The Novel Ceramide- and Phosphatidylcholine-Based Risk Score for the Prediction of New-Onset of Hypertension.

Ceramides and other sphingolipids are implicated in vascular dysfunction and inflammation. They have been suggested as potential biomarkers for hypertension. However, their specific association with hypertension prevalence and onset requires further investigation. This study aimed to identify specific ceramide and phosphatidylcholine species associated with hypertension prevalence and onset. The 2002 FINRISK (Finnish non-communicable risk factor survey) study investigated the association between coronary event risk scores (CERT1 and CERT2) and hypertension using prevalent and new-onset hypertension groups, both consisting of 7722 participants, over a span of 10 years. Ceramide and phosphatidylcholine levels were measured using tandem liquid chromatography-mass spectrometry. Ceramide and phosphatidylcholine ratios, including ceramide (d18:1/18:0), ceramide (d18:1/24:1), phosphatidylcholine (16:0/16:0), and the ratio of ceramide (d18:1/18:0)/(d18:1/16:0), are consistently associated with both prevalence and new-onset hypertension. Ceramide (d18:1/24:0) was also linked to both hypertension measures. Adjusting for covariates, CERT1 and CERT2 showed no-longer-significant associations with hypertension prevalence, but only CERT2 predicted new-onset hypertension. Plasma ceramides and phosphatidylcholines are crucial biomarkers for hypertension, with imbalances potentially contributing to its development. Further research is needed to understand the underlying mechanisms by which ceramides will contribute to the development of hypertension.

Serum ceramides in early pregnancy as predictors of gestational diabetes.

Ceramides contribute to the development of type 2 diabetes but it is uncertain whether they predict gestational diabetes (GDM). In this multicentre case-control study including 1040 women with GDM and 958 non-diabetic controls, early pregnancy (mean 10.7 gestational weeks) concentrations of four ceramides-Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)-were determined by a validated mass-spectrometric method from biobanked serum samples. Traditional lipids including total cholesterol, LDL, HDL and triglycerides were measured. Logistic and linear regression and the LASSO logistic regression were used to analyse lipids and clinical risk factors in the prediction of GDM. The concentrations of four targeted ceramides and total cholesterol, LDL and triglycerides were higher and HDL was lower among women with subsequent GDM than among controls. After adjustments, Cer(d18:1/24:0), triglycerides and LDL were independent predictors of GDM, women in their highest quartile had 1.44-fold (95% CI 1.07-1.95), 2.17-fold (95% CI 1.57-3.00) and 1.63-fold (95% CI 1.19-2.24) odds for GDM when compared to their lowest quartiles, respectively. In the LASSO regression modelling ceramides did not appear to markedly improve the predictive performance for GDM alongside with clinical risk factors and triglycerides. However, their adverse alterations highlight the extent of metabolic disturbances involved in GDM.

Ceramides and Phosphatidylcholines Associate with Cardiovascular Diseases in the Elderly.

BACKGROUND: The ceramide- and phospholipid-based cardiovascular risk score (CERT2) has been found to predict the risk for cardiovascular disease (CVD) events, especially cardiovascular mortality. In the present study, our aim was to estimate the predictive ability of CERT2 for mortality of CVD, coronary artery disease (CAD), and stroke in the elderly and to compare these results with those of conventional lipids. METHODS: We conducted a prospective study with an 18-year follow-up period that included a total of 1260 participants ages ≥64 years. Ceramides and phosphatidylcholines were analyzed using a LC-MS. Total cholesterol and triglycerides were performed by enzymatic methods and HDL cholesterol was determined by a direct enzymatic method. Concentrations of LDL-cholesterol were calculated according to the Friedewald formula. RESULTS: A higher score of CERT2 was significantly associated with higher CVD, CAD, and stroke mortality during the 18-year follow-up both in unadjusted and adjusted Cox regression models. The unadjusted hazard ratios (HRs) of CERT2 (95% CI) per SD for CVD, CAD, and stroke were 1.72 (1.52-1.96), 1.76 (1.52-2.04), and 1.63 (1.27-2.10), respectively, and the corresponding adjusted HRs (95% CI) per SD for CERT2 were 1.48 (1.29-1.69), 1.50 (1.28-1.75), and 1.41 (1.09-1.83). For conventional lipids, HRs per SD were lower than for CERT2. CONCLUSIONS: The risk score CERT2 associated strongly with CVD, CAD, and stroke mortality in the elderly, while the association between these events and conventional lipids was weak.

