Tumor necrosis factor-α enhances hypoxia–reoxygenation-mediated apoptosis in cultured human coronary artery endothelial cells: critical role of protein kinase C (original) (raw)

Abstract

Background: Ischemia and tumor necrosis factor-α (TNFα) released during ischemia both cause apoptosis and necrosis of myocardial tissues. Since endothelium may be critically important in determination of cardiac function, we examined the interaction between TNFα and hypoxia–reoxygenation with regard to induction of apoptosis and underlying signaling pathway in cultured human coronary artery endothelial cells (HCAECs). Methods and results: HCAECs were cultured and exposed to hypoxia alone, hypoxia–reoxygenation, TNFα alone, TNFα plus hypoxia–reoxygenation, or TNFα only during the period of reoxygenation. Apoptosis was evaluated by transmission electron microscopy, DNA nick-end labeling and DNA laddering. Hypoxia alone caused modest time-dependent apoptosis of cultured HCAECs, and reoxygenation increased the number of apoptotic cells (P<0.01 vs. hypoxia alone). TNFα induced concentration-dependent apoptosis, and enhanced reoxygenation-mediated apoptosis in cultured HCAECs (P<0.01 vs. hypoxia–reoxygenation alone). As expected, monoclonal antibody to TNFα significantly blocked the pro-apoptotic effect of TNFα-induced apoptosis (P<0.01). TNFα-induced apoptosis was found to be associated with marked activation of protein kinase C (PKC), and pretreatment of cells with a specific PKC inhibitor markedly reduced TNFα-mediated PKC activity and apoptosis. Conclusion: These observations indicate that hypoxia alone causes modest apoptosis, reoxygenation increases apoptosis beyond that caused by hypoxia in cultured HCAECs. TNFα alone causes apoptosis, and further enhances apoptosis caused by hypoxia–reoxygenation. The activation of PKC plays a critical role in TNFα-induced apoptosis of cultured HCAECs.

Time for primary review 21 days.

1 Introduction

Experimental and clinical studies have shown that the cytokine tumor necrosis factor-α (TNFα) plays a critical role in determination of myocardial injury and function after coronary artery occlusion [1, 2]. TNFα has been localized in atherosclerotic tissues [3] as well as in arterial tissues subjected to balloon injury [4]. This cytokine exerts a negative inotropic effect on myocardial tissues [5, 6], and recent studies [7] have suggested that circulating levels of TNFα and its receptors in the cardiac tissues are increased in the setting of congestive heart failure. This background has led to important questions regarding the mechanisms and signal conduction pathways involved in TNFα-mediated cardiac injury.

Early reperfusion of ischemic myocardium is a desired therapeutic goal; however, there is ample evidence that reperfusion itself causes additional myocardial injury [8]. The phenomenon of “reperfusion injury” is believed to be caused by release of a number of mediators [8–10]; among these are the pro-inflammatory cytokines, such as interleukins and TNFα [10]. Experimental studies have clearly shown evidence of endothelial injury/dysfunction upon reperfusion, mediated at least in part by oxygen free radicals [9], and cytokines [10]. Other studies have indicated that programmed cell death (apoptosis) plays a vital role in determining ultimate myocardial injury after ischemia or ischemia–reperfusion. The process of hypoxia and reoxygenation has been shown to induce apoptosis [11–13]. We hypothesized that TNFα and hypoxia–reoxygenation may in a cumulative fashion interact to enhance coronary endothelial cell injury.

In this study, we examined the degree of apoptosis in cultured human coronary artery endothelial cells (HCAECs) caused by hypoxia alone and its modulation by reoxygenation. We also examined the effect of TNFα alone and the interaction between TNFα and hypoxia–reoxygenation relative to the magnitude of apoptosis. Lastly, we determined signal conduction pathway of TNFα-induced apoptosis in cultured HCAECs.

