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Am J Physiol Heart Circ Physiol 276: H786-H792, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 3, H786-H792, March 1999

Proapoptotic effects of ANG II in human coronary artery endothelial cells: role of AT1 receptor and PKC activation

Dayuan Li, Baichun Yang, M. Ian Philips, and Jawahar L. Mehta

Departments of Medicine and Physiology, University of Florida and Department of Veterans Affairs Medical Center, Gainesville, Florida 32610


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Anoxia-reoxygenation, tumor necrosis factor-alpha (TNF-alpha ), and angiotensin II (ANG II) have been shown to induce apoptosis in myocytes. However, the role of these mediators in causing apoptosis of human coronary artery endothelial cells (HCAEC) is not known. This study was designed to examine the interaction of these mediators in induction of apoptosis in HCAEC. Cultured HCAEC were exposed to anoxia-reoxygenation, TNF-alpha , and ANG II. TNF-alpha enhanced apoptosis of HCAEC (determined by DNA nick-end labeling in situ and DNA laddering) caused by anoxia-reoxygenation. ANG II increased apoptosis beyond that caused by anoxia-reoxygenation and TNF-alpha . Apoptosis caused by ANG II was reduced by losartan, a specific ANG II type 1 receptor (AT1R) blocker, whereas PD-123,177, a specific ANG II type 2 receptor blocker, under identical conditions had minimal effect. The proapoptotic effects of ANG II were associated with the activation of protein kinase C (PKC). The importance of PKC activation as a signal transduction mechanism became evident in experiments wherein treatment of HCAEC with a specific inhibitor of PKC activation decreased ANG II-mediated apoptosis. Thus AT1R activation appears to be responsible for apoptosis caused by ANG II in HCAEC, and AT1R activation-mediated apoptosis involves activation of PKC.

angiotensin II; anoxia; reoxygenation; apoptosis; angiotensin II type 1 receptor; angiotensin II type 2 receptor; endothelial cell; signal transduction


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

ANGIOTENSIN II (ANG II), formed as a result of activation of the renin-angiotensin system, is well known to participate in the pathogenesis of myocardial ischemia-reperfusion injury (37). The critical role of the renin-angiotensin system is evident from significant cardioprotective effects of angiotensin-converting enzyme inhibitors in animals as well as in humans (30). The cardiovascular effects of ANG II are initiated when ANG II interacts with at least two pharmacological distinct subtypes of cell-surface receptors, ANG II type 1 (AT1R) and type 2 (AT2R) (10). AT1R and AT2R are found in both normal and failing cardiac tissue (22, 23). These receptors are found on myocytes (22), endothelial cells (28), fibroblasts (34), and vascular smooth muscle cells (7). Experimental studies have shown that AT1R mediates most of the known effects of ANG II in the heart (30, 36), even though cardiac tissues contain ~50% AT2R (31). In endothelial cells as well, it is the AT1R activation that mediates major functional response to ANG II (31).

Reperfusion injury after transient ischemia activates programmed cell death (apoptosis) in rabbit cardiomyocytes (12). The precise trigger of apoptosis during ischemia-reperfusion is unknown, but it has been speculated to be related to the generation of reactive oxygen species and release of cytokines and vasoactive peptides. Recent studies indicate that ANG II activates apoptosis in several models, such as neonatal rat ventricular myocytes (5) as well as rat kidney (4), pheochromocytoma (35), and granulosa cells (29). However, the role of different ANG II receptor subtypes in the induction of apoptosis in different issues remains controversial.

In the present study, we examined the relative distribution of AT1R and AT2R in cultured human coronary artery endothelial cells (HCAEC) and ANG II-induced apoptosis of cultured HCAEC during anoxia-reoxygenation. We also studied the role of AT1R (vs. AT2R) activation in ANG II-mediated apoptosis of HCAEC. Finally, we examined the contribution of protein kinase C (PKC) as a signal transduction mechanism in the effects of ANG II.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Cell Culture

HCAEC were purchased from Clonetics. Microvascular endothelium growth medium consisted of 500 ml endothelial cell basal medium, 5 ng human recombinant epidermal growth factor, 5 mg hydrocortisone, 25 mg gentamicin, 25 µg amphotericin B, 6 mg bovine brain extract, and 25 ml fetal bovine serum. HCAEC in 5 ml of medium were seeded in a 25-cm2 flask (4,000 cells/cm2), incubated at 37°C in 95% air-5% CO2. Fifth-generation HCAEC (1 × 106) were used in these experiments. The cells were examined under phase-contrast microscopy, and when ~85% confluent, culture medium was changed, and the cells were divided into several groups.

