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(Circulation. 1995;92:1494-1498.)
© 1995 American Heart Association, Inc.

Articles

Chronic Congestive Heart Failure Is Associated With a Phenotypic Shift of Intramyocardial Endothelial Cells

Monique M.H. Marijianowski, MSc; Marjolein van Laar, BSc; Johannes Bras, MD; Anton E. Becker, MD

From the Department of Cardiovascular Pathology, University of Amsterdam (the Netherlands), Academic Medical Center.

Correspondence to Anton E. Becker, MD, Department of Cardiovascular Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam-ZO, Netherlands.

	Abstract

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Background There is evidence that patients with chronic congestive heart failure have endothelial cell–related abnormalities of the peripheral circulation and the coronary microvasculature. For that reason, we have studied the phenotypic expression of endothelial cells in hearts of patients with congestive heart failure.

Methods and Results We studied cardiac explants (n=19) and autopsy hearts (n=5) of patients with chronic congestive heart failure caused by either a dilated cardiomyopathy (n=12) or ischemic heart disease (n=12) and compared them with normal hearts (n=12). The antigenic expression obtained with several endothelial cell markers (factor VIII–related antigen, EN-4, Ulex europaeus agglutinin–1 (UEA-1), PAL-E, endoglin, and endothelin) and adhesion molecules (intercellular adhesion molecule [ICAM], vascular cell adhesion molecule [VCAM], or E-selectin) was compared by use of immunohistochemical techniques. On the basis of the initial findings, the number of PAL-E– and EN-4–positive vessels was counted. The incidence of PAL-E–positive vessels per area was quantified and related to the percentage of heart muscle cells and the total number of vessels per area. In control hearts, endothelial cells rarely were positive for PAL-E. In hearts of patients with ischemic cardiomyopathies, there was distinct staining with this marker. Hearts of patients with dilated cardiomyopathies showed a marked increase in the number of PAL-E–positive endothelial cells. Vessels with a muscular media were PAL-E–negative. Two-sample analysis revealed a statistically significant difference between hearts with dilated cardiomyopathies and ischemic cardiomyopathies (P<.01), between hearts with dilated cardiomyopathies and control hearts (P<.01), and between hearts with ischemic cardiomyopathies and control hearts (P<.01). Endoglin and ICAM were positive but nondiscriminating. Endothelin, VCAM, and E-selectin were negative.

Conclusions A phenotypic shift in endothelial antigen expression of the coronary microvasculature occurs in both ischemic hearts and hearts with dilated cardiomyopathies, as revealed by PAL-E, compared with control hearts. The change may relate to compensatory mechanisms in long-standing chronic heart failure.

Key Words: dilated cardiomyopathy • endothelium • ischemic cardiomyopathy • immunology

	Introduction

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In patients with chronic congestive heart failure (CHF), the sympathetic nervous and the renin- angiotensin-aldosterone systems are activated as compensatory mechanisms. Moreover, there is evidence that these mechanisms induce secondary structural changes in the heart itself, which result in an increase of perivascular collagen.¹ The modulating role of endothelial cells is considered of prime importance in this respect.² Recently, it was claimed that endothelium-dependent dilation of the coronary microvasculature is impaired in patients with dilated cardiomyopathy (DCM) and that the phenomenon could be caused by endothelial dysfunction.^{3 4 5} Invasive studies in patients with DCM suggest a decrease in myocardial perfusion reserve, although a recent noninvasive study revealed no findings to this effect.⁶ Mosseri and colleagues⁷ demonstrated that hearts of patients with congestive cardiomyopathy contain enlarged capillaries but have a normal ratio of capillaries to myocytes. These are interesting observations in light of findings in the peripheral circulation in which it has been claimed that systemic vasoconstriction, itself an obligatory feature in advanced chronic CHF, is due to a reduced release of endothelium-derived relaxation factor or to raised plasma-endothelin–1 levels.^{8 9 10 11 12 13} Endothelin is produced by endothelial cells and acts as a potent inotropic agent.¹⁴ There is much evidence, therefore, that local changes in the function of endothelial cells play a role not only in the peripheral circulation in patients with CHF, regardless of the underlying cause, but also possibly in the intramyocardial circulation.

As part of our study of the connective tissues in hearts of patients with chronic CHF, we noticed variable expression with a particular endothelial cell marker, PAL-E.¹⁵ This particular antigen, expressed by endothelial cells, has not yet been characterized, but it seemed worthwhile to further explore these observations in light of the hypothesis of localized endothelial cell dysfunction.

The present study, therefore, was directed toward a possible change in the phenotypic expression of endothelial cells in hearts with chronic CHF.

