolved under negative selection, while the dim-light RH1 photopigment diversified. The evolutionary patterns of sw1 and of violet/ultraviolet sensitivity in birds suggest that avian ancestors had violet-sensitive vision. Additionally, we demonstrate an adaptive association between the RH2 opsin and the MC1R plumage color gene, suggesting that plumage coloration has been photic mediated. At the intra-avian level we observed some unique adaptive patterns. For example, barn owl showed early signs of pseudogenization in RH2, perhaps in response to nocturnal behavior, and penguins had amino acid deletions in RH2 sites responsible for the red shift and retinal binding. These patterns in the barn owl and penguins were convergent with adaptive strategies in nocturnal and aquatic mammals, respectively. CONCLUSIONS: We conclude that birds have evolved diverse opsin adaptations through gene loss, adaptive selection and coevolution with plumage coloration, and that differentiated selective patterns at the species level suggest novel photic pressures to influence evolutionary patterns of more-recent lineages.

Role of Stromal Cell-Derived Factor-1 in Neoangiogenesis in Endometriosis Lesions

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.

Altered pericyte-endothelial relations in the rat retina during aging: Implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of a-smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte-endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of

Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms.

The murine VEGF gene is alternatively transcribed to yield the VEGF120, VEGF164, and VEGF188 isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF164/164 mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF120/120 mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF188/188 mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF164, was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.

Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis.

Raman microscopy, based upon the inelastic scattering (Raman) of light by molecular species, has been applied as a specific structural probe in a wide range of biomedical samples. The purpose of the present investigation was to assess the potential of the technique for spectral characterization of the porcine outer retina derived from the area centralis, which contains the highest proportion of cone:rod cell ratio in the pig retina. METHODS: Retinal cross-sections, immersion-fixed in 4% (w/v) PFA and cryoprotected, were placed on salinized slides and air-dried prior to direct Raman microscopic analysis at three excitation wavelengths, 785 nm, 633 nm, and 514 nm. RESULTS: Raman spectra of each of the photoreceptor inner and outer segments (PIS, POS) and of the outer nuclear layer (ONL) of the retina acquired at 785 nm were dominated by vibrational features characteristic of proteins and lipids. There was a clear difference between the inner and outer domains in the spectroscopic regions, amide I and III, known to be sensitive to protein conformation. The spectra recorded with 633 nm excitation mirrored those observed at 785 nm excitation for the amide I region, but with an additional pattern of bands in the spectra of the PIS region, attributed to cytochrome c. The same features were even more enhanced in spectra recorded with 514 nm excitation. A significant nucleotide contribution was observed in the spectra recorded for the ONL at all three excitation wavelengths. A Raman map was constructed of the major spectral components found in the retinal outer segments, as predicted by principal component analysis of the data acquired using 633 nm excitation. Comparison of the Raman map with its histological counterpart revealed a strong correlation between the two images. CONCLUSIONS: It has been demonstrated that Raman spectroscopy offers a unique insight into the biochemical composition of the light-sensing cells of the retina following the application of standard histological protocols. The present study points to the considerable promise of Raman microscopy as a component-specific probe of retinal tissue.

Visual functioning and quality of life in the subfoveal radiotherapy study (SFRADS): SFRADS report 2

