Association of Primary Open-Angle Glaucoma with Diabetic Retinopathy among Patients with Type 1 and Type 2 Diabetes: A Large Global Database Study.

PURPOSE: To assess the correlation between primary open-angle glaucoma (POAG) and the risk of developing diabetic retinopathy (DR) in patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). DESIGN: A retrospective cohort study leveraging the global patient database of TriNetX Research Network. PARTICIPANTS: The study included 44 359 patients with diabetes mellitus (DM) with POAG and 4 393 300 patients with DM without any glaucoma ≥ 18 years of age. Propensity score matching harmonized the cohorts to 39 680 patients each, covering diagnoses from January 1, 2005, to January 1, 2023. METHODS: We analyzed data using specific International Classification of Diseases, 10th Revision (ICD-10) codes for DM and glaucoma. We matched the cohorts using propensity score matching, adjusting for age, sex, race/ethnicity, blood markers, relevant medical history, and ophthalmic service use. MAIN OUTCOME MEASURES: The primary outcome was the first-time occurrence of DR, including nonproliferative DR (NPDR) and proliferative DR (PDR), in patients with DM with and without glaucoma at 1-, 5-, and 10-year intervals from their individual index dates. RESULTS: At 10 years, patients with T1DM with POAG exhibited a heightened risk for any DR (adjusted risk ratios [RRs], 4.12; 95% confidence interval [CI], 3.05-5.57, P < 0.0001) and PDR (RR, 7.02; 95% CI, 3.62-13.61, P < 0.0001). Patients with T2DM and POAG also faced an increased 10-year risk for any DR (RR, 2.47; 95% CI, 2.28-2.68, P < 0.0001) and PDR (RR, 3.82; 95% CI, 3.09-4.70, P < 0.0001). The combined association of POAG on DR risk in those with T1DM and T2DM at 10 years was found to be significantly higher among patients with POAG (5.45%) compared with those without glaucoma (2.12%) (adjusted hazard ratio [aHR], 2.33; 95% CI, 2.14-2.53). The cumulative incidence of DR was significantly higher in the POAG group compared with nonglaucoma counterparts after a decade (log-rank P < 0.001). CONCLUSIONS: Our findings underscore a substantial association between POAG and DR development in both T1DM and T2DM patients, emphasizing the need for vigilant screening and comprehensive management in glaucomatous patients with DM to mitigate the risk of DR. Future research should delve into elucidating the causal mechanisms driving these observed associations. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Glaucomatous aqueous humor vesicles are smaller and differ in composition compared to controls.

Cells communicate with each other using vesicles of varying sizes, including a specific repertoire known as exosomes. We isolated aqueous humor (AH)-derived vesicles using two different methods: ultracentrifugation and an exosome isolation kit. We confirmed a unique vesicle size distribution in the AH derived from control and primary open-angle glaucoma (POAG) patients using various techniques, including Nanotracker, dynamic light scattering, atomic force imaging, and electron microscopy. Bonafide vesicle and/or exosome markers were present by dot blot in both control and POAG AH-derived vesicles. Marker levels differed between POAG and control samples, while non-vesicle negative markers were absent in both. Quantitative labeled (iTRAQ) proteomics showed a reduced presence of a specific protein, STT3B, in POAG compared to controls, which was further confirmed using dot blot, Western blot, and ELISA assays. Along the lines of previous findings with AH profiles, we found vast differences in the total phospholipid composition of AH vesicles in POAG compared to controls. Electron microscopy further showed that the addition of mixed phospholipids alters the average size of vesicles in POAG. We found that the cumulative particle size of type I collagen decreased in the presence of Cathepsin D, which normal AH vesicles were able to protect against, but POAG AH vesicles did not. AH alone had no effect on collagen particles. We observed a protective effect on collagen particles with an increase in artificial vesicle sizes, consistent with the protective effects observed with larger control AH vesicles but not with the smaller-sized POAG AH vesicles. Our experiments suggest that AH vesicles in the control group provide greater protection for collagen beams compared to POAG, and their increased vesicle sizes are likely contributing factors to this protection.

Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse.

