Cholesterol crystal formation is a unifying pathogenic mechanism in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: Hyper-reflective crystalline deposits found in retinal lesions have been suggested to predict the progression of diabetic retinopathy, but the nature of these structures remains unknown. METHODS: Scanning electron microscopy and immunohistochemistry were used to identify cholesterol crystals (CCs) in human donor, pig and mouse tissue. The effects of CCs were analysed in bovine retinal endothelial cells in vitro and in db/db mice in vivo using quantitative RT-PCR, bulk RNA sequencing, and cell death and permeability assays. Cholesterol homeostasis was determined using 2H2O and 2H7-cholesterol. RESULTS: We identified hyper-reflective crystalline deposits in human diabetic retina as CCs. Similarly, CCs were found in the retina of a diabetic mouse model and a high-cholesterol diet-fed pig model. Cell culture studies demonstrated that treatment of retinal cells with CCs can recapitulate all major pathogenic mechanisms leading to diabetic retinopathy, including inflammation, cell death and breakdown of the blood-retinal barrier. Fibrates, statins and α-cyclodextrin effectively dissolved CCs present in in vitro models of diabetic retinopathy, and prevented CC-induced endothelial pathology. Treatment of a diabetic mouse model with α-cyclodextrin reduced cholesterol levels and CC formation in the retina, and prevented diabetic retinopathy. CONCLUSIONS/INTERPRETATION: We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy.

2-Hydroxypropyl-ß-cyclodextrin mitigates pathological changes in a mouse model of retinal cholesterol dyshomeostasis.

CYP46A1 is a CNS-specific enzyme, which eliminates cholesterol from the brain and retina by metabolism to 24-hydroxycholesterol, thus contributing to cholesterol homeostasis in both organs. 2-Hydroxypropyl-ß-cyclodextrin (HPCD), a Food and Drug Administration-approved formulation vehicle, is currently being investigated off-label for treatment of various diseases, including retinal diseases. HPCD was shown to lower retinal cholesterol content in mice but had not yet been evaluated for its therapeutic benefits. Herein, we put Cyp46a1-/- mice on high fat cholesterol-enriched diet from 1 to 14 months of age (control group) and at 12 months of age, started to treat a group of these animals with HPCD until the age of 14 months. We found that as compared with mature and regular chow-fed Cyp46a1-/- mice, control group had about 6-fold increase in the retinal total cholesterol content, focal cholesterol and lipid deposition in the photoreceptor-Bruch's membrane region, and retinal macrophage activation. In addition, aged animals had cholesterol crystals at the photoreceptor-retinal pigment epithelium interface and changes in the Bruch's membrane ultrastructure. HPCD treatment mitigated all these manifestations of retinal cholesterol dyshomeostasis and altered the abundance of six groups of proteins (genetic information transfer, vesicular transport, and cytoskeletal organization, endocytosis and lysosomal processing, unfolded protein removal, lipid homeostasis, and Wnt signaling). Thus, aged Cyp46a1-/- mice on high fat cholesterol-enriched diet revealed pathological changes secondary to retinal cholesterol overload and supported further studies of HPCD as a potential therapeutic for age-related macular degeneration and diabetic retinopathy associated with retinal cholesterol dyshomeostasis.

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Selective LXR agonist DMHCA corrects retinal and bone marrow dysfunction in type 2 diabetes.

In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) make up the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N, N-dimethyl-3ß-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. In this study, we use a multisystem approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic (db/db) mice and human circulating angiogenic cells (CACs), which are hematopoietic progenitor cells with vascular reparative capacity. We found that DMHCA is sufficient to correct retinal and BM dysfunction in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis; rejuvenating membrane fluidity in CACs; hampering systemic inflammation; and correcting BM pathology. Using single-cell RNA sequencing on lineage-sca1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide potentially novel insights into hematopoiesis and reveal DMHCA's mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the beneficial effects of DMHCA treatment on diabetes-induced retinal and BM pathology.

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1ß, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 µM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Extracellular Vesicle-Induced Classical Complement Activation Leads to Retinal Endothelial Cell Damage via MAC Deposition.