2 Materials

2.1 Cell culture

HCAECs (initial batch from Clonetics) were pure based on morphology and staining for factor VIII and acetylated LDL. These cell were 100% negative for alpha actin smooth muscle expression. Microvascular endothelium growth medium consisted of 500 ml of endothelial cell basal medium, 5 ng of human recombinant epidermal growth factor, 5 mg of hydrocortisone, 25 mg of gentamycin and 25 μg of amphotericin B, 6 mg of bovine brain extract, and 25 ml fetal bovine serum. HCAECs were seeded in a 25-cm2 flask (4000 cells/cm2), incubated at 37°C in air–CO2 (95:5). Fifth generation HCAECs (1·106) were used in these experiments [14–16]. The cells were examined under phase-contrast microscopy, and when about 85% confluent, culture medium was changed and the cells were divided into different groups:

Control group — cells were incubated in air–CO2 (95:5)
Hypoxia alone group — cells were exposed to N2–CO2 (95:5) for different periods (3, 6, 12, 18 and 24 h);
TNFα alone group — TNFα (0, 5, 20, 40 and 100 ng/ml, Recombinant (E. coli) human TNFα, Sigma) was added to the culture medium and cells incubated in air–CO2 (95:5) the for 24 h;
Hypoxia–reoxygenation group — cells were exposed to hypoxia for 24 h followed by reoxygenation [air–CO2 (95:5) for 3 h];
TNFα plus hypoxia–reoxygenation group — cells were incubated with TNFα (20 ng/ml) and then exposed to hypoxia (24 h)–reoxygenation (3 h);
Hypoxia plus TNFα plus reoxygenation group — cells were exposed to N2–CO2 (95:5) for 24 h and then TNFα (20 ng/ml) was added to the culture medium before reoxygenation;
Anti-TNFα-monoclonal antibody plus TNFα group — anti-human TNFα monoclonal antibody (200 μg/ml, Sigma product no. T1549) was present in the HCAEC culture medium and cells were exposed to TNFα (20 ng/ml) and air–CO2 (95:5) for 24 h;
Protein kinase C (PKC) inhibitor plus TNFα group — PKC inhibitor (100 μM) was present in the HCAEC culture medium and cells were exposed to TNFα (20 ng/ml) and air–CO2 (95:5) for 24 h. The myristoylated PKC peptide inhibitor used in this study (Promega) is essentially 100% pure. With a PKC substrate concentration of 10 μM, maximum inhibition of PKC activity is obtained with 100 μM of the inhibitor peptide.

Cells were made hypoxic by exposure to N2–CO2 (95:5) in a specially designed chamber. The amount of dissolved oxygen in medium (_P_O2) declined from 150 mmHg at baseline to 30–40 mmHg within 1 h of hypoxia. This decrease in (_P_O2) remained stable over the course of the hypoxic period. Reoxygenation of cells was performed by transferring cells into an incubator maintained at air–CO2 (95:5).

2.2 Transmission electron microscopy

HCAECs exposed to hypoxia, hypoxia–reoxygenation and TNFα were fixed in cacodylate buffer containing 2.0% glutaraldehyde (pH 7.4) for 1 h. After three buffer washes, the cells were post-fixed in OsO4 in cacodylate buffer for 1 h. Subsequently, the pellets were dehydrated in ethanol and embedded in epoxy resin (Agar 100). Thin sections were stained in uranyl acetate and Reynolds lead citrate and viewed at 75 kV in a Hitachi electron microscope (model H 7000).

