Cultured interstitial cells from human heart valves express both specific skeletal muscle and non-muscle markers.

Cardiac valve interstitial cells are a phenotypically diverse and dynamic population, comprising myofibroblasts, fibroblasts and smooth muscle cells. To understand how these contribute to valve function and to optimize the choice of cells for seeding tissue-engineered valves, we are fingerprinting interstitial cells from all four human heart valves for useful phenotypic markers. We have begun by selecting markers indicated as of interest from previous work on myofibroblast-like cell lines. We show that interstitial cells express a variety of skeletal muscle contractile proteins and the skeletal muscle transcription factor myogenin, but not the related factors MyoD, myf-5 and MRF4, suggesting partial activation of the muscle programme in these cells. Expression of non-muscle isoforms of creatine kinase (CK-B) and AMP deaminase (AMPD2 and AMPD3) was found in contrast to muscle-restricted isoforms. Non-muscle isoforms of alpha- and beta-tropomyosins were detected specifically in contrast to skeletal muscle-specific isoforms. Several members of the Frizzled (FZD) family of Wnt receptors were also detected. In addition, intact cusps of all four valves from pig were capable of contacting to non-receptor and receptor-mediated stimulation in vitro. We conclude that interstitial cells from human heart valves express various sarcomeric proteins, and suggest that these cells have contractile potential due to a unique pattern of expression of both muscle-specific and non-muscle isoforms of metabolic and structural proteins. This may be under the control of myogenin, activated through specific Wnt/FZD signaling. Identifying such molecular markers could prove useful for engineering allogenic non-valve cell sources for seeding the synthetic valve.

In vitro endothelial cell activation and inflammatory responses in end-stage heart failure.

BACKGROUND: vascular endothelial cell activation and dysfunction are observed in patients with severe heart failure and may contribute to systemic manifestations of this syndrome. It remains unknown whether inflammatory activation of these cells occurs in these patients because of increased circulating proinflammatory mediators. AIM: to determine whether the serum from patients with heart failure possesses a net proinflammatory bioactivity to active proinflammatory pathways in cultured endothelial cells. METHODS: serum was obtained from stable patients with end-stage heart failure undergoing elective cardiac transplantation (Tx) and severely decompensated patients with heart failure requiring emergency left ventricular assist device (LVAD) implantation. Net proinflammatory bioactivity of serum was investigated by monitoring IkappaBalpha degradation and E-selectin expression in cultured human pulmonary artery endothelial cells (HPAEC) following incubation with serum samples. Serum cytokine concentrations were measured by ELISA and neutralizing antibodies were used to determine the role of specific factors in the observed bioactivity. RESULT: serum from both patient groups induced HPAEC IkappaBalpha degradation. Low basal HPAEC E-selectin expression significantly increased following treatment with Tx but not LVAD serum. Serum tumor necrosis factor-alpha (TNF-alpha) and IL-10 concentrations were higher in patients with LVAD than those with Tx, and soluble TNF-alpha receptor expression was high in both groups. Neither TNF-alpha nor IL-10 blocking experiments altered either bioassay result. CONCLUSION: activation of a specific profile of pro- and anti-inflammatory mediators is associated with heart failure resulting in HPAEC nuclear factor (NF)-kappaB activation. However, E-selectin expression is further regulated by unidentified factors. TNF-alpha is upregulated but appears to play no part in NFkappaB activation in these patients. These findings could have important therapeutic implications.

Redox regulation of p38 MAPK activation and expression of ICAM-1 and heme oxygenase-1 in human alveolar epithelial (A549) cells.

We have explored the potential role of redox events in p38 mitogen-activated protein kinase (MAPK) activation and their relevance to the inducible expression of intercellular adhesion molecule-1 (ICAM-1) and heme oxygenase-1 (HO-1) in A549 cells. Tumor necrosis factor-alpha (TNFalpha) and hydrogen peroxide (H2O2) both activated p38, but only TNFalpha activated nuclear factor-kappaB (NF-kappaB). N-Acetyl-L-cysteine (20 mM) inhibited both H2O2- and TNFalpha-induced p38 phosphorylation (14 +/- 7 and 37 +/- 4% of control, respectively). The mitochondrial complex I and III inhibitors, rotenone and antimycin A, and allopurinol partially inhibited H2O2- but not TNFalpha-induced p38 activation. However, rotenone and antimycin A augmented intracellular oxidative stress measured by dichlorofluorescein fluorescence. TNFalpha, but not H2O2, induced ICAM-1 in A549 cells, which was attenuated by a proteasome inhibitor, but not by the p38 MAPK inhibitor SB203580. In contrast, hemin and hemoglobin, but neither TNFalpha nor H2O2, caused efficient HO-1 expression. However, hemin had no effect on p38 activation and SB203580 did not influence hemin-induced HO-1 protein expression. Collectively, these data suggest that p38 is a cytokine- and oxidative stress-responsive pathway in A549 cells. Whereas NF-kappaB appears crucial in ICAM-1 induction, p38 activation itself is not sufficient to confer HO-1 expression and may not be involved in HO-1 and ICAM-1 induction in A549 cells.

