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WHAT’S NEW? The role of oxidative stress and low-grade endotoxemia is unclear in patients with coronary microvascular angina (MVA). This study aimed to evaluate NADPH oxidase-2 activation, serum lipopolysaccharide levels, as well as oxidative stress production, and endothelial dysfunction in MVA patients and controls. The results of this study show that MVA patients have high circulating levels of serum lipopolysaccharide, oxidative stress, and endothelin 1. NADPH oxidase-2 activation could contribute to provoking an imbalance between pro-oxidant and antioxidant systems, which could determine endothelial dysfunction and vasoconstriction in MVA. |
INTRODUCTION
Microvascular angina (MVA) is defined as microvascular dysfunction of the coronary arteries. It is characterized by typical chest pain, evidence of myocardial ischemia on an electrocardiogram, and the absence of obstructive coronary artery disease (CAD) [1]. MVA represents an increasingly growing problem when we consider that up to 50% of patients with angina who undergo coronary angiography have not any significant artery obstruction [2–4]. Despite the absence of significant coronary lesions, its prognosis is not benign, because MVA is an independent risk factor for major adverse cardiovascular events [5, 6].
Microvascular dysfunction is studied invasively with coronary angiography and use of pharmacological tests to evaluate the endothelial reactivity [7]. It has been demonstrated that it mainly embraces two aspects: firstly, inadequate vasodilatation to a stimulus, and secondly, exaggerated vasoconstriction (coronary microvascular spasm) [7]. Experimental studies suggested a major role of oxidative stress in the initiation and progression of microvascular dysfunction in MVA [8]. NADPH oxidase-2 (NOX2) is considered one of the main sources of superoxide anion in humans, a modulator of the arterial tone [9–12], and an enzyme directly implicated in microvascular dysfunction [13]. The role of NOX2 in MVA has not been yet elucidated.
Lipopolysaccharide (LPS) is an endotoxin derived from the membrane of gram-negative bacteria that, by binding to Toll-like receptor 4 (TLR4), activates intracellular transcription of several inflammatory mediators in the vessels [14]. An impairment of tight junction causes translocation of LPS from the gut to the systemic circulation where it initiates pro-inflammatory and pro-oxidant effects in the vessels [15]. Moreover, a recent experimental study showed that LPS from Escherichia coli was localized in human plaque and may contribute to atherosclerotic damage via TLR4-mediated oxidative stress and NOX2 activation [16, 17]. A relationship between LPS and NOX2 activation has been previously described in other clinical settings such as non-alcoholic fatty liver disease (NAFLD), pneumonia [18], and neurodegenerative disease [19], but no data have showed its role in MVA yet. Thus, in this study, we evaluated the potential role of low-grade endotoxemia by LPS in eliciting systemic Nox2 activation and the balance of pro-oxidant molecules, such as H2O2, and the antioxidant system, such as hydrogen peroxide breakdown activity (HBA), in MVA.
METHODS
The study was supported by the Sapienza University in cooperation with Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova, to strengthen institutional competencies and the management of partner universities, finalized to cardiovascular prevention [20].
Forty consecutive Moldovan patients with coronary MVA and 40 controls (CT) matched for age and sex agreed to participate in the study, which was performed between July 2018 and December 2020. These patients were recruited at the Moldavian Research Institute of Cardiology, Chisinau, Republic of Moldova.
Coronary MVA was diagnosed according to the criteria suggested by the Coronary Vasomotion Disorders International Study Group [21]; these criteria include (1) presence of symptoms suggestive of myocardial ischemia; (2) objective documentation of myocardial ischemia, as assessed by currently available techniques; (3) absence of obstructive CAD (<50% coronary diameter reduction and/or fractional flow reserve >0.80) documented by CT coronary scan or coronary angiography, as in our case; (4) confirmation of a reduced coronary blood flow reserve and/or inducible microvascular spasm [21]. Microvascular disorder was confirmed by using the angiographic flow assessment criteria based on the thrombolysis in myocardial infarction (TIMI) flow grade or TIMI frame count (TFC/cTFC). TIMI-2 flow grade, which means partial perfusion (i.e. requiring three or more beats to opacify the distal vessel) or a corrected TIMI frame count >27 frames, in at least one major coronary vessel, have been frequently used and were, therefore, preliminarily assumed. The latter is based upon images acquired at 30 frames/second and a correction factor of 1.7 for the left anterior descending artery (LAD). The speed with which the dye reaches the distal bifurcation of LAD from the ostium is measured in frame counts. In right coronary artery, the flow was measured from the ostium to the origin of the first posterolateral branch. In the left circumflex, the flow was measured from the ostium to the most distal branch of the last OM. A correction factor of 1.7 was used for LAD (for corrected TFC). The normal count was considered 21 ± 3 frames.