Aims: To determine whether or not self reported visual functioning and quality of life in patients with choroidal neovascularisation caused by age related macular degeneration (AMD) is better in those treated with 12 Gy external beam radiotherapy in comparison with untreated subjects. Methods: A multicentre single masked randomised controlled trial of 12 Gy of external beam radiation therapy (EBRT) delivered as 6x2 Gy fractions to the macula of an affected eye versus observation. Patients with AMD, aged 60 years or over, in three UK hospital units, who had subfoveal CNV and a visual acuity equal to or better than 6/60 (logMAR 1.0). Methods: Data from 199 eligible participants who were randomly assigned to 12 Gy teletherapy or observation were available for analysis. Visual function assessment, ophthalmic examination, and fundus fluorescein angiography were undertaken at baseline and at 3, 6, 12, and 24 months after study entry. To assess patient centred outcomes, subjects were asked to complete the Daily Living Tasks Dependent on Vision (DLTV) and the SF-36 questionnaires at baseline, 6, 12, and 24 months after enrolment to the study. Cross sectional and longitudinal analyses were conducted using arm of study as grouping variable. Regression analysis was employed to adjust for the effect of baseline co-variates on outcome at 12 months and 24 months. Results: Both control and treated subjects had significant losses in visual functioning as seen by a progressive decline in mean scores in the four dimensions of the DLTV. There were no statistically significant differences between treatment and control subjects in any of dimensions of the DLTV at 12 months or 24 months after study entry. Regression analysis confirmed that treatment status had no effect on the change in DLTV dimensional scores. Conclusions: The small benefits noted in clinical measures of vision in treated eyes did not translate into better self reported visual functioning in patients who received treatment when compared with the control arm. These findings have implications for the design of future clinical trials and studies.

Reduced vision in older adults with age related macular degeneration interferes with ability to care for self and impairs role as carer

Aim: To study the relation between visual impairment and ability to care for oneself or a dependant in older people with age related macular degeneration (AMD). Method: Cross sectional study of older people with visual impairment due to AMD in a specialised retinal service clinic. 199 subjects who underwent visual function assessment (fully corrected distance and near acuity and contrast sensitivity in both eyes), followed by completion of a package of questionnaires dealing with general health status (SF36), visual functioning (Daily Living Tasks Dependent on Vision, DLTV) and ability to care for self or provide care to others. The outcome measure was self reported ability to care for self and others. Three levels of self reported ability to care were identified—inability to care for self (level 1), ability to care for self but not others (level 2), and ability to care for self and others (level 3). Results: People who reported good general health status and visual functioning (that is, had high scores on SF36 and DLTV) were more likely to state that they were able to care for self and others. Similarly people with good vision in the better seeing eye were more likely to report ability to care for self and others. People with a distance visual acuity (DVA) worse than 0.4 logMAR (Snellen 6/15) had less than 50% probability of assigning themselves to care level 3 and those with DVA worse than 1.0 logMAR (Snellen 6/60) had a probability of greater than 50% or for assigning themselves to care level 1. Regression analyses with level of care as the dependent variable and demographic factors, DLTV subscales, and SF36 dimensions as the explanatory variables confirmed that the DLTV subscale 1 was the most important variable in the transition from care level 3 to care level 2. The regression analyses also confirmed that the DLTV subscale 2 was the most important in the transition from care level 3 to care level 1. Conclusions: Ability to care for self and dependants has a strong relation with self reported visual functioning and quality of life and is adversely influenced by visual impairment. The acuity at which the balance of probability shifts in the direction of diminished ability to care for self or others is lower than the level set by social care agencies for provision of support. These findings have implications for those involved with visual rehabilitation and for studies of the cost effectiveness of interventions in AMD.

Calcium signaling in ocular arterioles

Local control of blood flow to the photoreceptors and associated neurons in the retina is largely achieved through changes in tone within the choroidal and retinal arterioles. This is primarily achieved through changes in [Ca2+] within the smooth muscle of these vessels, which regulates cell contraction and vascular constriction. Here we review some aspects of the cell physiology involved in these Ca2+-signaling processes, with particular emphasis on the molecular mechanisms involved. Ca2+-influx across the plasma membrane can occur via a variety of Ca2+-channels, including voltage-operated, store-operated, and receptor-operated channels. Ca2+ may also be released from intracellular stores via RyR-, or IP3R-gated channels in the SR membrane. Using high-speed confocal Ca2+-imaging, we have also demonstrated that the resulting signals are far from homogeneous, with spontaneous activity in retinal arterioles being characterized by both localized Ca2+-sparks and more global Ca2+-waves and oscillations. These signals may be specifically and differentially targeted, for example, to Ca2+-sensitive ion channels (stimulus-excitation coupling), or pathways regulating contraction (stimulus-contraction coupling). Exploring the role of changes in such targeting in disease states will provide exciting opportunities for future research.

