Transition in the mechanism of flow-mediated dilation with aging and development of coronary artery disease

Abstract

In microvessels of patients with coronary artery disease (CAD), flow-mediated dilation (FMD) is largely dependent upon the endothelium-derived hyperpolarizing factor H₂O₂. The goal of this study is to examine the influence of age and presence or absence of disease on the mechanism of FMD. Human coronary or adipose arterioles (~150 µm diameter) were prepared for videomicroscopy. The effect of inhibiting COX [indomethacin (Indo) or NOS (L-NAME), eliminating H₂O₂ (polyethylene glycol-catalase (PEG-CAT)] or targeting a reduction in mitochondrial ROS with scavengers/inhibitors [Vitamin E (_mtVitamin E); phenylboronic acid (_mtPBA)] was determined in children aged 0-18 years; young adults 19-55 years; older adults >55 years without CAD, and similarly aged adults with CAD. Indo eliminated FMD in children and reduced FMD in younger adults. This response was mediated mainly by PGI₂, as the prostacyclin-synthase-inhibitor trans-2-phenyl cyclopropylamine reduced FMD in children and young adults. L-NAME attenuated dilation in children and younger adults and eliminated FMD in older adults without CAD, but had no effect on vessels from those with CAD, where mitochondria-derived H₂O₂ was the primary mediator. The magnitude of dilation was reduced in older compared to younger adults independent of CAD. Exogenous treatment with a sub-dilator dose of NO blocked FMD in vessels from subjects with CAD, while prolonged inhibition of NOS in young adults resulted in a phenotype similar to that observed in disease. The mediator of coronary arteriolar FMD evolves throughout life from prostacyclin in youth, to NO in adulthood. With the onset of CAD, NO-inhibitable release of H₂O₂ emerges as the exclusive mediator of FMD. These findings have implications for use of pharmacological agents, such as nonsteroidal anti-inflammatory agents in children and the role of microvascular endothelium in cardiovascular health.

Keywords: Coronary artery disease; Flow-mediated dilation; Microvasculature; Vasodilation.

Fig. 1

Mediator of flow-mediated dilation is changed throughout life and with onset of disease. Flow-mediated dilation (FMD) was compared in isolated atrial microvessels from a children age 0–18, b younger adults age 18–55, c older adults age >55 and d subjects with clinical diagnosed coronary artery disease independent of age. The mediator of FMD changed from PGI₂/NO in children to NO/PGI₂ in younger adults, to primarily NO in older adults, then to H₂O₂ in patients with CAD. ^#P < 0.05 vs. 18–55; *P < 0.05 two-way ANOVA RM Tukey’s post hoc analysis, N = 4–10

Fig. 2

Cyclooxygenase expression in human atrial sections. Representative image of IHC in atrial sections for COX1 (a-d) and COX2 (e-i) and Western blot quantification of COX1 and 2 in left ventricle tissue (j, k). COX1 expression was significantly increased in vessels from atrial sections (IHC) and whole left ventricle (WB) tissue from pediatric subjects and patients with CAD compared to adult subjects without CAD. COX2 expression is elevated in vessels from atrial sections (IHC) and whole left ventricle (WB) of CAD or children’s tissue vs. non-CAD controls but not in microvessels of subjects with CAD. d, h Control sections without primary antibody staining. Positive control (i) for COX2 is derived from subjects with autoimmune disease *P < 0.05 two-way t test with Tukey’s post hoc analysis, IHC, N = 4; WB, N = 5

Fig. 3

Prostacyclin receptor-and flow-mediated dilation in vessels from children age 0–18. Dilation was evaluated in isolated atrial microvessels in response to either the PGI₂ mimic iloprost, or to shear. Inhibition of NOS with L-NAME partly attenuated iloprost mediated dilation. The PGI₂ receptor antagonist TPC (100 μM) blocked FMD. *P < 0.05 two-way ANOVA RM Tukey’s post hoc analysis, N = 4–6

Fig. 4

Mitochondrial H₂O₂ mediates FMD in vessels from subjects with CAD. a, b The H₂O₂ specific fluorescent probe PYI targeted to mitochondria (mitoPYI) was used to quantify H₂O₂ in isolated adipose and atrial vessels. Due to the volume and cleanness of vessels needed, both adipose vessels were used. Specificity of the probe for H₂O₂ was confirmed using Peg-catalase. c FMD in atrial microvessels was reduced in the presence of mitochondrial targeted antioxidant _mtVitamine. e, d The mitochondrial targeted scavenger of H₂O₂ (_mtPBA) reduced FMD in atrial arterioles from patients with CAD. e, f PBA (10 mM), the cellular scavenger of H₂O₂ had no effect on FMD but blocked FMD at a tenfold higher concentration. *P < 0.05 two-way ANOVA RM (dose response curve) or t test (single point dilation and fluorescence data) with Tukey’s post hoc, N = 4–5

Fig. 5

NO at a concentration subthreshold for dilation suppresses FMD in atrial arterioles from patients with and without CAD. a FMD was abolished by incubation with NONOate in atrial arterioles from patients with but not without CAD. b, c Treatment of adipose arterioles with L-NAME for either 1 or 18 h shifted the mechanism of dilation from NO to H₂O₂ (Peg-catalase inhibitable). The dilation was abolished by treatment with DETANONOate. *P < 0.05 two-way ANOVA RM Tukey’s post hoc analysis, N = 3–6

Fig. 6

Proposed change in mediators of FMD throughout life. The mechanism of FMD in children is primarily mediated by PGI₂ with a small contribution by NO. With healthy aging NO becomes the dominant mediator of dilation changes with a small PGI₂ component. With onset of CAD NO mediated dilation is lost and H₂O₂ becomes the sole endothelial derived relaxing factor in response to shear