	Methods

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Tissue Specimens
Cardiac explants (n=19) and autopsy hearts (n=5) of patients with chronic CHF caused by either DCM (n=12; 4 women, 8 men) or ischemic heart disease (IHD; n=12; 2 women, 10 men) were used. The ages ranged from 36 to 60 years (mean, 51 years) for DCM hearts and from 32 to 70 years (mean, 50 years) for IHD hearts. All patients were in the New York Heart Association functional class III-IV, and all were treated with diuretics and inotropic drugs. An elevated blood pressure had been present in some of the patients with IHD, but none had been treated specifically for hypertension. None of the patients with DCM had presented evidence of an inflammatory process. Hearts from individuals who died of noncardiovascular causes (n=12; 5 women, 7 men) served as reference. They were age-matched with a mean of 47 years. All autopsy hearts became available within 10 hours after death.

A cross section of the heart was taken perpendicular to the left ventricular long axis immediately apical to the level of the base of both papillary muscles. Tissue blocks were taken from the lateral free wall of the left ventricle. In hearts with IHD, care was taken to sample tissues remote from the scarred areas. The samples were snap-frozen in isopentane and stored at -80°C; then 5-µm cryostat sections were cut on organosilane-coated glass, air-dried (±3 hours), and fixed in cold acetone (10 minutes, 4°C). For endothelin detection, formalin-fixed and paraffin-embedded samples also were used.

Immunohistochemistry
Endothelial cells were evaluated for the expression of factor VIII–related antigen, EN-4, PAL-E, the lectin Ulex europaeus agglutinin–1 (UEA-1), endoglin, and endothelin.¹⁶ In addition endothelial cells were screened for expression of the adhesive molecules with cellular adhesion markers (intercellular adhesion molecule [ICAM],¹⁷ vascular cell adhesion molecule [VCAM],¹⁸ and E-selectin¹⁹ ). Table 1 lists the sources of the antibodies and the working dilutions. In case of negative staining results, aortic samples were used as controls.

View this table: [in this window] [in a new window]   Table 1. Antibodies Used

Single Staining
Factor-VIII–related antigen and endothelin were detected by a three-step indirect immunohistochemical technique by applying the primary rabbit polyclonal antibody, biotinylated goat anti-mouse immunoglobulin, and horseradish peroxidase (HRP)–labeled streptavidin. EN-4, PAL-E, endoglin, ICAM, VCAM, and E-selectin were detected by a three-step indirect immunohistochemical technique by applying the mouse monoclonal antibody, biotinylated goat anti-mouse immunoglobulin, and HRP-labeled streptavidin. UEA-1 was detected by an indirect immunoperoxidase procedure that binds -fructose residues on endothelial cells. Rabbit anti–UEA-1 conjugated to HRP was used as a second step. HRP activity was detected with hydrogen peroxide (0.01%) as substrate and amino-ethyl-carbazole (AEC) (2.38 mmol/L) as dye (5 minutes, room temperature)²⁰ ; on paraffin sections, HRP activity was detected with 3'3-diaminobenzidine tetrachloride (10 minutes, room temperature). Sections were counterstained with hematoxylin.

Triple Staining
To evaluate whether endothelial cells were surrounded by a layer of smooth muscle cells (SMCs), the endothelial cell markers EN-4 and PAL-E were applied in the presence of an anti-SMC marker. For this purpose, an immunoenzyme triple staining was performed with three different enzyme labels.²¹ The triple staining was based on different subclasses of the primary antibodies.²² The primary antibodies were incubated (60 minutes, room temperature) in a mixture containing PAL-E (IgG2a), EN-4 (IgG1), and SMC (IgM) at working dilutions of 1:10, 1:100, and 1:20, respectively. Anti-mouse immunoglobulin isotype and subclass specific antibodies, all raised in goat, were applied (30 minutes, room temperature) in a mixture containing HRP-conjugated anti-IgG2a, alkaline phosphatase (AP)–conjugated anti-IgG1, and anti-IgM–biotinylated. Finally, ß-galactosidase (GAL)–conjugated streptavidin was applied (30 minutes, room temperature). GAL, AP, and HRP were detected successively in turquoise, blue, and red. Briefly, GAL activity was visualized according to Bondi et al²³ by use of bromo-chloro-indolyl-ß-galactoside (1.22 mmol/L) as substrate and potassium ferro/ferricyanide (both 3 mmol/L) as dyes (15 to 60 minutes, 37°C). HRP activity was visualized with AEC as a chromogen, and AP activity was visualized using fast blue BB (8 mg/50 mL) as azo dye and naphthol-AS-MX-phosphate (0.24 mmol/L) as substrate (10 to 20 minutes, room temperature).²⁴ Levamisole 1 mmol/L was added to the incubation medium for blocking endogenous AP activity.²⁵

To enhance the contrast, a counterstain was performed with fast green (0.1%) to visualize the cardiac myocytes. The microscopic slides were mounted with coverslips by use of gelatin-glycerin.

