Lysosomal Instability Research Articles

Mechanism of protective action of mangiferin on suppression of inflammatory response and lysosomal instability in rat model of myocardial infarction

Lysosomal instability has been suggested as a major factor in the development of cellular injury during myocardial necrosis through the formation of inflammatory mediators. The present study was designed to investigate the effect of mangiferin on lysosomal hydrolases and TNF-alpha production during isoproterenol (ISPH) induced myocardial necrosis in rats. The rats given ISPH (200 mg/kg body weight twice, subcutaneous) for 2 days showed a significant increase in plasma TNF-alpha production, serum and heart lysosomal hydrolases activity. ISPH administration to rats resulted in decreased stability of the membranes, which was reflected by the lowered activity of cathepsin-D and beta-glucuronidase in mitochondrial, nuclear, lysosomal and microsomal fractions. Pretreatment with mangiferin (100 mg/kg body weight, intraperitoneally) for 28 days, significantly prevented the alterations and restored the enzyme activities to near-normal status. These findings demonstrate that mangiferin could preserve lysosomal integrity through decrease in the inflammatory process and hence establish the cardioprotective effect of mangiferin.

Cross Dendritic Cells Anger T Cells after Kidney Injury

Nearly 30 yr ago, Stewart Cameron caused a minor revolution by pointing out that simple quantitation of proteinuria was much better at predicting outcome than histopathological diagnosis—then the gold standard prognostic marker.1 We now know that severe proteinuria does not simply reflect glomerular injury but is itself harmful.2 Reabsorption of large amounts of protein from the glomerular filtrate induces stress responses in proximal tubular epithelial cells that result in lysosomal instability and can even initiate epithelial-mesenchymal transition.3 Epithelial cells synthesize chemokines, cytokines, and complement components that recruit inflammatory cells and lymphocytes into the interstitium causing progressive fibrosis. A fascinating paper by Macconi et al. from Ariela Benigni's group in the current issue of JASN4 adds another layer of complexity by showing that albumin reabsorbed from proximal tubules induces the generation of albumin-specific, IFN-γ secreting, CD8 T cells that may contribute to progressive renal injury. Before discussing the results in detail, it is important to consider how proteins reabsorbed by the proximal tubule are presented to T cells. The interstitium of normal kidneys contain numerous resident monocytic myelocytes, traditionally believed to be macrophages because they express relevant markers such as F4/80 in mice and CD68 in man.5 It is now known they also express dendritic cell markers and can indeed present antigen—one of the defining features of renal dendritic cells with …

A role for p53 in the regulation of lysosomal permeability by Δ9‐tetrahydrocannabinol in rat cortical neurones: implications for neurodegeneration

The psychoactive ingredient of marijuana, Delta9-tetrahydrocannabinol (Delta9-THC), can evoke apoptosis in cultured cortical neurones. Whilst the intracellular mechanisms responsible for this apoptotic pathway remain to be fully elucidated, we have recently identified a role for the CB1 type of cannabinoid (CB) receptor and the tumour suppressor protein, p53. In the current study, we demonstrate the Delta9-THC promotes a significant increase in lysosomal permeability in a dose- and time-dependent manner. The increase in lysosomal permeability was blocked by the CB1 receptor antagonist, AM251. Delta9-THC increased the localization of phospho-p53Ser15 at the lysosome and stimulated the release of the lysosomal cathepsin enzyme, cathepsin-D, into the cytosol. The p53 inhibitor, pifithrin-alpha and small interfering RNA-mediated knockdown of p53 prevented the Delta9-THC-mediated increase in lysosomal permeability. Furthermore, the Delta9-THC -mediated induction of apoptosis was abrogated by a cell-permeable cathepsin-D inhibitor (10 microM). Thus, the study demonstrates that Delta9-THC impacts on the lysosomal system, via p53, to evoke lysosomal instability as an early event in the apoptotic cascade. This provides evidence for a novel link between the CB1 receptor and the lysosomal branch of the apoptotic pathway which is crucial in regulating neuronal viability following exposure to Delta9-THC.

Lysosomal dysfunction on hydrogen peroxide-induced apoptosis of osteoarthritic chondrocytes

The purpose of this study was to examine the mechanism of hydrogen peroxide-induced apoptosis in osteoarthritic chondrocytes. We evaluated the reactive oxygen species (ROS) formation, lysosomal staining and dysfunction of the mitochondrial membrane potential in these cells after exposure to hydrogen peroxide. Osteoarthritic chondrocytes were isolated, and divided into 4 dishes in which different concentrations (0.1 mM, 1 mM and 10 mM) of hydrogen peroxide, or no additive (control) was added. The cells were incubated for 1 or 4 h, then assayed for ROS formation, mitochondrial membrane potential and lysosomal staining. ROS formation was detected in chondrocytes after 1 h of exposure to hydrogen peroxide concentrations over 0.1 mM. Lysosomal swelling was detected after 1 h of exposure to hydrogen peroxide concentrations of 0.1 mM and over, possibly revealing lysosomal membrane instability. Moreover, indications of lysosomal rupture, including release of lysosomal enzymes, were apparent 1 h after addition of 10 mM of hydrogen peroxide. The addition of hydrogen peroxide to chondrocytes induces ROS formation and lysosomal dysfunction, revealed by swelling and rupture, prior to dysfunction of the mitochondrial membrane potential. Anti-oxidants may have a therapeutic application in the prevention of lysosomal dysfunction to inhibit chondrocyte apoptosis and degradation of the cartilage matrix.