Regulation of NF-kappaB and ICAM-1 expression in human airway epithelial cells.

The aim of this study was to elucidate the redox regulation of cytokine-induced NF-kappaB activation and NF-kappaB mediated gene induction in A549 cells and primary cultures of human airway epithelial cells. In A549 cells, Western blot analysis showed transient depletion of IkappaBalpha after 15 min IL-1beta treatment followed by its reappearance after 60 min, indicating efficient NF-kappaB-driven gene induction. A similar pattern was observed in primary epithelial cells however, the kinetics were slower and depletion was less. In primary airway epithelial cells IkappaBalpha levels were 59.8+/-8.5% of control following 30 min treatment with IL-1beta and in A549 cells 29.1+/-8.5% of control following 15 min IL-1beta treatment. Cytokine-induced IkappaBalpha depletion was associated with NF-kappaB nuclear accumulation and subsequent resynthesis of IkappaBalpha and upregulation of ICAM-1 in both cell types. The antioxidant, NAC (20 mM) had no effect on the kinetics of cytokine-induced IkappaBalpha depletion or NF-kappaB p65 nuclear translocation in either cell type and failed to influence kappaB dependent IkappaBalpha resynthesis. H2O2 treatment alone or in combination with cytokines had no significant effects on IkappaBalpha depletion, NF-kappaB p65 nuclear translocation or ICAM-1 expression in either cell type but did cause significant activation of p38 MAPK. These results suggest that cytokine-induced NF-kappaB activation in cultured human airway epithelial cells does not involve an NAC-sensitive oxidant stress and that H2O2-induced oxidant stress does not result in effective NF-kappaB activation and NF-kappaB mediated gene induction.

Off-pump coronary artery bypass surgery is associated with reduced neutrophil activation as measured by the expression of CD11b: a prospective randomized study.

BACKGROUND: Coronary artery bypass grafting (CABG) surgery is associated with systemic inflammation. Activation of neutrophils is a crucial step in inflammation and results in neutrophil sequestration within the tissues. One of the potential advantages of performing off-pump coronary artery bypass (OPCAB) surgery is the attenuation of the systemic inflammatory response. This prospective randomized study compares neutrophil activation in patients undergoing OPCAB versus those undergoing CABG with cardiopulmonary bypass (CPB). METHODS: Twenty patients undergoing primary isolated CABG were randomly divided prospectively into 2 groups: 1 group underwent CABG with CPB, and the other group underwent OPCAB. Central venous blood samples were obtained before skin incision and at 15 minutes, 60 minutes, 2 hours, 5 hours, and 24 hours following the initiation of CPB or application of the stabilization device. Differential white cell counts were measured with routine laboratory techniques. CD11b surface expression on neutrophils was measured by flow cytometry. Interleukin 8 levels in the plasma were measured by enzyme-linked immunosorbent assays. RESULTS: The 2 groups were matched with respect to preoperative and operative characteristics. White cell and neutrophil counts rose in both groups following the operation but were significantly higher in the OPCAB group at 5 hours (P < .001 and P = .002, respectively). Interleukin 8 concentrations were significantly higher in the CPB group at 5 hours following the initiation of CPB (P = .034). CD11b levels were significantly higher in the CPB group at 60 minutes (P = .002). CONCLUSION: This prospective randomized study demonstrates that the activation of circulating neutrophils as measured by CD11b expression is lower following OPCAB than in CPB. Although OPCAB is associated with significantly higher neutrophil counts, these neutrophils exhibit fewer activation markers. The lower postoperative neutrophil counts occurring in the CPB group may be explained by the activation and consequent sequestration of the neutrophils in the CPB circuit and tissues.

Antioxidants in myocardial ischemia-reperfusion injury: therapeutic potential and basic mechanisms.

Oxidative stress is a constant threat to all living organisms and an immense repertoire of cellular defense systems is being employed by most pro- and eukaryotic systems to eliminate or to attenuate oxidative stress. Ischemia and reperfusion is characterized by both a significant oxidative stress and characteristic changes in the antioxidant defense. By focusing on this antioxidant response of the cardiovascular system in the setting of ischemia-reperfusion injury, the aim of this review was threefold. First, based on recent animal experiments and clinical studies we shall discuss how endogenous antioxidants respond to oxidative stress during ischemia-reperfusion injury and highlight the results of recent trials on the ability of antioxidants to modulate ischemia-reperfusion injury. In this aspect, we will particularly focus on the emerging concept that various lines of antioxidant defenses do not act individually but are linked to each other in a systematic relationship as part of an antioxidant network. It is well known that enzymatic mechanisms are important components of the endogenous antioxidant repertoire; however, the relative importance of the different enzyme systems and isoforms has been much debated. The second part will focus on recent suggestions attributing a potentially key role of mitochondrial MnSOD in cardiac ischemia-reperfusion injury. Finally, the third part of the review will critically examine how endogenous antioxidants might regulate the complex signal transduction pathways of cellular activation with particular attention to the NF-kappaB and MAPK systems that appears to determine outcome of injury, survival, and adaptation.