Unexpected effect of proton pump inhibitors: elevation of the cardiovascular risk factor asymmetric dimethylarginine

Abstract

Background: Proton pump inhibitors (PPIs) are gastric acid-suppressing agents widely prescribed for the treatment of gastroesophageal reflux disease. Recently, several studies in patients with acute coronary syndrome have raised the concern that use of PPIs in these patients may increase their risk of major adverse cardiovascular events. The mechanism of this possible adverse effect is not known. Whether the general population might also be at risk has not been addressed.

Methods and results: Plasma asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. Elevated plasma ADMA is associated with increased risk for cardiovascular disease, likely because of its attenuation of the vasoprotective effects of endothelial nitric oxide synthase. We find that PPIs elevate plasma ADMA levels and reduce nitric oxide levels and endothelium-dependent vasodilation in a murine model and ex vivo human tissues. PPIs increase ADMA because they bind to and inhibit dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA.

Conclusions: We present a plausible biological mechanism to explain the association of PPIs with increased major adverse cardiovascular events in patients with unstable coronary syndromes. Of concern, this adverse mechanism is also likely to extend to the general population using PPIs. This finding compels additional clinical investigations and pharmacovigilance directed toward understanding the cardiovascular risk associated with the use of the PPIs in the general population.

Keywords: N,N dimethylarginine; dimethylarginine dimethylaminohydrolase; endothelium; nitric oxide; proton pump inhibitors.

Figure 1

The ADMA pathway. Asymmetric dimethylarginine (ADMA) is derived from proteins (largely nuclear) containing methylated arginine residues. ADMA is largely (80%) metabolized by dimethylarginine dimethylaminohydrolase (DDAH). ADMA is a competitive inhibitor of nitric oxide synthase (NOS). Endothelial NOS (eNOS) is highly regulated, and produces small amounts of NO locally to effect vascular homeostasis. Increased levels of ADMA (such as through possible inhibition by the PPIs) could impair eNOS activity, reducing NO generation while increasing superoxide anion generation. The vasoprotective action of eNOS is lost, increasing the risk for adverse vascular events. In this setting, inflammatory cells are attracted into the vessel wall, and express inducible NOS (iNOS), which generates superoxide anion and nitric oxide, which combine to form the cytotoxic free radical peroxynitrite anion.

Figure 2

Proton pump inhibitors (PPIs) inhibit DDAH activity. A) Colorimetric assay showing reduced production of L-citrulline from ADMA. B) Fluorimetric assay showing inhibited signal associated with DDAH enzymatic activity. In A) L-citrulline conc. was calculated from standard curve. In B) ebselen was used as a positive assay control . Data is from triplicate experiments (Mean ± SEM) at 50 μM final compound concentration. *p<0.05 when the PPIs are compared to the vehicle control by One-Way ANOVA followed by Bonferroni posttest correction.

Figure 3

The PPI omeprazole binds to DDAH. Surface plasmon resonance (SPR) sensorgram data indicating the interaction between omeprazole and human DDAH1. DDAH was coupled to a chip and omeprazole or vehicle was passed over the chip. Binding is shown by the peaks in the sensorgrams at the different concentrations tested (Green = 12.5 μM; Pink = 25 μM; Blue = 50 μM and Purple = 100 μM). Data is representative of duplicate experiments per group.

Figure 4

PPIs increase ADMA concentration and reduce NO levels in human endothelial cells. A) Human microvascular endothelial cells (HMVECs) were treated with the indicated small molecules at 20 μM final conc each. DMSO (solvent used to prepare the compounds) was used as a vehicle control. The intracellular [ADMA] was determined after 24 hours. B) The effect of omeprazole on total NO level was assessed by treating HMVECs with vehicle or various concentration of omeprazole for 24 hours. Total nitrite (NOx) was measured from lysates using Griess reaction and was normalized to total protein concentration. Data is Mean ± SEM from duplicate experiments. L-257 is a selective DDAH1 inhibitor and was included as a control. *p<0.05 when each PPI was compared to the vehicle group using unpaired student t-tests.

Figure 5

The PPI omeprazole reduces the expression of total and active endothelial NOS. Regulation of total endothelial NOS (eNOS) and active eNOS (phospho-eNOS) by omeprazole was studied by Western blot using endothelial cell (EC) lysate exposed to omeprazole or vehicle. ECs were treated with VEGF (50 ng/mL) as positive eNOS phosphorylation control. The expression of each protein was normalized to β-actin (ACTB).

Figure 6

The effect of PPI (Omeprazole) on nitric oxide production of human saphenous vein grafts (SVGs). SVGs were treated with vehicle or omeprazole (3–100 μM) for 24 hours A) at baseline level or B) upon stimulation with the calcium ionophore A23187 (0.5 μM) to increase NO production. Total nitrite (NOx) was measured in the conditioned medium using Griess reaction. Data is Mean ± SEM from duplicate experiments. *p<0.05 when the PPIs are compared to the vehicle control by One-Way ANOVA followed by Bonferroni posttest correction.

Figure 7

Omeprazole impairs vascular reactivity in response to the pharmacological agents acetylcholine (ACh) and phenylephrine (PE) on isolated mouse aorta. Concentration response curve to A) PE (10⁻⁸ to 10⁻⁴ M) and B) ACh (10⁻¹² to 10⁻⁴ M) are shown. In C) the percent relaxation in response to the endothelium-independent Sodium Nitroprusside (SNP; 10⁻¹² to 10⁻⁵ M) is shown. Drug concentrations in each data set were log transformed and the PE concentration response curve (in Figure A) is expressed as force (mN) applied to the transducer of the myograph for each dose of the compound added. The ACh and SNP induced relaxation (in Figure B and C) is expressed in the concentration response curve as a percentage of the contraction to the 80% of the maximal contraction reached with PE. Differences in best-fit values of selected parameters (force, % relaxation) were compared between the PPI and vehicle groups (n=6 vessels at each drug conc. in each group) using extra sum-of-squares F test and the data are expressed as Mean ± SEM in each panel.

Figure 8

PPI treatment increases plasma ADMA. Lansoprazole (LPZ) treatment caused a sustained and significant increase in serum ADMA levels (*p<0.05) in mice (n=8 animals per group). Data are from duplicate experiments (Mean ± SEM) at 5-weeks post-treatment.