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Microparticles from Patients Undergoing Percutaneous Coronary Intervention Impair Vasodilatation by Uncoupling Endothelial Nitric Oxide Synthase

Ye, Sha*; Shan, Xue-Feng; Han, Wen-Qi; Zhang, Qian-Rong*; Gao, Jie*; Jin, Ai-Ping*; Wang, Yi; Sun, Chao-Feng§; Zhang, Sui-Long*

doi: 10.1097/SHK.0000000000000823
Basic Science Aspects
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Objectives: Percutaneous coronary interventions (PCIs) save countless acute myocardial infarction (AMI) patients. However, endothelial injury is still an inevitable complication. Circulating microparticles (MPs) play important roles in vascular dysfunction. Whether PCI affects function of MPs remains unclear.

Methods: MPs were obtained from AMI patients (n = 38) both preoperatively and 24 h after PCI, and healthy subjects (n = 20). MPs origins were tested by flow cytometry. Rat thoracic aortas were incubated with MPs to determine the effects of MPs on phosphorylation of endothelial nitric oxide synthase (eNOS), caveolin-1 expression, eNOS association with heat shock protein 90 (Hsp90), generation of nitric oxide (NO) and superoxide anion (O2), and endothelial-dependent vasodilatation.

Results: Compared with healthy subjects, MP concentrations increased in AMI patients. Undergoing PCI had no further effect on MPs concentration, but it results in increased endothelial-derived MPs proportion and decreased platelet-derived MP ratio. MPs from AMI patients decreased eNOS phosphorylation at Ser1177, increased eNOS phosphorylation at T495 and caveolin-1 expression, decreased eNOS association with Hsp90, decreased NO production but increased (O2) generation, damaged endothelial-dependent vasodilatation. All of these effects of MPs were strengthened by PCI.

Conclusions: PCI further enhances the vascular injury effect of MPs. Circulating MPs may be a potential therapeutic target for patients undergoing PCI.

*Geriatric Vasculocardiology Department, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China

Department of Children's Cardiac Surgery, The First Affiliated Hospital of Xinjiang Medical University, Urumuqi, Xinjiang, China

Department of Cardiovascular Medicine, Shaanxi Provincial People's Hospital, Xi’an, Shaanxi, China

§Department of Cardiovascular Medicine, MOE, Ion Channel Disease Laboratory, MOE Key Laboratory of Environment and Genes Related to Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China

Address reprint requests to Sui-Long Zhang, MD, PhD, The Second Affiliated Hospital of Xi’an Jiaotong University, No. 157, West Five Road, Xi’an, Shaanxi 710004, P.R. China. E-mail: mss0392@126.com

Received 27 September, 2016

Revised 11 October, 2016

Accepted 13 December, 2016

The authors report no conflicts of interest.

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INTRODUCTION

Percutaneous coronary intervention (PCI) is effective in quickly increasing blood supply to ischemic myocardial tissues and is commonly used for the treatment of chronic coronary heart disease, ST-segment elevation myocardial infarction (STEMI), non-STEMI, and unstable angina pectoris. However, PCI can lead to a series of vascular complications, including vessel dissection, restenosis, and even death. All of these may result from post-procedural endothelial dysfunction and platelet activation (1–3). To improve the prognosis of PCI, investigation into the mechanisms of these effects is necessary.

Microparticles (MPs) contain complex procoagulant and proinflammatory properties and are shed from activated or apoptotic vascular or peripheral blood cells (4, 5). Our previous study reported that MPs were increased in acute coronary syndrome (ACS), and MPs impaired vasodilatation by inhibiting the Akt/eNOS- Hsp90 signaling pathway in rats (6). Recently, it has been reported that the elevated MPs were reduced after PCI (7, 8). However, the biological function of MPs after PCI has not been reported on until now, and the aim of the present study was to test the influence of PCI on MP function.