2.3 Quantification of apoptosis by nick-end labeling

To detect DNA fragmentation in situ, nick-end labeling was performed by FragEL™-Klenow DNA Fragmentation Detection system (Calbiochem) described by Gu et al. [17]. In brief, the cells were pelleted by gentle centrifugation (800 g) for 5 min at 4°C, resuspended in 4% buffered formaldehyde at a cell density of 1·106 cells/ml and incubated at room temperature (RT) for 10 min. The fixed cells were immobilized onto glass slides using a Cytospin®, and incubated at RT for 20 min with 20 μg/ml proteinase K to increase cell permeability. The cells were then incubated at RT for 5 min with 3% H2O2 to inactivate endogenous peroxidases. The cells were then incubated with Klenow Enzyme and biotinylated dNTP in reaction buffer in a humidified chamber at 37°C for 1.5 h. After rinsing with TBS, the cells were incubated with streptavidin–horse radish peroxidase at RT for 30 min, rinsed with TBS, and then stained with 3,3′- diaminobenzidine at RT for 10 min. Methyl green was used as counterstain. The negative control sample was generated by substituting distilled water for the Klenow enzyme in the labeling step. The positive control sample was generated by covering the entire sample with 1 μg/μl DNase I in 1×TBS–1 mM MgSO4 at RT for 20 min following proteinase K treatment. The negative control sample contained predominantly rounded cells that appeared counterstained with methyl green. A dark brown signal under microscopy indicates an apoptotic cell. At least 500 cells from randomly selected fields were counted to determine the percentage of apoptotic cells.

2.4 Analysis of DNA fragmentation in agarose gel

Cultured HCAECs (1·106) were removed from culture dishes, washed twice with phosphate buffered saline and pelleted by centrifugation. Cell pellets were then treated for 10 min with lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM Tris–HCl, pH 7.5). After centrifugation for 5 min at 1600 g, the supernatant was collected and the extraction process was repeated with the same amount of lysis buffer. The supernatants were brought to 1% SDS and treated for 2 h with RNase A (final concentration 5 μg/μl) at 56°C followed by digestion with proteinase K (final concentration 2.5 μg/μl) for 2 h at 37°C. After addition of 0.5 volume of 10 M ammonium acetate the DNA was precipitated with 2.5 volume of absolute ethanol. DNA was recovered by centrifugation at 12 000 g for 10 min and dissolved in gel loading buffer. DNA was separated by electrophoresis in 1% agarose gel with ethidium bromide [18].

2.5 Determination of protein kinase C activity

Cells were washed twice with PBS, and scraped into 0.5 ml of cold extraction buffer (in mM): 25 Tris (pH 7.4), 0.5 EDTA, 0.5 EGTA, 10 β-mercaptoethanol, 100 PMSF, 0.05% Triton X-100, 1 μg/ml leupeptin, 1 μg/ml aprotinin. The lysate was homogenized and centrifuged at 14 000 g at 4°C for 30 min and the supernatant saved for PKC assay. A specific assay system (Promega) was used for determination of PKC activity [19]. Results were expressed as pmol ATP/min/μg protein.

2.6 Data analysis

Cell counts for HCAECs under various conditions represent duplicate samples from six independently performed experiments, Data are presented as mean±S.D. Analysis of variance was used to evaluate the statistical significance. Statistical significance was assigned at the level of P<0.05.

3 Results

3.1 Transmission electron microscopy

Transmission electron microscopy was performed to further document that the light microscopic features of apoptosis were accompanied by appropriate ultrastructural morphology. Under control conditions, normal cellular structure was identified. In contrast, cells exposed to hypoxia, reoxygenation and TNFα showed typical features of apoptosis, including condensation of chromatin at the periphery of the nucleus and fragmentation of the nucleus, and vacuolization and shrinkage of the cytoplasm (Fig. 1).

Fig. 1

Transmission electron microscopy of cultured HCAECs exposed to hypoxia (24 h), reoxygenation (3 h) and TNFα. Whereas control cells show normal architecture, cells exposed to hypoxia, reoxygenation and TNFα show condensation of chromatin at the periphery and fragmentation of the nucleus, and vacuolization, shrinkage of cytoplasm. In these representative experiments, TNFα was present in the incubation medium throughout the period of hypoxia and reoxygenation. Original magnification — control cell ×8750, hypoxia–reoxygenation ×12 500, TNFα+hypoxia–reoxygenation ×10 000.

