Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium

Recent studies have implicated local inflammation and activation of complement amongst the processes involved in the pathogenesis of age-related macular degeneration (AMD). Several lines of investigation also indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment epithelial (RPE) cells, contribute to the disease process. In an investigation of a potential trigger for complement activation in AMD, we explored the notion that the complex mixture of products resulting from photooxidation of A2E might include a range of fragments that could be recognized by the complement system as "foreign" and that could serve to activate the complement system, leading to low-grade inflammation. To this end, we established an in vitro assay by using human serum as a source of complement, and we measured products of C3 activation by enzyme immunoassay. Accordingly, we found that the C3 split products inactivated C3b (iC3b) and C3a were elevated in serum, overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation. Precoating of microtiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated complement. We suggest that products of the photooxidation of bis-retinoid lipofuscin pigments in RPE cells could serve as a trigger for the complement system, a trigger than would predispose the macula to disease and that, over time, could contribute to chronic inflammation. These findings link four factors that have been posited as being associated with AMD: inflammation, oxidative damage, drusen, and RPE lipofuscin.     

Rpe65 Leu450Met variant is associated with reduced levels of the retinal pigment epithelium lipofuscin fluorophores A2E and iso-A2E

There is a growing body of evidence that the nondegradable fluorophores that accumulate as the lipofuscin of retinal pigment epithelium (RPE) are involved in mechanisms leading to the degeneration of RPE in macular degeneration. Most of the constituents of RPE lipofuscin are inadvertent products of the retinoid visual cycle, the enzymatic pathway by which the 11-cis-retinal chromophore of rhodopsin is generated. Indeed, a major constituent of RPE lipofuscin, the pyridinium bisretinoid A2E, is a diretinal conjugate that forms in photoreceptor cells and is deposited in RPE cells as a consequence of the phagocytosis of the outer segment membrane by RPE cells. Given the adverse effects of A2E, there is considerable interest in combating its deposition so as to protect against vision loss. These efforts, however, necessitate an understanding of factors that modulate its formation. Here we show that an amino acid variant in murine Rpe65, a visual-cycle protein required for the regeneration of 11-cis-retinal, is associated with reduced A2E accumulation.     

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.     

Carbonic anhydrase XIV is enriched in specific membrane domains of retinal pigment epithelium, Muller cells, and astrocytes

Carbonic anhydrases (CAs) are ubiquitous enzymes important to many cell types throughout the body. They help determine levels of H(+) and HCO(-)(3) and thereby regulate intracellular and extracellular pH and volume. CA XIV, an extracellular membrane-bound CA, was recently shown to be present in brain and retina. Here, we analyze the subcellular distribution of CA XIV in retina by high-resolution immunogold cytochemistry and show that the distribution in retina (on glial cells but not neurons) is different from that reported for brain (on neurons but not glia). In addition, CA XIV is strongly expressed on retinal pigment epithelium (RPE). The specific membrane domains that express CA XIV were endfoot and nonendfoot membranes on Muller cells and astrocytes and apical and basolateral membranes of RPE. Gold particle density was highest on microvilli plasma membranes of RPE, where it was twice that of glial endfoot and Muller microvilli membranes and four times that of other glial membrane domains. Neither neurons nor capillary endothelial cells showed detectable labeling for CA XIV. This enrichment of CA XIV on specific membrane domains of glial cells and RPE suggests specialization for buffering pH and volume in retinal neurons and their surrounding extracellular spaces. We suggest that CA XIV is the target of CA inhibitors that enhance subretinal fluid absorption in macular edema. In addition, CA XIV may facilitate CO(2) removal from neural retina and modulate photoreceptor function.     