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MATERIALS AND METHODS

Study population

A total of 38 patients (20 men and 18 women, but a power analysis has not been performed to get a suitable sample size) with ST-segment elevation myocardial infarction (STEMI) and non-STEMI (NSTEMI) without previous myocardial infarction (patients suffered percutaneous coronary intervention [PCI]) were recruited during 2014. All patients were admitted to the intensive care unit of The Second Affiliated Hospital, Xi’an Jiaotong University. Primary PCI was performed within 24 h of symptom onset for ongoing pain despite pharmacotherapy, including intravenously infused nitroglycerin trinitrate, orally administered prophylactic antianginal agents, and intravenous heparin. All of the patients with diseases that could have increased MPs were excluded, including infectious disease, hypertension, severe trauma, diabetes mellitus, multiple sclerosis, lupus anticoagulant, renal failure, and rheumatic diseases in the acute stage. In addition, 20 age- and sex-matched healthy subjects were enrolled as a control group. Informed consent was obtained from both the patients and the healthy subjects. This study was approved by The Second Affiliated Hospital Xi’an Jiaotong University Ethics Review Board.

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Blood sampling and MP isolation

Fasting peripheral blood samples were obtained on the day of hospitalization and 24 h post-procedure. Platelet-poor plasma was obtained from the blood samples by centrifugation (11,000 g, 2 min, 4°C, 50 μL platelet-poor plasma was used for flow cytometry analysis in the next step), and MPs were isolated by centrifugation at 13,000 g for 45 min (4°C) (9). Then, the precipitant (MPs) was obtained and resuspended with RPMI1640 (Gibco-Invitrogen, Carlsbad, Calif, 100 μL). A bicinchoninic acid protein assay (Merck, Kenilworth, NJ) was used to determine the concentrations of MPs. The MPs were stored at −80°C for further analysis. To perform a series of experiments with limited blood samples, we mixed several MPs from different patients into batches in the present study. To reduce the difference among batches, patients with different cardiac function and coronary artery stenosis were included in each batch. The MP concentrations we used for the experiments corresponded to the circulating plasma (9), as shown in Figure 1 (control: 2.65 ± 0.52 mg/mL; pre-operative: 4.70 ± 0.78 mg/mL; and postoperative: 4.88 ± 0.61 mg/mL). To test the hypothesis that the quality of MPs was impacted by PCI, the MP concentration for the following experiments was set at the same level (3 mg/mL).

Fig. 1

Fig. 1

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Flow cytometry analysis

The platelet-poor plasma (50 μL, reserved in the above procedure) was incubated with 5 μL of anti-CD31-PE and 5 μL of anti-CD41-FITC (Beckman Coulter, Brea, Calif) for 30 min at room temperature (10). Before the samples were analyzed, flow count calibrator beads (50 μL Beckman Coulter) were added to the antibody-labeled tubes. Endothelial-derived MPs (EMPs) were defined as CD31(+)/CD41(−) MPs, while CD31(+)/CD41(+) MPs were considered platelet-derived MPs (PMPs). The gate size was set <1 μm.

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Vasodilatation testing

Sprague–Dawley rats were purchased from the Animal Experimental Center of Xi’an Jiaotong University. After decapitation, the thoracic aortas were separated and immersed in ice-cold Krebs solution (mmol/L), Sigma-Aldrich (St. Louis, Mo): NaCl 119.0, NaHCO3 25.0, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, and MgSO4 1.2 (37°C, pH 7.4). After the tissue surrounding the aortas was carefully removed, the aortic rings were cut into four to five segments that were 2 to 3 mm wide and connected to an isometric force transducer (EMKA Technologies, Paris, France) as previously described (11). After equilibration in organ chambers filled with Krebs solution (aerated continuously with 95% O2 and 5% CO2) for 60 min, the aortic rings were exposed to 60 mmol/L KCl at least three times to test their stabilization. Then, the rings were treated for 30 min with MPs from the healthy patients or the AMI patients before and after PCI. Then, phenylephrine (PE, Sigma-Aldrich, 10−6 mol/L) was used to preconstrict the rings. With or without pre-incubation with NG-nitro-L-arginine methyl ester (L-NAME; 1 mmol/L, eNOS inhibitor, Sigma-Aldrich) for 30 min, endothelium-dependent relaxation to acetylcholine (Ach: 10−8–10−4 mol/L. Sigma-Aldrich) was performed after PE. Nitrovasodilator sodium nitroprusside (SNP; 10−8–10−4 mol/L, Sigma-Aldrich) was applied to detect endothelium-independent relaxations. All animal experiments were approved by The Second Affiliated Hospital Xi’an Jiaotong University Animal Ethics Committee. A “blank” group from the same thoracic aorta without MPs incubation was used to test the effect of manipulation on endothelial functions.

