Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.

Chronic exposure of pancreatic ß cells to high concentrations of free fatty acids leads to lipotoxicity (LT)-mediated suppression of glucose-stimulated insulin secretion. This effect is in part caused by a decline in mitochondrial function as well as by a reduction in lysosomal acidification. Because both mitochondria and lysosomes can alter one another's function, it remains unclear which initiating dysfunction sets off the detrimental cascade of LT, ultimately leading to ß-cell failure. Here, we investigated the effects of restoring lysosomal acidity on mitochondrial function under LT. Our results show that LT induces a dose-dependent lysosomal alkalization accompanied by an increase in mitochondrial mass. This increase is due to a reduction in mitochondrial turnover as analyzed by MitoTimer, a fluorescent protein for which the emission is regulated by mitochondrial clearance rate. Mitochondrial oxygen consumption rate, citrate synthase activity, and ATP content are all reduced by LT. Restoration of lysosomal acidity using lysosome-targeted nanoparticles is accompanied by stimulation of mitochondrial turnover as revealed by mitophagy measurements and the recovery of mitochondrial mass. Remarkably, re-acidification restores citrate synthase activity and ATP content in an insulin secreting ß-cell line (INS-1). Furthermore, nanoparticle-mediated lysosomal reacidification rescues mitochondrial maximal respiratory capacity in both INS-1 cells and primary mouse islets. Therefore, our results indicate that mitochondrial dysfunction is downstream of lysosomal alkalization under lipotoxic conditions and that recovery of lysosomal acidity is sufficient to restore the bioenergetic defects.-Assali, E. A., Shlomo, D., Zeng, J., Taddeo, E. P., Trudeau, K. M., Erion, K. A., Colby, A. H., Grinstaff, M. W., Liesa, M., Las, G., Shirihai, O. S. Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.

Elamipretide Promotes Mitophagosome Formation and Prevents Its Reduction Induced by Nutrient Excess in INS1 ß-cells.

Elamipretide is a tetrapeptide that restores defects in mitochondrial function, binds to cardiolipin, and is being tested in clinical trials for mitochondria-related diseases. However, whether elamipretide modulates mitochondrial quality control and dynamics, processes essential to preserve mitochondrial function, is unclear. Thus, we tested the effects of elamipretide on mitochondrial morphology, mitophagosome formation, and their early disruption induced by excess nutrients in INS1 ß-cells. Elamipretide treatment was sufficient to increase engulfment of mitochondria into autophagosomes in control INS1 ß-cells, without inducing widespread changes in mitochondrial morphology or membrane potential. In an early pathogenic context mimicked by short-term exposure to nutrient excess, elamipretide treatment prevented both mitochondrial fragmentation and defects in the engulfment of mitochondria into autophagosomes. On the other hand, elamipretide did not prevent lysosomal defects induced by nutrient excess. Accordingly, elamipretide treatment did not entail benefits on pathogenic p62 and LC3II accumulation or on insulin secretory function. In conclusion, our data show that elamipretide selectively stimulates the engulfment of mitochondria into autophagosomes and prevents its defects induced by nutrient excess. Thus, we propose that improved selectivity of mitochondrial quality control processes might contribute to the benefits stemming from elamipretide treatments in other disease models.

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis.

BAT-controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet-induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold-stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1-cre (BAT-Mfn2-KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold-stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender-specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP-synthesizing fat oxidation, whereas in BAT from males, complex I-driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT-Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole-body cold-stimulated thermogenesis.

Modulation of mTOR signaling as a strategy for the treatment of Pompe disease.

Mechanistic target of rapamycin (mTOR) coordinates biosynthetic and catabolic processes in response to multiple extracellular and intracellular signals including growth factors and nutrients. This serine/threonine kinase has long been known as a critical regulator of muscle mass. The recent finding that the decision regarding its activation/inactivation takes place at the lysosome undeniably brings mTOR into the field of lysosomal storage diseases. In this study, we have examined the involvement of the mTOR pathway in the pathophysiology of a severe muscle wasting condition, Pompe disease, caused by excessive accumulation of lysosomal glycogen. Here, we report the dysregulation of mTOR signaling in the diseased muscle cells, and we focus on potential sites for therapeutic intervention. Reactivation of mTOR in the whole muscle of Pompe mice by TSC knockdown resulted in the reversal of atrophy and a striking removal of autophagic buildup. Of particular interest, we found that the aberrant mTOR signaling can be reversed by arginine. This finding can be translated into the clinic and may become a paradigm for targeted therapy in lysosomal, metabolic, and neuromuscular diseases.

Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity.

In pancreatic ß-cells, liver hepatocytes, and cardiomyocytes, chronic exposure to high levels of fatty acids (lipotoxicity) inhibits autophagic flux and concomitantly decreases lysosomal acidity. Whether impaired lysosomal acidification is causally inhibiting autophagic flux and cellular functions could not, up to the present, be determined because of the lack of an approach to modify lysosomal acidity. To address this question, lysosome-localizing nanoparticles are described that, upon UV photoactivation, enable controlled acidification of impaired lysosomes. The photoactivatable, acidifying nanoparticles (paNPs) demonstrate lysosomal uptake in INS1 and mouse ß-cells. Photoactivation of paNPs in fatty acid-treated INS1 cells enhances lysosomal acidity and function while decreasing p62 and LC3-II levels, indicating rescue of autophagic flux upon acute lysosomal acidification. Furthermore, paNPs improve glucose-stimulated insulin secretion that is reduced under lipotoxicity in INS1 cells and mouse islets. These results establish a causative role for impaired lysosomal acidification in the deregulation of autophagy and ß-cell function under lipotoxicity.

Murine Mesenchymal Stem Cell Commitment to Differentiation Is Regulated by Mitochondrial Dynamics.

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105(+) CD90(+) CD73(+) CD29(+) CD34(-) mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells.

Measurement of mitochondrial turnover and life cycle using MitoTimer.

Current methodologies available to quantify changes in mitochondrial turnover are limited to pulse-chase assays or specific assays that quantify mitophagy. Accordingly, new tools that can assess mitochondrial turnover are needed for the study of cellular, subcellular, and spatial parameters of mitochondrial turnover and quality control. Recently, a group of studies described the use of the MitoTimer fluorescent probe to investigate various aspects of mitochondrial turnover, including changes to protein import, interorganelle protein sharing, and autophagy-mediated turnover. MitoTimer provides a fluorescent readout which directly relates to the mitochondrial turnover rate and allows quantification of relative changes to turnover. Importantly, MitoTimer can be used to investigate mitochondrial turnover on the subcellular level. Due to the fact that MitoTimer is a dual-emission probe and a number of factors can affect MitoTimer readout, certain considerations must be taken into account when using this tool both in experimental design and data interpretation. When used and interpreted appropriately, MitoTimer serves as a unique tool to understand pivotal aspects of mitochondrial turnover.

Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity.

AL amyloidosis is the consequence of clonal production of amyloidogenic immunoglobulin light chain (LC) proteins, often resulting in a rapidly progressive and fatal amyloid cardiomyopathy. Recent work has found that amyloidogenic LC directly initiate a cardio-toxic response underlying the pathogenesis of the cardiomyopathy; however, the mechanisms that contribute to this proteotoxicity remain unknown. Using human amyloidogenic LC isolated from patients with amyloid cardiomyopathy, we reveal that dysregulation of autophagic flux is critical for mediating amyloidogenic LC proteotoxicity. Restoration of autophagic flux by pharmacological intervention using rapamycin protected against amyloidogenic light chain protein-induced pathologies including contractile dysfunction and cell death at the cellular and organ level and also prolonged survival in an in vivo zebrafish model of amyloid cardiotoxicity. Mechanistically, we identify impaired lysosomal function to be the major cause of defective autophagy and amyloidogenic LC-induced proteotoxicity. Collectively, these findings detail the downstream molecular mechanisms underlying AL amyloid cardiomyopathy and highlight potential targeting of autophagy and lysosomal dysfunction in patients with amyloid cardiomyopathy.

MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age.

To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

Mitochondrial dysfunction and endoplasmic reticulum stress in diabetic retinopathy: mechanistic insights into high glucose-induced retinal cell death.

