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Cardiovascular Research 1999 41(3):737-745; doi:10.1016/S0008-6363(98)00287-9
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Copyright © 1999, European Society of Cardiology

Immunohistological and ultrastructural analysis of the intimal thickening in coarctation of human aorta

Maria Jimenez, Danièle Daret, Alain Choussat and Jacques Bonnet*

Institut National de la Santé et de la Recherche Médicale, Unité 441 de Cardiologie, Pessac, France

* Corresponding author. Tel: +33-5-5789-1975; Fax: +33-5-5636-8979

Received 16 January 1998; accepted 30 June 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. Methods: In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. Results: Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson’s trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: {alpha}-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only {alpha}-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. Conclusions: These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.

KEYWORDS Human aortic coarctation; Smooth muscle cell; Contractile apparatus; Congenital defects; Heart


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The etiology of aortic coarctation is a subject of debate. The hemodynamic theory proposed by Rudolph [1]accounts for the three features of the coarctation syndrome, the localized shelf, the associated intracardiac defects and the tubular hypoplasia by a reduced anterograde aortic flow. However, the fact that the isolated aortic coarctations in typical cases are located near the entrance of the ductus arteriosus has led several authors to suspect a possible relationship between coarctation and ductus arteriosus [2]. In foetal ductus arteriosus, an earlier smooth muscle cell (SMC) differentiation in comparison to other great arteries, has been observed and could participate in the ductus occlusion at birth [3–5]. The present study was designed to analyse the patterns of SMCs differentiation in human aortic coarctation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Study material
Samples were obtained from ten children admitted to hospital for surgical treatment of isolated aortic coarctation. Their ages ranged from 15 days to 4 years (mean age 15.8±12.2 months). None of the patients had complex intracardiac lesions or had received prostaglandin E1 (Table 1). After aortic resection and end-to-end aortic anastomoses, the coarcted shelf and the pre- and post-stenotic part of the upper descending aorta were examined.


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  		Table 1 Clinical details of the ten patients studied

 
The tissues were carefully rinsed in Nutrient Mixture HAM F10 (Gibco BRL, Gaithersburg, MD), and quick-frozen in isopentane prechilled in liquid nitrogen, then embedded in Tissue Tek (Miles Laboratories, Naperville, IN) for immunohistochemistry. In order to evaluate the histological appearance of the aortic narrowing, aortic segments were examined after hemalun–eosin staining. To identify connective tissue, adjacent sections were stained with Masson’s trichrome and Weigert’s resorcin–fuchsin for elastic fibers [6].

2.2 Immunohistochemistry
In each specimen of human aortic coarctation, a series of areas were analyzed: pre- and post-stenotic areas (normal or moderate stenotic part) and coarcted shelf (almost totally occluded part). Five-micron thick cryostat sections were mounted on precleaned and gelatin-coated glass slides. Serial sections were cold acetone-fixed for immunohistological analysis, and endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. Sections were first exposed to 5% bovine serum albumin, then to primary antibodies overnight at 4°C. Antibody binding was revealed using the horseradish peroxidase complex staining procedure described above using 3,3'-diaminobenzidine as chromogen. All sections were counterstained with Mayer’s hemalun, dehydrated and coverslipped with DPX mounting medium. The various manipulations were standardized in order to avoid differences in immunostaining. For example, slides were treated with the same antibody in one batch, and the staining development time was determined accurately and kept constant for all antibodies.

The primary mouse monoclonal antibodies used at appropriate dilutions against desmin and {alpha}-smooth muscle actin ({alpha}-SM actin IA4) were purchased from Immunotech (Marseille, France) and Sigma Chemical Co (St Louis, MO). The cell-type specific monoclonal antibodies against endothelial cells (anti-von Willebrand factor), human T-cells (anti-Leu-4) and human macrophages (HAM-56) were obtained from Dako (Carpinteria, CA), Becton Dickinson (Mountain View, CA) and Enzo Diagnostics (NY). The monoclonal antibodies against human smooth muscle-myosin heavy chain (SM-MHC) and h-caldesmon (high molecular weight form) were kindly provided by Dr. M. Glukhova [7]. The proliferative profile of cross-sectional areas was assessed by immunocytochemical labeling for the proliferation cell nuclear antigen (PCNA) using an anti-PCNA antibody (Dako) [8]. An irrelevant isotype-matched immunoglobulin (Sigma) was used as a negative control.

