The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles

Abstract

We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs) and their microvesicles (MVs). Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E) activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.

Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery

“…In a recent work, we demonstrated the existence of a functional machinery for ROS detoxification in HUVEC eMVs [ 27 ]; now, our present study demonstrates that these HUVEC eMVs, especially those derived from senescent cells, can synthesize NAD(P)H, the fuel that feeds the antioxidant machinery, used as precursors of different blood plasma metabolites. The combination of both results indicates that eMVs are subcellular structures with an autonomous capacity for ROS scavenging, as had been suggested by Soleti et al [ 31 ]; eMV protection against oxidative stress regulating eNOS/Akt signaling has also been recently demonstrated [ 32 ].…”

“…The contribution of ROS to aging has been widely documented, although the beneficial effects produced by ROS have led to a new interpretation of this traditional view [ 57 – 59 ]. An increase of ROS production has also been related with senescence, an experimental model widely used in aging studies [ 20 , 24 , 60 ] and also demonstrated in our previous work in senescent endothelial cells [ 27 ]. In accordance with the stronger capacity of ROS elimination demonstrated by senescent eMVs in our previous work, the present study demonstrates a strong increase of the machinery involved in NADPH synthesis in senescent eMVs.…”

“…The presence of some antioxidant enzymes in plasma MVs [ 33 – 35 ], in endothelial-derived MVs [ 36 ], and in HUVEC-derived MVs [ 31 ] had been already demonstrated. However, in our previous work [ 27 ], we demonstrated that HUVECs had a complete antioxidant machinery and that their MVs included a specific group of functional enzymes mainly involved in O 2 − and H 2 O 2 detoxification. Moreover, to date, the possibility of NADPH synthesis in MVs has not been considered in the literature.…”

“…This is the reason why NADPH is considered the “fuel” of the antioxidant machinery. We have recently demonstrated that endothelium-derived MVs (eMVs) have a well-developed and functional enzymatic team to eliminate ROS which, moreover, increases in senescence [ 27 ]. Apparently, if eMVs can eliminate ROS, they should (a) contain large amounts of NADPH, (b) obtain it from the plasma, or (c) be able to synthesize it.…”

Hypoxia-Inducible Factor-1α: The Master Regulator of Endothelial Cell Senescence in Vascular Aging

“…EVs are essential modulators of vascular cell functions relevant to vascular inflammation and atherosclerosis [126,127]. Furthermore, EVs have been identified as cell-to-cell communicators.…”