Hyperglycemia, a prominent characteristic of diabetes, has been implicated in the apoptotic death of vascular and neuronal cells in the retina. In diabetic retinopathy, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and subsequent breakdown of cellular homeostasis play a critical role in retinal cell death. In particular, changes in mitochondrial morphology, mitochondrial membrane potential heterogeneity, oxygen consumption rate and protein misfolding are beginning to be recognized as key players in the demise of retinal vascular cells in diabetes. Some of these key changes contribute to oxidative stress and influence ion transport, impacting overall cellular homeostasis. The primary objective of this review is to provide insight into the mechanisms in which high glucose influences two disparate cellular organelles, mitochondria and ER, in promoting apoptotic demise of retinal vascular and neuronal cells in diabetic retinopathy.

Downregulation of mitochondrial connexin 43 by high glucose triggers mitochondrial shape change and cytochrome C release in retinal endothelial cells.

PURPOSE: To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells. METHODS: MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with ß-glycerrhetinic acid (ß-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with ß-GA, and cytochrome c release assessed by Western blot. RESULTS: Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with ß-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with ß-GA showed increased cytochrome c release. CONCLUSIONS: HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.

A faster, high resolution, mtPA-GFP-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy.

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment. Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary. Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment. A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP). Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE. The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity. In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15 nM TMRE in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

High glucose induces mitochondrial morphology and metabolic changes in retinal pericytes.

PURPOSE: Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, a prominent lesion of diabetic retinopathy. High glucose (HG) has been reported to induce mitochondrial fragmentation and dysfunction in retinal endothelial cells, contributing to apoptosis. In this study, the effects of HG on mitochondrial morphology, membrane potential, and metabolic changes and whether they could contribute to HG-induced apoptosis in retinal pericytes were investigated. METHODS: Bovine retinal pericytes (BRPs) were grown in normal or HG medium for 7 days. Both sets of cells were double stained with mitochondrial membrane potential-independent dye and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and imaged by confocal microscopy. The images were analyzed for average mitochondria shape, by using form factor and aspect ratio values, and membrane potential changes, by using the ratio between the red and green dye. BRPs grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification with a flux analyzer and apoptosis by TUNEL assay. RESULTS: BRPs grown in HG media exhibited significant fragmentation of mitochondria and increased membrane potential heterogeneity compared with the BRPs grown in normal medium. Concomitantly, BRPs grown in HG showed reduced steady state and maximum oxygen consumption and reduced extracellular acidification. Number of TUNEL-positive pericytes was increased in HG condition as well. CONCLUSIONS: In HG condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. The detrimental effects of HG on mitochondrial function and cellular metabolism could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.

High glucose-induced altered basement membrane composition and structure increases trans-endothelial permeability: implications for diabetic retinopathy.

PURPOSE: The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy. MATERIALS AND METHODS: To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis. RESULTS: Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02). CONCLUSIONS: High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.

Fenofibric acid reduces fibronectin and collagen type IV overexpression in human retinal pigment epithelial cells grown in conditions mimicking the diabetic milieu: functional implications in retinal permeability.

PURPOSE: To determine whether fenofibric acid (FA) reduces high glucose (HG)-induced basement membrane component overexpression and hyperpermeability in human retinal pigment epithelial (RPE) cells. METHODS: Retinal pigment epithelial cells (ARPE-19) were cultured for 18 days in normal glucose (5 mM) or HG (25 mM) medium and studied for the effects of FA on fibronectin (FN) and collagen IV (Coll IV) expression. During last 3 days of the experiment, 100 µM FA was added to cells grown in HG medium or in HG medium plus IL-1ß (HG + IL-1ß) to mimic, at least in part, the inflammatory aspect of the diabetic milieu. Real-time RT-PCR was performed to determine FN and Coll IV mRNA levels, whereas protein levels were assessed by Western blot analyses. Cell monolayer morphology and barrier function were analyzed by confocal microscopy using specific antibodies against tight junction proteins, ZO-1, and claudin-1 and by measuring apical-basolateral movements of FITC-dextran, respectively. RESULTS: FN and Coll IV expression were significantly increased in RPE cells grown in HG or HG + IL-1ß medium compared with cells grown in normal medium. When cells grown in HG or HG + IL-1ß medium were treated with FA, significant reductions in FN and Coll IV expression were observed. In addition, exposure to FA decreased excess permeability in a dose-dependent manner in cells grown in HG + IL-1ß medium. This effect was unrelated to changes in tight junction protein content. CONCLUSIONS: Findings from this study suggest that the downregulation of basement membrane components by FA may have a protective effect against outer blood-retinal barrier leakage associated with diabetic retinopathy.