Immunohistochemistry analysis were performed by two investigators independently. Results were reported separately then compared.

2.3 Ultrastructural study
All specimens were prepared for ultrastructural examination by fixation in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated with alcohol, and embedded in Epon 812. Ultrathin sections were cut with a Reichert Om U3 ultramicrotome, double stained with uranyl acetate and lead citrate. The study was made on three different pieces of each sample examined with a Philips EM 201 transmission electron microscope.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Histology
In the first few months after birth, the normal thoracic aorta was easily visualized, being characterized by an aortic media with regular elastic lamellae alternating with smooth muscle cell layers. The aortic intima consisted of a simple flattened endothelium which directly covered the internal elastic lamina (Fig. 1A).


Figure 1
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  		Fig. 1 Light microscopic sections of aortic coarctation. A and B: Immunoreactivity with anti-{alpha}-SM actin in a normal part of the aorta (A) and in the coarctation shelf (B). C and D: Masson’s trichrome staining showed intimal thickening (i) with separation of endothelial cells from the internal elastic lamina (iel), by a widening subendothelial region (C). This figure was shot in the border of the thickening. D: Detail of fragmented internal elastic lamina with reoriented cells. E: Weigert’s staining demonstrated a lack of elastic fibers in the subendothelial region. F: The endothelial cells were stained with anti-von Willebrand factor. G: Immunoreactivity with anti-{alpha}-SM actin was localized in the media (m) and subendothelial region. H: No immunoreactivity was observed with anti-PCNA antibodies. (Figures A, C, E, F, G, H, magnificationx20, figure B, magnificationx10, figure D, magnificationx100).

 
Histological study of the coarctation shelf on hemalun eosin-stained cryosections evidenced a separation of endothelial cells from the internal elastic lamina with a widened subendothelial region. Staining with anti-von Willebrand factor antibodies demonstrated the presence of endothelial cells at the border of the lumen as well as in the narrowest part of the coarctation, with no extension of endothelial cells into the subendothelial region (Fig. 1F). Staining with Masson’s trichrome revealed a marked increase in both collagen and cell numbers in the intimal thickening (Fig. 1 C,D). However, Weigert’s staining evidenced scant elastic material, with no regular distribution of fine elastic fibers (Fig. 1E). In the outer subendothelial region, a fragmentation of the internal elastic lamina along with radially orientated smooth muscle cells, recognizable by their staining with anti-{alpha}-SM actin, were observed (Fig. 1G). No inflammatory cells were identified in the intima.

Radially oriented SMCs were seen in the inner media. They increased in number with the extent of thickening and appeared to cross the internal elastic lamina and protrude into the subendothelial region. The structural alterations in the inner media and outer subendothelial region fragmented the internal elastic lamina, which was less readily recognizable. The middle and outer media contained closely packed layers of circularly orientated SMCs. However, when the intima was quite thick, the media was enlarged with less organized SMCs. The internal elastic lamina was not readily identified. The media lacked the regularly spaced elastic lamellae of normal aorta, and mainly consisted of a loose arrangement of smooth muscle fibres in a connective tissue matrix. Very fine, wavy elastic fibers were discerned in the matrix.

3.2 Immunohistochemistry
The intima of control aorta consisted of a monolayer of endothelial cells closely apposed to the intimal elastic lamina. There was no evidence of intimal SMCs, and the media was immunostained by anti-{alpha}-SM actin monoclonal antibodies (Fig. 1A).