Ceramides and phospholipids in plasma extracellular vesicles are associated with high risk of major cardiovascular events after carotid endarterectomy.

Ceramides and phosphatidylcholines (PCs) are bioactive lipids and lipid bilayer membrane components. Distinct ceramides/PCs (ratios) predict cardiovascular outcome in patients with coronary artery disease. Extracellular vesicles (EVs) are proposed biomarkers for cardiovascular disease and contain ceramides/PCs. Ceramides/PCs have not been studied in patients undergoing carotid endarterectomy (CEA) nor in EVs. We therefore investigated whether levels of ceramides/PCs in plasma and EVs are associated with postoperative risk of major adverse cardiovascular events (MACE) following CEA. In 873 patients undergoing CEA of the Athero-Express biobank, we quantitatively measured seven ceramides/PCs in preoperative blood samples: Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) in plasma and two plasma EV-subfractions (LDL and TEX). We analyzed the association of ceramides, PCs and their predefined ratios with the three-year postoperative risk of MACE (including stroke, myocardial infarction and cardiovascular death). A total of 138 patients (16%) developed MACE during the three-year follow-up. In the LDL-EV subfraction, higher levels of Cer(d18:1/24:1) and Cer(d18:1/16:0)/PC(16:0/22:5) ratio were significantly associated with an increased risk of MACE (adjusted HR per SD [95% CI] 1.24 [1.01-1.53] and 1.26 [1.04-1.52], respectively). In the TEX-EV subfraction, three ratios Cer(d18:1/16:0)/Cer(d18:1/24:0), Cer(d18:1/18:0)/Cer(d18:1/24:0) and Cer(d18:1/24:1)/Cer(d18:1/24:0) were positively associated with MACE (adjusted HR per SD 1.34 [1.06-1.70], 1.24 [1.01-1.51] and 1.31 [1.08-1.58], respectively). In conclusion, distinct ceramides and PCs in plasma EVs determined in preoperative blood were independently associated with an increased 3-year risk of MACE after CEA. These lipids are therefore potential markers to identify high-risk CEA patients qualifying for secondary preventive add-on therapy.

Comparison of recent ceramide-based coronary risk prediction scores in cardiovascular disease patients.

AIM: Cholesterol-based risk prediction is often insufficient in cardiovascular disease (CVD) patients. Ceramides are a new kind of biomarkers for CVD. The Coronary Event Risk Test (CERT) is a validated cardiovascular risk predictor that uses only circulating ceramide levels, determined by coupled liquid chromatography-mass spectrometry, to allocate patients into one of four risk categories. This test has recently been modified (CERT2) by additionally including phosphatidylcholine levels. METHODS AND RESULTS: In this observational cohort study, we have recruited 999 Austrian patients with CVD and followed them for up to 13 years. We found that CERT and CERT2 both predicted cardiovascular events, cardiovascular mortality, and overall mortality. CERT2 had the higher performance compared to CERT and also to the recent cardiovascular risk score of the ESC/EAS guidelines (Systematic COronary Risk Evaluation (SCORE)) for low-risk European countries. Combining CERT2 with the ESC/EAS-SCORE, predictive capacity was further increased leading to a hazard ratio of 3.58 (2.02-6.36; P < 0.001) for cardiovascular events, 11.60 (2.72-49.56; P = 0.001) for cardiovascular mortality, and 9.86 (4.23-22.99; P < 0.001) for overall mortality when patients with very high risk (category 4) were compared to those with low risk (category 1). The use of the combined score instead of the ESC/EAS-SCORE significantly improved the predictive power according to the integrated discrimination improvement index (P = 0.004). CONCLUSION: We conclude that CERT and CERT2 are powerful predictors of cardiovascular events, cardiovascular mortality, and overall mortality in CVD patients. Including phosphatidylcholine to a ceramide-based score increases the predictive performance and is best in combination with classical risk factors as used in the ESC/EAS-SCORE.

