Impairment of vascular endothelial function following reperfusion therapy in patients with acute myocardial infarction

Abstract

Objective

To investigate vascular endothelial impairment as a result of reperfusion therapy in patients with acute myocardial infarction (AMI).

Methods

Patients with AMI underwent reperfusion therapy (percutaneous cardiac intervention [PCI] or thrombolytic therapy) or conservative drug therapy. Healthy control subjects were recruited. Endothelial impairment was assessed via endothelial nitric oxide (NO) synthase (eNOS), NO and endothelin-1 (ET-1) levels, 24 h after reperfusion or on enrolment, as appropriate.

Results

Patients who underwent PCI (n = 47) or thrombolytic therapy (n = 45) had significantly lower eNOS and NO levels, and higher ET-1 levels than those who received conservative drug therapy (n = 46). All patient groups had significantly lower eNOS and NO levels, and higher ET-1 levels, than controls (n = 45). There was a significant positive correlation between eNOS and NO, as well as significant negative correlations between eNOS/ET-1 and NO/ET-1 in all four groups.

Conclusions

Patients with AMI who underwent reperfusion therapy displayed low eNOS activity. This may result in impairment of endothelial function via downregulation of NO and upregulation of ET-1.

Introduction

The development of coronary heart disease is associated with many pathological factors. ¹ Acute myocardial infarction (AMI) is the main cause of sudden death throughout the world, ² and the timely and effective control of AMI occurrence and development is a primary focus of medical research. Reperfusion therapy is an effective and life-saving treatment for AMI ³ that results in improved cardiac vascular endothelial function. ⁴ Other studies have revealed impairment of general vascular endothelial function following reperfusion therapy, however. ⁵

Nitric oxide (NO) is an indicator of vascular endothelial function, ⁶ and a combination of NO and endothelin-1 (ET-1) is commonly used for clinical determination and analysis of vascular endothelial function. ⁷ NO and ET-1 interact such that when the NO level increases, the attendant ET-1 level correspondingly decreases and vice versa. ⁸ Abnormalities in this system can lead to disequilibrium in vascular endothelial function, resulting in pathological changes. ⁹ NO is synthesized by endothelial NO synthase (eNOS), which has been shown to play an important role in cardiovascular disease. ¹⁰

The aims of the present study were to investigate vascular endothelial impairment via eNOS, NO and ET-1, in patients with AMI who had undergone reperfusion therapy.

Patients and methods

Study population

The study recruited patients with AMI (diagnosed according to World Health Organization criteria ¹¹ ) who were treated in the Cardiology Department, Heilongjiang Provincial Hospital, Harbin, China, between June 2010 and June 2011. Exclusion criteria were: (i) vascular atherosclerotic plaque, thrombosis or irregular stenosis in bilateral carotid artery or bilateral lower extremity arteries on colour Doppler ultrasonography examination; (ii) clinical manifestation of ischaemic disease of the lower extremity, including intermittent claudication and ischaemic pain; (iii) history of cerebral infarction or visible cerebral ischaemic lesions on computed tomography or magnetic resonance imaging; (iv) family history of hypertension; (v) diabetes; (vi) any malignant disease, severe liver disease, nephritis, infectious disease, recent trauma or surgery (within 1 year), or severe mental illness. Patients were divided between three groups according to a computer-generated randomization schedule: reperfusion by percutaneous coronary intervention (PCI); reperfusion by thrombolytic therapy; conservative drug therapy.

The control group comprised healthy individuals free from the exclusion criteria (described above), attending the Health Examination Centre, Heilongjiang Provincial Hospital, Harbin, China for routine health screening.

Peripheral blood samples were obtained after a 12-h fast within 24 h after reperfusion therapy or at enrolment, as appropriate. Immediately after collection, samples for eNOS and NO quantification (3 ml each) were centrifuged at 3000  g for 15 min at 20℃, and the resulting serum was collected and stored at −20℃ until use. Blood samples for ET-1 analysis were collected at the same time: for these analyses, 3-ml samples of peripheral blood were collected in sterile tubes containing 15 IU/ml heparin.

The study was conducted in accordance with the Declaration of Helsinki, ¹² and was approved by the Ethics Committee of First Hospital, Jilin University, Changchun, China (D5395L00006). All participants provided written informed consent prior to enrolment.

eNOS activity

A colorimetric assay kit was used to quantify eNOS activity (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions.

NO quantification

Serum NO levels were quantified using an NO nitrate reductase assay kit (Nanjing Jiancheng Biochemical Co., Ltd., Nanjing, China), according to the manufacturer’s instructions, and expressed as µmol/l NO³⁻.

ET-1 quantification

Quantification of plasma ET-1 was performed using a radioimmunoassay kit (Nanjing Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Intra- and interassay variation were <10% and <15%, respectively.

Statistical analyses

Data were provided as mean ± SD, and non-normally distributed data were log-transformed. Differences between two groups were analysed with Student’s t-test or χ² test, as appropriate. Linear correlation analysis was performed to identify the correlations among the related indicators. Statistical analyses were performed using SPSS® software, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. A P-value <0.05 was considered statistically significant.

