Recent studies have documented evidence for the death of smooth muscle cells (SMCs) within advanced human atheroma. These lesions contain macrophages and T lymphocytes in addition to SMCs. We therefore investigated whether interferon-gamma (IFN-gamma), a cytokine secreted by T lymphocytes, or interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), two cytokines characteristically produced by activated macrophages, can trigger apoptosis of vascular SMCs. Simultaneous treatment with IFN-gamma and TNF-alpha and/or IL-1 beta but not with each cytokine alone promoted death of human and rat SMCs. Exposure for 48 hours to a combination of IFN-gamma (400 U/mL), TNF-alpha (400 U/mL), and IL-1 beta (100 U/mL) significantly (P < .001) increased the accumulation of oligonucleosomes comprising DNA fragments and histones in human SMCs. Electrophoresis of genomic DNA showed internucleosomal fragments of genomic DNA isolated from the cytokine-cotreated SMCs of both humans and rats. These cells exhibited morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. In situ 3' end labeling of DNA fragments with terminal transferase confirmed the fragmentation of genomic DNA in these cells. Simultaneous treatment with IFN-gamma and TNF-alpha or IL-1 beta induced elaboration of nitrite, an end product of nitric oxide, in rat but not human SMCs. NG-monomethyl-L-arginine inhibited nitrite accumulation and also partly blocked cytokine-induced apoptosis of rat SMCs but had little effect on human SMCs, suggesting operation of both nitric oxide-dependent and -independent mechanisms for cytokine-induced apoptosis in vascular SMCs. Production of immune cytokines by vascular cells and/or infiltrating leukocytes may regulate apoptotic death of SMCs during atherogenesis.

The inhibitor of apoptosis (IAP) proteins suppress cell death by inhibiting the catalytic activity of caspases. Here we present the crystal structure of caspase-7 in complex with a potent inhibitory fragment from XIAP at 2.45 A resolution. An 18-residue XIAP peptide binds the catalytic groove of caspase-7, making extensive contacts to the residues that are essential for its catalytic activity. Strikingly, despite a reversal of relative orientation, a subset of interactions between caspase-7 and XIAP closely resemble those between caspase-7 and its tetrapeptide inhibitor DEVD-CHO. Our biochemical and structural analyses reveal that the BIR domains are dispensable for the inhibition of caspase-3 and -7. This study provides a structural basis for the design of the next-generation caspase inhibitors.

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. Here we report the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180 degrees flipping of the N terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of induced fit.

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The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein.