Male rat hypothalamic extraretinal photoreceptor Opsin3 is sensitive to osmotic stimuli and light.

The light-sensitive protein Opsin 3 (Opn3) is present throughout the mammalian brain; however, the role of Opn3 in this organ remains unknown. Since Opn3 encoded mRNA is modulated in the supraoptic and paraventricular nucleus of the hypothalamus in response to osmotic stimuli, we have explored by in situ hybridization the expression of Opn3 in these nuclei. We have demonstrated that Opn3 is present in the male rat magnocellular neurones expressing either the arginine vasopressin or oxytocin neuropeptides and that Opn3 increases in both neuronal types in response to osmotic stimuli, suggesting that Opn3 functions in both cell types and that it might be involved in regulating water balance. Using rat hypothalamic organotypic cultures, we have demonstrated that the hypothalamus is sensitive to light and that the observed light sensitivity is mediated, at least in part, by Opn3. The data suggests that hypothalamic Opn3 can mediate a light-sensitive role to regulate circadian homeostatic processes.

Towards a population pharmacokinetic/pharmacodynamic model of anti-VEGF therapy in patients with age-related macular degeneration.

PURPOSE: To develop a population pharmacokinetic/pharmacodynamic model (popPKPD) of intravitreal bevacizumab treatment for neovascular age-related macular degeneration (nAMD) patients to learn about the PK/PD relationship and utilise it for dosing regimen decisions on future nAMD patients. METHODS: The Greater Manchester Avastin for Neovascularisation (GMAN) randomised clinical trial data was retrospectively utilised, and the best-corrected visual acuity (BCVA) and central macular retinal thickness (CRT, measured by optical coherence tomography) were the PD inputs to the model. Using the nonlinear mixed-effects method, the best PKPD structural model was investigated, and the clinical significance of the two different dosing treatment regimens (as-needed versus routine) was evaluated. RESULTS: A structural model to describe the change of BCVA from the baseline of nAMD patients was successfully obtained based on the turnover PD model concept (drug stimulates the "visual acuity response production"). The popPKPD model and simulation indicate that the routine regimen protocol improves patient visual outcome compared to the as-needed protocol. For the change in CRT, the turnover structural PKPD model was too demanding to fit to the given clinical data. CONCLUSIONS: This is the first popPKPD attempt in nAMD treatment that shows the potential of this strategy to understand/inform the dosing regimen. Clinical trials with richer PD data will provide the means to build more robust models.

Spatial distribution of metabolites in the retina and its relevance to studies of metabolic retinal disorders.

INTRODUCTION: The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. OBJECTIVES: To better understand the properties of different retinal areas we studied the differential distribution of metabolites across the retina. METHODS: We conducted an untargeted metabolomics analysis on full-thickness punches from three different regions (macula, temporal peri-macula and periphery) of healthy primate retina. RESULTS: Nearly half of all metabolites identified showed differential abundance in at least one comparison between the three regions. Furthermore, mapping metabolomics results from macula-specific eye diseases onto our region-specific metabolite distributions revealed differential abundance defining systemic metabolic dysregulations that were region specific. CONCLUSIONS: The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Our results may help to better understand the pathobiology of retinal diseases with region specificity.

Choosing transcatheter aortic valve replacement in porcelain aorta: outcomes versus surgical replacement.

OBJECTIVES: Porcelain aorta complicates aortic valve replacement and is an indication for transcatheter approaches. No study has compared surgical and transcatheter valve replacement in the setting of porcelain aorta. We characterize porcelain aorta patients undergoing aortic valve replacement and the association of aortic calcification and outcomes. METHODS: Patients undergoing aortic valve replacement with porcelain aorta were identified. Aortic calcium volume was determined using 3D computed tomography thresholding techniques. Propensity scoring was performed to assess the effect of surgical versus transcatheter approaches. Risk factors for composite major hospital complications (death, stroke and dialysis) were identified using random forest machine learning. RESULTS: From January 2006 to January 2015, 164 patients with porcelain aorta underwent aortic valve replacement [105 (64%) surgical replacement, 59 (36%) transcatheter replacement]. Propensity scoring matched 29 pairs (49% of transcatheter patients). Before matching, 5-year survival was 41% [(43% surgical, 35% transcatheter, P(log-rank) = 0.9]. After matching, mortality for surgical versus transcatheter replacement was 3.4% (n = 1) vs 10% (n = 3), stroke 14% (n = 4) vs 3.4% (n = 1) and dialysis 6.9% (n = 2) versus 11% (n = 3). Matched 5-year survival was 40% after surgical replacement and 29% after transcatheter replacement [P(log-rank) = 0.4]. Total aortic calcium volume was greater in transcatheter than surgical patients [18 (8.0) vs 17 (7.7) ml] and was associated with more major hospital complications after either approach. CONCLUSIONS: Surgical and transcatheter approaches are complementary options for aortic stenosis with porcelain aorta. Surgical valve replacement remains an effective treatment for patients requiring concomitant procedures. Quantifying aortic calcium volume is a helpful risk predictor in all patients with porcelain aorta.