Reactive oxygen species-producing site in radiation-induced apoptosis of human peripheral T cells: Involvement of lysosomal membrane destabilization

In our previous study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that the addition of 1 or 10 mM hydrogen peroxide induced reactive oxygen species (ROS) formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation was involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells. In contrast to the osteosarcoma cell line HS-Os-1, human peripheral T cells are considered to be easily susceptible to oxidative stress, because these cells lack peroxidase activity. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichloro-dihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently diffusely distributed in T cells oxidatively stressed with 0.1 mM hydrogen peroxide. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane destabilization, were observed in these cells. Based on the above results, there exists an apoptotic cascade involving early lysosomal membrane destabilization in the hydrogen peroxide-induced apoptosis of human peripheral T cells. Therefore, the possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in hydrogen peroxide-induced T cell apoptosis.

α-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability

Certain vitamin E analogues, such as α-tocopheryl succinate (α-TOS), exhibit in vivo anti-tumour activity and, in vitro, induce apoptosis of cultured tumour cells. In the present study we report that these effects may be explained, at least in part, by destabilization of lysosomal membranes. α-TOS, but not α-tocopheryl acetate or α-tocopherol (α-TOH), induced early lysosomal destabilization followed by apoptosis. Similar effects were observed with β-TOS, whereas β-TOH was inactive. Cathepsin D-deficient cells were more resistant to α-TOS than their normal counterparts, and featured delayed caspase activation. Possible detergent and lysosomotropic effects of α- and β-TOS were suggested by their haemolytic activity in an in vitro test and their release of β-galactosidase from isolated lysosomes, whereas the non-succinylated analogues were inactive. The pro-apoptotic activity of α-TOS was pH-dependent, being greater at lower pH, typical of the interstitium of solid tumours. These findings indicate that lysosomal destabilization may partially or fully explain the induction of apoptosis in cultured cells by α-TOS and the mechanism whereby this agent exerts in vivo anti-tumour effects.

Alpha-tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability.

Certain vitamin E analogues, such as alpha-tocopheryl succinate (alpha-TOS), exhibit in vivo anti-tumour activity and, in vitro, induce apoptosis of cultured tumour cells. In the present study we report that these effects may be explained, at least in part, by destabilization of lysosomal membranes. alpha-TOS, but not alpha-tocopheryl acetate or alpha-tocopherol (alpha-TOH), induced early lysosomal destabilization followed by apoptosis. Similar effects were observed with beta-TOS, whereas beta-TOH was inactive. Cathepsin D-deficient cells were more resistant to alpha-TOS than their normal counterparts, and featured delayed caspase activation. Possible detergent and lysosomotropic effects of alpha- and beta-TOS were suggested by their haemolytic activity in an in vitro test and their release of beta-galactosidase from isolated lysosomes, whereas the non-succinylated analogues were inactive. The pro-apoptotic activity of alpha-TOS was pH-dependent, being greater at lower pH, typical of the interstitium of solid tumours. These findings indicate that lysosomal destabilization may partially or fully explain the induction of apoptosis in cultured cells by alpha-TOS and the mechanism whereby this agent exerts in vivo anti-tumour effects.

Beneficial effects of ibuprofen in pacing-induced myocardial ischemia

Lysosomal membrane instability and platelet activation are both associated with acute myocardial ischemia. The effect of ibuprofen on cathepsin D as a marker of lysosomal membrane "leakiness" and thromboxane B2 as a marker of platelet activation was evaluated in 44 patients with angina pectoris. Samples of blood analyzed for cathepsin D, thromboxane B2, and lactate were withdrawn from the coronary sinus and brachial artery before and after pacing to 140 beats/min for 4 minutes. Myocardial ischemia was assessed by determination of transmyocardial lactate extraction or production. Ibuprofen (800 mg) or placebo was administered orally 2 hours before cardiac catheterization. Patients were classified into 4 groups on the basis of administration of placebo or ibuprofen and the presence or absence of myocardial ischemia as determined by demonstration of lactate extraction or production after atrial pacing. In patients with lactate extraction, no significant efflux of cathepsin D or thromboxane B2 occurred after pacing. In patients with lactate production given placebo, a 64 +/- 25% increase in the thromboxane B2 level and a 113 +/- 37% increase in cathepsin D activities occurred in the coronary sinus effluent sampled after pacing. In contrast, in patients with comparable coronary artery disease and comparable lactate production who were given ibuprofen, no release of thromboxane B2 (p = 0.05 compared with patients given placebo) or cathepsin D (p less than 0.01 compared with patients given placebo) occurred after pacing-induced ischemia. These findings suggest that ibuprofen stabilizes membranes and prevents platelet-activated release of thromboxane A2 in pacing-induced myocardial ischemia.