Progressive photoreceptor death occurs in blinding diseases such as retinitis pigmentosa. Myeloid differentiation primary response protein 88 (MyD88) is a central adaptor protein for innate immune system Toll-like receptors (TLR) and induces cytokine secretion during retinal disease. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased photoreceptor survival, which was associated with altered cytokines and increased neuroprotective microglia. However, the identity of additional molecular changes associated with MyD88 inhibitor-induced neuroprotection is not known. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling followed by LC-MS/MS for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. Quantitative proteomics using iTRAQ LC-MS/MS is a high-throughput method ideal for providing insight into molecular pathways during disease and experimental treatments. Forty-two proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti-apoptotic properties was identified in the MyD88-inhibited mice. These data suggest that inhibiting MyD88 enhances chaperone-mediated retinal protection pathways. Therefore, this study provides insight into molecular events contributing to photoreceptor protection from modulating inflammation.

Endogenous ocular lipids as potential modulators of intraocular pressure.

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open-angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.

Differentiation of soluble aqueous humor metabolites in primary open angle glaucoma and controls.

We report an analysis of the aqueous humor (AH) metabolome of primary open angle glaucoma (POAG) in comparison to normal controls. The AH samples were obtained from human donors [control (n = 35), POAG (n = 23)]. The AH samples were subjected to one-dimensional 1H nuclear magnetic resonance (NMR) analyses on a Bruker Avance 600 MHz instrument with a 1.7 mM NMR probe. The same samples were then subjected to isotopic ratio outlier analysis (IROA) using a Q Exactive orbitrap mass spectrometer after chromatography on an Accela 600 HPLC. Clusterfinder Build 3.1.10 was used for identification and quantification based on long-term metabolite matrix standards. In total, 278 metabolites were identified in control samples and 273 in POAG AH. The metabolites identified were fed into previously reported proteome and genome information and the OmicsNet interaction network generator to construct a protein-metabolite interactions network with an embedded protein-protein network. Significant differences in metabolite composition in POAG compared to controls were identified indicating potential protein/gene pathways associated with these metabolites. These results will expand our previous understanding of the impeded AH metabolite composition, provide new insight into the regulation of AH outflow, and likely aid in future AH and trabecular meshwork multi-omics network analyses.

Nuclear prelamin a recognition factor and iron dysregulation in multiple sclerosis.

Dysregulation of iron metabolism and aberrant iron deposition has been associated with multiple sclerosis. However, the factors that contribute to this pathological state remain to be understood. In this study, human multiple sclerosis and mice brain samples were analyzed through mass spectrometry as well as histological and immunoblot techniques, which demonstrated that iron deposition is associated with increased levels of nuclear prelamin A recognition factor (NARF). NARF is a protein associated with the mitochondria which has also been linked to mitochondrial defects in multiple sclerosis. We report NARF to be associated in multiple sclerosis pathology and aberrant iron deposition.

Tissue protein and lipid alterations in response to metallic impaction.

Tissue response to metallic contact and injury remains poorly understood at the level of proteins and lipid changes. We used corneas as a model system (bovine, porcine [n = 300 each], and human corneas [n = 6]) to characterize proteomic and lipidomic reactions to metallic exposure (impaction with iron). We also made a limited investigation into protein extractability and profile changes due to copper, and lead exposure as well. We identified selected proteins after trypsin digestion using an LCQ Deca XP and lipids using a TSQ Quantum Access Max mass spectrometer, respectively. Our findings indicate that iron impaction to corneal tissue results in cleavage of 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-2 variant (PLCB2; 134 kDa) into a 36 kDa species and presence of the epithelial layer is necessary for this cleavage. Penetration of the cornea with other metals (copper and lead) resulted in lower protein extractability from corneal tissue compared with controls but not distinct changes in PLCB2. The changes in protein profiles were unique to the type of metal used for impaction. The depth of injury negatively affected protein extractability and profile compared with controls. These changes were also dependent on several other factors in a complex manner. Iron impaction of corneal tissue for 24 hours results in cleavage of PLCB2 commensurate with significant changes in phosphatidylinositols but not phosphatidylcholines or other phospholipids.

Lung inflammation and disease: A perspective on microbial homeostasis and metabolism.