Brief Report: DARPP32, a target of hyperactive mTORC1 in the retinal pigment epithelium

The mechanistic target of rapamycin (mTOR) is assembled into signaling complexes of mTORC1 or mTORC2, and plays key roles in cell metabolism, stress response, and nutrient and growth factor sensing. Accumulating evidence from human and animal model studies has demonstrated a pathogenic role of hyperactive mTORC1 in age-related macular degeneration (AMD). The retinal pigment epithelium (RPE) is a primary injury site in AMD. In mouse models of RPE-specific deletion of Tuberous sclerosis 1 (Tsc1), which encodes an upstream suppressor of mTORC1, the hyperactivated mTORC1 metabolically reprogrammed the RPE and led to the degeneration of the outer retina and choroid (CH). In the current study, we use single-cell RNA sequencing (scRNA-seq) to identify an RPE mTORC1 downstream protein, dopamine- and cyclic AMP-regulated phosphoprotein of molecular weight 32,000 (DARPP-32). DARPP-32 was not found in healthy RPE but localized to drusen and basal linear deposits in human AMD eyes. In animal models, overexpressing DARPP-32 by adeno-associated virus (AAV) led to abnormal RPE structure and function. The data indicate that DARPP-32 is a previously unidentified signaling protein subjected to mTORC1 regulation and may contribute to RPE degeneration in AMD.     

Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: a model of age-related macular degeneration

Oxidative stress has long been linked to the pathogenesis of neurodegenerative diseases; however, whether it is a cause or merely a consequence of the degenerative process is still unknown. We show that mice deficient in Cu, Zn-superoxide dismutase (SOD1) have features typical of age-related macular degeneration in humans. Investigations of senescent Sod1(-/-) mice of different ages showed that the older animals had drusen, thickened Bruch's membrane, and choroidal neovascularization. The number of drusen increased with age, and exposure of young Sod1(-/-) mice to excess light induced drusen. The retinal pigment epithelial cells of Sod1(-/-) mice showed oxidative damage, and their beta-catenin-mediated cellular integrity was disrupted, suggesting that oxidative stress may affect the junctional proteins necessary for the barrier integrity of the retinal pigment epithelium. These observations strongly suggest that oxidative stress may play a causative role in age-related retinal degeneration, and our findings provide evidence for the free radical theory of aging. In addition, these results demonstrate that the Sod1(-/-) mouse is a valuable animal model to study human age-related macular degeneration.     

Relationship between gamma-irradiation, life span, metabolic rate and accumulation of fluorescent age pigment in the adult male housefly, Musca domestica

The patterns of fluorescent age pigment (FAP) accumulation in response to gamma-irradiation were examined in adult male houseflies under conditions of relatively high and low levels of physical activity. Flies were exposed to 0, 20, 40, and 66 kR and their level of physical activity was altered by permitting or restricting flight activity. Under conditions of high activity the mean life span of flies exposed to 20 kR and 40 kR was greater than in the controls. The mean life span of high activity flies exposed to 66 kR and all groups of low activity irradiated flies was less than the control life span. The force of mortality as indicated by Gompertz slope was greater in high activity controls than in unirradiated flies maintained under low activity conditions. Gompertz slope was similar in corresponding high and low activity groups exposed to radiation. Radiation decreased the rate of FAP accumulation in high activity groups, but increased the rate of FAP accumulation under low activity conditions. The low activity control and low activity flies exposed to 20 kR had a lower FAP accumulation than the corresponding high activity groups at 14 days of age. At this age, low activity groups of flies exposed to 40 kR and 66 kR had concentrations of FAP that were similar to the corresponding high activity groups. These results indicate that radiation exposure decreased longevity by causing damage rather than by increasing the rate of aging.     