Control group. Cells were incubated in 95% air-5% CO2 for 27 h.

Anoxia-reoxygenation group. Cells were exposed to anoxia for 24 h followed by reoxygenation (3 h).

Tumor necrosis factor-alpha plus anoxia-reoxygenation group. Cells were incubated with tumor necrosis factor-alpha (TNF-alpha ; 20 ng/ml) and then exposed to anoxia (24 h)-reoxygenation (3 h). The concentration of human recombinant TNF-alpha (Sigma) used in the present study was chosen on the basis of previously published literature (20).

ANG II plus anoxia-reoxygenation group. Cells were incubated with ANG II (0.3 µM) and then exposed to anoxia (24 h)-reoxygenation (3 h). The concentration of human sequence ANG II (Sigma) used in the present study was chosen on the basis of previously published literature (5).

ANG II plus TNF-alpha plus anoxia-reoxygenation group. Cells were incubated with ANG II and TNF-alpha and then exposed to anoxia (24 h)-reoxygenation (3 h).

Losartan plus ANG II plus TNF-alpha plus anoxia-reoxygenation group. Cells were incubated with the specific AT1R blocker losartan (10 µM) and ANG II and TNF-alpha and then exposed to anoxia (24 h)-reoxygenation (3 h).

PD-123,177 plus ANG II plus TNF-alpha plus anoxia-reoxygenation group. Cells were incubated with the specific AT2R blocker PD-123,177 (10 µM) and ANG II and TNF-alpha and then exposed to anoxia (24 h)-reoxygenation (3 h).

PKC inhibitor plus anoxia-reoxygenation or ANG II plus anoxia-reoxygenation groups. Cells were incubated with myristoylated PKC peptide inhibitor (8) and then exposed to anoxia (24 h)-reoxygenation (3 h) alone or ANG II plus anoxia (24 h)-reoxygenation (3 h).

Cells were made anoxic by exposure to 95% N2-5% CO2 in a specially designed chamber. The amount of dissolved oxygen in the incubation medium (PO2) declined from 150 mmHg at baseline to 30-40 mmHg within 1 h of exposure to 95% N2-5% CO2. This decrease in PO2 remained stable over the course of the anoxic period. Reoxygenation of cells was performed by transferring cells into an incubator maintained at normal atmospheric O2 and 5% CO2.

AT1R and AT2R Binding Assay in HCAEC

Cells (1 × 105) were seeded in 60-mm tissue-culture dishes. Cells were washed twice in ice-cold phosphate-buffered saline (PBS), pH 7.2, before addition of reaction mixture. Reaction mixture consisted of radiolabeled ANG II antagonist 125I-labeled [Sar1,Ile8]ANG II and 2% BSA in PBS, pH 7.2. Radiolabeled ANG II was added to each dish at a final concentration of 12.5, 25, 50, 100, 200, and 400 pM. Cells were incubated with 1 ml of reaction mixture in the absence and presence of 1 µM unlabeled ANG II, specific AT1 receptor antagonist losartan, or specific AT2 receptor antagonist PD-123,319 to determine total, nonspecific AT1 and AT2 receptor binding, respectively. Incubation was carried out at room temperature for 90 min. Cells were washed four times with ice-cold PBS, pH 7.2, containing 1% BSA. Cells were lysed at room temperature in 0.5 M NaOH solution. An aliquot of the cell lysate was counted to determine the amount of bound radiolabeled ANG II antagonist 125I-labeled [Sar1,Ile8]ANG II. Protein was then quantitated by bicinchoninic acid protein assay kit. ANG II receptor maximum binding (Bmax) values and dissociation constants (KD) were determined by Scatchard plot (9).

RT-PCR for Endothelial AT1R and AT2R mRNA in HCAEC

Total RNA (1 µg) extracted from cultured HCAEC was reverse transcribed with random hexamer using Super Script (GIBCO). Two microliters of the reverse-transcribed product were amplified with Taq DNA polymerase (Promega) using a primer pair specific to human AT1R (forward primer, 5'-TCATTTACTTTTATATGAA-3'; reverse primer, 5'-TGAATTTCATAAGCCTTCTT-3'). PCR product is 532 bp. For PCR, 40 cycles were used at 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min (25). A primer pair specific to human AT2R was as follows: 5'-AATATGAAGGGCAACTCCAC-3' (forward primer), 5'-TTAAGACACAAAGGTCTCCAT-3' (reverse primer). PCR product is 1,100 bp. For PCR, 35 cycles were used at 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min (31). The RT-PCR-amplified samples were visualized on 1.5% agarose gels using ethidium bromide. beta -Actin was amplified as a reference for quantitation of AT1R and AT2R mRNA. Relative intensities of bands of interest were analyzed by use of an MSF-300G Scanner (Microtek Lab) and Scan Analysis software (Biosoft) and expressed as the ratio to beta -actin (3).

