CONSORT flow diagram of the study

<p>CONSORT flow diagram of the study</p

Acute phase protein responses in CABG patients receiving HSA/GA vs general anesthetic alone.

<p>Individuals received HSA plus general anesthetic (open) or general anesthetic alone (solid). P values reflect statistical significance (Mann Whitney).</p

Preoperative patient characteristics (mean±SD).

<p>Preoperative patient characteristics (mean±SD).</p

Magnitude and duration of systemic pro-inflammatory cytokine responses in individuals receiving supplemental HSA.

<p>Median values are shown for groups receiving HSA plus general anesthetic (open bars) or general anesthetic alone (solid bars). P values reflect inter-group comparisions using Mann-Whitney at the time points indicated.</p

Intraoperative and postoperative patient characteristics (mean±SD).

<p>Intraoperative and postoperative patient characteristics (mean±SD).</p

Kinetics of in vivo chemokine, cytokine and acute phase protein responses following CABG.

<p>Serum obtained at baseline and the time points indicated was analysed as described at Materials and Methods to assess response kinetics for each biomarker individually. All levels are compared to baseline/study entry conditions. Median responses and statistical significance (Wilcoxon) of longitudinal comparisons relative to pre-induction baseline are shown.</p

High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

<div><p>Fibroblast growth factor 2 (FGF-2) is a multifunctional protein synthesized as high (Hi-) and low (Lo-) molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial) Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD) and 68% (±25 SD) of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs) expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II) up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-Ab^Hi-FGF-2) reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1β and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-Ab^Hi-FGF-2<i>in vitro</i>. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial) tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes deleterious (pro-fibrotic, pro-inflammatory, and pro-hypertrophic) responses <i>in vitro.</i> Selective targeting of Hi-FGF-2 production may, therefore, reduce pathological remodelling in the human heart.</p></div

Detection of Hi-FGF-2 in human atrial myofibroblasts.

<p><b>Panel (A)</b> shows two sets of western blots analyzing FGF-2 isoforms. The first set, (i), is a composite of two blots (separated by a broken line) and analyzes FGF-2 isoforms in hMF lysates (20 µg/lane), from atrial myofibroblast primary cultures obtained from 10 patients (patients 11–20), and correspondingly labeled as C11–20. The second set, (ii), also a composite of two blots separated by a broken line, analyzes FGF-2 isoforms in atrial tissue lysates from patients 11–20, and labelled T11–20 (50 µg/lane). The hMF blots or tissue blots were also probed for, respectively, β-tubulin (β-tub), or Troponin-T (TnT), as indicated. Following densitometry of the hMF blots, the % contribution of each FGF-2 isoform to the total FGF-2 signal was determined for each individual lane, and cumulative results (mean±SD) are included in graph form (n = 10). <b>In Panel (B)</b>, a western blot shows FGF-2 signals from 0.5 and 0.2 ng/lane of recombinant histidine tagged Lo-FGF-2 (FGF-2<i>^His</i>), atrial tissue lysates (T11, T15 and T17, loaded at 50 µg/lane), side by side with FGF-2 signals from lysates obtained from corresponding primary hMF cultures (C11, C15 and C17, loaded at 10 µg/lane). The graph shows comparisons between tissue and cell lysates for their relative total FGF-2 content, assessed by densitometry as optical density (O.D.) units (n = 3). Measurements corresponding to cell FGF-2 were multiplied by 5, to correct for the 5-fold difference in total protein loading. In both panels, comparisons between groups are indicated by brackets, where P>0.05 is marked as ns, while P<0.001, 0.01, are marked as ***, or **, respectively. <b>Panels C and D</b> show immunofluorescence images of hMFs stained for, (C), Hi-FGF-2 (green), as well as, (D), alpha smooth muscle actin (red) and nuclei (blue). White arrows point to cytosolic Hi-FGF-2; pink and pale- pink arrows arrows point to nuclear and nucleolar Hi-FGF-2, respectively. Grey sizing bars correspond to 20 µm.</p

Detection of Hi-FGF-2 in human atrial tissue.

<p><b>Panel (A)</b> shows representative western blot images of human atrial extracts (hA1, hA2, hA3, 50 µg/lane) probed for FGF-2 with an antibody detecting all FGF-2 isoforms. Expected migration of all human FGF-2 isoforms (34, 24, 22–22.5, and 18 kDa), corresponding to Hi- or Lo-FGF-2, is indicated by arrows; please note that the 34 kDa isoform is not detectable in tissue lysates. Western blots were also probed for cardiac troponin T (TnT) to verify equivalent loading of lanes. Samples hA1, hA2 were analyzed in small (8.3×5.5 cm²) 15% polyacrylamide gels, while hA3 was analyzed in a large (16×11.5 cm²) 15% polyacrylamide gel. The included graph shows percentage of each isoform over total FGF-2, where, n = 45; comparisons between groups are indicated by brackets, where *** and ** denote P<0.001, and P<0.01, respectively. <b>Panels (B) and (C)</b> show images from patient-derived serial atrial sections, subjected to (B) incubation with purified anti-Hi-FGF-2 antibodies followed by immunohistochemical visualization of antigen-antibody complexes (brown color) as well as nuclear staining (hematoxylin, blue), and (C) similar procedures as in B but without the anti-Hi-FGF-2 antibodies. Incubation with anti-Hi-FGF-2 antibody elicits extensive immunostaining, in what appears to be nuclear as well as cytosolic sites in cardiomyocytes; staining of non-cardiomyocytes located at the epicardium is indicated by arrows. <b>Panels (D) and (E)</b> are close-up images from human atrial tissue sections stained as in (B), showing cellular and subcellular distribution of Hi-FGF-2. <b>Panels (G) and (F)</b> show human atrial sections subjected to double-immunofluorescence staining for Hi-FGF-2 (green), and either vimentin (G, red), or desmin (F, red). In all images, yellow or pink arrows point, respectively, to nuclear or cytosolic sites within cardiomyocytes. Blue arrows in (D) point to small connective tissue cells, likely fibroblasts. Green arrows in (E) point to endothelial cells, lining a vessel. White arrows in (G) and (F) point to non-myocytes, found at or near the epicardial region. These cells are positive for vimentin, but not desmin. In (F), co-staining with desmin confirms presence of Hi-FGF-2 in atrial cardiomyocytes. Sizing bars in (B) or (D,E,F,G) correspond to 250 or 100 µM, respectively. Insets within panels G and F are shown in larger magnification in Fig S2.</p

Human Hi-FGF-2 exerts pro-hypertrophic effect.

<p><b>Panel A.</b> Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. <b>Panel B</b>. Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab^FGF-2), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. <b>Panel C.</b> Protein synthesis (³H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab^Hi-FGF-2). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. <b>D</b>. Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab ^Hi-FGF-2. Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. <b>E</b>. Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P<0.05, <0.01, <0.001, or P>0.05.</p