Intervention with the aldose reductase inhibitor,tolrestat, in renal and retinal lesions of streptozotocin-diabetic rats

Summary The progressive increase in urinary albumin excretion, which precedes the development of diabetic nephropathy, can be prevented in diabetic rats if the aldose reductase inhibitor, tolrestat, is administered at the initiation and throughout the duration of hyperglycaemia. We therefore determined the ability of tolrestat to intervene in the further progression of already established urinary albumin excretion of streptozotocin-diabetic female Wistar rats. Two months after streptozotocin injection, diabetic rats were grouped as low-urinary albumin excretion (0.2–1.0 mg albumin/day) or high-urinary albumin excretion (1.9–5.9 mg albumin/day), at which time tolrestat intervention (25 mg/kg per day) was begun for half of the diabetic rats in each urinary albumin excretion group. After six months of treatment tolrestat caused a significant reduction in the urinary albumin excretion rate of the low-urinary albumin excretion group only. The diabetes-induced rise of total urinary protein in both groups was significantly reduced by tolrestat. Furthermore, the diabetes-induced increase (49%) in the thickness of the basement membranes of retinal capillaries from the outer plexiform layer was significantly diminished by tolrestat administration. In conclusion, intervention therapy with the aldose reductase inhibitor, tolrestat, can reduce the progression of urinary albumin excretion and retinal basement membrane thickening in long-term diabetic rats.     

Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile,providing a viable mechanism for the emergence of protocells

Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells.     

Age-related antioxidant capacity of the vitreous and its possible relationship with simultaneous changes in photoreceptors, retinal pigment epithelium and Bruchs' membrane in human donors' eyes

It is well established that the retina undergoes several decremental functional and structural changes with age, and it has been suggested that most of these age-related changes may be mainly due to oxidative stress through light-induced mechanisms. While the cornea and the lens absorb part of the light radiation, the vitreous appears to be the main site of protection against radiation damage to the retina. Thus the purpose of this study was to explore possible interrelationships between the oxidative capacity of the vitreous and the number of photoreceptors, the fluorescent intensity of the retina, and the thickness of the Bruch's membrane in eyes from donors dying from the third to the eighth decade of life. The results showed that the total reactive antioxidant potential of the vitreous significantly decreased with the age of the donor's eyes, and that this decrease was directly correlated with decreases in the photoreceptors and inversely correlated with increases in the autofluorescent intensity of the retina and the thickness of the Bruch's membrane. Although these correlations did not prove a cause-effect relationship, these findings may provide new insights for the understanding and possible treatment of the age-dependent changes of the human eyes.     

A comparative evaluation of digital imaging, retinal photography and optometrist examination in screening for diabetic retinopathy.

AIMS: To compare the respective performances of digital retinal imaging, fundus photography and slit-lamp biomicroscopy performed by trained optometrists, in screening for diabetic retinopathy. To assess the potential contribution of automated digital image analysis to a screening programme. METHODS: A group of 586 patients recruited from a diabetic clinic underwent three or four mydriatic screening methods for retinal examination. The respective performances of digital imaging (n=586; graded manually), colour slides (n=586; graded manually), and slit-lamp examination by specially trained optometrists (n=485), were evaluated against a reference standard of slit-lamp biomicroscopy by ophthalmologists with a special interest in medical retina. The performance of automated grading of the digital images by computer was also assessed. RESULTS: Slit-lamp examination by optometrists for referable diabetic retinopathy achieved a sensitivity of 73% (52-88) and a specificity of 90% (87-93). Using two-field imaging, manual grading of red-free digital images achieved a sensitivity of 93% (82-98) and a specificity of 87% (84-90), and for colour slides, a sensitivity of 96% (87-100) and a specificity of 89% (86-91). Almost identical results were achieved for both methods with single macular field imaging. Digital imaging had a lower technical failure rate (4.4% of patients) than colour slide photography (11.9%). Applying an automated grading protocol to the digital images detected any retinopathy, with a sensitivity of 83% (77-89) and a specificity of 71% (66-75) and diabetic macular oedema with a sensitivity of 76% (53-92) and a specificity of 85% (82-88). CONCLUSIONS: Both manual grading methods produced similar results whether using a one- or two-field protocol. Technical failures rates, and hence need for recall, were lower with digital imaging. One-field grading of fundus photographs appeared to be as effective as two-field. The optometrists achieved the lowest sensitivities but reported no technical failures. Automated grading of retinal images can improve efficiency of resource utilization in diabetic retinopathy screening.     