Several studies have suggested that there is a link between membrane attack complex (MAC) deposition in the retina and the progression of diabetic retinopathy (DR). Our recent investigation demonstrated that circulating IgG-laden extracellular vesicles contribute to an increase in retinal vascular permeability in DR through activation of the complement system. However, the mechanism through which extracellular vesicle-induced complement activation contributes to retinal vascular cytolytic damage in DR is not well understood. In this study, we demonstrate that IgG-laden extracellular vesicles in rat plasma activate the classical complement pathway, and in vitro Streptozotocin (STZ)-induced rat diabetic plasma results in MAC deposition and cytolytic damage in human retinal endothelial cells (HRECs). Moreover, removal of the plasma extracellular vesicles reduced the MAC deposition and abrogated cytolytic damage seen in HRECs. Together, the results of this study demonstrate that complement activation by IgG-laden extracellular vesicles in plasma could lead to MAC deposition and contribute to endothelium damage and progression of DR.

The peroxisome proliferator-activated receptor-ß/δ antagonist GSK0660 mitigates retinal cell inflammation and leukostasis.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARß/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARß/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARß/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARß/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARß/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARß/δ inhibition is a potential therapeutic strategy against early DR pathology.

Micro-respirometry of whole cells and isolated mitochondria.

Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O2, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O2 consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O2 permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 vs. 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100× dynamic range for O2 consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O2 consumption rates at any desired [O2], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O2 uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.

Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy by Activating the Classical Complement Pathway.

Diabetic retinopathy (DR) is a microvascular complication of diabetes and is the leading cause of vision loss in working-age adults. Recent studies have implicated the complement system as a player in the development of vascular damage and progression of DR. However, the role and activation of the complement system in DR are not well understood. Exosomes, small vesicles that are secreted into the extracellular environment, have a cargo of complement proteins in plasma, suggesting that they can participate in causing the vascular damage associated with DR. We demonstrate that IgG-laden exosomes in plasma activate the classical complement pathway and that the quantity of these exosomes is increased in diabetes. Moreover, we show that a lack of IgG in exosomes in diabetic mice results in a reduction in retinal vascular damage. The results of this study demonstrate that complement activation by IgG-laden plasma exosomes could contribute to the development of DR.

ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability.

Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1ß-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.

The Mechanism of Diabetic Retinopathy Pathogenesis Unifying Key Lipid Regulators, Sirtuin 1 and Liver X Receptor.

Diabetic retinopathy (DR) is a complication secondary to diabetes and is the number one cause of blindness among working age individuals worldwide. Despite recent therapeutic breakthroughs using pharmacotherapy, a cure for DR has yet to be realized. Several clinical trials have highlighted the vital role dyslipidemia plays in the progression of DR. Additionally, it has recently been shown that activation of Liver X receptor (LXRα/LXRß) prevents DR in diabetic animal models. LXRs are nuclear receptors that play key roles in regulating cholesterol metabolism, fatty acid metabolism and inflammation. In this manuscript, we show insight into DR pathogenesis by demonstrating an innovative signaling axis that unifies key metabolic regulators, Sirtuin 1 and LXR, in modulating retinal cholesterol metabolism and inflammation in the diabetic retina. Expression of both regulators, Sirtuin 1 and LXR, are significantly decreased in diabetic human retinal samples and in a type 2 diabetic animal model. Additionally, activation of LXR restores reverse cholesterol transport, prevents inflammation, reduces pro-inflammatory macrophages activity and prevents the formation of diabetes-induced acellular capillaries. Taken together, the work presented in this manuscript highlights the important role lipid dysregulation plays in DR progression and offers a novel potential therapeutic target for the treatment of DR.

The role of dyslipidemia in diabetic retinopathy.

Diabetic retinopathy (DR) affects over 93million people worldwide and is the number one cause of blindness among working age adults. These indicators coupled with the projected rise of patients diagnosed with diabetes, makes DR a serious and prevalent vision threating disease. Data from recent clinical trials demonstrate that in addition to the well accepted role of hyperglycemia, dyslipidemia is an important, but often overlooked factor in the development of DR. The central aim of this review article is to showcase the critical role of dyslipidemia in DR progression as well as highlight novel therapeutic solutions that take advantage of the vital roles lipid metabolism plays in DR progression.

Epoxygenated Fatty Acids Inhibit Retinal Vascular Inflammation.

The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.