Quantitative Methods
The number of PAL-E– or EN-4–positive but SMC-negative vessels and the cardiac myocyte volume fraction (percentage of cardiac myocytes per area) were established in triple-stained sections counterstained with fast green with an objective of x40 and a square grid with 10 horizontal and 10 vertical lines (100 points). For each tissue sample, eight different fields were counted, which contained only cardiomyocytes in cross section, and the mean value for each sample was calculated. PAL-E positivity was related to the total number of vessels per area and to the percentage of heart muscle cells. In sections taken from hearts with IHD, microscopic areas of replacement fibrosis were excluded from the quantification.

The results are expressed as the percentage of PAL-E–positive vessels per area.

Statistical Analysis
The results were analyzed with one-way ANOVA as a first step, followed by two-sample analysis (t test). Thereafter, the Bonferroni procedure was used to adjust the probability values. Values of P<.05 were considered significant.

	Results

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There were no differences in results between cardiac explants and hearts obtained at autopsy.

Immunohistochemistry
The antibody against factor-VIII–related antigen showed a consistent and overall weak staining of endothelial cells. UEA-1 also labeled all endothelial cells of blood vessels. However, the staining intensity was extremely variable within one section and between sections. The EN-4 antibody showed a consistent and overall distinct staining of endothelial cells. There were no differences in incidence or intensity of staining among the different groups.

PAL-E was rarely positive in the reference hearts. In hearts with IHD, however, there was a distinct staining of endothelial cells with PAL-E. Moreover, in hearts with DCM, the increase in the number of PAL-E–positive endothelial cells was most marked (Fig 1). PAL-E positivity showed a patchy distribution in tissues of IHD and a more diffuse expression in DCM.

View larger version (12K): [in this window] [in a new window]   Figure 1. Line graph showing the percentage of PAL-E–positive vessels in dilated cardiomyopathy (DCM), ischemic heart disease (IHD), and reference hearts.

Triple staining with EN-4, PAL-E, and SMC revealed that PAL-E positivity and EN-4 positivity occurred in neighboring cells in one cross section. In reference hearts, the vast majority of endothelial cells stained with EN-4; only rarely did they stain with PAL-E. In hearts with both dilated and ischemic cardiomyopathies, however, the vast majority of cells expressed PAL-E (Fig 2). At the same time, the triple staining procedure revealed that none of the vessels with a muscular media showed PAL-E expression of the endothelial cells, regardless of the heart being "normal" or "diseased."

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrographs showing PAL-E expression (in red) in a reference heart (A) and a heart with dilated cardiomyopathy (DCM, B). Both panels are of the same magnification (original magnification x320) to emphasize the hypertrophic state of the myocardium in hearts with DCM vs reference hearts. An immunohistochemical triple stain was applied that shows EN-4 in blue, PAL-E in red, and smooth muscle cells in turquoise. The myocardium of the heart with DCM shows a distinct increase in the number of vessels expressing the PAL-E antigen. Note that a vessel with a muscular medium has no PAL-E activity (B).

Endoglin, an endothelial homodimeric membrane antigen used as a marker for adhesion receptors of the integrin family, showed positive staining of all endothelial cells in all three groups. The same applies to ICAM, which represents an adhesion molecule normally expressed on endothelial cells in most circumstances. On the other hand, both VCAM and E-selectin, which also represent adhesion molecules and usually are upregulated as part of an inflammatory process, were negative in all specimens studied. Endothelin, a potent vasoconstrictor considered to play an important role in regulating peripheral vascular resistance in CHF, showed no staining of endothelial cells in any of the three groups. Control sections of the aorta were positive.

Quantification
Table 2 summarizes the results. The total number of vessels varied among the three groups. One-way ANOVA revealed no significant differences between the three groups.

View this table: [in this window] [in a new window]   Table 2. DCM, IHD, Reference Hearts, Total Number of Vessels and Non–Muscle-Coated Vessels Expressing PAL-E and EN-4, and Cardiac Myocyte Volume Fraction in Cross Sections of Lateral Wall of Left Ventricle

There is a statistically significant increase of PAL-E–positive vessels in hearts with DCM and ischemic heart disease (Fig 2). The results of the two-sample analysis (t test) showed a level of significance between DCM and ischemic cardiomyopathies of P<.01. The level of significance between DCM and reference hearts was P<.01; between ischemic cardiomyopathy and controls, P<.01.

