Proton Pump Inhibitors Accelerate Endothelial Senescence

Abstract

Rationale: Proton pump inhibitors (PPIs) are popular drugs for gastroesophageal reflux, which are now available for long-term use without medical supervision. Recent reports suggest that PPI use is associated with cardiovascular, renal, and neurological morbidity.

Objective: To study the long-term effect of PPIs on endothelial dysfunction and senescence and investigate the mechanism involved in PPI-induced vascular dysfunction.

Methods and results: Chronic exposure to PPIs impaired endothelial function and accelerated human endothelial senescence by reducing telomere length.

Conclusions: Our data may provide a unifying mechanism for the association of PPI use with increased risk of cardiovascular, renal, and neurological morbidity and mortality.

Keywords: aging; cardiovascular diseases; proteostasis deficiencies; proton pump inhibitors.

Figure 1. Esomeprazole impairs proteostasis

(A) Intensity of pHrodo^™ Green AM fluorescence, which is inversely proportional to lysosomal pH (n=4). (B) Acid phosphatase assay (n=4). (C&E) Intracellular cathepsin-B activity assessed by Magic Red^® fluorescence dye (n=4). (D&F) Intracellular protein aggregates assessed by PROTEOSTAT^® assay (fluorescent staining in upper panel and corresponding phase-contrast image on lower panel) and quantification (n=4). *p< 0.05 vs vehicle (DMSO).

Figure 2. Esomeprazole impairs endothelial function

(A&C) Superoxide anion generation assessed by dihydroethidium staining (n=4). (B&D) Nitric oxide generation assessed by di-amino fluorescein 2-diacetate (DAF-2DA) staining (n=4). (E) Total nitrate/nitrite levels assessed by Greiss reaction (n=6). (F) Measurement of cell proliferation using real time cell analyzer which generates cell index (CI) values represented as area under curve (n=5). (G) Cell proliferation assessed by BrdU assay (n=8). (H) p21 mRNA expression using RT-PCR (n=4). (I–L) Angiogenic capacity of ECs reflected by network formation in growth factor depleted matrigel. *p< 0.05 vs vehicle (DMSO).

Figure 3. PPIs accelerate endothelial senescence

(A&D) Senescent cell number detected by staining for senescence associated-β-galactosidase (SA-β-gal; upper panel) and for SYTO-13 to detect cell nuclei for total cell count (lower panel). (B,C,E and F) Respective quantification for % positive SA-β-gal cells and average cell count per field (n=6). (G and H) PAI-1 protein expression by Western blot analysis (n=3). (I) PAI-1 mRNA expression quantified by RT-PCR (n=6). *p< 0.05 vs vehicle (DMSO).

Figure 4. PPIs reduce telomere length and expression of shelterin complex subunits

Relative telomere length assessed by mmqPCR (A) in human MVEC (n=6) (B–G) Expression of shelterin complex genes assessed by RT-PCR (n=6). *p< 0.05 vs vehicle (DMSO)