CircZNF532 knockdown protects retinal pigment epithelial cells against high glucose‐induced apoptosis and pyroptosis by regulating the miR‐20b‐5p/STAT3 axis

IntroductionThe loss of retinal pigment epithelial (RPE) cells is associated with the etiology of diabetic retinopathy (DR). This study investigated the effects of circular RNA ZNF532 (circZNF532) on apoptosis and pyroptosis of RPE cells.Materials and MethodsBlood samples were collected from patients with DR and healthy volunteers. A human RPE cell line ARPE‐19 was induced by high glucose (HG) and assayed for cell viability, apoptosis, and pyroptosis. The binding of miR‐20b‐5p with circZNF532 and STAT3 was confirmed by a luciferase activity assay. A mouse model of diabetic retinopathy was established.ResultsCircZNF532 and STAT3 were upregulated but miR‐20b‐5p was downregulated in the serum samples of patients with DR and HG‐induced ARPE‐19 cells. Elevated miR‐20b‐5p or CircZNF532 knockdown enhanced proliferation but reduced apoptosis and pyroptosis of ARPE‐19 cells. CircZNF532 sponged miR‐20b‐5p and inhibited its expression. STAT3 was verified as a target of miR‐20b‐5p. MiR‐20b‐5p modulated ARPE‐19 cell viability, apoptosis, and pyroptosis by targeting STAT3. Mice with STZ‐induced diabetes showed elevated expressions of circZNF532 and STAT3 but decreased the level of miR‐20b‐5p compared with the controls. Knockdown of circZNF532 inhibited apoptosis and pyroptosis in mouse retinal tissues.ConclusionCircZNF532 knockdown rescued human RPE cells from HG‐induced apoptosis and pyroptosis by regulating STAT3 via miR‐20b‐5p.     

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG– vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N– and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. J. L. Barth, Y. Yu and W. Song contributed equally to this paper.     

Increased serum pigment epithelium-derived factor is associated with microvascular complications, vascular stiffness and inflammation in Type 1 diabetes.

AIMS: To determine in Type 1 diabetes patients if levels of pigment epithelium-derived factor (PEDF), an anti-angiogenic, anti-inflammatory and antioxidant factor, are increased in individuals with complications and positively related to vascular and renal dysfunction, body mass index, glycated haemoglobin, lipids, inflammation and oxidative stress. METHODS: Serum PEDF levels were measured by ELISA in a cross-sectional study of 123 Type 1 diabetic patients (71 without and 52 with microvascular complications) and 31 non-diabetic control subjects. PEDF associations with complication status, pulse-wave analysis and biochemical results were explored. RESULTS: PEDF levels [geometric mean (95% CI)] were increased in patients with complications 8.2 (7.0-9.6) microg/ml, vs. complication-free patients [5.3 (4.7-6.0) microg/ml, P < 0.001] and control subjects [5.3 (4.6-6.1) microg/ml, P < 0.001; anova between three groups, P < 0.001], but did not differ significantly between control subjects and complication-free patients (P > 0.05). In diabetes, PEDF levels correlated (all P < 0.001) with systolic blood pressure (r = 0.317), pulse pressure (r = 0.337), small artery elasticity (r = -0.269), glycated haemoglobin (r = 0.245), body mass index (r = 0.362), renal dysfunction [including serum creatinine (r = 0.491), cystatin C (r = 0.500)], triglycerides (r = 0.367), and inflammation [including log(e)C-reactive protein (CRP; r = 0.329), and soluble vascular cell adhesion molecule-1 (r = 0.363)]. Age, blood urea nitrogen, systolic blood pressure, pulse pressure and log(e)CRP correlated with PEDF levels in control subjects (all P < 0.04). PEDF levels were not significantly correlated with measures of oxidative stress: isoprostanes, oxidized low-density lipoprotein or paraoxonase-1 activity. On stepwise linear regression analysis (all subjects), independent determinants of PEDF levels were renal function, triglycerides, inflammation, small artery elasticity and age (r(2) = 0.427). CONCLUSIONS: In Type 1 diabetes, serum PEDF levels are associated with microvascular complications, poor vascular health, hyperglycaemia, adiposity and inflammation.     