A Randomized Controlled Dietary Intervention Improved the Serum Lipid Signature towards a Less Atherogenic Profile in Patients with Rheumatoid Arthritis.

Diet is a major modifiable risk factor for cardiovascular disease (CVD). One explanation for this is its effect on specific lipids. However, knowledge on how the lipidome is affected is limited. We aimed to investigate if diet can change the new ceramide- and phospholipid-based CVD risk score CERT2 and the serum lipidome towards a more favorable CVD signature. In a crossover trial (ADIRA), 50 patients with rheumatoid arthritis (RA) had 10 weeks of a Mediterranean-style diet intervention or a Western-style control diet and then switched diets after a 4-month wash-out-period. Five hundred and thirty-eight individual lipids were measured in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipid risk scores were analyzed by Wilcoxon signed-rank test or mixed model and lipidomic data with multivariate statistical methods. In the main analysis, including the 46 participants completing ≥1 diet period, there was no significant difference in CERT2 after the intervention compared with the control, although several CERT2 components were changed within periods. In addition, triacylglycerols, cholesteryl esters, phosphatidylcholines, alkylphosphatidylcholines and alkenylphosphatidylcholines had a healthier composition after the intervention compared to after the control diet. This trial indicates that certain dietary changes can improve the serum lipid signature towards a less atherogenic profile in patients with RA.

Coffee, Atrial Fibrillation, and Circulating Ceramides in Patients with Chronic Heart Failure.

Ceramides are sphingolipids that play roles as structural lipids and as second messengers in biological processes. Circulating ceramides are influenced by diet/food and predict major cardiovascular (CV) events, such as atrial fibrillation (AF). In 1227 patients with symptomatic chronic heart failure (HF), an association between diet and ceramides was found for coffee consumption of ≥3 cups and Cer(d18:1/24:0). Increased Cer(d18:1/24:0) was associated with lower incident AF (24.3% vs 15.4% tertile 1 vs 3, P = 0.016) and lower CV mortality (28.4% vs 12.0% tertile 1 vs 3, P < 0.0001). For coffee consumption, only an association with incident AF was found (24.5% never, 5.2% ≥3 cups). These inverse associations with AF were confirmed in survival analyses corrected for biomarkers (Cer(d18:1/24:0) HR: 0.79, P = 0.018; coffee consumption HR: 0.22, P = 0.001). In conclusion, higher coffee intake was associated with a lower risk of incident AF and with higher concentrations of Cer(d18:1/24:0). Cer(d18:1/24:0) was inversely associated to risk of AF.

Lipid profiles of patients with manifest coronary versus peripheral atherosclerosis - Is there a difference?

