Albumin Redox Modifications Promote Cell Calcification Reflecting the Impact of Oxidative Status on Aortic Valve Disease and Atherosclerosis

Abstract

Calcific aortic valve disease (CAVD) and coronary artery disease (CAD) are related cardiovascular diseases in which common mechanisms lead to tissue calcification. Oxidative stress plays a key role in these diseases and there is also evidence that the redox state of serum albumin exerts a significant influence on these conditions. To further explore this issue, we used multimarker scores (OxyScore and AntioxyScore) to assess the global oxidative status in patients with CAVD, with and without CAD, also evaluating their plasma thiol levels. In addition, valvular interstitial cells were treated with reduced, oxidized, and native albumin to study how this protein and its modifications affect cell calcification. The differences we found suggest that oxidative status is distinct in CAVD and CAD, with differences in redox markers and thiol levels. Importantly, the in vitro interstitial cell model revealed that modified albumin affects cell calcification, accelerating this process. Hence, we show here the importance of the redox system in the development of CAVD, emphasizing the relevance of multimarker scores, while also offering evidence of how the redox state of albumin influences vascular calcification. These data highlight the relevance of understanding the overall redox processes involved in these diseases, opening the door to new studies on antioxidants as potential therapies for these patients.

Keywords: aortic stenosis; aortic valve; artery; calcification; interstitial cells; multimarker score; oxidative stress.

Figure 1

Workflow of the study. (A) Different markers of oxidative damage and antioxidant defense, including thiol levels, were measured in plasma samples from patients with CAVD, with and without CAD. (B) Human VICs were treated with oxidized, reduced, and native HSA. Subsequently, calcification of these cells was assessed by using Alizarin Red staining. Microscope images size is 665.6 µm × 665.6 µm.

Figure 2

Markers of oxidative status in plasma from the four groups of study. (A) Markers of oxidative damage, including protein carbonyls, 8-hydroxy-2′-deoxyguanosine (8-OHdG) xanthine oxidase (XOD) activity and oxidized LDL (oxLDL) (n = 15 subjects/each group). (B) Markers of antioxidants defense, including total antioxidant capacity (TAC), superoxide dismutase (SOD) activity and catalase (CAT) activity (n = 15 subjects/each group). (C) Free reduced thiols (n = 14 subjects/CAD group and n = 17 subjects/C, CAVD and CAVD + CAD group). Data are represented as the mean ± SD. AUC, Area under the curve; C, Controls; CAD, Coronary artery disease; CAVD, Calcific aortic valve disease. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Multimarker scores of (A) oxidative damage (OxyScore) and (B) antioxidant defense (AntiOxyscore) (n = 15 subjects/each group). C, Controls; CAD, Coronary artery disease; CAVD, Calcific aortic valve disease. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Results from in vitro model. (A) Immunoblot of modified HSA. Reduced HSA (RedHSA) was labeled with SulfoBiotics-PEG-PCMal, which produces a mobility shift corresponding to approximately 5 kDa for each molecule of PEG-PCMal bound to a free thiol group of the target protein. Thus, after the blotting, several bands corresponding to RedHSA are observed (from 60 to 150 kDa), while the lane of native HSA only shows one band. Oxidized HSA (OxHSA) was evaluated using OxyBlot Protein Oxidation Detection Kit. This kit allows the immunodetection of carbonyl groups through DNP-derivatization and subsequent detection of this DNP moiety with a specific primary antibody. It can be observed that the bands corresponding to OxHSA are more intense than the bands corresponding to native HSA. (B) Representative images of the Alizarin Red staining at a concentration of 1 mg/mL of modified or native HSA after 24 h of treatment. Microscope images size is 665.6 µm × 665.6 µm. (C) Calcification levels of cells cultured for 24 h in medium for fibroblast (FIBm) when supplemented with different concentrations of modified or native HSA. (D) Calcification levels of cells cultured for 24 h in osteogenic medium (OSTm) when supplemented with different concentrations of modified or native HAS was measured using Alizarin red staining. All experiments were performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Hypothesis about the effect of oxidized HSA (OxHSA) in medium for fibroblasts (FIBm) and reduced HSA (RedHSA) in osteogenic medium (OSTm). In both cases, an intense calcification is observed in valvular interstitial cells, but OxHSA is believed to induce proinflammatory cytokines and reactive oxygen species, while RedHSA may act as a carrier of the osteogenic components that have the OSTm.