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

Abstract

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.

Figure 1

Schematic diagram of an eMV including the proposed metabolic pathways according to the proteomic results. The blood plasma metabolites that can feed these routes and their plasma concentrations under control conditions are also included. 1,3BPG: 1,3-bisphosphoglycerate; 2PG: 2-phosphoglycerate; 3PG: 3-phosphoglycerate; 3PHP: 3-phosphohydroxypyruvate; 6PG: 6-phosphogluconate; 6PGL: 6-phosphoglucono-1,5-lactone; AKG: alpha-ketoglutarate; BCAA: branched-chain amino acids; DHAP: dihydroxyacetone 3-phosphate; F1,6BP: fructose 1,6-biphosphate; F6P: fructose 6-phosphate; G3P: glycerol 3-phosphate; G6P: glucose 6-phosphate; GADP: glyceraldehyde 3-phosphate; NAG: N-acetylglucosamine; NAG6P: N-acetylglucosamine-6-phosphate; OAA: oxaloacetate; PEP: phosphoenolpyruvate; P-Ser: phosphoserine; Ru5P: ribulose 5-phosphate.

Figure 2

Western blot analysis of 6PGL, GK, and PSPH and the corresponding normalized analysis. A representative pool is included in the figure: young (Y) and senescent (S). THP-1 cells were used as a positive control (not shown). Bands were located at the expected molecular weight of 6PGL (28 kDa), GK (61 kDa), and PSPH (25 kDa). Protein data for the eMVs was normalized against the intensity of Ponceau red staining. Bars represent mean ± SD (n = 4 pools). ^∗∗∗p < 0.001.

Figure 3

Effect of incubating eMVs with different blood plasma metabolites on the ratio of absorbance at 260 nm and 340 nm, absorption peaks of NAD(P) and NAD(P)H, respectively. No blood metabolites were added to control eMVs. Error bars represent SD; n = 3. To clarify the plot, only statistical significance of the Mann–Whitney–Wilcoxon rank-sum test is included. ^∗p < 0.05, ^∗∗p < 0.01. In the Kruskall-Wallis test for young eMVs, significant differences were observed in control versus lactate and pyruvate (p < 0.05) and between glucose versus lactate (p < 0.01). Significant differences were also observed for senescent eMVs in control versus glycerol and pyruvate (p < 0.05) and between glycerol versus glucose and lactate (p < 0.01).

Figure 4

eMVs observed with a multiphoton confocal microscope. (a) Young eMVs under control conditions; arrows indicate eMVs. The square delimited by a dotted line is a magnification of the two eMVs indicated by the two arrows. (b) Senescent eMVs after glycerol incubation. (c) Young eMVs after lactate incubation. (d) Senescent eMVs after pyruvate incubation. The different size of the eMVs in the image is due to the fact that the eMVs were floating in the buffer in different positions in the z-axis. eMVs were obtained after mixing four pools. Scale bar, 1 μm.

Figure 5

MS analysis of NADP⁺ (6.88 elution time) and NADPH (8.79 elution time) content in senescent eMVs. eMVs were obtained after mixing four pools. NADP⁺ MW: 742 D, NADPH MW: 744 D.

Figure 6

A young (a) and a senescent (b) eMV. AOM: antioxidant machinery; PPP: pentose phosphate pathway; GNG: gluconeogenesis; ROS: reactive oxygen species; DH: dehydrogenases; BCAA: branched-chain amino acids. Note that the senescent eMV has larger enzymatic machineries, uses more metabolites to feed them, and also has a higher capacity for ROS scavenging.