Results

The study recruited 45 control subjects (23 males/22 females; mean age 53.8 ± 6.4 years, range 44–72 years) and 138 patients with AMI. Treatment randomization of the patients meant that 47 participants underwent reperfusion via PCI (23 males/24 females; mean age 53.4 ± 6.3 years, range 45–72 years), 45 underwent reperfusion via thrombolytic therapy (23 males/22 females; mean age 53.7 ± 6.2 years old, range 45–73 years) and 46 received conservative drug therapy (23 males/23 females; mean age 54.1 ± 6.3 years, range 44–71 years). There were no significant between-group differences in sex distribution or patient age.

Data regarding eNOS, NO and ET-1 levels in the four study groups are given in Table 1. Levels of eNOS and NO were significantly lower, and ET-1 levels were significantly higher, in patients who underwent PCI or thrombolytic therapy compared with those who received conservative drug therapy or controls (P < 0.01 for each comparison; Table 1). In addition, eNOS and NO levels were significantly lower, whereas ET-1 levels were significantly higher, in the conservative drug therapy group compared with the control group (P < 0.01 for each comparison; Table 1). There were no significant differences in eNOS, NO or ET-1 levels between the PCI and thrombolytic therapy groups.

Table 1.

Endothelial nitric oxide (NO) synthase (eNOS) activity, and levels of NO (expressed as NO³⁻) and endothelin-1 (ET-1), in patients with acute myocardial infarction (randomized to receive treatment via percutaneous coronary intervention [PCI], thrombolytic therapy or conservative drug therapy) and healthy control subjects.

Parameter	PCI group n = 47	Thrombolytic therapy group n = 45	Conservative drug therapy group n = 46	Control group n = 45
eNOS, U/ml	4.57 ± 0.16ab	4.99 ± 0.20ab	6.35 ± 0.22 b	7.86 ± 0.19
NO3−, µmol/l	23.52 ± 15.48ab	24.08 ± 14.97ab	39.47 ± 12.18 b	60.43 ± 13.58
ET-1, pg/ml	22.45 ± 5.47ab	20.38 ± 5.16ab	13.27 ± 4.12 b	6.57 ± 2.19

Data presented as mean ± SD.

ab

P < 0.01 vs conservative drug therapy group, P < 0.01vs control group; Student’s t-test.

Linear correlation analysis revealed a significant positive correlation between eNOS and NO, as well as significant negative correlations between eNOS/ET-1 and NO/ET-1 in all four groups (P < 0.05 for each correlation; Table 2).

Table 2.

Correlation analysis of endothelial nitric oxide (NO) synthase (eNOS) activity, and levels of NO and endothelin-1 (ET-1). in patients with acute myocardial infarction (randomized to receive treatment via percutaneous coronary intervention [PCI], thrombolytic therapy or conservative drug therapy), and healthy control subjects.

Parameter	PCI group n = 47	Thrombolytic therapy group n = 45	Conservative drug therapy group n = 46	Control group n = 45
eNOS/NO	0.47	0.41	0.48	0.40
eNOS/ET-1	−0.46	−0.49	−0.45	−0.43
NO/ET-1	−0.49	−0.42	−0.52	−0.48

Data presented as correlation coefficient.

P < 0.05 for each correlation.

Discussion

Both AMI and reperfusion therapy are known to cause vascular endothelial function impairment, ⁵ resulting in decreased NO levels and increased ET-1 levels. In addition, early reperfusion therapy effectively improved coronary blood supply for patients with partial AMI, but did not improve NO and ET-1 levels in all patients. ¹³

Right brachial artery flow-mediated vasodilatation and nitroglycerin-mediated vasodilatation have been shown to predict vascular endothelial function accurately, ¹⁴ and have been used for clinical evaluation of endothelial function. ¹⁵ Nitroglycerin is an endothelium-independent vasodilator that directly generates NO, ¹⁶ therefore it is difficult to differentiate between the NO generated by nitroglycerin and that generated by the body. For this reason, the present study used NO and ET-1 for the indirect assessment of vascular endothelial function.

Patients with AMI had significantly lower eNOS and NO levels, and significantly higher ET-1 levels, than control subjects in the present study, regardless of treatment group. Correlation analysis found that eNOS and NO were inversely related to ET-1. A reduction in eNOS may result in lower NO secretion, and a reduction in NO could upregulate ET-1.

Levels of eNOS, NO and ET-1 were similar in the two reperfusion therapy groups (PCI and thrombolytic therapy) in the present study. This suggests that the intervention was not the main cause of endothelial impairment, and that ischaemia–reperfusion impairment remains an important factor.

The present study is limited by the fact that endothelial function was assessed only once, at 24 h after reperfusion therapy. Endothelial impairment caused by reperfusion therapy has been shown to disappear gradually over time, resulting in improved vascular endothelial function. ¹⁷

In conclusion, the present study found that patients with AMI who underwent reperfusion therapy displayed low eNOS activity. This may result in impairment of endothelial function via downregulation of NO and upregulation of ET-1.