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Cardiovascular Research 1999 43(2):492-499; doi:10.1016/S0008-6363(99)00108-X
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Copyright © 1999, European Society of Cardiology

The isoprostane, 8-epi-PGF2{alpha}, is accumulated in coronary arteries isolated from patients with coronary heart disease

Mohammad Reza Mehrabia,*, Cem Ekmekcioglub, Franz Tatzberc, Anthony Oguoghoc, Robert Ullrichd, Abdelmonem Morgana, Forouzan Tamaddona, Michael Grimme, Helmut D Glogara and Helmut Sinzingerc

aDepartment of Cardiology, University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna, Austria
bDepartment of Medical Physiology, University of Vienna, Vienna, Austria
cDepartment of Nuclear Medicine, University of Vienna, Vienna, Austria
dDepartment of Clinical Pathology, University of Vienna, Vienna, Austria
eDepartment of Surgery, University of Vienna, Vienna, Austria

* Corresponding author. Tel.: +43-140-4004614; fax: +43-140-81148 mmehrabi{at}pop3.kard.akh-wien.ac.at

Received 4 November 1998; accepted 9 February 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: In the present study we wanted to know whether 8-epi-PGF2{alpha}, which belongs to the class of isoprostanes formed by free radical-mediated peroxidation of arachidonic acid and arachidonyl-containing phospholipids, is enriched in isolated coronary arteries of patients suffering from coronary heart disease (CHD, n=23) who received allograft heart transplants as compared to vessels derived from patients with dilative cardiomyopathy (CMP, n=19) or from healthy heart donors (controls, n=6). Methods: Sections from the isolated coronary arteries were analysed by semiquantitative immunohistochemistry by determining the area and intensity of positive reaction for 8-epi-PGF2{alpha} in the vascular intima and media. In addition, the 8-epi-PGF2{alpha} content was determined using a specific immunoassay after extraction and purification. Results: The immunohistochemical results indicated that 8-epi-PGF2{alpha} is significantly enriched in arteries from patients suffering from CHD as compared to CMP (P<0.0001). In controls, significantly less immunostaining was observed. Furthermore, a significant positive correlation between semiquantitative immunohistochemistry and radioimmunological determination was observed too. Conclusions: From our findings we conclude that 8-epi-PGF2{alpha} is especially accumulated in coronary arteries from CHD patients and therefore is likely to be involved in atherogenesis.

KEYWORDS Atherosclerosis; Coronary disease; Free radicals; Cardiomyopathy; Prostaglandins


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Elevated LDL-cholesterol is a key risk factor for the development of atherosclerosis in coronary arteries [1,2]. Recently, increasing evidence was accumulated that its arterial deposition and oxidative modification is a key event during atherogenesis facilitating unlimited cholesterol uptake [3,4]. Vascular cell lipid deposits finally lead to the excessive formation of arterial foam cells being a marker of the atherosclerotic process. Free oxygen radicals provided by white blood cells, platelets and endothelial cells may promote this process by leading to free radical-induced peroxidation injury in vivo. Morrow’s group [5–7] was the first to report that prostaglandin F2-like compounds, the so called F2-isoprostanes, are generated independently from cyclooxygenase by a free radical catalysed mechanism. These authors also claimed that the in vivo modification of F2-isoprostane production could be a promising measure to examine oxidative stress in vivo. In particular, for smoking, an increased urinary excretion has been reported [8]. Recently, in vascular tissue [9,10], blood and urine of hypercholesterolemic patients [11], an increased amount of the major isoprostane, 8-epi-PGF2{alpha}, has been detected [12]. This compound via its potent proliferative and vasoconstrictory action [13] may exert a significant pathogenetic action. It was therefore the aim of this study to determine 8-epi-PGF2{alpha}, the major isoprostane in the arterial wall, immunochemically and by immunohistochemistry in human coronary arteries of CHD and CMP patients, who received heart allograft transplantation, as compared to controls.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Coronary arteries
The material was collected in accordance with the principles outlined in the Declaration of Helsinki [14].

Coronary arteries isolated from human hearts after orthotopic heart transplantation of patients with dilative (mean age 48.7±13.9, n=19) or ischaemic (mean age 58.2±8.4, n=23) cardiomyopathy with left ventricular ejection fraction <30% (for patients characteristics, see Table 1) were used in this study. Patients with an anamnestic history of myocardial infarction in combination with effective occlusion(s) of one or more coronary arteries, as documented by coronary angiography, were included in the coronary heart disease group (CHD group). In contrast, patients without a history of coronary artery disease and normal findings during coronary catheterization were classified as cardiomyopathy patients (CMP patients). The control group consisted of coronary arteries isolated from healthy donor hearts, which were primarily intended for transplantation, but not used afterwards (mean age 22.0±3.5 S.D., n=6) for several reasons, e.g. such as the donors died during accidents with severe thoracic trauma leading to endocardial bleeding with an additional increased risk of infection.