The percentage of heart muscle cells per surface area was variable within all patient groups, suggesting variable degrees of myocyte hypertrophy. The volume fraction of heart muscle cells expressed as a percentage showed no correlation with the expression of PAL-E (Table 2).

	Discussion

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The present study documents a change in the phenotypic expression of the endothelial cells within the myocardium of hearts of patients with chronic CHF. There is a shift in endothelial antigen expression in both ischemic and dilated cardiomyopathies, as revealed by PAL-E, compared with reference hearts. This change, moreover, affects only the microvasculature because vessels with a muscular media did not express PAL-E. The importance of this observation remains uncertain but adds evidence to the concept that local functional changes of endothelial cells occur in chronic CHF.

The fact that endothelial cells can modulate vascular tone is well established,^{26 27} and a regulatory role in cardiac contractility was reported recently.²⁸ These data, together with findings indicating functional changes of both the peripheral and the intramyocardial microcirculation,^{3 4 5 6 8 9 10} suggest that endothelial cells play an active role in the compensatory mechanisms initiated by chronic CHF. Moreover, perivascular fibrosis in hearts of patients with chronic CHF is considered to be due to effects of the renin-angiotensin-aldosterone systems,¹ in which endothelial cells are considered to play a key role.² It was suggested recently that endothelial cells modulate both cardiac fibroblast collagen synthesis and degradation.²⁹ The finding of increased expression of PAL-E, therefore, is worthy of further investigation.

How Does the PAL-E Finding Relate to the Literature?
Leenstra and coworkers³⁰ found PAL-E reactivity of endothelial cells in brain tumors, in diseases with a developmental etiology such as primitive tumors and congenital vascular malformations, and in the developing human brain. They suggest that PAL-E expression relates to the specialized endothelial cell function as part of the blood-brain barrier.

Leenstra et al³⁰ also suggested that PAL-E expression relates to vascular neogenesis. We have looked into this option using EN-4, a membrane marker for all endothelial cells, but could not find such a relation.

Furthermore, we found no correlation between PAL-E reactivity and expression of adhesion molecules, endothelin, or endoglin. The membrane-associated endoglin molecule is a transforming growth factor-ß–binding protein that is expressed most abundantly on endothelial cells in tissues with inflammatory characteristics. Endoglin expression is also found in tumors and granulation tissue in which neovascularization can be found. In our study, we found no difference in the staining intensity with endoglin between reference hearts and hearts with DCM or IHD. Lack of activation of endothelial cells is also confirmed by the lack of expression of the adhesion molecules VCAM and E-selectin. All three represent markers of endothelial cell activation related to leukocyte adhesion and migration in the setting of inflammatory processes. In addition, there were no differences among the three groups.

Finally, endothelin expression was not found within the myocardium, whereas specimens of the aorta, which served as control, showed distinct endothelin expression of endothelial cells and of intimal and medial SMC as reported in the literature.¹⁶ This negative finding is of interest because plasma endothelin levels are increased in patients with chronic CHF.^{31 32} It seems unlikely, therefore, that the increased expression of PAL-E has a relation with endothelin production by endothelial cells.

Study Limitations
The principal limitation in interpreting the observations of this study is twofold. First, the antigen recognized by PAL-E is uncharacterized as yet. This simple fact makes it extremely difficult to speculate on its functional significance. Second, one has to take into account that all patients with chronic CHF have been treated with inotropic drugs for a long time. Whether these drugs may induce a phenotypic change of endothelial cells remains a matter of speculation. Moreover, when the phenotypic change is drug-induced, the question as to its functional significance remains. Is it part of the overall compensatory mechanisms in CHF, or could it have a deleterious effect? Whatever the case, the option of a drug-induced phenotypic change has to be taken into account.

The present observations clearly indicate that some sort of change in the phenotypic expression of intramyocardial endothelial cells occurs in hearts of patients with CHF. Hence, further studies of the intramyocardial coronary vessels in those patients are justified.

	Acknowledgments

 
We are indebted to Dr Jessica Mann, Department of Histopathology, St George's Hospital Medical School, London, UK, for providing material of cardiac explants. Dr Chris M. van der Loos gave technical advice regarding the immunohistochemical triple-stain method. Marsha Schenker provided excellent secretarial assistance.

Received February 13, 1995; accepted March 17, 1995.

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