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Superoxide (O2) detecting

Briefly, the aortic rings were split vertically and incubated in Krebs solution for 30 min for equilibration. Then, the rings were pre-incubated with or without L-NAME (1 mmol/L) for another 30 min in this solution. After pre-incubation, the vessels were treated with or without MPs from healthy subjects or AMI patients for 30 min. Then, the rings were washed twice with PBS (Gibco-Invitrogen, Carlsbad, Calif). PBS with hydroethidine (HE; Ana Spec, Fremont, Calif, 10 μmol/L) was added for another 30 min after washing. Fluorescence images were obtained using a laser scanning confocal microscope (excitation 488 nm, emission 530 nm). H2O2 (0.5 mol/L) was used as a positive control. The range of fluorescent intensities was assessed using NIH image J software. Superoxide anion generation was reported in terms of the relative changes in pixel intensity in arbitrary units as described previously (12).

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Measurement of nitric oxide (NO) generation

The aortic rings were split vertically and underwent equilibrium for 30 min in 12-well plates with Krebs solution. Then, the rings were treated with or without MPs from the healthy subjects or AMI patients. Thirty minutes after the MPs incubation, NO concentration in the supernatant was detected by No assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Vascular endothelial growth factor (VEGF, 50 ng/mL, Sigma-Aldrich) was used as a positive control. The dry weights of the aortic rings were recorded to calculate NO generation (nmol/mg protein) (10).

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Western blot analysis

The rat thoracic aortas were treated with or without MPs from the healthy subjects or AMI patients for 1 h (12 h for caveolin-1 detecting) before being washed three times with PBS. Then, the aortic proteins were harvested and immunoblotting was performed as previously described (10). Antibodies of endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Santa Cruz, Calif), phosphorylated eNOS at Ser1177 (Cell Signaling Technology, Danvers, Mass), phosphorylated eNOS at T495 (Cell Signaling Technology), caveolin-1 (Cell Signaling Technology), and GAPDH (Cell Signaling Technology) were used for western blot analysis.

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Immunoprecipitation

After being treated for 1 h with or without MPs from the healthy subjects or AMI patients, the aortas were washed three times with PBS. Then, the aortic proteins were harvested on ice. Aortic protein lysates were incubated for 24 h with the anti-eNOS antibody (Santa Cruz Biotechnology, sc-136977) to immunoprecipitate eNOS. Then, the eNOS immunocomplex was mixed with Laemmli buffer (Cell Signaling Technology), heated (95°C, 5 min), mixed, and stored on ice for at least 2 min before centrifugation (2,400 rpm, 2 min, 4°C). The separated proteins (supernatant) were immunoblotted for association of eNOS (Santa Cruz Biotechnology, sc-654) and heat shock protein 90 (Hsp90, Santa Cruz Biotechnology), as previously described (10).

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Statistical analysis

All data are presented as the means ± standard deviation, and data were analyzed using Graph Pad Prism, version 5.0. Independent-samples t test was used for normally distributed continuous variables comparing, and Mann–Whitney U test was used for non-normally distributed continuous variables. Comparison between two-groups was performed using an independent-samples t test, and a one-way analysis of variance was used for multigroup comparison. Differences were considered significant when P <0.05.

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RESULTS

Clinical data

As shown in Table 1, all of the clinical characteristics were similar between the healthy subjects and patients, except for the Killip classification and medications.

Table 1

Table 1

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Plasma MPs concentrations

As shown in Figure 1, when compared with the control group (2.65 ± 0.52 mg/mL, n = 20), the plasma MP concentrations were elevated in the AMI patients before they underwent PCI (4.70 ± 0.78 mg/mL, n = 38). PCI can slightly increase the concentrations of the MPs 24 h post-procedure (5.46 ± 0.61 mg/mL, n = 38).

