The most effective way to limit myocardial ischemic necrosis is reperfusion, but reperfusion itself may result in tissue injury, which has been difficult to separate from ischemic injury. This report identifies elements of apoptosis (programmed cell death) in myocytes as a response to reperfusion but not ischemia. The hallmark of apoptosis, nucleosomal ladders of DNA fragments (-200 base pairs), was detected in ischemic/reperfused rabbit myocardial tissue but not in normal or ischemic-only rabbit hearts. Granulocytopenia did not prevent nucleosomal DNA cleavage. In situ nick end labeling demonstrated DNA fragmentation predominantly in myocytes. The pattern of nuclear chromatin condensation was distinctly different in reperfused than in persistently ischemic tissue by transmission electron microscopy. Apoptosis may be a specific feature of reperfusion injury in cardiac myocytes, leading to late cell death. (J. Clin. Invest. 1994. 94:1621-1628.

Background — The angiogenic response to myocardial ischemia can be augmented in animal models by gene transfer with the use of a replication defective adenovirus (Ad) containing a human fibroblast growth factor (FGF) gene. Methods and Results — The objectives of the Angiogenic GENe Therapy (AGENT) trial were to evaluate the safety and anti-ischemic effects of 5 ascending doses of Ad5-FGF4 in patients with angina and to select potentially safe and effective doses for subsequent study. Seventy-nine patients with chronic stable angina Canadian Cardiovascular Society class 2 or 3 underwent double-blind randomization (1:3) to placebo (n=19) or Ad5-FGF4 (n=60). Safety evaluations were performed at each visit and exercise treadmill testing (ETT) at baseline and at 4 and 12 weeks. Single intracoronary administration of Ad5-FGF4 seemed to be safe and well tolerated with no immediate adverse events. Fever of <1-day duration occurred in 3 patients in the highest-dose group. Transient, asymptomatic elevations in liver enzymes occurred in 2 patients in lower-dose groups. Serious adverse events during follow-up (mean, 311 days) were not different between placebo and Ad5-FGF4. Overall, patients who received Ad5-FGF4 tended to have greater improvements in exercise time at 4 weeks (1.3 versus 0.7 minutes, P =NS, n=79). A protocol-specified, subgroup analysis showed the greatest improvement in patients with baseline ETT ≤10 minutes (1.6 versus 0.6 minutes, P =0.01, n=50). Conclusions — Results show evidence of favorable anti-ischemic effects with Ad5-FGF4 compared with placebo, and it appears to be safe. Angiogenic gene transfer with Ad5-FGF4 shows promise as a new therapeutic approach to the treatment of angina pectoris.

Recent evidence indicates that leukocytes (LEU) are large, stiff, viscous cells that naturally adhere to vascular endothelium. Their broad role in the early myocardial microvascular response to acute ischemia was suggested by 1) the role of leukocyte capillary plugging in the no-reflow phenomenon, 2) resistance increases in skeletal muscle with LEU infusions, and 3) salvage of ischemic myocardium by anti-LEU agents. We perfused the coronary circulation under matched, controlled conditions with whole blood or granulocyte-depleted whole blood. During 1 h of ischemia (left anterior descending occlusion) circumflex perfusion pressure was servocontrolled to a constant value. In whole blood-perfused hearts, flow measured by the radiolabeled microsphere method decreased in endocardium from 0.12 +/- 0.05 at 5 min of ischemia to 0.09 +/- 0.04 ml X min-1 X g-1 at 60 min of ischemia and in epicardium from 0.27 +/- 0.17 to 0.21 +/- 0.16 ml X min-1 X g-1, both P less than 0.05. In granulocyte-depleted blood-perfused hearts, flow increased over the same period from 0.18 +/- 0.15 to 0.29 +/- 0.18 ml X min-1 X g-1 in endocardium (P less than 0.05) and did not change significantly in epicardium (0.36 +/- 0.22 to 0.41 +/- 0.24 ml X min-1 X g-1). The LEU-depleted blood perfusate contained less than 33 granulocytes/microliter, whereas control perfusate contained 4,265/microliter. Reperfusion at normal pressures with carbon suspension allowed for histologic evaluation of the no-reflow phenomenon. With whole blood perfusion the no-reflow phenomenon in the endocardium was present with 27% of capillaries occluded, compared with nearly complete reperfusion in LEU-depleted animals (1% of capillaries occluded, P less than 0.05). Furthermore, LEU depletion prevented the increases in tissue water content seen in control hearts and decreased the incidence of ventricular arrhythmias. These studies demonstrate the significant participation of granulocytes in the unfavorable responses of flow, edema formation, and arrhythmias to the 1st h of myocardial ischemia and further document their role in the no-reflow phenomenon.

Neutrophils in tissue culture spontaneously undergo programed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H+-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H+-ATPase and that intracellular acidification is an early event in the apoptosis program.The properties of apoptotic cells have been studied extensively, but little is known about how apoptosis is initiated. There is, however, some evidence suggesting that intracellular acidification may play a role in this process. For example, intracellular acidification increases the susceptibility of cells to killing by heat and chemotherapeutic agents (1-3) and occurs in HL-60 cells undergoing apoptosis in response to etoposide (4). Cell shrinkage, an event characteristic of apoptosis, is also typically seen in cells undergoing intracellular acidification (5). A role for acidification in the DNA breakdown that occurs during apoptosis is suggested by the otherwise perplexing presence in many tissues of an endonuclease that is active only at pH values below 6.6-6.8 (6, 7). In neutrophils, this endonuclease is the only DNase that can be detected (ref. 8; R.A.G., H.A.G., and B.M.B., unpublished data).In this study, we have examined the relationship between intracellular acidification and apoptosis in cultured human neutrophils. Our results suggest that acidification may be causally related to the destruction of the genome that occurs in these cells when they undergo apoptosis. MATERIALS AND METHODSIsolation of Neutrophils and Cell Culture. Purified neutrophils (90-95% pure) were obtained from volunteer donors by 6% dextran sedimentation followed by purification over a discontinuous Percoll (9) or Ficoll/Hypaque (10) gradient, then suspended at 5 x 106 cells per ml in calcium-, magnesium-, and bicarbonate-free Hanks' balanced salt solution supplemented with 20 mM Hepes (pH 7.4) plus 0.25% autologous plasma (HBSS), and cultured for 24 hr at 370C (11). Where indicated, cultures contained granulocyte colonystimulating factor (G-CSF; 104 units/ml) and/or bafilomycin A1 {50-200 nM final concentration, added in dimethyl sulfoxThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in (14). Ten thousand events were collected and data were plotted as forward scatter (size) versus fluorescence ratio (pH).In Situ Nick End Labeling. Cells were labeled by a modification of the method ofWijsman et al (...

Circadian variation of the onset of acute myocardial infarction has been noted in many studies and may carry important pathophysiologic implications. However, only a few previous studies have attempted subgroup analyses. In 4,796 patients with documented acute myocardial infarction, the time of symptom onset was recorded. As in other studies, the peak of onset occurred in the morning from 6:01 AM to 12:00 noon, and 28% of the population (1.16 times the average percentage for the other time periods) experienced symptom onset in that period (p<0.001). There was a second, lower peak (25%) in the evening between 6:01 PM and 12:00 midnight, which was also observed in some previous studies. We sought to determine whether or not the presence of subgroups with specific clinical characteristics would exhibit different patterns and thereby contribute to these peaks in the overall population. In patients with a history of congestive heart failure (n=606) or with non-Q wave infarction (n=832), a pronounced peak (29%o) occurred only in the evening. Two nearly equal peaks were observed in patients older than 70 years of age (n = 1,422), smokers (n=2,057), diabetics (n =767), women (n = 1,213), and patients taking ,B-blocking drugs (n = 847). Finally, in patients with a previous myocardial infarction (n = 1,104), no peaks were observed. In a subgroup of patients (n = 1,084) free of the most important modifying factors, there was a single very pronounced late morning peak (32%, 1.39 times the average percentage for the other time periods, p<0.001) without evidence of a second evening peak. It is concluded that marked differences in diurnal patterns of myocardial infarction onset occur in subgroups of patients with modifying factors, particularly non-Q wave infarction, smoking, ,B-blocker use, diabetes, prior congestive heart failure, and prior myocardial infarction. The circadian pattern observed in a given total population reflects the contributions of these subgroups. (Circulation 1989;80:267-275) A circadian variation in the frequency of onset of acute myocardial infarction has been described in a number of studies during the past 25 years.1-8 Most show an increased onset in the morning with a peak incidence between 6:00 AM and 12:00 noon, although a secondary peak in the late evening has also been reported in some studies.1-3,5-7 A circadian variation in onset of other *All editorial decisions for this article, including selection of reviewers and the final decision, were made by a guest editor. This procedure applies to all manuscripts with authors from the