Lipid peroxidation and lysosomal enzymes in D-galactosamine hepatitis and its protection by vitamin E

Role of lipid peroxidation on lysosomal instability in liver tissue was investiaged in an experimental model of D-galactosamine hepatitis in rats fed on vitamin E (V.E) deficient diet. Administration of D-galactisamine to V.E deficient rats resulted in a sudden increase of serum glutamic oxaloacetic transaminase (sGOT), glutamic pyruvic transaminase (sGPT), lipid peroxide value, as well as beta-glucuronidase and acid phosphatase activity examined as markers of lysosomal enzymes, when compared with control rats fed on V.E supplemented diet. Lipid peroxide in the liver tissue also showed significant increase in V.E deficient rats. In contrast, beta-glucuronidase and acid phosphatase in the liver tissue were found to decrease in V.E deficient rats by the administration of D-galactosamine, indicating that the enzymes in the lysosome were entirely released outside the liver cells as a result of cell destruction. It is concluded that the increase of lipid peroxide causes the instability of lysosomal membranes and releases various kinds of hydrolytic enzymes to lead further to cell damage. V.E might act on inhibiting lipid peroxidation to stabilize lysosomal membranes.

Studies on the Pathogenesis of Rheumatoid Arthritis

Sera and synovial fluids from patients with rheumatoid arthritis (RA), and with osteoarthritis (OA) as a control, were investigated for lipid peroxide level, β-glucuronidase activity, acid phosphatase activity, and α-tocopherol level. Lipid peroxide level β-glucuronidase activity and acid phosphatase activity were found to increase in synovial fluids of RA patients, suggesting that the generation of lipid peroxide in the rheumatoid joints plays a role in the liberation of lysosomal enzymes into the synovial fluids. Signiflcant correlation was demonstrated between lipid peroxide and β-glucuronidase, and or acid phosphatase in synovial fluids from RA patients. Increased α-tocopherol level in RA synovial fluids might be explained by the increased vascular permiability in the rheumatoid joints since there was no difference in α-tocopherol level between RA and OA sera. The data led us to conclude that the increase of lipid peroxide in the synovial fluids in the rheumatoid joints played an important role in lysosomal instability of either synovial lining cells or influxed polymorphonuclear leukocytes.

Nutrition and lysosomal activity. The influence of vitamin E deficiency and its duration on the stability of lysosomes in the kidneys of rats.

1. Experiments on rats were made to find whether the increased liability of the kidney-cortex tubules to autolysis post mortem, which is a well-established abnormality in vitamin E deficiency, can be correlated with changes in lysosomal activity. Parallel observations were made on the development of certain other abnormalities characteristic of avitaminosis E. 2. In rats killed after long periods (8-10 months) of subsistence on a standard vitamin E-deficient diet, containing lard, both the rate of kidney autolysis post mortem and the enzyme activity of lysosome preparations from the fresh tissues were much greater than in controls. A greater percentage difference was usually found in the ;free' enzyme fraction than in ;bound' or ;total' activity. 3. In rats killed after graded periods (3-8 months) of deficiency, two abnormalities (decreased resistance of the erythrocytes to haemolysis, and brown discoloration of the uterus) were already evident at a stage (3-4 months) when the liability to rapid kidney autolysis had not begun. At this point the enzymic activity of kidney extracts differed little between deficient animals and controls given alpha-tocopherol. As the duration of deficiency advanced, parallel increases occurred in the rate of kidney autolysis and in lysosomal instability. 4. When cod-liver oil, rich in polyunsaturated fatty acids but freed from vitamin A, was substituted for lard in the diet, the time (1(1/2) months) required for the inducement of both rapid kidney autolysis and decreased lysosomal stability was greatly shortened. The time for the inducement of brown discoloration of the uterus was not shortened and the kidney abnormalities appeared while the uterus was still normal. 5. Confirmation was thus obtained for the view that the various tissues of the rat respond differently to the relationship between the adequacy of the vitamin E status and the intake of polyunsaturated fatty acids. The kidney-cortex tubules, as evidenced by autolysis post mortem and the corresponding decrease in lysosomal stability, may be classed among those tissues that are most sensitive to the effect of high intakes of polyunsaturated acids.