AIM: Peripheral arterial disease (PAD) and coronary artery disease (CAD) are both caused by atherosclerosis. Serum lipids and lipoproteins are predictive of the development of atherosclerosis but it is not clear if they differ in the two manifestations, PAD and CAD. We tested whether a more detailed characterization of the lipid and lipoprotein patterns of PAD and CAD allows a clear differentiation between the two atherosclerotic phenotypes. METHODS: A cohort of 274 statin-naïve patients with either newly diagnosed imaging proven PAD (n = 89) or stable CAD (n = 185) was characterized using nuclear magnetic resonance- and liquid chromatography-tandem mass spectrometry-based advanced lipid and lipoprotein analysis. An independent cohort of 1239 patients with PAD and CAD was used for validation. RESULTS: We found a significant difference in markers of inflammation as well as ceramide and phosphatidylcholine levels between patients with PAD and CAD. In contrast, basic lipid markers including total cholesterol, LDL cholesterol, HDL cholesterol, lipoprotein(a) or detailed lipoprotein profiles did not differ significantly between patients with PAD and CAD. Applying ratios and scores derived from ceramides and phosphatidylcholines further improved the discrimination between PAD and CAD. These significant differences were independent of body composition, from the status of smoking or type 2 diabetes mellitus, and also from apolipoprotein C-III and other inflammatory parameters which were different between CAD and PAD. CONCLUSION: The present study clearly suggests that PAD and CAD differ in terms of their ceramide- and phosphatidylcholine-based lipid patterns but not in lipoprotein characteristics.

A Novel Two-Lipid Signature Is a Strong and Independent Prognostic Factor in Ovarian Cancer.

Epithelial ovarian cancer (EOC) generally responds well to oncological treatments, but the eventual development of a refractory disease is a major clinical problem. Presently, there are no prognostic blood-based biomarkers for the stratification of EOC patients at the time of diagnosis. We set out to assess and validate the prognostic utility of a novel two-lipid signature, as the lipidome is known to be markedly aberrant in EOC patients. The study consisted of 499 women with histologically confirmed EOC that were prospectively recruited at the university hospitals in Turku (Finland) and Charité (Berlin, Germany). Lipidomic screening by tandem liquid chromatography-mass spectrometry (LC-MS/MS) was performed for all baseline serum samples of these patients, and additionally for 20 patients of the Turku cohort at various timepoints. A two-lipid signature, based on the ratio of the ceramide Cer(d18:1/18:0) and phosphatidylcholine PC(O-38:4), showed consistent prognostic performance in all investigated study cohorts. In the Turku cohort, the unadjusted hazard ratios (HRs) per standard deviation (SD) (95% confidence interval) were 1.79 (1.40, 2.29) for overall and 1.40 (1.14, 1.71) for progression-free survival. In a Charité cohort incorporating only stage III completely resected patients, the corresponding HRs were 1.59 (1.08, 2.35) and 1.53 (1.02, 2.30). In linear-mixed models predicting progression of the disease, the two-lipid signature showed higher performance (beta per SD increase 1.99 (1.38, 2.97)) than cancer antigen 125 (CA-125, 1.78 (1.13, 2.87)). The two-lipid signature was able to identify EOC patients with an especially poor prognosis at the time of diagnosis, and also showed promise for the detection of disease relapse.

Absolute and relative risk prediction in cardiovascular primary prevention with a modified SCORE chart incorporating ceramide-phospholipid risk score and diabetes mellitus.

Aims: A risk score, CERT2, based on distinct ceramide and phosphatidylcholine lipid species, has shown robust performance in predicting cardiovascular risk in secondary prevention. Here, our aim was to investigate the predictive value of CERT2 in primary prevention compared to classical lipid biomarkers and its compatibility with clinical characteristics used in the SCORE risk chart. Methods and results: Four ceramides [Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1)] and three phosphatidylcholines [PC(14:0/22:6), PC(16:0/22:5), PC(16:0/16:0)] were analysed by targeted tandem liquid chromatography-mass spectrometry method in FINRISK 2002, which is a population-based risk factor survey investigating men and women aged 25-74 years. Primary prevention subjects (N = 7324) were followed up for 10 years for the following outcomes: incident coronary heart disease (CHD), cardiovascular disease (CVD), major adverse cardiovascular event (MACE), stroke, and heart failure. Hazard ratios per standard deviation obtained from adjusted Cox proportional hazard models were significant for all these endpoints, and the highest for fatal ones, i.e. fatal CHD [1.45 (95% confidence interval 1.07-1.97)], CVD [1.39 (1.06-1.83)], and MACE [1.39 (1.07-1.80)]. The categorical net reclassification improvement was 0.051 for the 10-year risk of incident CVD. Incidence of fatal events was over 10-fold more frequent in the highest CERT2 category compared to the lowest risk category and modified SCORE risk charts, utilizing CERT2 and diabetes mellitus, increased granularity of risk assessment compared to a chart utilizing total cholesterol. Conclusion: CERT2 is a significant predictor of incident cardiovascular outcomes and risk charts utilizing this score provide an easy tool to estimate relative and absolute risk for incident CVD.