Vascular basement membrane thickening in diabetic retinopathy.

Vascular basement membrane (BM) thickening is a fundamental structural alteration of small blood vessels in diabetes. Over two decades of research has established hyperglycemia as the primary causal factor mediating this alteration. Various high glucose-induced mechanisms have been investigated and excess synthesis of BM components has been identified as a major contributing factor to BM thickening. Although BM thickening has been long hailed as the histological hallmark of diabetic microangiopathy, the consequences of BM thickening on the functionality of target organs of diabetes remain elusive even today. This review presents an overview of our current understanding of the BM structure and function, and focuses on how capillary BM thickening develops, its effect on retinal vascular function, and potential strategies for preventing the development of BM thickening in diabetic retinopathy.

High glucose disrupts mitochondrial morphology in retinal endothelial cells: implications for diabetic retinopathy.

Mitochondrial dysfunction has been implicated in diabetic complications; however, it is unknown whether hyperglycemia affects mitochondrial morphology and metabolic capacity during development of diabetic retinopathy. We investigated high glucose (HG) effects on mitochondrial morphology, membrane potential heterogeneity, cellular oxygen consumption, extracellular acidification, cytochrome c release, and apoptosis in retinal endothelial cells. Rat retinal endothelial cells grown in normal (5 mmol/L) or HG (30 mmol/L) medium and double-stained with MitoTracker Green and tetramethylrhodamine-ethyl-ester-perchlorate were examined live with confocal microscopy. Images were analyzed for mitochondrial shape change using Form Factor and Aspect Ratio values, and membrane potential heterogeneity, using deviation of fluorescence intensity values. Rat retinal endothelial cells grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification using an XF-24 flux analyzer, cytochrome c release by Western blot, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Rat retinal endothelial cells grown in HG medium exhibited increased mitochondrial fragmentation concurrent with membrane potential heterogeneity. Metabolic analysis showed increased extracellular acidification in HG with reduced steady state/maximal oxygen consumption. Cytochrome c and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were also increased in HG. Thus, HG-induced mitochondrial fragmentation with concomitant increase in membrane potential heterogeneity, reduced oxygen consumption, and cytochrome c release may underlie apoptosis of retinal endothelial cells as seen in diabetic retinopathy.

Reduced connexin 43 expression and its effect on the development of vascular lesions in retinas of diabetic mice.

PURPOSE. To examine whether diabetes-induced connexin 43 downregulation promotes retinal vascular lesions characteristic of diabetic retinopathy (DR). METHODS. Two animal models, streptozotocin-induced diabetic mice and Cx43 heterozygous knockout (Cx43(+/-)) mice, were studied to directly assess whether diabetes reduces the expression of retinal Cx43, which, in turn, contributes to retinal vascular cell loss by apoptosis. Retinal Cx43 protein levels were assessed in nondiabetic control mice, diabetic mice, and Cx43(+/-) mice by Western blot analysis, and Cx43 localization and distribution in the retinal vascular cells were studied by immunostaining of retinal trypsin digests (RTDs). In parallel, RTDs were stained with hematoxylin and periodic acid Schiff to determine pericyte loss (PL) and acellular capillaries (AC), and TUNEL assays were performed to determine retinal vascular cell apoptosis. RESULTS. Western blot analysis indicated significant reductions in retinal Cx43 protein levels in diabetic mice and Cx43(+/-) mice compared with those of nondiabetic mice. Similarly, a significant reduction in Cx43 immunostaining was observed in the retinal capillaries of diabetic mice and Cx43(+/-) mice compared with those of control mice. Both diabetic and age-matched Cx43(+/-) mice exhibited increased amount of PL, AC, and TUNEL-positive cells compared with control mice. CONCLUSIONS. Diabetes-induced inhibition of Cx43 expression contributes to vascular cell apoptosis in retinas of diabetic mice. This suggests that reduced Cx43 expression plays a critical role in the development of AC and PL associated with DR.