Ruthenium Complex Improves the Endothelial Function in Aortic Rings From Hypertensive Rats

<div><p>Abstract Background: The endothelium is a monolayer of cells that extends on the vascular inner surface, responsible for the modulation of vascular tone. By means of the release of nitric oxide (NO), the endothelium has an important protective function against cardiovascular diseases. Objective: Verify if cis- [Ru(bpy)2(NO2)(NO)](PF6)2 (BPY) improves endothelial function and the sensibility of conductance (aorta) and resistance (coronary) to vascular relaxation induced by BPY. Methods: Normotensive (2K) and hypertensive (2K-1C) Wistar rats were used. For vascular reactivity study, thoracic aortas were isolated, rings with intact endothelium were incubated with: BPY(0.01 to10 µM) and concentration effect curves to acetylcholine were performed. In addition, cumulative concentration curves were performed to BPY (1.0 nM to 0.1 µM) in aortic and coronary rings, with intact and denuded endothelium. Results: In aorta from 2K-1C animals, the treatment with BPY 0.1µM increased the potency of acetylcholine-induced relaxation and it was able to revert the endothelial dysfunction. The presence of the endothelium did not modify the effect of BPY in inducing the relaxation in aortas from 2K and 2K-1C rats. In coronary, the endothelium potentiated the vasodilator effect of BPY in vessels from 2K and 2K-1C rats. Conclusion: Our results suggest that 0.1 µM of BPY is able to normalize the relaxation endothelium dependent in hypertensive rats, and the compound BPY induces relaxation in aortic from normotensive and hypertensive rats with the same potency. The endothelium potentiate the relaxation effect induced by BPY in coronary from normotensive and hypertensive rats, with lower effect on coronary from hypertensive rats.</p></div

High Treg activity and reduced levels of IFN-γ correlate with normal LVEF.

<p>Correlation analyses were performed considering the values of LVEF and the levels IFN-γ in PBMC culture of patients (<b>A</b>) or <i>in vitro</i> suppressive activity of Treg (<b>B</b>). In <b>C</b> is showed the correlation between the frequency of CD4⁺CD25^highFoxp3⁺ and CD3⁺CD4⁺IL-17⁺ T cells in PBMC from patients with the different clinical forms of Chagas' disease (free/mild cardiomyopathy patients, patients with moderate/severe cardiomyopathy and chagasic chronic patients treated with benznidazol after <i>in vitro</i> stimulation with <i>T. cruzi</i> antigens. The p values as long as the correlation coefficient are shown for each graph.</p

Increased frequency of CD4⁺IL-17⁺ Tcells in PBMC from free/mild cardiomyopathy patients.

<p>To examine the existence of Th17 lymphocytes in chronic Chagas' disease patients, PBMC (5×10⁶ cells/ml in a 48 plate well) from patients were cultured by 48 h with trypomastigote antigen (10 µg/mL) and the intracellular expression of IL-17 determined in CD3⁺CD4⁺ T cells by flow cytometry. PBMC from control and Chagas' disease patients in these analyses were gated on lymphocytes via their forward (FSC) and side scatter (SSC) properties, and CD3⁺CD4⁺IL-17⁺ were analyzed to determine the Th17 population. Representatives flow cytometry analysis of CD3⁺CD4⁺IL-17⁺ T cells gated lymphocytes from, Healthy individuals, Free/Mild cardiomyopathy patients, Moderate/Severe cardiomyopathy patients and Bz-treated patients are shown in <b>A</b>., while <b>B</b> shows the grouped analyses of all subjects in each group. Chagas' disease patients were grouped as: Group 1 (n = 10): Patients not treated with benznidazole and free/mild cardiomyopathy, group 2 (n = 11): Patients not treated with benznidazole but with moderate/severe cardiomyopathy, group 3 (n = 8): Patients previously treated with benznidazole free/mild cardiomyopathy. Healthy Individuals (n = 10) from the same endemic areas were included in this study as controls, composing the group 4, as described in Materials and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001630#s2" target="_blank">Methods</a>.</p

Higher IL-10 and lower TNF-α and IFN-γ secretion in free/mild <i>vs.</i> moderate/severe cardiomyopathy patients' PBMC.