Quantification of Apoptosis by Nick-End Labeling

To detect DNA fragmentation in situ, nick-end labeling was performed by a DNA fragmentation detection system (Calbiochem), as described by Gu et al. (13). Briefly, the cells plated on slides were fixed in 4% formaldehyde at room temperature for 10 min. The slides were incubated at room temperature for 20 min with 20 µg/ml proteinase K to increase cell permeability. The slides were then incubated at room temperature for 5 min with 3% H2O2 to inactivate endogenous peroxidases. The slides were covered with Klenow enzyme and biotinylated dNTP in reaction buffer in a humidified chamber at 37°C for 1.5 h. After rinse with Tris-buffered saline, the cells were incubated with streptavidin-horseradish peroxidase at room temperature for 30 min and then stained with 3,3'-diaminobenzidine at room temperature for 10 min. Methyl green was used for counterstain. The negative control sample was generated by substituting distilled water for the Klenow enzyme in the reaction mixture during the labeling step. The positive control sample was generated by covering slide with 1 µg/µl DNase I at room temperature for 20 min after proteinase K treatment. At least 500 cells from randomly selected fields were counted to determine the percentage of apoptotic cells.

Analysis of DNA Fragmentation in Agarose Gels

HCAEC (1 × 106) were removed from culture dishes, washed twice with PBS, and pelleted by centrifugation. Cell pellets were then treated for 10 min with lysis buffer (1% Nonidet P-40 in 20 mM EDTA and 50 mM Tris · HCl, pH 7.5). After centrifugation for 5 min at 1,600 g, the supernatant was collected, and the extraction 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 one-half volume of 10 M ammonium acetate, the DNA was precipitated with 2.5 vol absolute ethanol. DNA was recovered by centrifugation at 12,000 g for 10 min and dissolved in Tris-EDTA buffer (10 mM Tris · HCl, 1 mM EDTA). DNA fragmentation was separated by electrophoresis in 1.6% agarose gel with ethidium bromide (16).

PKC Activity

Cells were washed twice with PBS and scraped into 0.5 ml of cold extraction buffer containing the following (in mM): 25 Tris (pH 7.4), 0.5 EDTA, 0.5 EGTA, 10 beta -mercaptoethanol, 100 phenylmethylsulfonyl fluoride, 0.05% Triton X-100, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. The lysate was homogenized and centrifuged at 14,000 g at 4°C for 30 min, and the supernatant was saved for PKC assay. Promega's SignaTECT PKC assay system was used for determination of PKC activity (2). Results were expressed as picomoles of ATP per minute per microgram of protein.

Data Analysis

All data represent means of duplicate samples from at least four independently performed experiments. Data are presented as means ± SD. Statistical significance was determined in multiple comparisons among independent groups of data, in which ANOVA and the F-test indicated the presence of significant differences. A P value of <= 0.05 was considered significant.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Identification of ANG II Receptor Types in HCAEC

mRNA for AT1R and AT2R was detected in all fifth- generation cultured HCAEC (n = 6). Expression of AT1R mRNA was consistently higher than that of AT2R mRNA (Fig. 1). This observation was supported by binding assays that showed that HCAEC possessed high-affinity ANG II binding sites, as determined by a triplicate reciprocal plot of the data. Scatchard analysis indicated that the KD values of AT1R and AT2R for HCAEC were 168 and 172 pM, respectively. Scatchard plots indicated that the Bmax values of AT1R and AT2R for HCAEC were 2.21 and 1.19 fmol/mg cell protein, respectively (Fig. 2).


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  		Fig. 1.   ANG II type 1 receptor (AT1R) and ANG II type 2 receptor (AT2R) mRNA by RT-PCR in human coronary artery endothelial cells (HCAEC). See text for details of methodology. This gel is representative of 6 separate experiments. AU, arbitrary units.


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  		Fig. 2.   Identification of ANG II AT1R and AT2R in HCAEC by 125I-labeled [Sar1,Ile8]ANG II binding. KD, dissociation constant; Bmax, maximum binding.

