Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease

Abstract

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.

Keywords: Fabry disease; HUVEC; aging; endothelial cells; miR-126-3p; microRNA; senescence; small extracellular vesicles.

Figure 1

Representative Nanoparticle Tracking Analysis (NTA) analysis of small extracellular vesicles (sEVs) collected by ultracentrifugation method; sEVs isolated from plasma of different age classes of CTRs and FD EVs had a mean diameter of 138.9 +/− 1.9 nm and mode of 111.8 +/− 4.5 nm, here called small extracellular vesicles (sEVs).

Figure 2

MiR-126-3p levels in sEVs of CTRs are modulated by aging. Scatterplots showing relative miR-126-3p expression according to age in CTRs (a). Box and Whiskers indicate median, minimum and maximum levels of miR-126-3p in sEVs from plasma of healthy CTRs divided in age class (24–44 years, n = 20), adult (53–68 years, n = 20) and old (81–96 years, n = 20) (b). CTs (cycle thresholds) resulting from qRT-PCR analysis were normalized with mir-30a; levels were calculated with 2-ΔCT method and expressed as folds, with respect to lowest value registered. Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test. * p < 0.01.

Figure 3

MiR-126-3p levels in sEVs of FD patients are not modulated by aging. Scatterplots showing relative miR-126-3p expression according to age in FD patients (a); miR-126-3p levels variation in sEVs of FD patients grouped in age class. Box and Whiskers indicate median, minimum, and maximum levels of miR-126-3p in sEVs from different age class of FD patients (30) (b). Data were calculated by qRT-PCR and represent mean ± SD of three different experiments analyzed in triplicate. CTs (cycle thresholds) resulting from qRT-PCR analysis were normalized with mir-30a; levels were calculated with 2-ΔCT method and expressed as folds, with respect to lowest value registered. Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test.

Figure 4

Comparisons of miR-126-3p levels among CTRs and FD patients. Box and Whiskers indicate median, minimum, and maximum levels of miR-126-3p in sEVs from plasma of all FD patients (30) and all CTRs (60) (a). Comparisons of miR-126-3p levels among CTRLs and FD patients subdivided into three age classes: young, adult, and old (b). CTs (cycle thresholds) resulting from qRT-PCR analysis were normalized with miR-30a; levels were calculated with 2-ΔCT method and expressed as folds with respect to lowest value registered. * p from t-test < 0.01.

Figure 5

Human umbilical vein endothelial cells (HUVECs) senescence status. Senescence status was estimated by SA-β-Gal assay in different passage numbers; images are representative of three independent experiments (a). Replicative senescence. Data are expressed as % of SA-β-Gal positive cells and are the means ± SD (b). Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test. * p < 0.01.

Figure 6

MiR-126a-3p levels variation in HUVECs undergoing senescence. Bar charts show sEVs (a) and intracellular (b) miR-126-3p levels in young, intermediate, and senescent HUVECs. Data were calculated by qRT-PCR and represent mean ± SD of three different experiments analyzed in triplicate. CTs (cycle thresholds) resulting from qRT-PCR analysis were normalized with miR-30a (EVs) and RNU6b (intracellular); levels were calculated with 2-ΔCT method and expressed as folds, with respect to lowest value registered. Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test. * p < 0.01.

Figure 7

Lyso-Gb3 and Gb3 effect on HUVECs. Intracellular ROS levels and cell viability were assayed in HUVECs treated with TBH 60 µM, Lyso-Gb3 25 nM, and Gb3 10 µM. Intracellular ROS were measured as fluorescence intensity, using a DCFH-DA probe. The results, in arbitrary units (au), are presented as mean ± SD of three different experiments analyzed in triplicate (a). Cell viability was evaluated with MTS assay. The results presented as mean % of viable cells ± SD of three different experiments analyzed in triplicate (b). Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test. * p < 0.01.

Figure 8

Lyso-Gb3 and GB3 effect on HUVECs senescence. Senescence status was estimated by SA-β-Gal assay in HUVECs treated with TBH 60 µM, Lyso-Gb3 25 nM, and Gb3 10 µM. Images are representative of ten random fields (a). Data are expressed as % of SA-b-Gal positive cells and are the means ± SD (b). Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test. * p < 0.01.

Figure 9

Variation in miR-126a-3p levels in HUVEC treated with TBH 60 µM, Lyso-Gb3 25 nM, and Gb3 10 µM. Bar charts show intracellular (a) and sEVs miR-126-3p levels (b). Data were calculated by qRT-PCR and represent mean ± SD of three different experiments analyzed in triplicate. CTs (cycle thresholds) resulting from qRT-PCR analysis were normalized with miR-30a (EVs) and RNU6b (intracellular); miR-126a-3p levels were calculated with 2-ΔCT method and expressed as folds, with respect to lowest value registered. Comparison among multiple groups was analyzed by one-way analysis of variance, followed by Bonferroni’s post hoc test. * p < 0.01.