Considering the COVADIS (Coronary Vasomotion Disorders International Study Group) criteria, our cohort of patients had a definitive diagnosis of MVA.
From all patients included in the study, blood samples for analysis of oxidative stress, LPS, and zonulin levels were collected after a fasting period of 8 hours.
Subjects were excluded from the study if they had liver insufficiency, advanced chronic kidney disease, acute cerebrovascular disease, acute myocardial infarction, recent abdominal surgery or were taking antioxidants. We also excluded patients with active cancer, uncontrolled blood pressure or diabetes, inflammatory bowel disease, and those taking or with a recent intake of antibiotics.
Informed written consent was obtained from all subjects: the study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethical Committee of the Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova (no. 3-9-19/41/49).
Serum sNOX2-dp detection
NOX2 activation was measured as soluble NOX2-derived peptide (sNOX2-dp) with an ELISA method, as previously described [22]. Briefly, the peptide is recognized by binding to a specific monoclonal antibody against the amino acid sequence (224–268), the extra membrane portion of NOX2. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation were 8.95% and 9.01%, respectively.
H2O2 production
The H2O2 was evaluated by a Colorimetric Detection Kit (ArborAssay, Ann Arbor, MI, US) and expressed as μM. Intra-assay and inter-assay coefficients of variation were 2.1% and 3.7%, respectively.
Determination of % HBA in serum
Serum hydrogen peroxide (H2O2) HBA was measured with an HBA assay kit (Aurogene, Rome, Italy; code HPSA-50). The percentage of HBA was calculated according to the following formula: % of HBA = [(Ac–As) / Ac] × 100, where Ac is the absorbance of H2O2 1.4 mg/ml, and As is the absorbance in the presence of the serum sample.
NO bioavailability
A colorimetric assay kit (Abcam, Cambridge, UK) was used to determine NO bioavailability as previously described [23]. Intra-assay and inter-assay coefficients of variation were 2.9% and 1.7%, respectively.
Serum zonulin
Serum zonulin was used as an intestinal permeability assay. Serum zonulin levels were measured using a commercial ELISA kit (Elabscience, Houston, TX, US). Antibody specific for zonulin has been pre-coated onto a microplate and 100 μl of standards, and patient sera samples were added and incubated for 90 min at 37°C. Then, a biotinylated detection antibody specific for zonulin and Avidin-Horseradish Peroxidase (HRP) conjugate was added to each microplate. Values were expressed as ng/ml; both intra-assay and inter-assay coefficients of variation were within 10%.
Serum LPS assay
LPS levels in serum were measured using a commercial ELISA kit (Cusabio, Houston, TX, US). The standards and samples were plated for 2 hours at room temperature into a micro-plate pre-coated with the antibody specific for LPS. After incubation, samples were read at 450 nm. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation were <10%.
Serum endothelin 1 assay
Serum endothelin 1 (ET-1) levels were measured using a commercial ELISA kit (Thermo Fisher Scientific, Waltham, MA, US).
Statistical analysis
Statistical analyses were undertaken using SPSS 25.0 software for Windows (IBM, Armonk, NY, US). The Kolmogorov–Smirnov test was used to determine whether variables were normally distributed. Normally distributed data are described as means and standard deviations (SD). Between-group differences were analyzed by Student t-test. Differences between percentages were assessed by the χ2 test. Bivariate analysis was performed by Spearman correlation; the variables with evidence of an association P <0.10 were included in a multivariable linear regression using an automated procedure. The results of the multivariable linear regression analysis were expressed as standardized coefficient beta (β) with standard error (SE). Moreover, the coefficient of determination was provided (R2). A P-value of <0.05 was considered statistically significant.
Sample size determination
We computed the minimum sample size with respect to a two-tailed, one-sample Student t-test considering, on the basis of data from a previous pilot study (data not shown): a difference of 7.3 pg/ml for sNOX2-dp levels between patients and controls (mean patients, 32; mean controls, 24.2 pg/ml), 10 as SD, 0.05 (α) as type-I error probability and 0.95 as power 1−β. The sample size was 40 patients/group.