In order to characterize SMC phenotypes in human aortic coarctation, we analyzed the pattern of expression of smooth muscle markers ({alpha}-SM actin, SM-MHC, h-caldesmon, desmin) in the normal descending aorta (Fig. 2A,B,C) and coarcted shelf. In coarctation with moderate intimal thickening (Fig. 2D,E,F), at least two distinct smooth muscle phenotypes for contractile and cytoskeletal protein expression were identified. In the outer region of intimal thickening, cells in the juxtamedial part of the intima strongly expressed three smooth muscle markers, SM-MHC, h-caldesmon and desmin. Faint reactivity was frequently observed in the subendothelial part of the intimal thickening, although most intimal cells were only labeled by the anti-{alpha}-SM actin monoclonal antibody, and did not express SM-MHC, h-caldesmon or desmin. Cells within the inner media were negative for all the smooth muscle markers tested except {alpha}-SM actin. In the outer media, SMCs were well differentiated, expressing all smooth muscle markers.


Figure 2
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  		Fig. 2 Immunohistochemistry of normal human aorta (A, B, C) and coarctation with moderate intimal thickening (D, E, F). A, B: Immunoreactivity with SM-MHC and caldesmon was localized in normal aortic media layer. C: Desmin-negative SMCs were found in normal aorta. D, E, F: SM-MHC, caldesmon and desmin antibodies labeled the outer region of intimal thickening with a weak reactivity in the subendothelial part of the thickening (magnificationx20). ii: inner intima, oi: outer intima, m: media.

 
In the almost totally occluded parts of the coarcted aorta, the distribution pattern of specific proteins markers in the intima was heterogeneous (Fig. 3). A strong expression of SM-MHC and h-caldesmon in the subendothelial part showed the presence of strongly differentiated SMCs in the occluded part of the aorta (Fig. 3C,D). However, these cells were characterized by a third smooth muscle phenotype slightly expressing desmin (Fig. 3E). The underlying media in these regions of marked intimal thickening, characterized by disorganized SMCs, was also abnormal. The media was heterogeneously stained by the different smooth muscle markers (Fig. 3B,C,D). Results were summarized in Table 2.


Figure 3
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  		Fig. 3 Immunochemistry in marked aortic coarctation. A: Endothelial cells (e) immunoreacted with anti-von Willebrand factor. B, C, D: Intimal thickening and media, different pattern of staining with SM-MHC (C) and caldesmon (D) to that with anti-{alpha}-SM actin antibody (B). E: Low immunoreactivity with desmin antibody in marked coarctation (magnificationx20).

 

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  		Table 2 Summarized results of immunohistochemical stainings

 
No PCNA-positive cells were observed in intimal thickening of the aortic narrowing (Fig. 1H).

No difference according to the age of children was observed in the SMC phenotype in coarcted segments. In the same way, no difference was demonstrated between the SMC phenotype in pre- or post-stenotic areas.

3.3 Ultrastructural microscopy
Several ultrathin sections of each sample were analyzed. Electron microscopic examination revealed a variety of SMC phenotypes in the intimal thickening of human coarctation, SMCs appeared as ‘modulated’ SMCs in the synthetic state but graduated from luminal to the deeper region adjacent to the medial part. In the superficial layer of the thickening, polygonal SMCs in a synthetic state were observed, characterized by a marked development of rough endoplasmic reticulum, a clear-cut Golgi apparatus and well developed surface vesicles (Fig. 4B). In the deeper layer of the thickening, synthetic along with contractile components were identified in the SMCs (Fig. 4A). These small cells appeared to be migrating from the media, frequently disrupting the internal elastic lamina. In many SMCs of this deeper part, the nucleus occupied much of the cell surface, while the cytoplasm contained Golgi apparatus, endoplasmic reticulum with aggregated or free ribosomes. Microfilaments and mitochondria surrounded by an accumulation of polysomes were also observed. The cells were enveloped with abundant extracellular matrix, collageneous fibers and proteoglycans. In the normal medial region, elongated spindly SMCs in a contractile state were observed between the elastic fibers. These well differentiated SMCs contained a large number of myofilaments, mitochondria and an electron-dense basement membrane adjacent to the plasma membrane.