Proliferative retinopathy and proteinuria predict mortality rate in type 1 diabetic patients from Fyn County, Denmark

Aims/hypothesis We evaluated the effect of diabetic retinopathy on 25 year survival rate among a population-based cohort of type 1 diabetic patients from Fyn County, Denmark. Methods In 1973 all diabetic patients from Fyn County, Denmark with onset before the age of 30 years as of 1 July 1973 were identified (n = 727). In 1981, only 627 patients were still alive and resident in Denmark. Of these, 573 (91%) participated in a clinical baseline examination, in which diabetic retinopathy was graded and other markers of diabetes measured. Mortality rate was examined in a 25 year follow-up and related to the baseline examination. Results Of the 573 patients examined at baseline in 1981 and 1982, 297 (51.8%) were still alive in November 2006. Of the others, 256 (44.7%) had died, three (0.5%) had left Denmark and 17 (3%) were of unknown status. Age- and sex-adjusted HRs of mortality rate were 1.01 (95% CI 0.72–1.42) and 2.04 (1.43–2.91) for patients with non-proliferative and proliferative retinopathy respectively at baseline compared with patients with no retinopathy. After adjusting for proteinuria, HR among patients with proliferative retinopathy lost statistical significance, but still remained 1.48 (95% CI 0.98–2.23). The 10 year survival rate of patients who had proliferative retinopathy as well as proteinuria at baseline was 22.2% and significantly lower (p < 0.001) than in patients with proteinuria only (70.3%), proliferative retinopathy only (79.0%) or neither (86.6%). Conclusions/interpretation Proliferative retinopathy and proteinuria predict mortality rate in a population-based cohort of type 1 diabetic patients. In combination they act even more strongly. Non-proliferative diabetic retinopathy did not affect survival rate.     

Caring for individuals with dementia and cognitive impairment, not dementia: findings from the aging, demographics, and memory study

OBJECTIVES: To compare the characteristics and outcomes of caregivers of adults with dementia with those of caregivers of adults with cognitive impairment, not dementia (CIND). DESIGN: Cross‐sectional. SETTING: In‐home assessment for cognitive impairment and self‐administered caregiving questionnaire. PARTICIPANTS: One hundred sixty‐nine primary family caregivers of participants in the Aging, Demographics, and Memory Study (ADAMS). ADAMS participants were aged 71 and older drawn from the nationally representative Health and Retirement Study. MEASUREMENTS: Neuropsychological testing, neurological examination, clinical assessment, and medical history were used to assign a diagnosis of normal cognition, CIND, or dementia. Caregiving measures included caregiving time, functional limitations, depressive symptoms, physical and emotional strain, caregiving rewards, caregiver health, and demographic characteristics. RESULTS: Dementia caregivers spent approximately 9 hours per day providing care, compared with 4 hours per day for CIND caregivers (P=.001). Forty‐four percent of dementia caregivers exhibited depressive symptoms, compared with 26.5% of CIND caregivers (P=.03). Physical and emotional strains were similar in both groups of caregivers. Regardless of the strains, nearly all caregivers reported some benefits from providing care. Behavioral problems (P=.01) and difficulty with instrumental activities of daily living (P=.01) in persons with CIND partially explained emotional strain experienced by CIND caregivers. For those with dementia, behavioral problems predicted caregiver emotional strain (P<.001) and depressive symptoms (P=.01). CONCLUSION: Although support services are available to dementia caregivers, CIND caregivers also expend considerable time and experience strains. The real caregiver burden of cognitive impairment in the U.S. population may therefore be greatly underestimated if people who have reached the diagnostic threshold for dementia are focused on exclusively.