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Detecting the origin of the MPs

As shown in Figure 2A, EMPs (CD31(+)/CD41(−), yellow area, 38.97 ± 2.17%, n = 38), and PMPs (CD31(+)/CD41(+), red area, 35.84 ± 2.49%, n = 38) accounted for the majority of the MPs before PCI. Among the MPs collected 24 h post-procedure, many more EMPs (yellow area, 43.53% ± 2.35%, n = 38, Fig. 2B) were detected than PMPs (red area, 31.71% ± 3.11%, n = 38, Fig. 2B). In healthy subjects, few EMP and PMP were detected (Fig. 2C).

Fig. 2

Fig. 2

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Effects of MPs on endothelium-dependent vasodilatation

Compared with the MPs from the healthy subjects, the MPs from the patients with AMI impaired endothelium-dependent relaxation (Fig. 3A) and this effect were enhanced by the postoperative MPs (Fig. 3A). However, the endothelium-independent vasodilatation responses to SNP were unaltered (Fig. 3B). L-NAME completely blocked Ach-induced vasodilatation treated by MPs, both from the control group and the AMI patients (data not listed).

Fig. 3

Fig. 3

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Effects of MPs on NO and O2 generation

To detect whether MPs from the AMI patients after PCI impaired vascular function by increasing oxidative stress, we tested the effects of MPs on NO and O2 generation. Compared with the MPs from the healthy subjects, the MPs from the AMI patients decreased NO (but no statistical differences, Fig. 4C) production but increased O2 generation (Fig. 4, A and B). Additionally, PCI enhanced the influence of the MPs on NO and O2 generation (Fig. 4).

Fig. 4

Fig. 4

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Effects of MPs on the eNOS and caveolin-1 expression

To investigate the mechanism by which the increased MPs impaired vascular function, their effects on eNOS, phosphorylation of eNOS, and caveolin-1 expression were detected by western blotting. Compared with the MPs from the healthy subjects, the MPs collected from AMI patients before PCI reduced eNOS phosphorylation at the Ser1177 site (Fig. 5A) and increased caveolin-1 and eNOS phosphorylation at the T495 site (Figs. 5B and 6A). All of these effects were enhanced by the MPs from the AMI patients who underwent PCI (Figs. 5 and 6A).

Fig. 5

Fig. 5

Fig. 6

Fig. 6

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Effects of MPs on the association of eNOS with Hsp90

To detect whether the MPs break the balance between NO and O2 by affecting the association of eNOS with Hsp90, immunoprecipitation was performed. MPs from the patients both with and without PCI significantly decreased the association of eNOS with Hsp90 (Fig. 6B).

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DISCUSSION

Our study demonstrated that plasma MPs were increased in AMI patients before and after PCI. Of the MPs that were obtained preoperatively, the majority were EMPs and PMPs. However, PCI increased the generation of EMPs but decreased the production of PMPs. MPs from the AMI patients, especially the MPs measured 24 h after PCI, impaired endothelium-dependent vasodilatation by inhibiting eNOS phosphorylation site at Ser1177, enhancing caveolin-1 expression and eNOS phosphorylation site at T495, uncoupling eNOS correlation with Hsp90, and increasing O2 production but reducing NO generation.

MPs are increased in a number of diseases, and nearly all of these diseases are harmful to the vascular system (13–18). Both our team and other investigators have demonstrated that plasma MPs are increased in patients with ACS (6, 19–21), and we also found that MPs from ACS patients can impair endothelial-dependent vasodilatation by inhibiting the AKT/eNOS-Hsp90 pathway (6). Goligorsky et al. found that EMPs directly affect the endothelium and aggravate pre-existing endothelial cell dysfunction by increasing O2 generation (22). Endothelium plays a key role in the maintenance of vascular homeostasis by secreting vasoactive factors (23). In contrast to the findings of Min et al. (8), we found that total MP concentrations increased in patients after PCI (Fig. 1). Maybe, Min et al. (8) detected MP levels in culprit coronary arteries, but we detected MP levels from the peripheral vasculature. What is more, the EMP proportion was increased but the PMP proportion was decreased post-PCI.

