Aging-associated oxidized albumin promotes cellular senescence and endothelial damage

Figures & data

Table 1 AOPPs and levels of endothelial microparticles in the plasma of healthy young subjects compared with healthy elderly subjects

Subjects	Age	Sex	AOPP (ng/mL)	Median ± SD	EMPs (µL)	Mean ± SD
1	29	Male	11.84	8.33±4.70	4	14.86±9.32
2	30	Male	13.47		6
3	28	Female	13.17		16
4	29	Male	9.68		29
5	30	Female	9.27		8
6	31	Female	9.82		18
7	28	Male	5.23		23
8	75	Male	28.95	30.64±12.74*	99	109.1±42.96*
9	82	Female	21.53		62
10	81	Female	47.33		122
11	79	Female	51.95		72
12	77	Male	39.27		83
13	76	Female	22.16		104
14	80	Female	14.30		143
15	79	Male	23.61		203
16	81	Male	26.59		94

Notes: Concentration of AOPPs is expressed as ng/mL of chloramine-T equivalents and EMPs amount is expressed as number of microparticles per µL (EMPs/µL).

* P<0.001 versus young subjects. In the case of AOPPS, all the values are median ± SD and mean ± SD for amount of EMPs (n=7–9 per group, respectively).

Abbreviations: AOPPs, advanced oxidation protein products; EMPs, endothelial microparticles; SD, standard deviation.

Figure 1 Confirmation of the albumin oxidation.

Notes: (A) OxyBlot technique for detection of carbonyl groups. The carbonyls generated by CuSO₄ were detected by reaction with DNPH and anti-DNP immunostaining (OxyBlot). The analyses show one major band corresponding to Ox Alb. (B) Two-dimensional gels of native and oxidized albumin produced from TCA precipitation. IEF separation of proteins was achieved using 7 cm IPG strips, pH 3–10, and the second dimension was run on a 10% polyacrylamide gel. (Upper) Proteome of the Nat Alb of the TCA precipitation and (lower) proteome of oxidized albumin. Images are representative of experiments performed on native and oxidized albumin samples (n=2).
Abbreviations: DNPH, 2,4-dinitrophenylhydrazine; DNP, 2,4-dinitrophenyl; TCA, Trichloroacetic acid; Mr, relative molecular mass; IPG, immobilized pH gradient; Ox Alb, oxidized albumin; Nat Alb, native albumin.

Figure 2 Flow cytometer analysis of endothelial adhesion molecules, (A) VCAM-1 and (B) ICAM-1, expression in HUVEC incubated with vehicle CuSO₄ (10 µmol/L) + 0.1 mmol/L EDTA (gray bar), Nat Alb (white bars), or Ox Alb (black bars).

Notes: HUVEC were treated with different doses (mg/mL) of native and oxidized albumin for 24 hours. MFI are presented as mean ± SD, n=3, in duplicate. *P<0.05 versus vehicle; ^#P<0.05 versus at all Nat Alb doses.
Abbreviations: Ox Alb, oxidized albumin; Nat Alb, native albumin; MFI, median fluorescence intensity; HUVECs, human umbilical vein endothelial cells; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; EDTA, ethylenediaminetetraacetic acid; SD, standard deviation.

Figure 3 Flow cytometer determination of (A) cellular apoptosis and (B) endothelial microparticles expression in the supernatants of albumin-treated cells.

Notes: (A) Endothelial cells were treated with different doses of Nat Alb (white bars), Ox Alb (black bars), and vehicle CuSO₄ (10 µmol/L) + 0.1 mmol/LEDTA; (gray bar) for 24 hours. After incubation, apoptosis was determined in PI-stained cells and analyzed by flow cytometry. Data are expressed as mean ± SD, n=3 in duplicate. *P<0.05 versus vehicle; ^#P<0.001 versus at all Nat Alb doses. (B) Endothelial cells were treated with native (Nat Alb; white bars), oxidized albumin (Ox Alb; black bars), and vehicle CuSO₄ (10 µmol/L) + 0.1 mmol/LEDTA; (gray bar) for 24 hours at different concentrations. Total microparticles from HUVECs were characterized by flow cytometry. Size-selected events were plotted as a function of their size and complexity, and also near of 1 µm flow count calibrator beads. Graphic shows the EMPs production per µL with the vehicle, native, and oxidized albumin treatment. Data are presented as mean ± SD, n=3 in duplicate. *P<0.05 versus vehicle, ^#P<0.05 versus Nat Alb.
Abbreviations: Ox Alb, oxidized albumin; Nat Alb, native albumin; HUVEC, human umbilical vein endothelial cells; EMPs, endothelial microparticles; PI, propidium iodide; SD, standard deviation.

Figure 4 (A) Flow cytometer analysis of intracellular ROS production in HUVEC incubated with vehicle (white), Nat Alb (gray), or Ox Alb (black). HUVEC were treated with native and oxidized albumin (2 mg/mL) for 4 hours, (B) the histogram shows ROS as the percentage of HE-positive cells, and (C) MFI with vehicle (gray bar) and different doses of Nat Alb (white bars) and Ox Alb (black bars) for 4 hours.

Notes: Data are presented as mean ± SD, n=4 in duplicate. *P<0.05 versus vehicle; ^#P<0.05 versus at all Nat Alb doses.
Abbreviations: Ox Alb, oxidized albumin; Nat Alb, native albumin; HUVEC, human umbilical vein endothelial cells; ROS, reactive oxygen species; HE, hidroethidine; MFI, median fluorescence intensity; SD, standard deviation.

Figure 5 Senescent-associated β-galactosidase activity staining of native and oxidized albumin treatments.

Notes: HUVEC were treated with Nat Alb and Ox Alb (2 mg/mL) for 24 hours. (A) Representative pictures of cells stained with β-galactosidase (10×). A zoom of each picture is showed. (B) Graphic shows n-fold increase in β-galactosidase-positive cells. Data are presented as mean ± SD, n=3. *P<0.05 versus vehicle; ^#P<0.05 versus Nat Alb.
Abbreviations: Ox Alb, oxidized albumin; Nat Alb, native albumin; HUVEC, human umbilical vein endothelial cells; SD, standard deviation.

Figure 6 HUVEC with Nat Alb and Ox Alb (2 mg/mL) were scratched and wound margins were imaged from 0 up to 24 hours later.

Notes: Experiments were performed as in (A), and the extent of wound closure was quantified by measuring the wound area and comparing with the initial wound area (n=3) (B). *P<0.05 versus vehicle and Ox Alb at the same time point.
Abbreviations: Ox Alb, oxidized albumin; Nat Alb, native albumin; HUVEC, human umbilical vein endothelial cells.