The transcription factor AP-1 is a mediator of hypertrophic growth and apoptosis in cardiomyocytes. This puts AP-1 in the center of two important processes found in the failing heart and implies that ...variations (i) in the AP-1 composition itself or (ii) in additional, interacting transcription factors are responsible for the diverse actions of AP-1. To test this hypothesis, we performed studies on isolated ventricular cardiomyocytes of rat under hypertrophy- or apoptosis-inducing conditions.
The NO donor SNAP (100 microM), which is a pro-apoptotic stimulus in cardiomyocytes, activated AP-1 within 2 h. c-Jun, JunB and FosB are identified as the main components of this AP-1 complex. This complex formation is identical to the composition of AP-1 found under hypertrophic growth stimulation by phenylephrine (PE, 10 muM). Analysis of other transcription factors able to interact with AP-1 revealed activation of SMAD activity only during stimulation with SNAP to 131+/-9.6% (p < 0.05 vs. control, n = 9). The SMAD complex is formed from SMAD4 and 3. Intracellular scavenging of SMAD proteins by transformation of cardiomyocytes with SMAD decoy oligonucleotides or inhibition of SMAD4 synthesis using SMAD4 antisense oligonucleotides reduced the number of apoptotic cells under stimulation with SNAP from 13.3 +/- 1.2% to control levels (8 +/- 1%, p < 0.05, n = 6). TGFbeta, which is a known stimulator of SMAD proteins, is also shown to stimulate apoptosis in cardiomyocytes. Again, simultaneous activation of AP-1 and SMAD is needed for this apoptosis induction.
In conclusion, AP-1/SMAD signaling has been identified as a common pathway in cardiomyocyte apoptosis. In contrast, SMAD proteins are dispensable for AP-1-mediated hypertrophic growth. This finding characterizes SMAD proteins as potential candidates for proteins that shift AP-1 signaling from hypertrophy to apoptosis.
Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects ...of the organic nitrates glyceryl trinitrate (100 mu mol/L), pentaerythritol tetranitrate (10 mu mol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors S-nitroso-N-acetyl-d,1-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 mu mol/L) caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8 plus minus 6.7% (SNAP) and 80.9 plus minus 6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 mu mol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5 plus minus 14.0%). By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6 plus minus 6.4%). A similar effect was observed with a low concentration (1 mu mol/L) of SNAP and DEA/NO. All increases in CR induced by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 mu mol/L). The positive contractile effect of isoprenaline was enhanced by 1 mu mol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrates are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of cGMP-dependent protein kinase.(Circ Res. 1996;78:91-101.)
Aims Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular growth and recruitment of perivascular mural cells. The purpose of the present study is to investigate ...the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB. Methods and results PDGF-BB increased vascular sprouting and branching of capillary-like structures in embryoid bodies as evaluated by computer-assisted analysis of CD31-positive cell structures. It also activated extracellular-regulated kinase 1,2 (ERK1,2) and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinase or PI 3-kinase. Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular Ca2+ levels in differentiating ES cells expressing the PDGF receptor β, an effect that was abolished in the presence of the intracellular Ca2+ chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels in embryoid bodies as evaluated using the redox-sensitive dye H2DCF-DA. ROS generation was blunted in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin as well as in the presence of BAPTA, suggesting that ROS generation is regulated by intracellular Ca2+ transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzenesulfonylfluoride and VAS2870, the free radical scavengers vitamin E, and N-(2-mercaptopropionyl)glycin as well as by BAPTA. Conclusion Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca2+-induced ROS generation, resulting in the activation of an ERK1,2-mediated signal transduction cascade.
Anti-neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In ...this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor alpha induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng-2.5 microg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab)2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen-antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis-related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3-induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG.
Endothelial cells (EC) contribute to the control of local vascular diameter by formation of an endothelium derived relaxant factor (EDRF) (1). Whether nitric oxide (NO) is identical with (EDRF) or ...might represent only one species of several EDRFs has not been decided as yet (2-5). Therefore, we have directly compared in cultured EC the kinetics of NO formation determined in a photometric assay with the vasodilatory effect of EDRF and NO in a bioassay. Basal release of NO was 16, 4 pmol/min/ml packed EC column. After stimulation with bradykinin (BK) and ATP onset of endothelial NO release and maximal response preceded the EDRF-mediated relaxation. Concentrations of NO formed by stimulated EC were quantitatively sufficient to fully explain the smooth muscle relaxation determined in the bioassay. Our data provide convincing evidence that under basal, BK and ATP-stimulated conditions 1. endothelial cells release nitric oxide as free radical, 2. nitric oxide is solely responsible for the vasodilatory properties of EDRF.
Key points
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Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation accompanied by a rise in intracellular Ca2+, an increase in myosin light chain (MLC) ...phosphorylation, and RhoA/Rho kinase (Rock) signalling and an inactivation of Rac1.
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Neither inhibition of RhoA/Rock signalling nor antagonising Ca2+ increase could protect against this hypoxia–reoxygenation‐induced loss of barrier function.
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Inhibition of MLC kinase (MLCK) abrogates hypoxia–reoxygenation‐induced MLC phosphorylation and partially protects against hypoxia–reoxygenation‐induced endothelial hyperpermeability.
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Activation of Rac1 using a cAMP analogue, 8‐CPT‐O′‐Me‐cAMP, which specifically activates Epac/Rap1 signalling abrogated reoxygenation‐induced hyperpermeability. The data help us to better understand the role of Rho GTPases and contractile machinery in the regulation of endothelial barrier function during hypoxia–reoxygenation.
Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho‐GTPases under hypoxia–reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia–reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia–reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE‐cadherin from cellular junctions. Inhibition of contractile activation with ML‐7 partially protected against hypoxia–reoxygenation‐induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia–reoxygenation‐induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8‐CPT‐O′‐Me‐cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE‐cadherin at cellular junctions and abrogated reoxygenation‐induced hyperpermeability. Similar results were reproduced in isolated saline‐perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation‐induced loss of barrier function.
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