RESULTS
Clinical characteristics of MVA patients and controls are reported in Table 1. There was no significant difference in age, sex, and classic cardiovascular risk factors between the two groups (Table 1).
|
Patients with microvascular angina (MVA) n = 40 |
Patients without microvascular angina (control group) n = 40 |
P-value |
|
|
Age, years |
62.7 (8.1) |
61.7 (5.7) |
0.56 |
|
Male sex |
14 (35) |
20 (50) |
0.17 |
|
Diabetes |
10 (25) |
4 (10) |
0.11 |
|
Dyslipidemia |
36 (90) |
38 (95) |
0.39 |
|
Hypertension |
37 (92) |
36 (90) |
0.69 |
|
Current smokers |
6 (15) |
8 (20) |
0.46 |
|
Obesity |
15 (37) |
18 (45) |
0.49 |
|
Anticoagulants |
3 (7.5) |
2 (5) |
0.74 |
|
Βeta-blockers |
34 (85) |
19 (47) |
<0.01 |
|
Calcium antagonists |
28 (70) |
10 (35) |
<0.01 |
|
ACE inhibitors |
23 (57) |
15 (37) |
0.14 |
|
Renin-angiotensin-aldosterone system inhibitors |
12 (30) |
2 (5) |
0.01 |
|
Statins |
33 (82) |
25 (62) |
0.11 |
|
Diuretics |
17 (42) |
3 (7.5) |
<0.01 |
|
Anti-platelet drugs |
35 (87) |
21 (52) |
<0.01 |
LPS and zonulin
MVA patients had increased levels of LPS (mean [SD], 37.5 [12.9] pg/ml vs. 20.7 [10.1] pg/ml; P <0.001) and zonulin (3.0 [0.9] ng/ml vs. 1.57 [0.5] ng/ml; P <0.001) compared to the control group (Figure 1A and B).
Oxidative stress
Compared with CT, MVA patients had significant higher mean values of sNOX2-dp (57.4 [13.1] pg/ml vs. 27.4 [11.5] pg/ml; P <0.001) and H2O2 production (47.7 [21.4] µM vs. 22.1 [10.3] µM; P <0.001) (Figure 2A and B). Conversely HBA were significantly lower in patients with MVA compared to controls (27.8 [10.8] vs. 56.1 [21.2] %; P <0.001) (Figure 2C).
Endothelial dysfunction and NO bioavailability
MVA patients had significant lower NO bioavailability (28.1 [10.5] µM vs. 35.6 [14.4] µM; P = 0.01) and higher levels of ET-1 (15.6 [6.2] pg/ml vs. 10.2 [5.6] pg/ml; P <0.001) (Figure 3A and B).
Associations among the studied variables
Univariate analysis by Spearman correlation test showed that sNOX2 was associated with serum LPS (Rs = 0.629; P <0.001), serum zonulin (Rs = 0.641; P <0.001), H2O2 (Rs = 0.590; P <0.01), and ET-1 (Rs = 0.410; P <0.001). Furthermore, an inverse correlation between sNOX2 and HBA (Rs = –0.646; P <0.001) and nitric oxide bioavailability (Rs = –0.312; P <0.01) was observed. LPS was also associated with serum ET-1 (Rs = 0.243; P = 0.03), zonulin (Rs = 0.474; P <0.001), and oxidative stress, as shown by its correlation with H2O2 (Rs = 0.490; P <0.001) and HBA (Rs = –0.560; P <0.001).
Multiple linear regression analysis, adjusted for statins, beta-blockers, calcium antagonists, diabetes and hypertension, showed that LPS (SE, 0.129; standardized coefficient β, 0.335; P =0.001) and zonulin (standardized coefficient β, 0.279; SE, 1.828; P =0.006) emerged as the only independent predictive variables associated with sNOX2 (R2 = 61%). Furthermore, sNOX2 (standardized coefficient β, 0.469; SE, 0.099; P = 0.001) and HBA (standardized coefficient β, –0.258; SE, 0.082; P = 0.04) were the independent predictive variables associated with LPS (R2 = 44%).
