GW27-e0262 A study on the mechanism of the Caveolin-1 in Ginsenoside Rb1 against the replicative senescence of endothelial cells

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) “young” and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

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Sulodexide reduces glucose induced senescence in human retinal endothelial cells

Scientific Reports ◽

10.1038/s41598-021-90987-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

A. Gericke ◽

K. Suminska-Jasińska ◽

A. Bręborowicz

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Glucose Concentration ◽

Chronic Exposure ◽

Replicative Senescence ◽

Population Doubling ◽

Population Doubling Time ◽

High Glucose Concentration ◽

Retinal Endothelial Cells

AbstractChronic exposure of retinal endothelium cells to hyperglycemia is the leading cause of diabetic retinopathy. We evaluated the effect of high glucose concentration on senescence in human retinal endothelial cells (HREC) and modulation of that effect by Sulodexide. Experiments were performed on HREC undergoing in vitro replicative senescence in standard medium or medium supplemented with glucose 20 mmol/L (GLU) or mannitol 20 mnol/L (MAN). Effect of Sulodexide 0.5 LRU/mL (SUL) on the process of HREC senescence was studied. Glucose 20 mmol/L accelerates senescence of HREC: population doubling time (+ 58%, p < 0.001) β-galactosidase activity (+ 60%, p < 0.002) intracellular oxidative stress (+ 65%, p < 0.01), expression of p53 gene (+ 118%, p < 0.001). Senescent HREC had also reduced transendothelial electrical resistance (TEER) (− 30%, p < 0.001). Mannitol 20 mmol/L used in the same scenario as glucose did not induce HREC senescence. In HREC exposed to GLU and SUL, the senescent changes were smaller. HREC, which became senescent in the presence of GLU, demonstrated higher expression of genes regulating the synthesis of Il6 and VEGF-A, which was reflected by increased secretion of these cytokines (IL6 + 125%, p < 0.001 vs control and VEGF-A + 124% p < 0.001 vs control). These effects were smaller in the presence of SUL, and additionally, an increase of TEER in the senescent HREC was observed. Chronic exposure of HREC to high glucose concentration in medium accelerates their senescence, and that process is reduced when the cells are simultaneously exposed to Sulodexide. Additionally, Sulodexide decreases the secretion of IL6 and VEGF-A from senescent HREC and increases their TEER.

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Abstract 776: Protein Disulfide Isomerase Is Involved In Redox Regulation Of Nitric Oxide Output During Laminar Shear Stress In Endothelial Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_149-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Denise C Fernandes ◽

Celio X Santos ◽

Hanjoong Jo ◽

Francisco R Laurindo

Keyword(s):

Endothelial Cells ◽

Nadph Oxidase ◽

Oxidase Activity ◽

Membrane Fraction ◽

Protein Disulfide Isomerase ◽

Disulfide Isomerase ◽

Nadph Oxidase Activity ◽

Caveolin 1 ◽

Protein Disulfide ◽

Laminar Shear

While anti-atherogenic effects of sustained laminar shear (LS) involve NO release from eNOS, increases in LS trigger transient superoxide production via NADPH oxidase. Recently, we showed that NADPH oxidase undergoes thiol-dependent regulation by the thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to promote NO internalization via trans-nitrosation reactions. We hypothesized that PDI-dependent support of NADPH oxidase activity affects NO output during sustained LS. Cultured rabbit aortic endothelial cells (RAEC) submitted to LS (15 dynes/cm 2 ) in a cone-plate system for 18h exhibited (vs. static controls): Decreased (~50%) superoxide production (HPLC analysis of DHE oxidation); Decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay); Decreased mRNA expression of Nox1 (67%) and Nox4 (45%) (real-time QPCR); Increased eNOS expression (~50%, western blot) and nitrite levels in culture medium (Δ = 7.1±2.5[SD] μM, NO Analyzer and Griess reaction); Decrease in total and membrane fraction PDI protein expression (~20%) without changes in membrane fraction/total ratio of PDI. RAEC were transfected with c-myc -tagged plasmid coding for wild-type (WT) PDI or PDI mutated in 4 thioredoxin-motif cysteine residues. Forced expression (2-fold) of mutated but not WT PDI led to increase in nitrite output after LS (18h) (Δmutated = 17.2±3.3 μM vs. ΔWT = 7.0±1.9 μM, n=3, p<0.02). Confocal microscopy indicated similar subcellular localization between WT and mutated PDI. PDI co-imunoprecipitated with p22phox NADPH oxidase subunit, but not with eNOS or caveolin-1, either in static condition or after LS. Fractionation studies in sucrose gradients showed that PDI is distributed throughout several fractions in static conditions, including caveolin-1-enriched fractions, but migrates to higher-density fractions, not containing caveolin-1, during sustained LS. These results suggest that PDI is involved in regulation of NO output during LS via its effects on NADPH oxidase activity.

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