Identification of Apoptosis

Determination of apoptosis by fragmented DNA nick-end labeling. Because a small number of cells normally die during culture or are damaged during processing, 1-6% (3.3 ± 2.4%) of control cells stained positive. Three hours of reoxygenation after 24 h of anoxia increased the number of apoptotic cells to 27.1 ± 7.3% of all cells in cultured HCAEC (P < 0.01 vs. control). TNF-alpha further increased the number of apoptotic cells (P < 0.05 vs. anoxia-reoxygenation). Presence of ANG II significantly enhanced the number of apoptotic cells (P < 0.05 vs. anoxia-reoxygenation alone group or TNF-alpha  + anoxia-reoxygenation group). Losartan, the specific AT1R blocker, reduced the increase in the number of apoptotic cells caused by ANG II (P < 0.05 vs. ANG II + TNF-alpha  + anoxia-reoxygenation group), whereas PD-123,177, the AT2R blocker, did not significantly affect the number of apoptotic cells. Data from multiple experiments are summarized in Fig. 3.


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  		Fig. 3.   Summary of data on number of apoptotic HCAEC as percentage of all cells. Reoxygenation for 3 h after anoxia for 24 h causes significant increase in number of apoptotic cells. Presence of tumor necrosis factor-alpha (TNF-alpha ) or ANG II further increases number of apoptotic cells. Presence of both TNF-alpha and ANG II exerts a cumulative effect on number of apoptotic cells. Losartan decreases, whereas PD-123,177 has no effect on, number of apoptotic cells. Each bar reflects data from 6 separate experiments in means ± SD. A-R, anoxia-reoxygenation.

Detection of DNA fragmentation in gel electrophoresis. HCAEC cultured under normoxic conditions showed no DNA laddering at 27 h. Under anoxia-reoxygenation conditions, cultured HCAEC contained fragmented DNA that produced a ladder of DNA bands representing the cleavage of genomic DNA into nucleosomal size of 180- to 200-bp fragments, indicating apoptotic cell death of HCAEC during anoxia-reoxygenation. The proportion of the fragmented DNA was increased by ~50% in ANG II plus anoxia-reoxygenation group compared with the anoxia-reoxygenation alone group. The amount of fragmented DNA was further increased by ~50% in ANG II plus TNF-alpha plus anoxia-reoxygenation group as compared with TNF-alpha plus anoxia-reoxygenation group. ANG II-mediated effect was blocked by losartan, but not by PD-123,177. Results of a representative experiment are shown in Fig. 4.


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  		Fig. 4.   DNA laddering evidence of apoptosis in HCAEC exposed to anoxia-reoxygenation, TNF-alpha , and ANG II. There is only modest DNA laddering in cells exposed to 3 h of reoxygenation after anoxia alone for 24 h. Presence of ANG II increases apoptosis. Presence of both TNF-alpha and ANG II causes marked DNA fragmentation. Losartan decreases, whereas PD-123,177 has no effect on, DNA fragmentation. This gel is representative of 6 separate experiments.

ANG II-Induced PKC Activation

Anoxia-reoxygenation increased PKC activity in cultured HCAEC compared with that in control cells (P < 0.01). ANG II alone also increased PKC activity in cultured HCAEC, and it further increased PKC activity in cells exposed to anoxia-reoxygenation (P < 0.01). The effect of ANG II was completely abolished by the PKC inhibitor (Fig. 5).


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  		Fig. 5.   Protein kinase C (PKC) signal transduction during ANG II-mediated apoptosis in HCAEC. Note that ANG II increases PKC activity in HCAEC and also enhances effect of anoxia-reoxygenation. ANG II-mediated increase in PKC activity is blocked by PKC inhibitor. Data are from 6 separate experiments.

Critical Role of PKC Activation in Apoptosis

To determine the role of PKC activation in apoptosis, HCAEC were treated with myristoylated PKC peptide inhibitor and then exposed to anoxia-reoxygenation, ANG II, or ANG II plus anoxia-reoxygenation. As shown in Fig. 6, PKC inhibitor significantly decreased apoptosis in HCAEC exposed to ANG II alone or with anoxia-reoxygenation. PKC inhibitor treatment also reduced apoptosis in HCAEC exposed to anoxia-reoxygenation alone.


