Clinical and hemodynamic characteristics of the HF population.

<p>Data are presented as the mean ± SD or number or percentage of subjects. CAD, coronary artery disease; DCM, dilated cardiomyopathy; LVEF, left ventricular ejection fraction; PCWP, pulmonary capillary wedge pressure; MPAP, mean pulmonary artery pressure; PVR, pulmonary vascular resistance; ACE, angiotensin converting enzyme; ARB, angiotensin II receptor blocker.</p

Toll-Like Receptor 9 Mediated Responses in Cardiac Fibroblasts

<div><p>Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and –C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation <i>in vitro</i>. Finally, results from <i>in vivo</i> TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our <i>in vitro</i> data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.</p></div

Levels of inflammatory mediators in pulmonary and femoral artery and after stimulation of alveolar macrophages.

<p>A. Plasma levels of ET-1, TNFα, IL-6 and MCP-1 in femoral and pulmonary artery in HF patients (<i>n</i> = 15) and controls (<i>n</i> = 10). Plasma levels of the various cytokines were measured by EIA. B. Release of ET-1, TNFα and IL-6 in alveolar macrophages from healthy controls (<i>n</i> = 5) that were cultured for 5 and 24 hours (h) with or without LPS (100 ng/mL), Pam₃Cys (1 µg/mL), isoproterenol (20 µM) and IL-1β (5 ng/mL). Cytokine levels in supernatants were measured by EIA. Data are mean±SEM. *p<0.05, **p<0.01 and ***p<0.001 versus controls or unstimulated (US) cell, respectively.</p

Kaplan-Meier curves for the primary end point (panel A), as well as for all-cause (B) and CV (C) mortality according to tertile sFRP3 concentration.

<p>T1, lowest tertile serum sFRP3; T3, highest tertile serum sFRP3. Patients with T2 sFRP3 showed a markedly better outcome than patients in T1 and T2; <i>p</i><0.001 for the primary end point and all-cause mortality, <i>p</i><0.002 for CV mortality.</p

Kaplan Meier plots showing the association between tertiles of sFRP3 and all-cause mortality in the GISSI-HF-HF trial stratified according to age and presence of ischemic heart disease.

<p><b>A.</b> ischemic HF <70 years of age <b>B.</b> ischemic HF >70 years of age <b>C.</b> non-ischemic HF > 70 years <b>D.</b> all-cause mortality in CORONA using cut-off derived from the GISSI-HF-HF trial.</p

Lung function, exhaled NO value and differential sputum cell count in the induced sputum population.

<p>FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; DLCO, diffusion capacity for carbon monoxide; eNO, exhaled nitric oxide; ppb, parts per billion. Data are mean ± SEM,</p>*<p>p<0.05 versus controls.</p

Comparative analysis of TLR9-stimulated responses between cardiac fibroblasts and bone-marrow derived -macrophages and –dendritic cells.

<p>Dose-response relationships of (100 ng/ml) CpG A (panels A–B), CpG B (panels C–D) and CpG C (panels E–F) stimulated release of CXCL2 (left panels) and TNFα (right panels) were examined in murine cardiac fibroblasts (CFs; circles) compared to bone marrow derived –macrophages (triangles) and –dendritic cells (DC; squares). Analysis was performed after 18 hours stimulation. Each data point represents the mean ± SEM of 3 experiments.</p

<i>In vivo</i> cardiac TLR9-stimulated cellular responses.

<p>Male C57BL/6 mice were injected i.p. with 100 µl CpG B (50 µg; n = 6, black bars) or vehicle (n = 6, white bars) and euthanized after 24 hours, with subsequent isolation of cardiac myocytes (CM), CD45⁺, CD31⁺ and non-CM/non-CD45⁺/non-CD31⁺ (denominated CF). TLR9-mediated responses in the cell-fractions were analyzed by mRNA expression levels of CXCL2 (panels A–D) and TNFα (panels E–H). Uncorrected data are shown in panels A and E. CXCL2 and TNFα are also presented as corrected for GAPDH (panels B and F), β-actin (panels C and G) and 18S (panels D and H). Data presented as mean ± SEM. *<i>p</i><0.05 vs. sham, **<i>p</i><0.01 vs. sham.</p

Primer sequences used in qPCR assays.

<p>GAPDH was chosen as housekeeping gene.</p

Possible mechanism linking sFRP3 release during LV wall stress and non-linear association with survival.

<p>Increased wall stress [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133970#pone.0133970.ref001" target="_blank">1</a>] may induce the release of sFRP3 from fibroblasts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133970#pone.0133970.ref002" target="_blank">2</a>]. Depending on concentration of sFRP3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133970#pone.0133970.ref003" target="_blank">3</a>], this may lead to insufficient, balanced or excess inhibition of the Wnt [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133970#pone.0133970.ref004" target="_blank">4</a>] in the presence of inflammation and lead to a non-linear association with survival [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133970#pone.0133970.ref005" target="_blank">5</a>].</p