An acute rise in intraluminal pressure shifts the mediator of flow-mediated dilation from nitric oxide to hydrogen peroxide in human arterioles

Abstract

Endothelial nitric oxide (NO) is the primary mediator of flow-mediated dilation (FMD) in human adipose microvessels. Impaired NO-mediated vasodilation occurs after acute and chronic hypertension, possibly due to excess generation of reactive oxygen species (ROS). The direct role of pressure elevation in this impairment of human arteriolar dilation is not known. We tested the hypothesis that elevation in pressure is sufficient to impair FMD. Arterioles were isolated from human adipose tissue and cannulated, and vasodilation to graded flow gradients was measured before and after exposure to increased intraluminal pressure (IILP; 150 mmHg, 30 min). The mediator of FMD was determined using pharmacological agents to reduce NO [N(G)-nitro-l-arginine methyl ester (l-NAME), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)], or H2O2 [polyethylene glycol (PEG)-catalase], and mitochondrial (mt) ROS was quantified using fluorescence microscopy. Exposure to IILP decreased overall FMD (max %dilation: 82.7 ± 4.9 vs. 62 ± 5.6; P < 0.05). This dilation was abolished by treatment with l-NAME prepressure and PEG-catalase after IILP (max %dilation: l-NAME: 23.8 ± 6.1 vs. 74.8 ± 8.6; PEG-catalase: 71.8 ± 5.9 vs. 24.6 ± 10.6). To examine if this change was mediated by mtROS, FMD responses were measured in the presence of the complex I inhibitor rotenone or the mitochondrial antioxidant mitoTempol. Before IILP, FMD was unaffected by either compound; however, both inhibited dilation after IILP. The fluorescence intensity of mitochondria peroxy yellow 1 (MitoPY1), a mitochondria-specific fluorescent probe for H2O2, increased during flow after IILP (%change from static: 12.3 ± 14.5 vs. 127.9 ± 57.7). These results demonstrate a novel compensatory dilator mechanism in humans that is triggered by IILP, inducing a change in the mediator of FMD from NO to mitochondria-derived H2O2.

Keywords: flow-mediated dilation; mitochondria; reactive oxygen species; vascular endothelium.

Fig. 1.

Increased intraluminal pressure (IILP) in microvessels from healthy individuals decreases flow-mediated dilation (FMD). The mechanism of FMD changed from nitric oxide (NO) [inhibited by N^G-nitro-l-arginine methyl ester (l-NAME) prepressure; A] to hydrogen peroxide (inhibited by catalase postpressure; B). IILP had no effect on endothelial-independent dilation to papaverine (C). *P < 0.05 vs. vehicle control; #P < 0.05 vs. prepressure by two-way ANOVA repeated measures (RM).

Fig. 2.

IILP increases mitochondrial free radical production in response to intraluminal flow. A: mitochondrial H₂O₂ generation in the presence or absence of flow was measured using mitochondria peroxy yellow 1 (MitoPY1), a cell-permeable fluorescent dye that is avidly taken up by the mitochondria. B: quantification of MitoPY1 signal in response to flow compared with baseline (0→100 cmH₂O). C and D: FMD in the presence of complex 1 inhibitor rotenone or mitochondrial free radical scavenger mitoTempol. *P < 0.05 vs. 0 flow control t-test (B); #P < 0.05 vs. prepressure two-way ANOVA RM (C and D).

Fig. 3.

In vessels from healthy subjects NO is the mediator of FMD. The NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) was used to differentiate involvement of NO or the dependency on nitric oxide synthase (NOS). *P < 0.05 vs. prepressure two-way ANOVA RM.

Fig. 4.

Sepiapterin preserves NO as the mechanism of FMD after exposure to IILP. In the presence of sepiapterin, FMD is similarly abrogated before and after IILP by both the NOS inhibitor l-NAME (A and B) and the NO-scavenger c-PTIO (C and D). Polyethylene glycol (PEG)-catalase had no effect on FMD in vessels treated with sepiapterin (E and F). Sepiapterin control curves are the same in pre- and postpressure. *P < 0.05 vs. prepressure two-way ANOVA RM. #P < 0.05, EC₅₀ vs. treated.

Fig. 5.

Mechanism of FMD. Mechanical shear (flow)-induced dilation in human microvessels under baseline (A) and after exposure to acute increase intraluminal pressure (B).