Table 1.
Baseline characteristics of patients with aortic stenosis (AS) and healthy controls
| Variable | Patients with AS (n=13) | Controls (n=9) | |
|---|---|---|---|
| Age, years | 66 [60–70] | 59 [58–65] | 0.148 |
| Male, n (%) | 10 (76.9) | 7 (77.8) | 0.991 |
| Body mass index, kg m−2 | 31.2 ±5.4 | 24 ±6.5 | 0.002 |
| Risk factors, n (%) | |||
| Arterial hypertension | 12 (92.3) | 0 | ND |
| Current smoking | 1 (7.7) | 0 | ND |
| Medications, n (%) | |||
| Beta-blockers | 11 (84.6) | 0 | ND |
| Acetylsalicylic acid | 7 (53.8) | 0 | ND |
| Angiotensin converting enzyme inhibitors | 9 (69.2) | 0 | ND |
| Statins | 10 (76.9) | 0 | ND |
| Echocardiographic parameters | |||
| Aortic valve area, cm2 | 0.86 [0.8–0.9] | ND | |
| Mean gradient, mmHg | 48 ±10.0 | ND | |
| Maximum gradient, mmHg | 80 ±15.4 | ND | |
| Peak transvalvular velocity, m/s | 4.5 ± 0.4 | ND | |
| Left ventricular ejection fraction, % | 52.3 ±12.5 | ND | |
| Laboratory investigation | |||
| White blood cells, 103/µl | 6.2 ±1.4 | 6.5 ±1.7 | 0.655 |
| Neutrophils, 103/µl | 3.5 [2.9–4.0] | 3.2 [2.3–3.6] | 0.189 |
| Creatinine, µmol/L | 96 [86–100] | 67 [48–76] | 0.003 |
| C-reactive protein, mg/L | 1.5 [1.0–2.5] | 0.99 [0.99–1.46] | 0.015 |
| Glucose, mmol/L | 5.2 [5.0–5.6] | 5.0 [4.7–5.4] | 0.115 |
| Total cholesterol, mmol/L | 4.2±1.1 | 4.9±0.9 | 0.131 |
| Low density lipoprotein cholesterol, mmol/L | 2.9±1.0 | 3.0±1.1 | 0.827 |
| High density lipoprotein cholesterol, mmol/L | 1.1±0.3 | 1.4±0.3 | 0.032 |
| Triglycerides, mmol/L | 1.7±0.7 | 1.5±0.7 | 0.518 |
[i] Data presented as numbers (percentages), mean ± SD or median [interquartile range] ND: non determined
Figure 1.
Serum levels of NET-specific biomarkers in AS patients and healthy controls
Single dot represents individual result. Horizontal central lines express median values. Boxes express interquartile (Q1–Q3) range
Figure 2.
Representative microphotograph of NETosis generated after ionomycin-stimulation of isolated neutrophils from healthy volunteers
Immunolabelling for citH3 (green). The red arrows indicate the NET-releasing neutrophils. Original magnification 40×
Figure 3.
NETosis examination in flow cytometry
a. Healthy person from the control group, ionomycin-stimulated neutrophils. Density plot, FSC lin/SSC log (SSC data presented in logarithmic scale) display. Neutrophils gated as elliptic gate. Arrows indicate: eosinophils (green arrow), disintegrated neutrophils (debris, red arrow) and mononuclears (blue arrow).
b. The same sample as a. Gated neutrophils displayed as MPO FITC / citH3 Alexa Fluor700 dot plot. Markers inserted according to the control sample (stained with secondary antibodies, ionomycin-stimulated). NET-releasing neutrophils are MPO+citH3+ events in the upper right quadrant (6.7%).
c. In comparison to the control, ionomycin stimulated neutrophils in AS patient. High percentage of NET-releasing neutrophils (22.1%).
d. NET-releasing neutrophils in unstimulated (ionomycin-free) sample (baseline NETs formation in 3.7% neutrophils) from AS patient.
e. The cellular debris (disintegrated neutrophils) gated. They contain the major part of positive MPO+citH3+ staining in the sample but they are not NET-releasing neutrophils.
f. The percentage of NET-releasing (MPO+citH3+) neutrophils in peripheral blood of AS patients compared to healthy controls. Single dot represents individual result. Horizontal central lines express median values. Boxes express interquartile (Q1–Q3) range.