PCI has been associated with a series of vascular complications because it directly and violently squeezes the coronary artery endothelium and plaques (1–3). Thus, patients undergoing PCI are very likely to suffer complications related to endothelial dysfunction: recurrent myocardial infarction, a high risk of in-stent stenosis, and even death. Since there is an overlap of PCI and MPs on endothelium and plaques, we evaluated whether PCI could further damage endothelial function in the present study. Both the total quantity of MPs and the proportions were influenced by PCI (Fig. 2). The increase in EMPs may have resulted from the violent squeeze to the coronary artery endothelium during PCI. To our surprise, the PMP ratio decreased after PCI (Fig. 2B), this may be explained by the administration of anticoagulants (Aspirin or Clopidogrel) (24).

Vasodilatation plays an important role in the regulation of hemoperfusion and arterial blood pressure, and the main participant is the endothelium (25). Cipolla and Huang reported that the imbalances in pro- and anti-inflammatory, and pro- and anti-thrombotic molecules result in endothelial dysfunction (26, 27). eNOS plays important role in maintaining endothelial function (28). Our previous study demonstrated that MPs from ACS patients impaired vasodilatation by blocking eNOS phosphorylation at site Ser1177 (6). In the present study, postoperative MPs from the AMI patients who underwent PCI further impaired vasodilatation by inhibiting eNOS phosphorylation at site Ser1177 and increasing eNOS phosphorylation at site T495 (Figs. 3 and 5).

Caveolin-1 can regulate NO production by interaction with eNOS to decrease its activity (29, 30). Gross et al. found that homocysteine could lead to endothelial dysfunction by promoting the correlation of eNOS and caveolin-1, which resulted in decreased NO (31). Balligand et al. reported that hypercholesterolemia could induce impairment of NO production through the modulation of caveolin abundance in endothelial cells (32). In the present study, we found that MPs from AMI patients, especially those who underwent PCI, increased the expression of caveolin-1 (Fig. 6A). This may result in the inactivity of eNOS, impairment of NO production, and vasodilatation.

NO can reduce the generation of reactive oxygen species by inhibiting the association of NAD(P)H oxidase subunits. However, when excessively produced, NO reacts with O2 resulting in the formation of peroxynitrite, which is a free radical that impairs endothelial function (33). Increased oxidative stress has been reported to impair vascular tone and increase platelet aggregation, pro-inflammatory cell adhesion, and vascular smooth muscle cell proliferation (34). Our previous work demonstrated that NO was generated mainly when eNOS connected with Hsp90, while the uncoupling of eNOS with Hsp90 led mostly to the production of O2(6). Thus, we tested the effect of MPs from AMI patients before and 24 h after PCI on oxidative stress and the correlation of eNOS with Hsp90 in the present study. MPs from AMI patients decreased NO generation (but no statistical differences, Fig. 4C) and eNOS correlated with Hsp90 (Fig. 6B), and undergoing PCI further strengthened these effects. However, MPs, especially the MPs collected postoperatively, increased O2 generation. The increased O2 could be inhibited by L-NAME. These data indicated that MPs increase oxidative stress by uncoupling eNOS.

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Limitations of study

Since currently there is no available method to isolate single-origin MPs from plasma or serum in humans, the MPs we used were a mixture of EMPs, PMPs, and others. Since aortic vasculature was used to detect the influence of MPs on endothelial function, the differences between aortic vascular and coronary artery on reactivity, physiological, and anatomical are one limitation of the present study. What is more, as the aortic vasculature we used was derived from rats, the influence of species was another limitation. The small-study population numbers were another limitation. Although we predicted that MPs produced after PCI may influence prognosis by impair vascular function, whether its further consequences are the result of acute conditions associated with myocardial infarction or the effect of MPs is unclear. Above all, the clinically meaningful of our conclusion is required verification.

In summary, postoperative MPs further impaired endothelial-dependent vasodilatation by decreasing eNOS phosphorylation at site Ser1177, eNOS correlation with Hsp90, and NO production; increasing caveolin-1 expression, eNOS phosphorylation at site T495, and O2 generation. The endothelial dysfunction resulting from MPs may result in the poor prognosis of patients with AMI who undergo PCI.

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Keywords:

Circulation microparticles; endothelial dysfunction; percutaneous coronary intervention; vasodilatation

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