Characteristics of the patient groups and routine blood analysis.

<p>SAP: stable angina pectoris, ACS: acute coronary syndrome patients.</p><p>Values of continuous variables are means ± SD, values of categorical variables are n (%).</p><p>Exceptions for sample numbers in lipid panel: total cholesterol, triglycerides (SAP: n = 19, ACS: n = 16); HDL-cholesterol (SAP: n = 18, ACS: n = 16); LDL-cholesterol (SAP: n = 18, ACS: n = 15). <i>p</i><0.05 values (significant differences between the two groups) are denoted by asterisks (*).</p

ACTH- and Cortisol-Associated Neutrophil Modulation in Coronary Artery Disease Patients Undergoing Stent Implantation

<div><p>Background</p><p>Psychosocial stress and activation of neutrophil granulocytes are increasingly recognized as major risk factors of coronary artery disease (CAD), but the possible relationship of these two factors in CAD patients is largely unexplored. Activation of neutrophils was reported to be associated with stenting; however, the issue of neutrophil state in connection with percutaneous coronary intervention (PCI) is incompletely understood from the aspect of stress and its hypothalamic-pituitary-adrenal axis (HPA) background. Thus, we aimed to study cortisol- and ACTH-associated changes in granulocyte activation in patients undergoing PCI.</p><p>Methodology/Principal Findings</p><p>Blood samples of 21 stable angina pectoris (SAP) and 20 acute coronary syndrome (ACS) patients were collected directly before (pre-PCI), after (post-PCI) and on the following day of PCI (1d-PCI). Granulocyte surface L-selectin, CD15 and (neutrophil-specific) lactoferrin were analysed by flow cytometry. Plasma cortisol, ACTH, and lactoferrin, IL-6 were also assayed. In both groups, pre- and post-PCI ratios of lactoferrin-bearing neutrophils were relatively high, these percentages decreased substantially next day; similarly, 1d-PCI plasma lactoferrin was about half of the post-PCI value (all <i>p</i>≤0.0001). Post-PCI ACTH was reduced markedly next day, especially in ACS group (SAP: <i>p</i><0.01, ACS: <i>p</i>≤0.0001). In ACS, elevated pre-PCI cortisol decreased considerably a day after stenting (<i>p</i><0.01); in pre-PCI samples, cortisol correlated with plasma lactoferrin (<i>r</i>∼0.5, <i>p</i><0.05). In 1d-PCI samples of both groups, ACTH showed negative associations with the ratio of lactoferrin-bearing neutrophils (SAP: <i>r</i> = −0.601, <i>p</i><0.005; ACS: <i>r</i> = −0.541, <i>p</i><0.05) and with plasma lactoferrin (SAP: <i>r = </i>−0.435, <i>p</i><0.05; ACS: <i>r = </i>−0.609, <i>p</i><0.005).</p><p>Conclusions/Significance</p><p>Pre- and post-PCI states were associated with increased percentage of activated/degranulated neutrophils indicated by elevated lactoferrin parameters, the 1d-PCI declines of which were associated with plasma ACTH in both groups. The correlation of plasma cortisol with plasma lactoferrin in the extremely stressed ACS before stenting, however, suggests an association of cortisol with neutrophil activation.</p></div

Plasma levels of lactoferrin and IL-6 in CAD patients undergoing PCI.

<p>Blood samples were collected from stable angina pectoris (SAP) and from acute coronary syndrome (ACS) patients directly before (Pre), directly after (Post) and on the following day (1d) of PCI. Data are presented as mean ± SEM; in case of SAP, n = 21 (IL-6: n = 18), and for ACS, n = 20 (IL-6: n = 19, except: Post: n = 18). Significant differences: <i>p</i>*** <0.001, <i>p</i>**** ≤0.0001.</p

Flow cytometry analysis of granulocytes in CAD patients undergoing PCI.

<p>Percentages of marker-bearing granulocytes and mean fluorescence intensities (MFI) of granulocytes reflect the cell surface appearance of activation markers directly before, directly after and on the following day of PCI (Pre, Post, 1d) in patients with stable angina pectoris (SAP) or with acute coronary syndrome (ACS). Data are mean ± SEM; in case of SAP, n = 21, and for ACS, n = 20 (ACS exception: 1d values, n = 19). Significant differences: <i>p</i>* <0.05, <i>p</i>** <0.01, <i>p</i>*** <0.001, <i>p</i>**** ≤0.0001.</p

Correlations between stress hormones and selected inflammatory markers.

<p>SAP: stable angina pectoris, ACS: acute coronary syndrome patients; pre-PCI: directly before, and 1d-PCI: on the following day of PCI (stenting).</p><p><i>r:</i> Spearman’s rank correlation coefficient; asterisks (*) denote significancy of correlations (<i>p</i><0.05).</p><p>SAP: n = 21, ACS n = 20, except for IL-6 (ACS pre- and 1-d-PCI: n = 19) and for percentage of surface lactoferrin-bearing cells (in ACS patients 1-day after PCI: n = 19).</p

Plasma levels of ACTH and cortisol in CAD patients undergoing PCI.

<p>Blood samples were collected from stable angina pectoris (SAP) and from acute coronary syndrome (ACS) patients directly before (Pre), directly after (Post) and on the following day (1d) of PCI. Data are presented as mean ± SEM; in case of SAP, n = 21, and for ACS, n = 20. Significant intragroup differences: Pre/1d, Post/1d; <i>p</i>* <0.05, <i>p</i>** <0.01, <i>p</i>**** ≤0.0001; significant intergroup differences (SAP vs ACS) for ACTH: 1d, <i>p</i>x** = <i>p</i>** <0.01; for cortisol: Pre, <i>p</i>* <0.05.</p

Effects of 5f and 5g (10 μM) on the expression of phosphorylated (upper panel) and total (lower panel) stathmin protein in HeLa cells after incubation for 48 h, determined by western blot analysis.

<p>Results are mean values ± SEM of the data on two separate measurements, n = 6. ns indicates p > 0.05, *** indicates p < 0.001 as compared with the untreated control cells. Panels below are representative membrane pictures.</p

Induction of caspase-3, caspase-8 and caspase-9 activities after incubation with compounds 5f and 5g for 24 h.

<p>White, gray and black columns denote 3, 10 and 30 μM of the given agent. * and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control condition.</p

Effects of compounds 5f, 5g, 5h and 4i on the HeLa cell cycle distribution after incubation for 24 (left panels) and 48 h (right panels).

<p>Gray and black columns indicate 3 and 10 μM, respectively. * and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control cells.</p

Expression of CDK1, cyclin B1, cyclin B2 and cdc25B at the mRNA level after incubation with 3 (gray columns) or 10 (black columns) μM of compounds 5f or 5g.

<p>*, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively, as compared with the control condition.</p