Comparison of Capillary and Schirmer Strip Tear Fluid Sampling Methods Using SWATH-MS Proteomics Approach.

Purpose: The purpose of this study was to examine the protein profile differences between capillary and Schirmer strip tear fluid samples. Methods: Both capillary and Schirmer strip tear samples were collected from 31 healthy participants at the same visit, and the samples were analyzed with nanoflow liquid chromatography coupled with time-of-flight mass spectrometer (NanoLC-MSTOF), implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). Sample type-specific and combined spectral libraries were used to evaluate the differences between the sample types in protein expression levels and biological functions. Results: In proportion, more extracellular proteins connected to immune response were quantified from the capillary samples while Schirmer strip samples contained more intracellular proteins. The sample types yielded similar counts of quantified proteins when a combined spectral library including both sample types was implemented. The differential expression analysis between the sample types identified proteins increased in the capillary samples (e.g., immunoglobulins) and Schirmer strip samples (e.g., heat-shock proteins, annexins, and S100 proteins). Conclusions: Tear proteomics data originating from the same participants vary depending on whether the sample is collected with capillary or Schirmer strip, although there is also overlap between the two sample types when a combined spectral library is implemented in the SWATH-MS analysis. In discovery-based proteomics research of tear fluid, appropriate sampling method should be chosen carefully based on the research focus. Translational Relevance: Currently, there is no consensus on how the tear fluid sampling methods affect the resulting proteomics data, and hence, identification of the most suitable sampling methods for clinical researchers with varying research interests is important.

In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prediction of Residual Risk by Ceramide-Phospholipid Score in Patients With Stable Coronary Heart Disease on Optimal Medical Therapy.

Background Identification of patients with stable coronary heart disease who are at significant residual risk could be helpful for targeted prevention. Our aim was to determine the prognostic value of the recently introduced ceramide- and phospholipid-based risk score, the Cardiovascular Event Risk Test (CERT2), in patients with stable coronary heart disease on optimal medical therapy and to identify biological processes that contribute to the CERT2 score. Methods and Results Plasma samples (n=11 222) obtained from the STABILITY (Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy) trial were analyzed using a tandem liquid chromatography-mass spectrometry method. STABILITY was a trial in patients with stable coronary heart disease randomized to the lipoprotein-associated phospholipase A2 inhibitor darapladib or placebo on optimized medical therapy at baseline, with a median follow-up of 3.7 years. Hazard ratios per SD for the CERT2 risk score were 1.32 (95% CI, 1.25-1.39) for major adverse cardiovascular event, 1.47 (95% CI, 1.35-1.59) for cardiovascular death, 1.32 (95% CI, 1.16-1.49) for stroke, 1.23 (95% CI, 1.14-1.33) for myocardial infarction, and 1.56 (95% CI, 1.39-1.76) for hospitalization due to heart failure, when adjusted for traditional cardiovascular risk factors. CERT2 showed correlation (P<0.001, r>0.2) with inflammatory markers high-sensitivity C-reactive protein, interleukin 6, the heart failure marker N-terminal pro-B-type natriuretic peptide, and low-density lipoprotein cholesterol. After also adjusting for levels of other prognostic biomarkers, the CERT2 score was still independently related to the risk of cardiovascular death but not to nonfatal events. Conclusions The CERT2 risk score can detect residual risk in patients with stable coronary heart disease and is associated with biomarkers indicating inflammation, myocardial necrosis, myocardial dysfunction, renal dysfunction, and dyslipidemia. REGISTRATION URL: https://www.clini​caltr​ials.gov. Unique identifier: NCT00799903.