<p>Levels of cytokines IL-17 (<b>A</b>), IL-10 (<b>B</b>), TNF-α (<b>C</b>) and IFN-γ (<b>D</b>) as examined by enzyme-linked immunosorbent assay in PBMC culture supernatants (5×10⁶ cells/mL in a 48 plate well) from patients, after 48 h of antigenic stimulation with trypomastigote antigen (100ηg/well) and independent of the stimuli (Medium). The Chagas' disease patients were grouped as: Group 1 (n = 10): Patients not treated with benznidazole and free/mild cardiomyopathy, group 2 (n = 11): Patients not treated with benznidazole but with moderate/severe cardiomyopathy, group 3 (n = 8): Patients previously treated with benznidazole free/mild cardiomyopathy. Healthy Individuals (n = 10) from the same endemic areas were included in this study as controls, composing the group 4, as described in Materials and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001630#s2" target="_blank">Methods</a>. The results are expressed in picograms per milliliter. Statistical differences are represented by letters: a and b, <i>P</i><0.05 (Spearman).</p

Patients with severe Chagas' disease cardiomyopathy exhibit deficient suppressor activity of Treg.

<p>PBMC (5×10⁶ cells/mL in a 48 plate well) from Chagas' disease patients were cultured with trypomastigote antigen (10 µg/mL) after 48 h of antigenic stimulation the mean intensity of fluorescence of CTLA-4 in CD4⁺CD25^high T cells (<b>A</b>) were performed. Free/Mild cardiomyopathy patients displayed high levels of CTLA-4 in CD4⁺CD25^high T cells (free/mild cardiomyopathy <i>vs.</i> moderate/severe P = 0.0352). For functional characterization of CD4⁺CD25⁺ regulatory T cells in Chagas' disease patients magnetic bead-sorted CD4⁺CD25⁺ T cells purified from PBMC from free/mild cardiomyopathy patients (<i>n</i> = 5), severe cardiomyopathy (<i>n</i> = 5), and healthy individuals (<i>n</i> = 5), were tested for their ability to suppress the proliferation of allogeneic PBMC. The CD4⁺CD25⁺ T cells were harvested and suppressor activity determined as % of proliferation inhibition in culture from PBMC/CD4⁺CD25⁺ T cells 1∶5 (<b>B</b>) and 1∶10 (<b>C</b>) proportion. Allogeneic PBMCs (2×10⁵ cells/well in a 96 plate well) CFSE stained were cultured during 72 h with medium only, CD4⁺CD25⁺ (2×10⁴ and 4×10⁴ cells/well, ratio of 1∶5), PHA (10 ηg/well), PHA plus CD4⁺CD25⁺ (ratio of 1∶10 and 1∶5) from Chagas' disease patients (free/mild cardiomyopathy patients and severe cardiomyopathy patients) or healthy controls. <b>a</b> and <b>b</b> indicate statistical differences with P<0.05 (healthy <i>vs.</i> free/mild cardiomyopathy P = 0.547; healthy patients <i>vs.</i> moderate/severe P = 0.0159; free/mild cardiomyopathy <i>vs.</i> moderate/severe P = 0.0189).</p

Characterization of CD4⁺CD25⁺ Treg in patients with different clinical manifestations of Chagas disease.

<p>Representative flow cytometry analysis of CD4⁺CD25^high, CD4⁺CD25⁺Low, CD4⁺CD25⁻ gated lymphocytes is shown in <b>A</b>. PBMC from control and Chagas' disease patients in these analyses were gated on lymphocytes via their forward (FSC) and side scatter (SSC) properties, and CD4⁺CD25^high (<b>B</b>), CD4⁺CD25⁺Low (<b>C</b>), CD4⁺CD25⁻ (<b>D</b>) were performed to determine the regulatory T cell population. CD4⁺CD25^high (<b>E</b>), CD4⁺CD25⁺Low (<b>F</b>), CD4⁺CD25⁻ (<b>G</b>) cells were analyzed for their expression of membrane CTLA-4, GITR, CD103, and intracellular Foxp3. PBMC (5×10⁶ cells/mL in a 48 plate well) from Chagas' disease patients were cultured with trypomastigote antigen (10 µg/mL) after 48 h of antigenic stimulation the expression of surface markers (CD4, CD25, CD103, CTLA-4, GITR) and transcriptional factor (Foxp3) were determined. The results are expressed as means ± standard errors. <b>a</b> and <b>b</b> indicate statistical differences with P<0.05.</p

Demographic and clinical characteristics of chronic chagasic subjects included in this investigation.

*<p>Subjects 22, 23, 24, 25, 26, 27, 28 and 29 = with previous etiologic treatment (3 to 35 years ago); Bz = benznidazole.</p><p>Cardiomyopathy-free patients: asymptomatic, normal physical examination, normal ECG, normal chest X-rays and normal 2D-echocardiogram. LVEF>50%.</p><p>Patients with mild cardiomyopathy : positive symptoms or physical abnormalities, or abnormal ECG and/or chest X-rays and abnormal 2D-echocardiogram but with preserved global left ventricular function (LVEF>50%); moderate cardiomyopathy : impaired global LV function but EF still >35%; severe cardiomyopathy : LVEF≤35%.</p