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  		Fig. 6.   Effect of PKC inhibitor (Inh) treatments on degree of apoptosis caused by anoxia-reoxygenation (A-R), ANG II, and ANG II plus A-R, as measured by nick-end labeling. Note that PKC inhibitor treatment decreases apoptosis in HCAEC in response to A-R. Pretreatment with both PKC inhibitors decreases apoptosis in HCAEC in response to ANG II plus A-R. Each bar reflects data from 6 separate experiments in means ± SD.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

This study for the first time shows expression of both AT1R and AT2R in cultured HCAEC with a predominance of AT1R. We also show that ANG II enhances anoxia-reoxygenation- and TNF-alpha -mediated apoptosis, and this process is triggered by activation of AT1R. As direct evidence, losartan, the specific blocker of AT1R, reduced ANG II-mediated apoptosis. In this process, activation of PKC appears to play a critical role as a signal transduction mechanism.

Anoxia-Reoxygenation, TNF-alpha , and Apoptosis

It is well known that reperfusion following ischemia leads to additional tissue injury, such as cell necrosis (18, 19) and apoptosis (12). Previous studies (18, 19) showed that ischemia-reperfusion causes free radical release and intracellular calcium overload. Both free radicals and calcium overload result in cell necrosis (18, 19) and apoptosis (5, 35). Work by Gottlieb et al. (12) showed that apoptosis is a specific feature of reperfusion injury in cardiac myocytes, leading to late cell death. In this study, we demonstrate that anoxia-reoxygenation also leads to apoptosis in HCAEC.

Myocardial ischemia-reperfusion injury is often associated with release of cytokines, such as TNF-alpha (14). Expression of TNF-alpha has also been observed in atherosclerotic human coronary arteries and in animal models of arterial injury (6). It is known that TNF-alpha induces expression of genes of apoptosis and triggers a suicide program and signal transduction system (16, 20). Several investigators have shown a direct proapoptotic effect of TNF-alpha in bovine pulmonary artery (21) and human umbilical vein (26) endothelial cell. On the basis of the observations that 1) ischemia-reperfusion injury is associated with apoptosis, 2) TNF-alpha is released during ischemia-reperfusion, and 3) TNF-alpha per se induces apoptosis, we hypothesized that an interaction between TNF-alpha and anoxia-reoxygenation relative to induction of apoptosis may exist. In the present study, we indeed observed that TNF-alpha enhances apoptosis in HCAEC exposed to anoxia-reoxygenation.

ANG II and Apoptosis

ANG II is well known to have cardiotoxic effects. Work done almost 20 years ago indicated that large doses of ANG II cause focal myocardial injury (11). An increasing body of evidence suggests that ANG II induces apoptosis in different tissues. Cigola et al. (5) reported that ANG II induced apoptosis of neonatal rat ventricular myocytes that were exposed to 10-9 M ANG II for 24 h in vitro. ANG II resulted morphologically in a 2.5-fold increase in the percentage of myocytes with double-strand cleavage of the DNA and formation of DNA fragments equal in size to mono- and oligonucleosomes. Other studies showed that ANG II mediates apoptosis in different tissues such as PC12W (rat pheochromocytoma) and R3T3 (mouse fibroblast) cells (36), rat ovarian granulosa cells (29), and rabbit vascular smooth muscle cells (20). In the present study, we found that ANG II significantly augmented apoptosis in HCAEC exposed to anoxia-reoxygenation alone or with TNF-alpha .

Role of ANG II Receptor Types in Apoptosis

AT1R and AT2R are found in both normal and failing cardiac tissues (22, 23). Sadoshima and Izumo (24) observed the molecular phenotype of cultured cardiac cells from neonatal rats in response to ANG II and the effects of selective ANG II receptor subtype antagonists in mediating the biological effects of ANG II. They found that ANG II causes a rapid induction of many immediate-early genes (c-fos, c-jun, jun B, Egr-1, and c-myc) in myocyte and nonmyocyte cultures. The overexpression of these genes may have a facilitatory effect on the induction of apoptosis by ANG II. Induction of these genes by ANG II was fully blocked by an AT1R antagonist, but not by an AT2R antagonist. All biological effects of ANG II in their work were mediated primarily by the AT1R activation.

Experimental studies from several laboratories in intact animals have shown that AT1R activation mediates most of the known effects of ANG II in cardiac tissues (10, 31, 37), even though cardiac tissues contain a significant number of AT2R (31). A recent study (15) in an AT1R knock-out mice shows total absence of arrhythmias following a period of ischemia-reperfusion, further confirming the critical role of AT1R in ischemia-reperfusion.