Development and validation of a ceramide- and phospholipid-based cardiovascular risk estimation score for coronary artery disease patients.

AIMS: Distinct ceramide lipids have been shown to predict the risk for cardiovascular disease (CVD) events, especially cardiovascular death. As phospholipids have also been linked with CVD risk, we investigated whether the combination of ceramides with phosphatidylcholines (PCs) would be synergistic in the prediction of CVD events in patients with atherosclerotic coronary heart disease in three independent cohort studies. METHODS AND RESULTS: Ceramides and PCs were analysed using liquid chromatography-mass spectrometry (LC-MS) in three studies: WECAC (The Western Norway Coronary Angiography Cohort) (N = 3789), LIPID (Long-Term Intervention with Pravastatin in Ischaemic Disease) trial (N = 5991), and KAROLA (Langzeiterfolge der KARdiOLogischen Anschlussheilbehandlung) (N = 1023). A simple risk score, based on the ceramides and PCs showing the best prognostic features, was developed in the WECAC study and validated in the two other cohorts. This score was highly significant in predicting CVD mortality [multiadjusted hazard ratios (HRs; 95% confidence interval) per standard deviation were 1.44 (1.28-1.63) in WECAC, 1.47 (1.34-1.61) in the LIPID trial, and 1.69 (1.31-2.17) in KAROLA]. In addition, a combination of the risk score with high-sensitivity troponin T increased the HRs to 1.63 (1.44-1.85) and 2.04 (1.57-2.64) in WECAC and KAROLA cohorts, respectively. The C-statistics in WECAC for the risk score combined with sex and age was 0.76 for CVD death. The ceramide-phospholipid risk score showed comparable and synergistic predictive performance with previously published CVD risk models for secondary prevention. CONCLUSION: A simple ceramide- and phospholipid-based risk score can efficiently predict residual CVD event risk in patients with coronary artery disease.

Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium.

BACKGROUND: Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. RESULTS: Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Nav subtypes. Importantly, we show that the Nav channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process. CONCLUSIONS: Taken together, our study shows that Nav channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.

Age-associated changes in human tear proteome.

BACKGROUND: Prevalence of many eye and ocular surface diseases increases with age. While the clinical characteristics and pathophysiologic mechanisms of these conditions are often either known or extensively studied, the effects of normal aging on tear film and ocular surface have not been as widely researched. METHODS: In order to examine the effects of aging on tear fluid proteomics, tear fluid samples were collected preoperatively from 115 subjects undergoing strabismus or refractive surgery using glass microcapillary tubes. In addition to their refractive error or strabismus, the subjects did not have any other current, known eye diseases. The non-pooled samples were analysed using NanoLC-TripleTOF implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry, resulting in quantified data of 849 proteins. RESULTS: According to correlation results, 17 tear proteins correlated significantly with increased age and many of these proteins were connected to inflammation, immune response and cell death. According to enrichment analysis, growth and survival of cells decreased while immune response and inflammation increased with aging. We also discovered several well-known, activated and inhibited upstream regulators, e.g. NF-κB, which has been previously connected to aging in numerous previous studies. CONCLUSIONS: Overall, the results show the common age-dependent alterations in tear fluid protein profile, which demonstrate similar age-associated alterations of biological functions previously shown in other tissue and sample types.

Comparison of iTRAQ and SWATH in a clinical study with multiple time points.