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  		Table 1 Patients characteristicsa

 
2.2 Sample processing
2.2.1 Immunoperoxidase staining.
The explanted hearts were immediately put into a cardioplegic physiological solution (containing a lipid free chemical composition, Celsior, Pharmacia & Upjohn, Yvelines, France) at 4°C. Most of the coronary arteries from the explanted hearts were isolated within 3–4.5 h at 4°C. This time interval was necessary for the transport and isolation procedure of the vessels. Only in six cases the time range was between 7–10 h, respectively. However these vessels showed no significantly higher accumulation in 8-epi-PGF2{alpha} as compared to the vessels isolated earlier assuming that the ex vivo formation of 8-epi-PGF2{alpha}was most probably moderate. At least three sections from similar locations of the left coronary artery (within 3 cm from the origin from the aorta) were analysed from each vessel. The mean values of these determinations were used for further calculations. Tissue samples for immunohistochemical analysis were fixed immediately after removal in 7.5% formaldehyde (in phosphate buffered saline, pH 7.2), using standard methods. Tissue embedded in paraffin wax was cut in 3–5-µm thick sections, dried at 55°C for 2 h and then deparaffinized in xylene for 20 min followed by dehydration through graded alcohols. The endogenous peroxidase activity was blocked with 3% H2O2 in methanol and afterwards tissue proteolysis was performed by pretreatment with 0.1% protease (protease XIV, EC 3.4.24.3 [EC] 1; Sigma, Vienna, Austria) in 0.05 mol/l Tris–HCl, pH 7.6. Sections were then immersed in Tris-buffered saline (0.15 mol/l sodium chloride, 0.05 mol/l Tris–HCl, pH 7.6), and incubated with anti-isoprostane [dilution 1:400 (rabbit anti-8-epi-PGF2{alpha}, Assay Designs, Inc. Ann Arbor, MI, USA)]. To exclude unspecific binding, IgG-control tissues were treated overnight at 4°C with polyclonal rabbit anti-mouse interleukin-3 (Genzyme, Cambridge, UK), followed by the addition of biotinylated goat-anti-rabbit antibody (Dako, Glostrup, Denmark) for 15 min at room temperature to stop the reaction. According to the manufacturer of the antibody (Assay Designs, Inc. Ann Arbor, U.S.A.) cross reactivities with PGF2{alpha}, whose structure is very similar to 8-epi-PGF2{alpha}, was below 2%, while cross reactivities with other important prostaglandins were below 0.2%.

Considering these values we can assume that the cross-reactivity of the antibody only insignificantly contributed to the 8-epi-PGF2{alpha} immunohistochemical staining.

After repeated washes with Tris–HCl buffer (pH 7,4), sections were incubated with streptABComplex/HRP (Dakopatts A/S, Glostrup,Denmark) for 15 min at room temperature. Afterwards, the reaction product was developed using aminoethylcarbazole (Dako, Glostrup, Denmark, AEC substrate system) to visualize the red-stained immunoreactive sites. Counterstaining was performed with hematoxylin; finally, sections were coverslipped in Aquatex (Merck, Darmstadt, Germany) and used for semiquantitative analysis.

2.2.2 Immunohistochemical analysis
The immunohistochemical sections were analysed by light microscopy. The positively stained areas for 8-epi-PGF2{alpha} assessed by planimetry were expressed in percent, while 0% indicates no reaction and 100% complete positivity of the respective segment with the isoprostane. In addition, the intensity of the positive areas was analysed using a score, ranging from 0–4 estimated units (eU), (0=no activity,1=minimal, 2=slight, 3=intensive, and 4=very intensive staining). The enrichment and intensity of 8-epi-PGF2{alpha} in the intima and media of the coronary arteries were determined and presented. The investigations both concerning immunochemistry and immunohistochemistry were performed by double-blind procedures.