Mast cell infiltration of the choroid and protease release are early events in age-related macular degeneration associated with genetic risk at both chromosomes 1q32 and 10q26.

Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometry­based quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cell­specific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from low­genetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.

GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition.

BACKGROUND: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. METHODS: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. RESULTS: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. CONCLUSIONS: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.

No association between breast pain and breast cancer: a prospective cohort study of 10 830 symptomatic women presenting to a breast cancer diagnostic clinic.

BACKGROUND: Women with breast pain constitute >20% of breast clinic attendees. AIM: To investigate breast cancer incidence in women presenting with breast pain and establish the health economics of referring women with breast pain to secondary care. DESIGN AND SETTING: A prospective cohort study of all consecutive women referred to a breast diagnostic clinic over 12 months. METHOD: Women were categorised by presentation into four distinct clinical groups and cancer incidence investigated. RESULTS: Of 10 830 women, 1972 (18%) were referred with breast pain, 6708 (62%) with lumps, 480 (4%) with nipple symptoms, 1670 (15%) with 'other' symptoms. Mammography, performed in 1112 women with breast pain, identified cancer in eight (0.7%). Of the 1972 women with breast pain, breast cancer incidence was 0.4% compared with ∼5% in each of the three other clinical groups. Using 'breast lump' as reference, the odds ratio (OR) of women referred with breast pain having breast cancer was 0.05 (95% confidence interval = 0.02 to 0.09, P<0.001). Compared with reassurance in primary care, referral was more costly (net cost £262) without additional health benefits (net quality-adjusted life-year [QALY] loss -0.012). The greatest impact on the incremental cost-effectiveness ratio (ICER) was when QALY loss because of referral-associated anxiety was excluded. Primary care reassurance no longer dominated, but the ICER remained greater (£45 528/QALY) than typical UK National Health Service cost-effectiveness thresholds. CONCLUSION: This study shows that referring women with breast pain to a breast diagnostic clinic is an inefficient use of limited resources. Alternative management pathways could improve capacity and reduce financial burden.

Intravitreal administration of recombinant human opticin protects against hyperoxia-induced pre-retinal neovascularization.

Opticin is an extracellular glycoprotein present in the vitreous. Its antiangiogenic properties offer the potential for therapeutic intervention in conditions such as proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the hypothesis that intravitreal administration of recombinant human opticin can safely protect against the development of pathological angiogenesis and promote its regression. We generated and purified recombinant human opticin and investigated its impact on the development and regression of pathological retinal neovascularization following intravitreal administration in murine oxygen-induced retinopathy. We also investigated its effect on normal retinal vascular development and function, following intravitreal injection in neonatal mice, by histological examination and electroretinography. In oxygen-induced retinopathy, intravitreal administration of human recombinant opticin protected against the development of retinal neovascularization to similar extent as aflibercept, which targets VEGF. Opticin also accelerated regression of established retinal neovascularization, though the effect at 18 h was less than that of aflibercept. Intravitreal administration of human recombinant opticin in neonatal mice caused no detectable perturbation of subsequent retinal vascular development or function. In summary we found that intraocular administration of recombinant human opticin protects against the development of pathological angiogenesis in mice and promotes its regression.

Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells.

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.

Beyond factor H: The impact of genetic-risk variants for age-related macular degeneration on circulating factor-H-like 1 and factor-H-related protein concentrations.

Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10-10; FHR-2, p = 6.0 × 10-10; FHR-3, p = 1.5 × 10-5; FHR-4, p = 1.3 × 10-3; FHR-5, p = 1.9 × 10-4) and FHL-1 (p = 4.9 × 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.

FHL-1 interacts with human RPE cells through the α5ß1 integrin and confers protection against oxidative stress.

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch's membrane, are essential for RPE cell health and function, but the signals induced by Bruch's membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch's membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and ß1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

Prevalence and phenotype associations of complement factor I mutations in geographic atrophy.