Cochlin in the eye: Functional implications

Aqueous humor is actively produced in the ciliary epithelium of the anterior chamber and has important functions for the eye. Under normal physiological conditions, the inflow and outflow of the aqueous humor are tightly regulated, but in the pathologic state this balance is lost. Aqueous outflow involves structures of the anterior chamber and experiences most resistance at the level of the trabecular meshwork (TM) that acts as a filter. The modulation of the TM structure regulates the filter and its mechanism remains poorly understood. Proteomic analyses have identified cochlin, a protein of poorly understood function, in the glaucomatous TM but not in healthy control TM from human cadaver eyes. The presence of cochlin has subsequently been confirmed by Western and immunohistochemical analyses. Functionally, cochlin undergoes multimerization induced by shear stress and other changes in the microenvironment. Cochlin along with mucopolysaccharide deposits have been found in the TM of glaucoma patients and in the inner ear of subjects affected by the hearing disorder DNFA9, a late onset, progressive disease that also involves alterations in fluid shear regimes. In vitro, cochlin induces aggregation of primary TM cells suggesting a role in cell adhesion, possibly in mechanosensation, and in modulation of the TM filter.

Prediction and Verification of miRNA Expression in Human and Rat Retinas.

PURPOSE: MicroRNAs (miRNAs) play a global role in regulating gene expression and have important tissue-specific functions. Little is known about their role in the retina. The purpose of this study was to establish the retinal expression of those miRNAs predicted to target genes involved in vision. METHODS: miRNAs potentially targeting important "retinal" genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The presence of candidate miRNAs in human and rat retinal RNA was assessed by RT-PCR. cDNA levels for each miRNA were determined by quantitative PCR. The ability to discriminate between miRNAs varying by a single nucleotide was assessed. The activity of miR-124 and miR-29 against predicted target sites in Rdh10 and Impdh1 was tested by cotransfection of miRNA mimics and luciferase reporter plasmids. RESULTS: Sixty-seven miRNAs were predicted to target one or more of the 320 retinal genes listed herein. All 11 candidate miRNAs tested were expressed in the retina, including miR-7, miR-124, miR135a, and miR135b. Relative levels of individual miRNAs were similar between rats and humans. The Rdh10 3'UTR, which contains a predicted miR-124 target site, mediated the inhibition of luciferase activity by miR-124 mimics in cell culture. CONCLUSIONS: Many miRNAs likely to regulate genes important for retinal function are present in the retina. Conservation of miRNA retinal expression patterns from rats to humans supports evidence from other tissues that disruption of miRNAs is a likely cause of a range of visual abnormalities.

Advanced glycation of fibronectin impairs vascular repair by endothelial progenitor cells: Implications for vasodegeneration in diabetic retinopathy

PURPOSE. Vascular repair by marrow-derived endothelial progenitor cells (EPCs) is impaired during diabetes, although the precise mechanism of this dysfunction remains unknown. The hypothesis for the study was that progressive basement membrane (BM) modification by advanced glycation end products (AGEs) contributes to impairment of EPC reparative function after diabetes-related endothelial injury.

METHODS. EPCs isolated from peripheral blood were characterized by immunocytochemistry and flow cytometry. EPC interactions on native or AGE-modified fibronectin (AGE-FN) were studied for attachment and spreading, whereas chemotaxis to SDF-1 was assessed with the Dunn chamber assay. In addition, photoreactive agent-treated monolayers of retinal microvascular endothelial cells (RMECs) produced circumscribed areas of apoptosis and the ability of EPCs to “endothelialize” these wounds was evaluated.