2.2.3 Radioimmunoassay of 8-epi-PGF2{alpha}
The concentration of the isoprostane 8-epi-PGF2{alpha}, was determined in the coronary vessels of all the three groups by RIA. For this purpose freshly isolated samples were rinsed with ice-cold buffer and then immediately placed into an isotonic NaCl solution containing 1 mM EDTA and 1 mM 4-hydroxy-tempo, a free radical scavenger. Afterwards the samples were homogenized by Ultraturax and then stored at –70°C. Addition of EDTA in combination with antioxidative substances and storage at –70°C were described to prevent ex vivo formation of 8-epi-PGF2{alpha} [10,15,16]. Total lipids were extracted with an ice-cold Folch solution, chloroform/methanol (2:1, v/v). Afterwards 8-epi-PGF2{alpha} was liberated in the organic phase after evaporation under N2 and hydrolysis with KOH. Subsequently the RIA procedure was performed according to Wang et al. [15]. The detection limit of the RIA was 2 pg/mg wet weight. The interassay variability was 8.3±2.4% while the intraassay variability amounted 5.0±2.3%.

2.3 Statistical analysis and data presentation
Values are presented as mean (±S.D.). Analysis of variance (ANOVA) with the Scheffé procedure as a post-hoc test or the Student’s t-test were used to compare the means. A P value of <0.05 was considered to be significant. Linear regression analysis was used to assess the correlation between radiommunoassay and immunohistochemical findings. Student’s t-test or the Mann–Whitney U-test were performed to evaluate the influence of risk factors (smoking, hypertension, and diabetes mellitus) and also alcohol consumption on the accumulation of 8-epi-PGF2{alpha} in CHD and CMP patients.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Patient characteristics
The anamnestic data of the patients and their characteristics are presented in Table 1. A total of 48 hearts derived from patients with dilative (n=19) or ischaemic cardiomyopathy (n=23) and the control group (n=6) were examined.

3.2 Anti-8-epi-PGF2{alpha}-staining
The area stained positively for 8-epi-PGF2{alpha} in the intima of the coronary arteries was highest in ischaemic (46.7±13.4%) as compared to dilative CMP (25.3±8.6%) and control vessels (15.0±6.3%), the differences being significant at different levels (statistics and data, see Fig. 1; photographs, see Fig. 2a–c). In patients with advanced atherosclerotic plaque formations we found distinctly higher rates of 8-epi-PGF2{alpha} accumulation around the plaque areas (Fig. 2e).


Figure 1
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  		Fig. 1 The percent positive areas (A) and intensity (B) of the isoprostane 8-epi-PGF2{alpha} in the intima of coronary arteries from control, CMP and CHD patients. Data are given as mean±S.D. * P<0.0001; ** P<0.0001; *** n.s.

 

Figure 2
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  		Fig. 2 Immunohistochemical staining for 8-epi-PGF2{alpha} in representative histological sections from similar locations of the left coronary artery in a CHD (a), CMP (b) and control (c) patient. The negative control in a CHD-derived vessel (d) consisted of nonimmune isotype-matched mouse IgG or monoclonal anti-IL3 as a substitute for the primary antibody. (e) Shows a CHD patient with advanced atherosclerotic plaque formation. The sections of the control patient showed the thickest intima of all controls in order to better visualize the accumulation of 8-epi-PGF2{alpha}. The thin arrows exemplify positive 8-epi-PGF2{alpha} staining in the intima and media of the vessel sections. The thick arrow indicates atherosclerotic plaques. The asterisk marks the lumen of the vessels. Original magnifications (a–e): x200.

 
The intensity of isoprostane staining in the intima showed comparable results with the greatest intensity in ischaemic (2.8±0.8 eU) followed by dilative CMP (1.6±0.5 eU) and control (1.2±0.4 eU) vessels (statistics and data, see Fig. 1).

When determining the area involved with isoprostane staining in the media of the coronary arteries we observed that 8-epi-PGF2{alpha} was again highest in the ischaemic group (7.6±4.0%) followed by the dilative CMP vessels (5.8±2.5%) and the controls (5.0±3.2%) (Fig. 3). The differences for the three groups, however, were not statistically significant. The intensity values in the media showed a similar finding (Fig. 3).


Figure 3
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  		Fig. 3 The percent involvement enrichment (A) and intensity (B) of anti 8-epi-PGF2{alpha} staining in the media of coronary arteries from control, CMP, and CHD patients. Results are presented as mean±S.D. Values are not significant between groups.

 
Comparing the area positively stained and the intensity between the intima and media in all the three groups we found that the values were significantly higher in the intima (Table 2).


