Myeloperoxidase deficiency preserves vasomotor function in humans

Abstract

Aims: Observational studies have suggested a mechanistic link between the leucocyte-derived enzyme myeloperoxidase (MPO) and vasomotor function. Here, we tested whether MPO is systemically affecting vascular tone in humans.

Methods and results: A total of 12 135 patients were screened for leucocyte peroxidase activity. We identified 15 individuals with low MPO expression and activity (MPO(low)), who were matched with 30 participants exhibiting normal MPO protein content and activity (control). Nicotine-dependent activation of leucocytes caused attenuation of endothelial nitric oxide (NO) bioavailability in the control group (P < 0.01), but not in MPO(low) individuals (P = 0.12); here the MPO burden of leucocytes correlated with the degree of vasomotor dysfunction (P = 0.008). To directly test the vasoactive properties of free circulating MPO, the enzyme was injected into the left atrium of anaesthetized, open-chest pigs. Myeloperoxidase plasma levels peaked within minutes and rapidly declined thereafter, reflecting vascular binding of MPO. Blood flow in the left anterior descending artery and the internal mammary artery (IMA) as well as myocardial perfusion decreased following MPO injection when compared with albumin-treated animals (P < 0.001). Isolated IMA-rings from animals subjected to MPO revealed markedly diminished relaxation in response to acetylcholine (P < 0.01) and nitroglycerine as opposed to controls (P < 0.001).

Conclusion: Myeloperoxidase elicits profound effects on vascular tone of conductance and resistance vessels in vivo. These findings not only call for revisiting the biological functions of leucocytes as systemic and mobile effectors of vascular tone, but also identify MPO as a critical systemic regulator of vasomotion in humans and thus a potential therapeutic target.

Figure 1

Assessment of myeloperoxidase protein expression and enzyme activity in human neutrophils. (A) Densitometric analysis of myeloperoxidase protein expression of all participants (MPO^low: n = 15; control n = 30). (B) Quantification of peroxidase activity in isolated polymorphonuclear neutrophils from all participants.

Figure 2

Leucocyte activation at baseline and following nicotine administration in humans with normal and reduced myeloperoxidase activity. (A) Matrix metalloproteinase-9 plasma levels before and after stimulation with nicotine. (B) Myeloperoxidase plasma levels before and following nicotine administration are shown. Data are displayed for the 15 subjects with reduced myeloperoxidase expression and/or activity (MPO^low) subjects and for the 30 matched controls.

Figure 3

Vascular function in humans with low and normal myeloperoxidase activity in response to nicotine. (A) Flow-mediated and (B) nitroglycerine-mediated dilation before and after nicotine stimulation. Data are displayed for all 15 subjects with diminished myeloperoxidase expression and/or activity (MPO^low) subjects and for the 30 matched controls. (C) Spearman's correlation between flow-mediated dilation following leucocyte activation and MPO activity. Myeloperoxidase-dependent consumption of nitric oxide. (D) Polymorphonuclear neutrophils derived from MPO^low subjects exhibited significantly less nitric oxide consumption when compared with controls. Exogenous addition of MPO (344 pmol/L) to myeloperoxidase-deficient polymorphonuclear neutrophils restored nitric oxide consumption. (E) MPO^low subjects also displayed significantly higher baseline plasma levels of nitrite when compared with controls with baseline nitrite plasma levels being inversely correlated to circulating myeloperoxidase (r = −0.40, P = 0.034). Nicotine treatment did not significantly impact on nitrite plasma levels.

Figure 4

Myeloperoxidase plasma levels following injection of human myeloperoxidase vs. human serum albumin in an open-chest pig model. Injection of human myeloperoxidase resulted in a rapid increase in MPO levels in pig plasma (n= 9, as assessed by ELISA), and decreased thereafter when compared with HSA-treated (n = 6) animals (*P < 0.01 vs. baseline and ^#P < 0.01 vs. 1 min).

Figure 5

Effect of myeloperoxidase on blood flow of conductance vessels and myocardial blood flow in anaesthetized pigs. (A) Mean internal mammary artery and left artery descends flow in the myeloperoxidase group decreased significantly during the course of the experiment (P < 0.001) and when compared with human serum albumin-treated animals (P < 0.001). (B) Myocardial blood flow and perfusion as measured by fluorescent microspheres and fluorescent imaging in myeloperoxidase and human serum albumin-treated animals. Mean blood flow decreased significantly in the myeloperoxidase group (P for trend 0.009) as opposed to human serum albumin-treated animals (P for trend 0.026). The difference between the groups was also statistically significant (P = 0.006). (C) In accordance, myocardial perfusion (slope of fluorescent intensity) decreased in myeloperoxidase-treated animals (P for trend <0.001) and remained unchanged in human serum albumin-treated animals (P for trend 0.890). Slope of fluorescent intensity was significantly different between the groups (P = 0.002).

Figure 6

Nitric oxide-dependent relaxation of explanted pig vessel segments and vascular myeloperoxidase deposition. (A) Relaxation of explanted segments of the internal mammary artery in response to acetylcholine. There was a significant attenuation of acetylcholine-dependent relaxation in vessel segments from animals treated with myeloperoxidase (56.9 ± 3% in myeloperoxidase-treated pigs vs. 89.4 ± 4% in rings from human serum albumin-treated animals, P < 0.01). Addition of H₂O₂ further decreased acetylcholine-dependent vasorelaxation in rings explanted from myeloperoxidase-treated animals (45.4 ± 4%, *P = 0.021), whereas vessel segments from human serum albumin-treated animals showed no H₂O₂-dependent impairment of vessel relaxation (89.5 ± 5%). (B) Nitroglycerine-induced relaxation was significantly reduced in explanted segments of the internal mammary artery derived from myeloperoxidase-treated animals (P < 0.001). (C) Heparin-dependent liberation of myeloperoxidase after ex vivo perfusion of explanted pig internal mammary artery. Vessels from myeloperoxidase-treated animals revealed increased levels of myeloperoxidase in the eluate when compared with human serum albumin-treated pigs. (D) Immunofluorescent imaging displayed vascular myeloperoxidase deposition in explanted internal mammary artery segments of myeloperoxidase-treated pigs when compared with human serum albumin-treated animals (red, PECAM; green, MPO; blue, DAPI; magnification ×200).