Rare variants in the complement factor I (CFI) gene, associated with low serum factor I (FI) levels, are strong risk factors for developing the advanced stages of age-related macular degeneration (AMD). No studies have been undertaken on the prevalence of disease-causing CFI mutations in patients with geographic atrophy (GA) secondary to AMD. A multicenter, cross-sectional, noninterventional study was undertaken to identify the prevalence of pathogenic rare CFI gene variants in an unselected cohort of patients with GA and low FI levels. A genotype-phenotype study was performed. Four hundred and sixty-eight patients with GA secondary to AMD were recruited to the study, and 19.4% (n = 91) demonstrated a low serum FI concentration (below 15.6 µg/ml). CFI gene sequencing on these patients resulted in the detection of rare CFI variants in 4.7% (n = 22) of recruited patients. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. Of the total patients recruited, 3.2% (n = 15) expressed a CFI variant classified as pathogenic or likely pathogenic. The presence of reticular pseudodrusen was detected in all patients with pathogenic CFI gene variants. Patients with pathogenic CFI gene variants and low serum FI levels might be suitable for FI supplementation in therapeutic trials.

Neurotrophic effects of Botulinum neurotoxin type A in hippocampal neurons involve activation of Rac1 by the non-catalytic heavy chain (HCC/A).

Botulinum neurotoxins (BoNTs) are extremely potent naturally occurring poisons that act by silencing neurotransmission. Intriguingly, in addition to preventing presynaptic vesicle fusion, BoNT serotype A (BoNT/A) can also promote axonal regeneration in preclinical models. Here we report that the non-toxic C-terminal region of the receptor-binding domain of heavy chain BoNT/A (HCC/A) activates the small GTPase Rac1 and ERK pathway to potentiate axonal outgrowth, dendritic protrusion formation and synaptic vesicle release in hippocampal neurons. These data are consistent with HCC/A exerting neurotrophic properties, at least in part, independent of any BoNT catalytic activity or toxic effect.

Primary squamous cell carcinoma of the thyroid gland successfully treated with surgical resection and adjuvant chemoradiotherapy.

A 57-year-old woman presented with a 3-month history of an enlarging thyroid mass causing breathlessness and dysphagia. Cross-sectional imaging showed a thyroid tumour infiltrating the trachea and abutting the oesophagus. She underwent panendoscopy, total thyroidectomy and planned tracheal resection, but due to intraoperative findings, a staged procedure was planned instead. Histological analysis revealed a poorly differentiated squamous cell carcinoma (SCC) which was positive on paired box gene 8 (PAX8) immunostaining, suggesting a diagnosis of primary thyroid SCC. She subsequently underwent total laryngectomy and bilateral neck dissections followed by radiotherapy with concurrent cisplatin chemotherapy. The patient remains disease-free 22 months after treatment. Median overall survival is 10 months for macroscopically completely resected tumours. PAX8 immunostaining is a novel technique which helps with the diagnostic challenge of distinguishing between primary thyroid SCC and SCC metastatic to the thyroid from extrathyroidal sites. Complete surgical resection with adjuvant chemoradiotherapy may result in a favourable outcome despite conflicting reports in the literature.

Phosphorylation of Syntaxin-1a by casein kinase 2α regulates pre-synaptic vesicle exocytosis from the reserve pool.

The t-soluble NSF-attachment protein receptor protein Syntaxin-1a (Stx-1a) is abundantly expressed at pre-synaptic terminals where it plays a critical role in the exocytosis of neurotransmitter-containing synaptic vesicles. Stx-1a is phosphorylated by Casein kinase 2α (CK2α) at Ser14, which has been proposed to regulate the interaction of Stx-1a and Munc-18 to control of synaptic vesicle priming. However, the role of CK2α in synaptic vesicle dynamics remains unclear. Here, we show that CK2α over-expression reduces evoked synaptic vesicle release. Furthermore, shRNA-mediated knockdown of CK2α in primary hippocampal neurons strongly enhanced vesicle exocytosis from the reserve pool, with no effect on the readily releasable pool of primed vesicles. In neurons in which endogenous Stx-1a was knocked down and replaced with a CK2α phosphorylation-deficient mutant, Stx-1a(D17A), vesicle exocytosis was also increased. These results reveal a previously unsuspected role of CK2α phosphorylation in specifically regulating the reserve synaptic vesicle pool, without changing the kinetics of release from the readily releasable pool.

Homozygous deletion of CDKN2A in malignant mesothelioma: Diagnostic utility, patient characteristics and survival in a UK mesothelioma centre.