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  		Table 2 Comparison between the% area or intensity (eU) from the isoprostane 8-epi-PGF2{alpha} in the intima vs. media in all three patient groups

 
3.3 RIA of 8-epi-PGF2{alpha}
Determining 8-epi-PGF2{alpha} radioimmunologically in extracts of the respective vessels, the by far highest values were found in vessels from ischaemic CHD (Fig. 4). The 8-epi-PGF2{alpha} measured in CMP-derived vessels was significantly (P<0.0001) lower, but, on the other hand, significantly higher as in the controls (P=0.0002).


Figure 4
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  		Fig. 4 Radioimmunological determination of the isoprostane, 8-epi-PGF2{alpha},in coronary arteries from control, CMP and CHD patients. Values are presented as mean±S.D. * P<0.0001; ** P<0.0001; *** P=0.0002.

 
3.4 Correlation between RIA vs. immunohistochemistry of 8-epi-PGF2{alpha}
Our results indicate a significant correlation between the two variables in the intima and to a lesser extent in the media (Table 3).


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  		Table 3 Correlation between RIA vs. immunohistochemistry of 8-epi-PGF2{alpha}a

 
3.5 Influence of risk factors and alcohol
Subgrouping accounting to risk factors revealed that in CHD no difference in isoprostane was found between smokers and non-smokers. The non-smokers in this group, however, were only three. 8-Epi-PGF2{alpha} in the intima of hypertensive CHD patients was significantly higher (P=0.04, Student’s t-test) than in normotensives while there were no significant differences in CMP patients. Diabetes mellitus did not result in a difference of 8-epi-PGF2{alpha} in either of the subgroups evaluated. Alcohol was of no significant influence as well, however, in almost all the three subgroups the 8-epi-PGF2{alpha} accumulation was lower in those people having consumed higher amounts of alcohol (data not presented).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Isoprostanes are a new family of compounds fast gaining relevance for the understanding of atherosclerosis [10,17,18]. Derived as a consequence from free-radical attack of LDL, they are generated in situ and subsequently cleared in blood and urine [15,19,20].

The powerful proliferative and vasoconstrictory action of the most prominent member so far, 8-epi-PGF2{alpha} makes it an interesting parameter for both pathophysiological understanding and eventual diagnostic assessment of oxidation injury [21] in tissue extracts, plasma and/or urine [22]. So far very few data on the accumulation of isoprostanes in atherosclerotic lesions are available. Previously Pratico et al. [10] have shown that the isoprostanes, 8-epi-PGF2{alpha} and IPF2{alpha}-I, are accumulated in atherosclerotic lesions from human carotid arteries derived from CHD patients as compared to vessels from heart transplant CMP patients. In addition Gniwotta et al. [9] also reported the enrichment of isoprostanes in atherosclerotic areas of various human arteries (except for coronary arteries). Although we are aware that our paper is not the first investigating isoprostanes in atherosclerotic lesions we believe that it presents additional new valuable data in this study field. The intention of our investigation was not only to demonstrate 8-epi-PGF2{alpha} accumulation in atherosclerotic lesions but to illuminate the role of this isoprostane in coronary arteries of CHD patients as compared to vessels with modest or minimal atherosclerotic lesions of CMP or control patients. Therefore we used for the first time freshly isolated intact coronary arteries from CHD, CMP and control hearts in contrast to the previous manuscripts [9,10]. Although a significant correlation between for example carotid and coronary artery atherosclerosis is well known [23,24], previous studies [25] have also shown that the relationship between carotid intima-media thickness, as an indicator of atherosclerosis, and coronary artery disease is weak with a poor correlation. Therefore it is doubtless better to directly demonstrate 8-epi-PGF2{alpha} accumulation in coronary arteries especially, like in our case, when investigating coronary atherosclerosis. In our study we observed a smaller accumulation of 8-epi-PGF2{alpha} between CHD vs. CMP patients than in the study of Practico et al. [10]. Reasons for these differences between both studies could be that 8-epi-PGF2{alpha} accumulation in carotid vessels probably differs from that of coronary vessels. Furthermore, in contrast to the previous studies, we investigated vascular samples from similar locations in all the three patient groups (CHD, CMP, and control patients), which could also be one more explanation for the smaller differences in isoprostane accumulation between CHD vs. CMP or control vessels. We also included coronary arteries from healthy young heart donors in this study. The findings obtained between control vs. CHD or CMP vessels may be age dependent to a certain extent, because age of controls differed significantly from that of CMP and CHD patients. However, it should be noted that there were also significant differences in staining between CMP and CHD patients of comparable age. The CMP patient group had no hemodynamic effective coronary artery stenosis, as verified by coronary angiography, and therefore it was justified to compare these vessels with those of CHD patients. The reason to also include a younger health control group was the consideration to also investigate oxidative stress markers in intact young healthy coronary arteries which are very difficult to obtain. Due to the high frequency of heart allograft transplantations at our clinic, we have access to unique tissues required for such investigations, especially control materials. Our results had clearly shown that 8-epi-PGF2{alpha} is also present in young health vessels to a remarkable extent. However, due to the small number and lack of anamnestic data of these patients, like for example smoking behavior, one cannot generalize these results to the whole population.