3.2 Apoptosis in cultured HCAECs by nick-end labeling

Since a small number of cells normally die during culture or are damaged during processing, 1 to 5% (3.8±1.6%) of control cells stained positive. Hypoxia alone caused a time-dependent increase in typical features of apoptosis, and the number of apoptotic cells increased to 24.0±5.2% after 24 h of hypoxia. A 3-h period of reoxygenation following hypoxia significantly enhanced the number of apoptotic cells (P<0.01 vs. hypoxia alone group) (Fig. 2).

Fig. 2

Summary of data on the number of apoptotic cells as percent of all cells, as determined by nick-end labelling. Hypoxia alone causes modest time-dependent apoptosis. Reoxygenation for 3 h causes additional increase in the number of apoptotic cells (P<0.01 vs. hypoxia alone group). Presence of TNFα further increases the number of apoptotic cells (P<0.05 vs. hypoxia–reoxygenation group). Data from six separate experiments. Each point reflects data from six experiments expressed as mean±S.D.

TNFα alone induced concentration-dependent apoptosis in cultured HCAECs (Fig. 3). Presence of TNFα during the entire period of hypoxia–reoxygenation further increased the number of apoptotic cells (P<0.01 vs. hypoxia alone or hypoxia plus reoxygenation groups) in a cumulative fashion (Figs. 2 and 4). Presence of TNFα during the period of reoxygenation only also significantly increased apoptosis compared with hypoxia–reoxygenation without TNFα (P<0.05), but the magnitude of apoptosis was less as compared to when TNFα was present in the culture medium throughout the period of hypoxia and reoxygenation (Fig. 4).

Fig. 4

Apoptosis in cultured HCAECs exposed to hypoxia–reoxygenation, TNFα+hypoxia–reoxygenation, and only during the period of reoxygenation. Note that TNFα enhances the pro-apoptotic effect of hypoxia–reoxygenation (P<0.05 vs. hypoxia–reoxygenation group). Apoptosis is less marked when TNFα is present only during the period of reoxygenation. Each point reflects data from six experiments expressed as mean±S.D.

Concentration-dependent apoptosis in response to TNFα alone in cultured HCAECs. Each bar reflects data from six experiments expressed as mean±S.D.

Fig. 3

Concentration-dependent apoptosis in response to TNFα alone in cultured HCAECs. Each bar reflects data from six experiments expressed as mean±S.D.

3.3 Determination of DNA fragmentation in agarose gel

HCAECs cultured in control conditions showed no DNA laddering at 27 h. Under conditions of hypoxia–reoxygenation alone or TNFα alone, cultured HCAECs exhibited fragmented DNA that produced a ladder of DNA bands representing integer multiples of the internucleosomal DNA length (about 180 bp), indicating apoptotic cell death of HCAECs during hypoxia–reoxygenation. The proportion of the fragmented DNA in TNFα+hypoxia–reoxygenation group was increased by about 50% compared with hypoxia–reoxygenation alone or TNFα alone groups of HCAECs. The proportion of fragmented DNA in cells exposed to TNFα during reoxygenation alone group was also increased as compared to hypoxia–reoxygenation alone group. A representative example of DNA laddering in each group of HCAECs is shown in Fig. 5. These findings corroborated the data on apoptosis obtained from nick-end labeling described above.

DNA laddering evidence of apoptosis in HCAECs exposed to hypoxia–reoxygenation and TNFα. There is only modest DNA laddering in cells exposed to hypoxia, reoxygenation or TNFα alone. Combination of TNFα and hypoxia–reoxygenation causes additional DNA laddering. MAb to TNFα and the PKC inhibitor both reduce TNFα-mediated DNA laddering.

Fig. 5

3.4 Role of PKC signal pathway in TNFα-mediated apoptosis of HCAECs

TNFα markedly activated PKC activity of cultured HCAECs compared with control cells (P<0.01). The presence of myristoylated PKC peptide inhibitor in the HCAEC culture medium before the cells were exposed to TNFα for 24 h significantly inhibited TNFα-mediated activation of PKC (P<0.01) (Fig. 6) and apoptosis of HCAECs (P<0.05) (Fig. 7).