BACKGROUND: Advances in mass spectrometry have accelerated biomarker discovery in many areas of medicine. The purpose of this study was to compare two mass spectrometry (MS) methods, isobaric tags for relative and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), for analytical efficiency in biomarker discovery when there are multiple methodological constraints such as limited sample size and several time points for each patient to be analyzed. METHODS: A total of 140 tear samples were collected from 28 glaucoma patients at 5 time points in a glaucoma drug switch study. Samples were analyzed with iTRAQ and SWATH methods using NanoLC-MSTOF mass spectrometry. RESULTS: We discovered that even though iTRAQ is faster than SWATH with respect to analysis time per sample, it loses in sensitivity, reliability and robustness. While SWATH analysis yielded complete data of 456 proteins in all samples, with iTRAQ we were able to quantify 477 proteins in total but on average only 125 proteins were quantified in a sample. 283 proteins were common in the datasets produced by the two methods. Repeatability of the methods was assessed by calculating percent relative standard deviation (% RSD) between replicate MS analyses: SWATH was more repeatable (56% of proteins < 20% RSD), compared to iTRAQ (43% of proteins < 20% RSD). Despite the overall benefits of SWATH, both methods showed less than 1 log fold change difference in the expression of 74% common proteins. In addition, comparison to MS/MS peptide results using 8 isotopically labeled peptide standards, SWATH and iTRAQ showed similar results in terms of accuracy. Moreover, both methods detected similar trends in a longitudinal analysis of protein expression of two known tear biomarkers. CONCLUSIONS: Overall, we conclude that SWATH should be preferred for biomarker discovery studies when analyzing limited volumes of clinical samples collected at multiple time points. TRIAL REGISTERATION: The study was approved by the Ethics Committee at Tampere University Hospital and was registered in EU clinical trials register (EudraCT Number: 2010-021039-14).

Patient stratification in clinical glaucoma trials using the individual tear proteome.

Glaucoma patients are prone to concomitant ocular surface diseases; however, switching from preserved to preservative-free medication can often alleviate these symptoms. The objective of this study was to examine how the adverse effects and tear proteome change for glaucoma patients (n = 28) during a 12-month drug switch from preserved latanoprost (Xalatan) to preservative-free tafluprost (Taflotan). We hypothesized that patient stratification could help identify novel recovery patterns in both tear proteomics and clinical data. In order to accomplish patient stratification, we implemented sequential window acquisition of all theoretical mass spectrometry (SWATH-MS) as a tool for quantitative analysis of individual tear protein profiles. During each visit (baseline and four follow-up visits), the patients' tears were sampled and the state of their ocular surface was evaluated clinically. Altogether 785 proteins were quantified from each tear sample using SWATH strategy and as these protein expression levels were compared between baseline and 12-month follow-up, three distinct patient groups were identified. We evaluated how these patient groups differed in their protein expression levels at baseline and discovered that the patients with increased levels of pro-inflammatory proteins and decreased levels of protective proteins benefitted most from the medication switch.

SWATH-MS Proteomic Analysis of Oxygen-Induced Retinopathy Reveals Novel Potential Therapeutic Targets.

Purpose: Oxygen-induced retinopathy (OIR) is the most widely used model for ischemic retinopathies such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). The purpose of this study was to perform the most comprehensive characterization of OIR by a recently developed technique, sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. Methods: Control and OIR retina samples collected from various time points were subjected to SWATH-MS and detailed data analysis. Immunohistochemistry from mouse retinas as well as neovascular membranes from human PDR and RVO patients were used for the detection of the localization of the proteins showing altered expression in the retina and to address their relevance to human ischemic retinopathies. Results: We report the most extensive proteomic profiling of OIR to date by quantifying almost 3000 unique proteins and their expression differences between control and OIR retinas. Crystallins were the most prominent proteins induced by hypoxia in the retina, while angiogenesis related proteins such as Filamin A and nonmuscle myosin IIA stand out at the peak of angiogenesis. Majority of the changes in protein expression return to normal at P42, but there is evidence to suggest that proteins involved in neurotransmission remain at reduced level. Conclusions: The results reveal new potential therapeutic targets to address hypoxia-induced pathological angiogenesis taking place in number of retinal diseases. The extensive proteomic profiling combined with pathway analysis also identifies novel molecular networks that could contribute to the pathogenesis of retinal diseases.