It is now well appreciated that the human microbiome plays a significant role in a number of processes in the body, significantly affecting its metabolic, inflammatory, and immune homeostasis. Recent research has revealed that almost every mucosal surface in the human body is associated with a resident commensal microbiome of its own. While the gut microbiome and its role in regulation of host metabolism along with its alteration in a disease state has been well studied, there is a lacuna in understanding the resident microbiota of other mucosal surfaces. Among these, the scientific information on the role of lung microbiota in pulmonary diseases is currently severely limited. Historically, lungs have been considered to be sterile and lung diseases have only been studied in the context of bacterial pathogenesis. Recently however, studies have revealed a resilient microbiome in the upper and lower respiratory tracts and there is increased evidence on its central role in respiratory diseases. Knowledge of lung microbiome and its metabolic fallout (local and systemic) is still in its nascent stages and attracting immense interest in recent times. In this review, we will provide a perspective on lung-associated metabolic disorders defined for lung diseases (e.g., chronic obstructive pulmonary disease, asthma, and respiratory depression due to infection) and correlate it with lung microbial perturbation. Such perturbations may be due to altered biochemical or metabolic stress as well. Finally, we will draw evidence from microbiome and classical microbiology literature to demonstrate how specific lung morbidities associate with specific metabolic characteristics of the disease, and with the role of microbiome in this context. © 2018 IUBMB Life, 71(1):152-165, 2019.

A novel myelin basic protein transcript variant in the murine central nervous system.

Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 months of age was harvested and used for mRNA extraction. mRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel separation. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence analysis demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clinical significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.

Phospholipid secretions of organ cultured ciliary body.

Homeostasis of intraocular pressure (IOP) is important for the maintenance of anterior eye anatomic integrity, minimizing pressure-associated damage to the optic nerve, and maintaining a pressure gradient for blood flow to the eye. IOP is regulated by equilibrium between aqueous humor (AH) production and its outflow. The ciliary body (CB) is thought to actively secrete AH. However, whether AH composition and in particular, its phospholipids are entirely due to CB secretion remains uncertain. Comparison of phospholipids released by cultured CB, phospholipids present within CB tissue, within AH, and within blood and serum are consistent with release of most phospholipids into the AH by the CB. Treatment of CB in culture with timolol, a non-specific beta-adrenergic antagonist, alters the release of phospholipids by CB into the media. However, dorzalamide, a carbonic anhydrase inhibitor that reduces production of AH, does not affect phospholipid release thereby suggesting timolol, which also decreases IOP through decreased AH outflow, affects other physiological homeostatic mechanisms regulating aqueous outflow. These outflow changes also affect the composition of secreted phospholipids. We present evidence that release of lipids by the CB has a prolonged survival effect on cultured primary TM cells and TM tissue.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.

Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90ß/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90ß]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.

Phospholipidomic Studies in Human Cornea From Climatic Droplet Keratopathy.

Climatic droplet keratopathy (CDK) is an acquired degenerative disease predominantly affecting males over 40 years old. It results in progressive corneal opacities usually affecting both eyes. CDK is multifactorial and its etiology remains unknown. Our recent findings are consistent with CDK pathology being driven by environmental factors with oxidative stress playing an important role (e.g.,, contributing to lipid peroxidation) rather than climate factors. The changes in corneal lipid composition affected by environmental factors remain understudied. The purpose of this study was to systematically investigate phospholipids profile (phosphatidylcholine [PC] and phosphatidylserine [PS]) in corneas from CDK patients using tandem mass spectrometry. Samples from CDK areas and from non-affected areas were obtained from patients diagnosed with CDK who underwent cataract surgery, were subjected to lipid extraction using a modified Bligh and Dyer method; protein concentrations were determined using the Bradford's method. Lipids were identified and subjected to ratiometric quantification using TSQ Quantum Access Max triple quadrupole mass spectrometer, using appropriate class specific lipid standards. All phospholipid classes showed lower total amounts in affected areas compared to control areas from CDK's corneas. Comparative profiles of two phospholipid classes (PC, PS) between CDK areas and control areas showed several common species between them. We also found a few unique lipids that were absent in CDK areas compared to controls and vice versa. Lower amount of phospholipids in CDK areas compared to control areas could be attributed to the lipid peroxidation in the affected corneal regions as a consequence of increased oxidative stress. J. Cell. Biochem. 118: 3920-3931, 2017. © 2017 Wiley Periodicals, Inc.

Segmental outflow of aqueous humor in mouse and human.