Influence of monoclonal antibody to TNFα and the specific PKC inhibitor on TNFα-mediated apoptosis in cultured HCAECs. TNFα-mediated apoptosis is significantly attenuated by monoclonal antibody to TNFα as well as the specific PKC inhibitor. Each bar reflects data from six experiments expressed as mean±S.D.

Fig. 7

Influence of TNFα on protein kinase C (PKC) activity in cultured HCAECs. TNFα markedly increases PKC activity, and this increase is blocked by monoclonal antibody to TNFα as well as the specific PKC inhibitor. Each bar reflects data from six experiments expressed as mean±S.D.

Fig. 6

3.5 Specificity of the effect of TNFα

The presence of anti-human TNFα-monoclonal antibody in the HCAEC culture medium before the cells were exposed to TNFα for 24 h markedly reduced the PKC activity as well as the number of TNFα-mediated apoptotic HCAECs (Fig. 7).

4 Discussion

The process of apoptosis is a distinct mode of cell death with distinct morphologic features, mechanisms and significance [20]. There is ample evidence that apoptosis is induced by stimuli that are also capable of producing necrosis. The examples of these stimuli include hypoxia [11], hyperthermia [21], toxic agents [21], and ischemia–reperfusion [12]. The present study for the first time shows that hypoxia alone causes a modest, but significant, time-dependent apoptosis in cultured HCAECs. A brief period of reoxygenation (3 h) following hypoxia markedly enhances the number of apoptotic cells and DNA fragmentation. TNFα which is released during the process of ischemia, also induces a concentration-dependent apoptosis, and it significantly increases hypoxia–reoxygenation-mediated apoptosis of HCAECs. Lastly, this study demonstrates that PKC activation plays a critical role as a signaling mechanism in TNFα-induced apoptosis of cultured HCAECs.

4.1 TNFα and apoptosis

TNFα has been implicated in the pathogenesis of various cardiac disease states, including acute myocardial infarction, reperfusion injury, congestive heart failure, acute viral myocarditis and cardiac allograft rejection [2, 3, 19, 22, 23]. Several studies suggest that TNFα exerts both immediate and delayed negative inotropic effects on myocardial contractility [1, 5, 6]. Gurevitch et al. [1] found that the isolated rat myocardium synthesizes and releases TNFα during ischemia–reperfusion, and TNFα levels correlate with the post-ischemic deterioration in myocardial mechanical performance and the amount of cellular necrosis. Yokoyoma et al. [5] showed that treatment with TNFα produces a 20–30% decrease in the extent of cell shortening and a 40% decrease in peak levels in intracellular calcium. Murray and Freeman [6] found that TNFα infusion in conscious dogs causes precipitous decline in systolic dysfunction (about 25% decline in left ventricular performance), which persists for over 24 h.

Various studies have shown that TNFα is involved both in the development of atherosclerosis [3] and formation of thrombus [24]. TNFα facilitates coagulation by inhibiting the anticoagulant pathways [25], and increasing plasminogen activator inhibitor-1 synthesis [26]. TNFα also affects endothelial cell function, and facilitates the binding of neutrophils to the endothelium [27]. Recent work [28] shows that TNFα may directly induce apoptosis in bovine pulmonary artery endothelial cells.

In the present study, we describe a concentration-dependent pro-apoptotic effect of TNFα on cultured HCAECs. The specificity of the effect of TNFα became apparent with the use of specific monoclonal antibody to human TNFα, which prevented apoptosis. In this process, activation of PKC appears to play an important role as a signaling mechanism, since the PKC inhibitor markedly blocked TNFα-induced apoptosis. These findings imply an important role of TNFα in the apoptotic process that has been characterized in patients with atherosclerosis and myocardial ischemia–reperfusion. Notably, these disease states are associated with endothelial dysfunction.