DISCUSSION
The study shows for the first time that MVA patients have increased LPS serum levels. To find if gut permeability may account for LPS increase in patients with MVA, we measured the circulating levels of zonulin, which modulates gut permeability by disassembling the intercellular tight junctions [24]. Experimental and clinical studies demonstrated that zonulin up-regulation increases gut permeability [25]. The increased serum levels of zonulin in patients with MVA and its correlation with serum LPS could provide evidence that gut permeability is enhanced in this cohort and may be responsible for the high circulating levels of LPS.
LPS is a pro-inflammatory molecule that may favor endothelial dysfunction with an oxidative stress-mediated mechanism. In particular, LPS binds to TLR4 complex and activates a series of proteins and kinases in endothelial cells (as nuclear factor-kappa B [NF-κB]), interleukin 1 receptor-associated kinase (IRAK), tumor necrosis factor receptor-associated factor 6, NF-kB-inducing kinase, and inhibitor kappa B, which increase the production of proinflammatory molecules (as cytokines/chemokines) [26].
In accordance with this, LPS directly correlated with NOX2, suggesting a role for LPS as a trigger for oxidative stress [8]. The high levels of H2O2 and the close relation with sNOX2 observed in this study suggest that systemic oxidative stress in MVA could derive from LPS-induced NADPH oxidase activation as hypothesized by previous papers [8, 27].
NOX2 is considered an important modulator of artery vasodilation in endothelial cells; previous studies showed that NOX2 activation increases oxidative stress and impaired NO biosynthesis determining vasoconstriction [9, 11, 28].
No description of an association between LPS, NOX2, and endothelial dysfunction in MVA has been reported. Previously, in other settings, such as neurodegenerative diseases and neuropsychiatric disorders, associations among increased LPS, NOX2, oxidative stress, and endothelial dysfunction were reported [19, 29]. Experimental studies in animals showed that LPS could induce cardiac microvascular dysfunction [30]. Mice treated with LPS showed cardiac endothelial dysfunction and higher mortality that could be related to the loss of pericytes [30]. Other studies showed that LPS impaired endothelial dysfunction by reducing vasodilatory response to acetylcholine and eNOS phosphorylation [31, 32].
Our report supports and extends these previous findings in patients with MVA by demonstrating that NOX2 activation could increase oxidative stress and could determine endothelial dysfunction by reduction of NO bioavailability [33] and by increased ET-1 production. Reactive oxygen species generated by NOX2 activation could stimulate ET-1 secretion and provoke microvascular dysfunction [34]. In line with several previous studies, we found increased serum ET-1 levels in MVA patients [8, 35]; however, we cannot exclude that genetic dysregulation of ET-1 could be implicated [36].
The study has some limitations and implications. We did not perform all the invasive tests to evaluate the coronary function, including measurements of CFR, microvascular resistance, acetylcholine, or adenosine provocation test. We did not evaluate other NADPH isoforms, such as NOX1 and NOX4, and other antioxidant systems such as catalase, SOD, and glutathione peroxidase that could also contribute to increased oxidative stress in MVA. The mechanism accounting for LPS translocation from the gut microbiota to systemic circulation was not addressed by the present study. However, changes in gut permeability might be a plausible mechanism as increased serum zonulin, which reflects enhanced gut permeability, is significantly correlated with blood LPS. Nevertheless, zonulin is an indirect marker of gut permeability, and we cannot exclude that serum LPS could also be derived from other sources, thereby a further study is necessary to elucidate this issue. Furthermore, some sources of chronic inflammation such as periodontitis [37] that may have affected LPS levels were not analyzed in this study.
Future studies are necessary to assess if improvement of gut permeability and eventually lowering of low-grade endotoxemia improve artery dysfunction in MVA.
In conclusion, this study provides the first report attesting that patients with MVA have high LPS levels, NOX2 activation, and an imbalance between pro-oxidant and antioxidant systems, in favor of the oxidizing molecules that could be potentially implicated in the endothelial dysfunction and vasoconstriction of this disease. These results could open new therapeutic strategies to modulate gut microbiota and oxidative stress to reduce the cardiovascular risk in MVA patients.
Article information
Conflict of interest: None declared.
Funding: This study was supported by a grant from the University of Rome “La Sapienza” (Università degli Studi di ROMA “la Sapienza” Progetti di COOPERAZIONE E SVILUPPO 2017 n.: 000324_18_RS; funds to LL).
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