In this study we did not find it to be necessary to also investigate the accumulation of IPF2{alpha}-I (now known as IPF2{alpha}-IV), which is especially more abundant in oxidized LDL and human atherosclerotic plaques. Previously it has already been described [10] that there is a highly significant correlation (P<0.0001) in the accumulation between both isoprostanes in human atherosclerotic vascular tissue.

Although it is known that diabetes mellitus increases oxidative stress we found no significant difference in 8-epi-PGF2{alpha} accumulation between patients with as compared to patients without diabetes. Reasons for the lack of influence could be the low number of diabetic patients (six in CHD and only one in CMP) in both groups. Furthermore all the diabetic patients suffered from type II diabetes, diagnosed 2–4 years before heart transplantation and treated successfully with a dietary regimen in four cases and with drugs in two cases. Therefore we did not really expected a relevant difference in 8-epi-PGF2{alpha} accumulation between diabetic and non-diabetic patients in the subgroups. In contrast, however, we found a significantly higher 8-epi-PGF2{alpha} accumulation in the intima of hypertensive CHD patients as compared to normotensives. Most of the hypertensives suffered from the disease since at least 5 years which most probably favoured the formation of atherosclerotic lesions. Except for hypertension in CHD patients the results concerning the risk factors were in accordance with the findings of Pratico et al. [10].

In the present study we did not performed staining for other prostaglandins generated largely via the cyclooxygenase pathway. Previously it was reported that blocking nonspecifically COX with aspirin did not impaired urinary 8-epi-PGF2{alpha} excretion as compared to untreated patients [15,18]. Furthermore Pratico et al. [10] reported that there was no significant difference in the 8-epi-PGF2{alpha} levels between CHD patients taking aspirin versus those who did not. Twelve of our patients were treated with aspirin. We also did not find any difference in the accumulation of 8-epi-PGF2{alpha} between these patients and the ones not receiving aspirin. Therefore it can be assumed that 8-epi-PGF2{alpha} accumulation in atherosclerotic lesions derives mainly from non-enzymatic, free radical mediated processes.

We detected higher accumulations of 8-epi-PGF2{alpha} around atherosclerotic plaques. This is in accordance with the study of Pratico et al. [10]. In so far unpublished immunohistochemical experiments, we also found significant amounts of oxidized LDL around atherosclerotic plaques in CHD patients. Therefore it is likely that these areas are associated with substantial amounts of oxidative stress which in turn would favour the generation of 8-epi-PGF2{alpha}. The relationship between LDL oxidation and generation of isoprostanes was previously described by Pratico and Fitzgerald [26].

In conclusion our findings clearly show a remarkable enrichment of 8-epi-PGF2{alpha} in CHD patients which therefore is likely to be associated with the most severe oxidation injury, followed by CMP and controls. Immunohistochemical studies in the future, eventually at the ultrastructural level, may allow to gain more insights into the local oxidation injury involved in atherogenesis. Our results and previous investigations [9,17] suggest that 8-epi-PGF2{alpha} plays an important role in this process. However, it has to be considered that this isoprostane is only a minor component of the large isoprostane family. Therefore it is thinkable that other isoprostanes could also be involved significantly in the process of atherosclerosis. Most recently, Mallat et al. [27] were able to demonstrate elevated levels of 8-iso-PGF2{alpha} in pericardial fluid of patients with heart failure. Their finding of a strong correlation between functional severity of heart failure and 8-iso-PGF2{alpha} confirms the role of isoprostanes in the diseased heart. Oxidation products of arachidonic acid metabolism seem to contribute to atherogenesis and most likely they are also involved as initiators of the pathogenetic events. Further studies are necessary to clarify their role in atherogenesis.

Time for primary review 23 days.


   Acknowledgements
 
The authors acknowledge the expert technical assistance by Karina Plesch.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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