4.2 Hypoxia and apoptosis

Myocardial ischemia is generally caused by fixed or dynamic coronary artery narrowing and increased myocardial oxygen demand. Chronic ischemia or repetitive brief episodes of ischemia induce dysfunction of coronary endothelial cells [29]. Clinical studies indicate that ischemia induces apoptosis in human cardiac smooth muscle cells [30]. Kajstura et al. [13] clearly showed that while apoptosis and necrosis both contribute to cell death, apoptosis or programmed cell death is the major form of damage produced by occlusion of a major epicardial coronary artery. Experimental studies [11, 31] suggest that hypoxia rapidly induces the expression of several early genes, including c-myc, c-fos and Fas, and bifunction of redox protein/endonuclease; these genes may have a facilitatory effect on the induction of apoptosis. Our observations of hypoxia-mediated apoptosis in cultured human coronary endothelial cells are in keeping with the previous observations in human cardiac smooth muscle cells [30]. Importantly, we observed that the effects of hypoxia were time-dependent and about 24% of endothelial cells showed changes consistent with apoptosis after 24 h of hypoxia.

4.3 Hypoxia–reoxygenation and apoptosis

The most effective method of limiting cell death is restoration of blood flow; however, there is evidence that reperfusion per se leads to additional tissue injury [8], caused by release of oxygen free radicals [9], infiltration and activation of neutrophils [32], release of cytokines [10, 32], and calcium overload [33]. Indeed, there is evidence of loss of endothelial function upon reoxygenation of previously ischemic tissues, and the endothelial function can be preserved by pretreatment of animals with free radical scavengers [34]. Direct exposure of vascular tissues to xanthine–xanthine oxidase leads to endothelial disruption and dysfunction [35]. Recent studies indicate that free radicals can induce apoptosis, both physiologically and pathologically [36]. In the present study, we present direct evidence for reoxygenation-mediated enhancement of the effect of hypoxia in human coronary endothelial cells.

4.4 TNFα, hypoxia–reoxygenation and apoptosis

Since hypoxia–reoxygenation and TNFα independently induce apoptosis and TNFα is released during ischemia in cardiac tissues, we hypothesized that an interaction between the two relative to the magnitude of apoptosis must exist. Indeed, we observed that in the presence of TNFα in the culture medium during hypoxia–reoxygenation, apoptosis was markedly enhanced. This observation implies that reperfusion injury to the coronary artery endothelium results not just from hypoxia–reoxygenation, but from release of TNFα as well. Importantly, presence of TNFα during the period of reoxygenation only also significantly increased apoptosis compared with hypoxia–reoxygenation without TNFα; however, the magnitude of apoptosis was less as compared to when TNFα was present in the culture medium throughout the entire period of hypoxia and reoxygenation (Fig. 4). These observations suggest a time-dependent pro-apoptotic effect of TNFα. These phenomena are perhaps representative of coronary artery endothelial cell death/dysfunction in patients with acute myocardial infarction undergoing pharmacological or catheter-based reperfusion therapy.

4.5 Signal transduction pathway of apoptosis

Apoptotic cell death can result either from developmentally controlled activation of endogenous execution programs or from transduction of death signals triggered by a wide variety of exogenous stimuli [37]. It is known that endothelial cell death induced by TNFα is mediated via PKC pathway, and the bcl-2 family member A1 can inhibit TNFα-mediated endothelial cell death [38]. In the present study, we show that TNFα-mediated apoptosis of cultured HCAECs is also associated with significant increase in PKC activity. Furthermore, PKC inhibitor dramatically blocks TNFα-mediated apoptosis of HCAECs. These findings strongly indicate that PKC is involved in signal conduction of TNFα-mediated apoptosis of cultured HCAECs.

5 Conclusion

We have demonstrated that hypoxia per se causes a modest time-dependent apoptosis in cultured HCAECs. Reoxygenation and TNFα are important additional mediators which enhance the suicide program in HCAECs. PKC activation plays a critical role in this process.

Acknowledgements

Supported by grant-in-aid from the American Heart Association-Florida Affiliate, St. Petersburg, FL, and a Merit Review Award from the VA Central Office.

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