The main and only modifiable risk factor in glaucoma, the group of usually late onset progressive and irreversible blinding optic neuropathies, is elevated intraocular pressure (IOP). The increase in IOP is due to impeded aqueous humor (AH) outflow through the conventional pathway. The aberrant increased resistance at the trabecular meshwork (TM), the filter-like region in the anterior eye chamber is the major contributory factor in causing the impeded outflow. In normal as well as in glaucoma eyes the regions of the TM are divided into areas of high and low flow. The collector channels and distal outflow regions are now increasingly being recognized as potential players in contributing to impede AH outflow. Structural and molecular make-up contributing to the segmental blockage to outflow is likely to provide greater insight. Establishing segmental blockage to outflow in model systems of glaucoma such as the mouse in parallel to human eyes will expand our repertoire of tools for investigation. Further study into this area of interest has the potential to ultimately lead to the development of new therapeutics focused on lowering IOP by targeting the various components of segmental blockage of outflow in the TM and in the distal outflow region.

Deimination level and peptidyl arginine deiminase 2 expression are elevated in astrocytes with increased incubation temperature.

Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100ß, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.

Mass spectrometric analyses of phospholipids in the S334ter-3 rat model of retinal degeneration.

PURPOSE: The purpose of this study was to profile the endogenous phospholipid species in the retinal tissue of the S334ter-3 rat model of retinal degeneration. Retinal tissue was collected from S334ter-3 rats at postnatal day (P) 20, P30, and P60, while control retinal samples were collected from Sprague-Dawley (SD) rats at the same time points for comparison. METHODS: Lipids were extracted using the Bligh and Dyer method, and resuspended in an acetonitrile/isopropanol (1:1) solution. For lipid analyses, a positive ion-mode precursor ion scan (PIS) was used for phosphatidylcholine (PC; product m/z of 184), a negative ion-mode neutral loss scan (NLS) was used for phosphatidylserine (PS; product m/z of 87.1), and a negative ion-mode PIS was used for phosphatidylinositol (PI; product m/z of 241) and phosphatidylethanolamine (PE; product m/z of 196); the analyses were carried out using a TSQ Quantum Access Max mass spectrometer. The samples were directly infused with a Triversa Nanomate using 1.6 kV and 0.4 psi of pressure for the positive ion mode, and 1.3 kV and 0.6 psi of pressure for the negative ion mode, and scanned for 2 min between 200 m/z and 1000 m/z. Ratiometric quantification was performed using quantitative standards for each lipid class. RESULTS: The comparative profiles of PC, PE, PS, and PI between S334ter-3 and control rats showed that there were several lipid species common to both groups, as well as several that were unique to the S334ter-3 group and vice versa. CONCLUSIONS: It was found that the proportions of PC and PS were higher in the control retina compared to S334ter-3, and that the proportions of PE and PI were higher in the S334ter-3 retina compared to control.

The role of deimination in ATP5b mRNA transport in a transgenic mouse model of multiple sclerosis.

Deimination refers to conversion of protein-bound arginine into citrulline. An mRNA carrier, RNA binding export factor (REF), present on mitochondria undergoes loss of deimination with impaired ATP5b mRNA transport in ND4 mice (model of multiple sclerosis) compared with the controls. We present evidence of (1) reduced ATP5b mRNA binding strength of non-deiminated REF compared with deiminated REF, (2) impaired ATP5b mRNA transport in ND4 mice and (3) reduced mitochondrial ATP synthase activity on inhibition of deimination in PC12 cells. Impaired deimination of REF and defect in mitochondrial mRNA transport are critical factors in mitochondrial dysfunction in ND4 mice.

Appropriate patient population for future visual system axon regeneration therapies.

A number of blinding diseases caused by damage to the optic nerve result in progressive vision loss or loss of visual acuity. Secondary glaucoma results from traumatic injuries, pseudoexfoliation or pigmentary dispersion syndrome. Progressive peripheral vision loss is common to all secondary glaucoma irrespective of the initial event. Axon regeneration is a potential therapeutic avenue to restore lost vision in these patients. In contrast to the usual approach of having the worst possible patient population for initial therapies, axon regeneration may require consideration of appropriate patient population even for initial treatment trials. The current state of axon regeneration therapies, their potential future and suitable patient population when ready is discussed in this perspective. The selection of patients are important for adoption of axon regeneration specifically in the areas of central nervous system regenerative medicine. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology Neurological Diseases > Biomedical Engineering Metabolic Diseases > Molecular and Cellular Physiology.

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies.

Interconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.