In the study by Cigola et al. (5), ANG II-induced apoptosis of ventricular myocytes was inhibited by the AT1R antagonist losartan, whereas the selective AT2R blocker PD-123,319 did not reduce myocyte apoptosis. Pollman et al. (20) also observed that the proapoptotic effect of ANG II in rabbit aortic smooth muscle cells was mediated by activation of AT1R and not AT2R.

On the other hand, some studies have shown that AT2R activation is also capable of inducing apoptosis in some tissues. Tanaka et al. (29) examined the change in AT2R expression during differentiation and apoptosis of rat ovarian granulosa cells in culture, which express abundant AT2R. Their work showed that ANG II-induced apoptosis of granulosa cells was suppressed by the AT2R-selective antagonist PD-123,319 but not by DuP-753 (losartan). This study indicated that AT2R activation may induce apoptosis of granulosa cells. Yamada et al. (36) observed that AT2R activation mediates apoptosis in PC12W (rat pheochromocytoma) and R3T3 (mouse fibroblast) cells, which express abundant AT2R, but not the AT1R. These observations (29, 36) were established in cells that express abundant AT2R, but little or no AT1R, and suggest that tissues that express primarily AT2R exhibit AT2R-mediated apoptosis, and tissues that express primarily AT1R exhibit AT1R-mediated apoptosis.

The ANG II receptor types and their functional significance have not yet been defined in HCAEC. In this study, we documented by RT-PCR and binding assays that HCAEC express both AT1R and AT2R with predominance of AT1R. In accordance with the concept discussed above, ANG II-mediated apoptosis of HCAEC was significantly blocked by the AT1R blocker losartan and not by the AT2R blocker PD-123,177.

Signaling Pathways of ANG II-Mediated Apoptosis in Cultured HCAEC

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 (27). Signaling pathways of ANG II-mediated apoptosis in vascular endothelial cells, smooth muscle cells, and cardiomyocytes are not well defined. Vascular endothelial cells possess G protein-coupled AT1R for ANG II that couple to activation of multiple signaling pathways, such as phospholipase C-beta , which causes the subsequent release of calcium from intracellular pools and activation of PKC, protein tyrosine kinase, and mitogen-activated protein kinase (33). Mechanisms by which AT1R activates multiple signaling pathways are unclear, although the receptor might interact at some level with integrins and cytokine receptors. Different signaling pathways of the AT1R may serve to evoke different cellular responses. Signal transduction is initiated by receptor tyrosine kinases, G protein-coupled seven transmembrane-spanning receptor (i.e., AT1R), cytokines (i.e., TNF-alpha ), and integrins in cardiac fibroblasts (1). In this study, we examined PKC activation in HCAEC and found that ANG II-mediated apoptosis was associated with an increase in PKC activity. Furthermore, we observed that pretreatment of HCAEC with myristoylated PKC peptide inhibitor significantly reduced PKC activation as well as apoptosis in response to ANG II. This PKC inhibitor is cell permeable and inhibits calcium- and phospholipid-dependent PKC activation (8). These findings strongly indicate that PKC activation is involved in signal transduction of ANG II-mediated apoptosis of cultured HCAEC. There may well be activation of other signaling pathways in HCAEC upon exposure to ANG II. Cross talk among multiple signal conduction pathways in different tissues and models may determine the ultimate fate of the cell exposed to anoxia, cytokines, and ANG II.

Apoptosis is now recognized as a principal cause of cardiac muscle and vascular endothelial cell death in ischemia-reperfusion injury or during the early hours of acute myocardial infarction and as a potential contributor to congestive heart failure. Therefore, investigations of mechanisms and specific signaling pathway inhibitors of apoptosis may have important clinical significance.

In conclusion, we have demonstrated that both AT1R and AT2R are expressed in cultured HCAEC (AT1R > AT2R). Anoxia-reoxygenation per se causes apoptosis in cultured HCAEC. TNF-alpha enhances anoxia-reoxygenation-mediated apoptosis, and this process is further amplified in the presence of ANG II. Our study also indicates that the proapoptotic effects of ANG II in cultured HCAEC are triggered by the activation of AT1R. The activation of AT1R involves the intracellular PKC pathway as a signal transduction mechanism.


    ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid from the American Heart Association, Florida Affiliate (St. Petersburg, FL), a Department of Veterans Affairs Central Office merit review award, and a National Institutes of Health merit award.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. L. Mehta, Dept. of Medicine, Univ. of Florida College of Medicine, 1600 Archer Rd., PO Box 100277 JHMHC, Gainesville, FL 32610.

Received 13 August 1998; accepted in final form 22 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 276(3):H786-H792
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