RESULTS. EPC attachment and spreading on AGE-FN was reduced compared with control cells (P < 0.05–0.01) but was significantly restored by pretreatment with Arg-Gly-Asp (RGD). Chemotaxis of EPCs was abolished on AGE-FN but was reversed by treatment with exogenous RGD. On wounded RMEC monolayers, EPCs showed clustering at the wound site, compared with untreated regions (P < 0.001); AGE-FN significantly reduced this targeting response (P < 0.05). RGD supplementation enhanced EPC incorporation in the monolayer, as determined by EPC participation in tight junction formation and restoration of transendothelial electric resistance (TEER).

CONCLUSIONS. AGE-modification of vascular substrates impairs EPC adhesion, spreading, and migration; and alteration of the RGD integrin recognition motif plays a key role in these responses. The presence of AGE adducts on BM compromises repair by EPC with implications for vasodegeneration during diabetic microvasculopathy.

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

Advanced glycation end product (AGE) accumulation on Bruch's membrane: links to age-related RPE dysfunction.

PURPOSE: Advanced glycation end products (AGEs) accumulate during aging and have been observed in postmortem eyes within the retinal pigment epithelium (RPE), Bruch's membrane, and subcellular deposits (drusen). AGEs have been associated with age-related dysfunction of the RPE-in particular with development and progression to age-related macular degeneration (AMD). In the present study the impact of AGEs at the RPE-Bruch's membrane interface was evaluated, to establish how these modifications may contribute to age-related disease. METHODS: AGEs on Bruch's membrane were evaluated using immunohistochemistry. A clinically relevant in vitro model of substrate AGE accumulation was established to mimic Bruch's membrane ageing. Responses of ARPE-19 growing on AGE-modified basement membrane (AGE-BM) for 1 month were investigated by using a microarray approach and validated by quantitative (q)RT-PCR. In addition to identified AGE-related mRNA alterations, lysosomal enzyme activity and lipofuscin accumulation were also studied in ARPE-19 grown on AGE-BM. RESULTS: Autofluorescent and glycolaldehyde-derived AGEs were observed in clinical specimens on Bruch's membrane and choroidal extracellular matrix. In vitro analysis identified a range of dysregulated mRNAs in ARPE-19 exposed to AGE-BM. Altered ARPE-19 degradative enzyme mRNA expression was observed on exposure to AGE-BM. AGE-BM caused a significant reduction in cathepsin-D activity in ARPE-19 (P

Homocysteine, Methylenetetrahydrofolate Reductase C677T Polymorphism, and Risk of Retnal Vein Occlusion: A Meta-analysis

Objective: To assess the role of plasma total homocysteine (tHcy) concentrations and homozygosity for the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) C677T gene as risk factors for retinal vascular occlusive disease.

Design: Retinal vein occlusion (RVO) is an important cause of vision loss. Early meta-analyses showed that tHcy was associated with an increased risk of RVO, but a significant number of new studies have been published. Participants and/or Controls: RVO patients and controls.

Methods: Data sources included MEDLINE, Web of Science, and PubMed searches and searching reference lists of relevant articles and reviews. Reviewers searched the databases, selected the studies, and then extracted data. Results were pooled quantitatively using meta-analytic methods.

Main Outcome Measures: tHcy concentrations and MTHFR genotype.

Results: There were 25 case-control studies for tHcy (1533 cases and 1708 controls) and 18 case-control studies for MTHFR (1082 cases and 4706 controls). The mean tHcy was on average 2.8 mol/L (95% confidence
interval [CI], 1.8 –3.7) greater in the RVO cases compared with controls, but there was evidence of between-study heterogeneity (P0.001, I2 93%). There was funnel plot asymmetry suggesting publication bias. There was no evidence of association between homozygosity for the MTHFR C677T genotype and RVO (odds ratio [OR] 1.20; 95% CI, 0.84–1.71), but again marked heterogeneity (P 0.004, I2 53%) was observed.

Conclusions: There was some evidence that elevated tHcy was associated with RVO, but not homozygosity for the MTHFR C677T genotype. Both analyses should be interpreted cautiously because of marked heterogeneity between the study estimates and possible effect of publication bias on the tHcy findings.

Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.