Replicative Endothelial Cell Senescence May Lead to Endothelial Dysfunction by Increasing the BH2/BH4 Ratio Induced by Oxidative Stress, Reducing BH4 Availability, and Decreasing the Expression of eNOS

Abstract

Vascular aging is associated with the development of cardiovascular complications, in which endothelial cell senescence (ES) may play a critical role. Nitric oxide (NO) prevents human ES through inhibition of oxidative stress, and inflammatory signaling by mechanisms yet to be elucidated. Endothelial cells undergo an irreversible growth arrest and alter their functional state after a finite number of divisions, a phenomenon called replicative senescence. We assessed the contribution of NO during replicative senescence of human aortic (HAEC) and coronary (CAEC) endothelial cells, in which accumulation of the senescence marker SA-β-Gal was quantified by β-galactosidase staining on cultured cells. We found a negative correlation in passaged cell cultures from P0 to P12, between a reduction in NO production with increased ES and the formation of reactive oxygen (ROS) and nitrogen (ONOO^-) species, indicative of oxidative and nitrosative stress. The effect of ES was evidenced by reduced expression of endothelial Nitric Oxide Synthase (eNOS), Interleukin Linked Kinase (ILK), and Heat shock protein 90 (Hsp90), alongside a significant increase in the BH2/BH4 ratio, inducing the uncoupling of eNOS, favoring the production of superoxide and peroxynitrite species, and fostering an inflammatory environment, as confirmed by the levels of Cyclophilin A (CypA) and its receptor Extracellular Matrix Metalloprotease Inducer (EMMPRIN). NO prevents ES by preventing the uncoupling of eNOS, in which oxidation of BH4, which plays a key role in eNOS producing NO, may play a critical role in launching the release of free radical species, triggering an aging-related inflammatory response.

Keywords: Cyclophilin A (CypA); endothelial nitric oxide synthase (eNOS) uncoupling; endothelial senescence (ES); extracellular matrix metalloprotease inducer (EMMPRIN); inflammation; nitric oxide (NO); oxidative stress; tetrahydrobiopterin (BH4).

Figure 1

Passage-induced senescence in cultured HAEC and CAEC. Representative Beta-Galactosidase staining of cultured HAEC and CAEC from Passage P1 to P12. N = 3 by triplicate, Mean ± SD. * p < 0.05 P5 vs. P1. ** p < 0.01 P8, P10, P12 vs. P1 (HAEC&CAEC). Scale bars, 50 µm.

Figure 2

ES promotes the expression of inflammatory markers in ECs. Representative immunoblot detection of Cyclophilin A (A) and its receptor EMMPRIN (high glycosylated (HG) and low glycosylated (LG) forms) (B). N = 3 by triplicate. Mean ± SD * p < 0.01, ** p < 0.001, *** p < 0.0001 P1 vs. selected passages. (C,D) Representative expression of matrix metalloproteinases MM9 (C) and MMP13 (D) in the same cells. N = 3 by triplicate. Mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 P1 vs. indicated passages.

Figure 3

ES reduces the expression of eNOS and promotes oxidation of BH4 in HAEC and CAEC. (A) Representative expression of eNOS from P1 to P12 (N = 3 by triplicate. Mean ± SD. * p < 0.01 selected passages vs. P1). (B) Representative ratio BH2/BH4 in cultured HAEC and CAEC from P1 to P12 (N = 3 by triplicate. Mean ± SD. * p < 0.01, ** p < 0.001). (C,D) Representative expression of GTP cyclohydrolase and dihydrofolate reductase (DHFR), respectively, in cultured HAEC and CAEC from P1 to P12 (N = 3 by triplicate. Mean ± SD. * p < 0.01 vs. selected passages).

Figure 4

Cell passaging induces eNOS uncoupling from Hsp90 in senescent cells. Representative confocal microscopy of eNOS (FITC, green) and Hsp90 (Alexa 647, red) in HAEC and CAEC from P1 to P12. Merged panels show co-localization of both signals (yellow). N = 3 by triplicate. Mean ± SD. * p < 0.01 vs. selected passages (HAEC). * p < 0.05, ** p < 0.01 vs. selected passages (CAEC). Scale bars, 25 µm.

Figure 5

Cell passaging induces eNOS uncoupling from ILK in senescent cells. Representative confocal microscopy of eNOS (FITC, green) and ILK (Alexa 647, red) in HAEC and CAEC from P1 to P12. Merged panels show co-localization of both signals (yellow). N = 3 by triplicate. Mean ± SD. * p < 0.05 P1 vs. P8. ** p < 0.01 P1 vs. P10, P12 (HAEC). * p < 0.01 P1 vs. P5, P8. ** p < 0.001 P1 vs. P10, P12 (CAEC). Scale bars, 25 µm.

Figure 6

ES induces oxidative and nitrosative stress. Representative confocal microscopy detection of superoxide (A) and peroxynitrite (B) radicals in HAEC and CAEC from P1 to P12. (C) Representative immunoblot detection NOX in HAEC and CAEC from P1 to P12 (N = 3 mean by triplicate ± SD. * p < 0.05 P1 vs. indicated passages. ^# p < 0.01 P1 vs. indicated passages). Scale bars, 25 µm.