BACKGROUND: Detection of homozygous deletion of the p16 gene (CDKN2A) by fluorescence in situ hybridization (FISH) has been investigated as an ancillary technique in the diagnosis of malignant mesothelioma. METHOD: This retrospective study reviewed the results of all p16 FISH tests performed at a regional mesothelioma centre from February 2012 to November 2019 in cases of possible mesothelioma to examine the diagnostic utility of this test as well as patients characteristics and survival in p16 FISH positive mesothelioma versus p16 FISH negative mesothelioma. RESULTS: P16 FISH testing was requested in 216 pathological samples in the study period. The test failure rate was 4% (10/216). Median time from request to result was 10 days (IQR 7-13, range 1-30). The sensitivity, specificity, NPV and PPV were 60 %, 100 %, 39 % and 100 % respectively. There were no false positive results and this genetic aberration was only detected in cases of mesothelioma. The prevalence of p16 FISH positive mesothelioma was higher in cytological specimens compared to histological specimens (75 % vs 58 %, p = 0.03) and lower in women compared to men (33 % vs 66 %, p = 0.003). P16 FISH positive mesothelioma was associated with significantly worse survival (median overall survival 285 vs 339 days, p = 0.0018). This remained significant after adjusting for confounding variables (OR 4.4, 95 %CI 1.84-11.14, p = 0.001). CONCLUSIONS: In this study, 60 % of mesotheliomas harbour a homozygous deletion of CDKN2A and can be accurately, reliably and efficiently identified by p16 FISH testing. This test can be embedded within routine practice in mesothelioma pathways to enhance diagnostic accuracy.

Association of plasma trace element levels with neovascular age-related macular degeneration.

Although the triggers causing angiogenesis in the context of neovascular age-related macular degeneration (nAMD) are not fully understood, oxidative stress is likely involved. Oxidative stress in the eye can occur through exposure of macular tissues to sunlight and local or systemic exposure to oxidative stressors associated with environmental or lifestyle factors. Because trace elements have been implicated as regulators of oxidative stress and cellular antioxidant defense mechanisms, we hypothesized that they may play a role as a risk factor, modifying the progression toward nAMD. Herein, we determined whether levels of human plasma trace elements are different in 236 individuals with nAMD compared to 236 age-matched controls without AMD. Plasma levels of 16 trace elements including arsenic, barium, calcium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, lead, antimony, selenium, vanadium and zinc were measured using inductively coupled plasma mass spectrometry. Associations of trace elements with demographic, environmental and lifestyle factors and AMD-associated genetic variants were assessed. Elevated levels of barium and cadmium and reduced levels of chromium were observed in nAMD patients compared to controls. Mean plasma concentrations of barium were 1.35 µg/L (standard deviation [SD] 0.71) in nAMD and 1.15 µg/L (SD 0.63) in controls (P = 0.001). Mean levels of chromium were 0.37 µg/L (SD 0.22) in nAMD and 0.46 µg/L (SD 0.34) in controls (P = 0.001). Median levels for cadmium, which were not normally distributed, were 0.016 µg/L (interquartile range [IQR] 0.001-0.026) in nAMD and 0.012 µg/L (IQR 0.001-0.022) in controls (P = 0.002). Comparison of the Spearman's correlation coefficients between nAMD patients and controls identified a difference in correlations for 8 trace elements. Cadmium levels were associated with the smoking status (P < 0.001), while barium levels showed a trend of association with the usage of antihypertensive drugs. None of the AMD-associated genetic variants were associated with any trace element levels. In conclusion, in this case-control study we detected elevated plasma levels of barium and cadmium and reduced plasma levels of chromium in nAMD patients. An imbalance in plasma trace elements, which is most likely driven by environmental and lifestyle factors, might have a role in the pathogenesis of AMD. These trace elements may be incorporated as biomarkers into models for prediction of disease risk and progression. Additionally, population-based preventive strategies to decrease Cd exposure, especially by the cessation of smoking, could potentially reduce the burden of nAMD. Future studies are warranted to investigate whether supplementation of Cr would have a beneficial effect on nAMD.

Analysis of opticin binding to collagen fibrils identifies a single binding site in the gap region and a high specificity towards thin heterotypic fibrils containing collagens II, and XI or V/XI.

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.

Intravitreal Pharmacokinetic Study of the Antiangiogenic Glycoprotein Opticin.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.

Increased circulating levels of Factor H-Related Protein 4 are strongly associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10-6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10-56), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.