Chronic hypoxia induces apoptosis in cardiac myocytes: a possible role for Bcl-2-like proteins.

The effect of prolonged hypoxia as well as the molecular mechanisms on cardiac cell death is not well established. A possible role of Bcl-2 and Bax in hypoxia-induced apoptosis in different cell types has been proposed. Here we demonstrate the effect of hypoxia on the induction of apoptosis and the expression of Bcl-2-like proteins in vivo and in vitro. Hearts from rats exposed to chronic hypoxia (n = 4) showed an increased rate of apoptosis compared to normoxic hearts (n = 4). The induction of apoptosis in hypoxic hearts correlated with a significant decrease of Bcl-2 protein level, whereas Bax protein expression was increased. Exposure of isolated neonatal rat cardiac myocytes to hypoxia also resulted in a significant increase in apoptosis. However, Bcl-2 and Bax protein levels essentially remained unchanged. Our results may suggest a different molecular mechanism of hypoxia-induced apoptosis in vivo and in vitro.

Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway.

OBJECTIVE: Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS: Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

Novel polymer substrates for SFM investigations of living cells, biological membranes, and proteins.

Extended studies were performed to prepare substrates for scanning force microscopy of biological samples with a surface roughness below 1 nm rms over an area of 500 x 500 nm2. The substrate smoothness and the lack of tip obscuring material are indispensable in order to visualize detailed structures of membrane surfaces, particularly when scanning the lamellipodia of mechanically sensitive goldfish glial cells where peripheral lamellipodia are only 20-30 nm in thickness. Appropriate substrates are poly(vinyl phenyl ketone) or furan polymers with corrugations of 0.3 and 0.15 nm rms, respectively, to which the growth-promoting protein laminin adsorbs directly with an acceptably increased roughness. Cells show normal growth behavior on these substrates and stick to the substrates in a stable fashion during several scans. Thus details of the membrane's surface may be resolved and are not obscured by the substrate texture. The uncoated furan polymer substrates are suitable for immobilization of membrane preparations such as purified membrane fragments containing bacteriorhodopsin or Na, K-ATPase and protein preparations such as antibodies.

Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy.

We have developed a new method for observing cell/substrate contacts of living cells in culture based on the optical excitation of surface plasmons. Surface plasmons are quanta of an electromagnetic wave that travel along the interface between a metal and a dielectric layer. The evanescent field associated with this excitation decays exponentially perpendicular to the interface, on the order of some hundreds of nanometers. Cells were cultured on an aluminum-coated glass prism and illuminated from below with a laser beam. Because the cells interfere with the evanescent field, the intensity of the reflected light, which is projected onto a camera chip, correlates with the cell/substrate distance. Contacts between the cell membrane and the substrate can thus be visualized at high contrast with a vertical resolution in the nanometer range. The lateral resolution along the propagation direction of surface plasmons is given by their lateral momentum, whereas perpendicular to it, the resolution is determined by the optical diffraction limit. For quantitative analysis of cell/substrate distances, cells were imaged at various angles of incidence to obtain locally resolved resonance curves. By comparing our experimental data with theoretical surface plasmon curves we obtained a cell/substrate distance of 160 +/- 10 nm for most parts of the cells. Peripheral lamellipodia, in contrast, formed contacts with a cell substrate/distance of 25 +/- 10 nm.

Angiotensin II induces apoptosis of human endothelial cells. Protective effect of nitric oxide.

Angiotensin II (Ang II) importantly contributes to the pathobiology of atherosclerosis. Since endothelial injury is a key event early in the pathogenesis of atherosclerosis, we tested the hypothesis that Ang II may injure endothelial cells by activation of cellular suicide pathways leading to apoptosis. Human umbilical venous endothelial cells (HUVECs) were incubated with increasing doses of Ang II for 18 hours. Apoptosis of HUVECs was measured by ELISA specific for histone-associated DNA fragments and confirmed by DNA laddering and nuclear staining. Ang II dose-dependently induced apoptosis of HUVECs. Simultaneous blockade of both the AT1 and AT2 receptor prevented Ang II-induced apoptosis, whereas each individual receptor blocker alone was not effective. Selective agonistic stimulation of the AT2 receptor also dose-dependently induced apoptosis. Ang II-mediated as well as selective AT2 receptor stimulation-mediated apoptosis was associated with the activation of caspase-3, a central downstream effector of the caspase cascade executing the cell death program. Specific inhibition of caspase-3 activity abrogated Ang II-induced apoptosis. In addition, the NO donors sodium nitroprusside and S-nitrosopenicillamine completely inhibited Ang II-induced apoptosis and eliminated caspase-3 activity. Thus, Ang II induces apoptosis of HUVECs via activation of the caspase cascade, the central downstream effector arm executing the cell death program. NO completely abrogated Ang II-induced apoptosis by interfering with the activation of the caspase cascade.

Effects of redox-related congeners of NO on apoptosis and caspase-3 activity.

Nitric oxide has been shown to inhibit apoptosis of human umbilical venous endothelial cells (HUVEC). Therefore we investigated the effect of different NO donors, PAPA NONOate (NOC-15; NO.) and nitrosodium tetrafluoroborate (NOBF4, NO+), and the reaction product of NO and O2-, peroxynitrite (ONOO- ), on TNF-alpha- or serum depletion-induced apoptosis of HUVEC. TNF-alpha-induced DNA fragmentation, determined by ELISA, was inhibited by NOC-15, NOBF4, and ONOO- in a concentration-dependent manner (maximal effects with 10 microM NO. and ONOO- and 100 microM NO+). The inhibition of apoptosis correlated with a protective effect on cell viability. The caspases, a cysteine protease family, play an important role in apoptotic processes. To determine whether the different NO donors and ONOO- regulate this enzyme, caspase-3-like activity was measured in homogenates of TNF-alpha-treated HUVEC. The TNF-alpha-induced enzyme activity was abrogated by NO., NO+, and ONOO-. Furthermore, caspase-3 activity was determined in vitro by reconstitution of the separately cloned, bacterially expressed, and purified active p17 and p12 subunits. The reconstituted caspase-3 exhibited enzyme activity, which was suppressed by the different NO donors and ONOO- with an IC50 of 50 microM for NOC-15, 1 mM for NOBF4, and 50 microM for ONOO-. The inhibition of caspase-3 activity correlated with a S-nitrosylation of the reactive cysteine residue and was reversed by further addition of dithiothreitol. This study suggests that the cellular regulatory processes of NO to protect cells from apoptosis may be independent of the redox state and that low concentrations of NO and ONOO- inhibit the cellular suicide program in HUVEC via S-nitrosylation of members of the caspase family.

Expression of an L1-related cell adhesion molecule on developing CNS fiber tracts in zebrafish and its functional contribution to axon fasciculation.

E587 antigen, an L1-related cell adhesion molecule, is expressed by growing axons and has previously been shown to enhance axon growth and to mediate fasciculation of axons from newborn retinal ganglion cells in goldfish. In zebrafish, the monoclonal antibody E17 against E587 antigen stains all axons in the primary tracts and commissures from 17 h postfertilization (pf) onward and axons which are added subsequently to this scaffold. Moreover, Fab fragments of an E587 antiserum (E587 Fabs) injected into the ventricle of 30-h pf zebrafish embryos caused a marked defasciculation of distinct axon bundles in the posterior commissure, in hindbrain commissures, and in longitudinal tracts of the hindbrain, where they also caused increased crossings between fascicles. The regulated expression of E587 antigen by all developing axons and the effects caused by E587 Fabs show that E587 antigen contributes to the formation of tight and orderly fascicles in the developing CNS.