sirolimus and Cataract

Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation.. A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest.. In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy.. We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

Topics: Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Cataract; Diabetes Mellitus; Epithelial-Mesenchymal Transition; Glucose; Humans; Intracellular Signaling Peptides and Proteins; Mammals; Platelet Aggregation Inhibitors; Rats; Sirolimus; TOR Serine-Threonine Kinases

The mechanistic target of rapamycin (mTOR) signal pathway plays a critical regulating role in the occurrence and development of cataract. However, the role of mTORC1 downstream proteins, including ribosomal protein S6K (RP-S6K), eukaryotic initiation factor 4E-binding protein (EIF4EBP), eukaryotic initiation factor 4G (EIF-4G), eukaryotic initiation factor 4E (EIF-4E), and eukaryotic initiation factor 4A (EIF-4A), in regulating cataract development is still unknown. Herein, we conducted a mendelian randomization (MR) study to understand the function of mTORC1 signaling in the process of cataract development.. The causal estimate was evaluated with inverse-variance weighted (IVW) estimate, weighted median estimator, MR-Egger and MR robust adjusted profile score (MR. RAPS). The single-nucleotide polymorphisms (SNPs), P<5 × 10. The MR study suggests that EIF4EBP is a high-risk factor for cataract development. There may be a potential causal association between the mTORC1/EIF4EBP axis and cataract. This research highlights the potential mechanism for cataract development and a genetic target to prevent as well as treat cataracts.

Topics: Cataract; Eukaryotic Initiation Factor-4A; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Genome-Wide Association Study; Humans; Mechanistic Target of Rapamycin Complex 1; Mendelian Randomization Analysis; Polymorphism, Single Nucleotide; Sirolimus; TOR Serine-Threonine Kinases

Autophagic dysfunction and abnormal oxidative stress are associated with cataract. The purpose of the present study was to investigate the changes of cellular autophagy and oxidative stress and their association in lens epithelial cells (LECs) upon exposure to high glucose.. Autophagy and oxidative stress-related changes were detected in streptozotocin-induced Type 1 diabetic mice and normal mouse LECs incubated in high glucose conditions. Rapamycin at a concentration of 100 nm/l or 50 μM chloroquine was combined for analysis of the relationship between autophagy and oxidative stress. The morphology of LECs during autophagy was observed by transmission electron microscopy. The expressions of autophagy markers (LC3B and p62) were identified, as well as the key factors of oxidative stress (SOD2 and CAT) and mitochondrial reactive oxygen species (ROS) generation.. Transmission electron microscopy indicated an altered autophagy activity in diabetic mouse lens tissues with larger autophagosomes and multiple mitochondria. Regarding the expressions, LC3B was elevated, p62 was decreased first and then increased, and SOD2 and CAT were increased before a decrease during 4 months of follow-up in diabetic mice and 72 h of culture under high glucose for mouse LECs. Furthermore, rapamycin promoted the expressions of autophagy markers but alleviated those of oxidative stress markers, whereas chloroquine antagonized autophagy but enhanced oxidative stress by elevating ROS generation in LECs exposed to high glucose.. The changes in autophagy and oxidative stress were fluctuating in the mouse LECs under constant high glucose conditions. Autophagy might attenuate high glucose-induced oxidative injury to LECs.

Topics: Animals; Autophagy; Cataract; Cells, Cultured; Chloroquine; Culture Media; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Epithelial Cells; Glucose; Humans; Lens Capsule, Crystalline; Male; Mice; Mitochondria; Oxidative Stress; Primary Cell Culture; Reactive Oxygen Species; Sirolimus; Streptozocin

Rapamycin increases lifespan in mice, but whether this represents merely inhibition of lethal neoplastic diseases, or an overall slowing in multiple aspects of aging is currently unclear. We report here that many forms of age-dependent change, including alterations in heart, liver, adrenal glands, endometrium, and tendon, as well as age-dependent decline in spontaneous activity, occur more slowly in rapamycin-treated mice, suggesting strongly that rapamycin retards multiple aspects of aging in mice, in addition to any beneficial effects it may have on neoplastic disease. We also note, however, that mice treated with rapamycin starting at 9 months of age have significantly higher incidence of testicular degeneration and cataracts; harmful effects of this kind will guide further studies on timing, dosage, and tissue-specific actions of rapamycin relevant to the development of clinically useful inhibitors of TOR action.

Topics: Adrenal Gland Neoplasms; Aging; Animals; Cataract; Endometrium; Female; Liver; Longevity; Male; Mice; Mice, 129 Strain; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Motor Activity; Myocytes, Cardiac; Organ Specificity; Sirolimus; Tendons; Testis; TOR Serine-Threonine Kinases

Posterior capsular opacification (PCO) is caused by the proliferation and migration of residual lens epithelium cells (LECs) after extracapsular cataract extraction (ECCE). Rapamcin (RAPA) is known to be a potent immunosuppressive drug with anti-inflammatory and anti-proliferative effects. The aim of this study was to investigate the safety and efficacy of rapamycin sustained release from modified intraocular lens (IOLs) in the prevention of PCO in rabbits.. Three types of IOLs were used, including the original IOL without modification, IOL with polylactide-glycoli acid (PLGA) coating (PLGA-IOL), and RAPA-loaded PLGA-IOL (RAPA-PLGA-IOL). Sixty New Zealand albino rabbits undergoing phacoemulsification in left eyes were randomly and equally divided into three groups. Group A was implanted with the original IOLs, group B was implanted with the PLGA-IOLs, and group C was implanted with the RAPA-PLGA-IOLs. All of the 60 treated left eyes were examined by a slit-lamp microscope. The concentrations of RAPA in the aqueous humor and blood were determined by high-performance liquid chromatography (HPLC), indicating an vivo release of drug from the polymer carrier. Anterior segment tissue was histologically examined, and wet posterior capsules were weighed. Six months after intervention the PCO was graded.. The mean concentrations of RAPA in the aqueous humor from group C at 2 h, 1 days, 3 days, and 7 days after operation were 12.81 +/- 1.27 microg/ml, 14.57 +/- 0.99 microg/ml, 6.39 +/- 0.95 microg/ml, and 1.10 +/- 0.32 microg /ml respectively. The concentrations of RAPA in blood were undetectable. During the early days after the operation, the reactions of the anterior chamber from groups A and B were more severe than from group C. Our findings showed that the initial appearance of PCO in group C was much later than in the other two groups. The wet posterior capsules were weighed to be 0.3735 +/- 0.0943 g (group A), 0.3754 +/- 0.1093 g (group B), and 0.0432 +/- 0.0089 g (group C). Histological observation showed a similar phenomenon, that there was remarkably less accumulation of lens materials on the posterior capsules in group C than in the other two groups.. Our findings suggest that the designed RAPA-PLGA-IOL effectively prevented formation and development of PCO for a relatively long duration.

Topics: Animals; Aqueous Humor; Cataract; Cell Proliferation; Chromatography, High Pressure Liquid; Coated Materials, Biocompatible; Drug Carriers; Epithelial Cells; Immunosuppressive Agents; Lactic Acid; Lens Capsule, Crystalline; Lens Implantation, Intraocular; Lenses, Intraocular; Phacoemulsification; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Postoperative Complications; Rabbits; Sirolimus