Figure 4
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  		Fig. 4 Transmission electron microscopy of SMCs in the deeper region (A: SMC with synthetic and contractile components) and subendothelial part of intimal thickening (B: synthetic state of SMC) of aortic coarctation (magnificationx15000). g: golgi apparatus, n: nucleus, rer: rough endoplasmic reticulum.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In contrast, to investigations on the clinical and surgical management of coarctation, less has been known about morphological and phenotypic characteristics of coarctation muscle cells. No study has characterized the mechanism of intimal thickening and the phenotypic nature of SMCs in aortic coarctation.

The present study of human coarctation led us to suspect that the intimal thickening was associated with two processes: (1) accumulation of extracellular matrix in the subendothelial region, (2) migration of SMCs into the subendothelial space from the inner media, causing fragmentation of the internal elastic lamina. In our study, we did not find any proliferation. However, we can not exclude local SMC proliferation in fetal life. The separation of endothelial cells from the internal elastic lamina was akin to the formation of the ‘intimal cushions’ in human ductus arteriosus [2, 9–12]. In common with ductus arteriosus, the subendothelial region of coarctation contains less elastin than the regions rich in circularly orientated medial SMCs [13], which is indicative of a dedifferentiation of intimal SMCs. It is thus not inconceivable that coarctation and obliteration of ductus arteriosus share a common mechanism [2, 11, 14, 15]. In order to analyse such a hypothesis it was important to define SMC differentiation in human coarctation.

Differentiation of vascular SMCs involves phenotypic transitions of the cytoskeletal and cytocontractile apparatus [16, 17], and it can be tracked from the appearance of coordinately regulated cytoskeletal and contractile proteins. It should, however, be borne in mind that the expression of a given protein may be regulated by its own specific mechanism, and it is thus important to use a set of smooth muscle-specific markers rather than rely on a single one to assess the state of differentiation. In the present study, we examined the expression of SM-MHC, h-caldesmon and desmin as markers of differentiated SMCs [7, 18–25].

In the course of various vascular diseases (arterial hypertension, atherosclerosis, restenosis after angioplasty), SMCs involved in neointimal development, temporarily display a less differentiated phenotype resembling that seen in the embryo [16, 17]. The expression of SM-MHC, h-caldesmon, and desmin is down-regulated (or even lost) in proliferating smooth muscle during development of arteriosclerosis, and there is a concomitant induction of expression of non-muscle myosin heavy chain and low molecular weight caldesmon [25–29].

In our study, the intimal thickening of moderate stenosis areas was stained by a variety of smooth muscle markers, with numerous dedifferentiated SMCs in the inner region of the intima and more differentiated SMCs, expressing the three smooth muscle markers, in the juxtamedial part of the intima. A similar process has been described in neonatal ductus arteriosus: well differentiated SMCs in the media and outer intima comparable to the SMCs of normal aorta [3, 4]. Dedifferentiated SMCs, with a low expression of contractile and cytoskeletal proteins, have been found in the inner part of the intimal thickening of the ‘intimal cushions’ formed during closure of the ductus arteriosus [4, 9]. However, the occluded part of the coarcted aorta was characterized by the presence of more differentiated or redifferentiated SMCs strongly expressing SM-MHC and h-caldesmon in the subendothelial part of the intima. A majority of cells did not express desmin and appeared to differ from the juxtamedial differentiated, desmin-expressing SMCs in the regions of moderate intimal thickening. These findings suggest that SMCs with an intermediate phenotype are present in the narrowest part of coarctation. Giuriato et al. [10]have also noted a particularly low desmin content and heterogeneous staining with anti SM-MHC antibody in the ‘intimal cushions’ of ductus arteriosus.

The pattern of differentiation of SMCs in the medial layer of coarctation was also non-uniform. A number of authors [10, 30, 31]have observed within the aortic media of several mammalian species, a variety of SMC phenotypes for the expression of contractile and cytoskeletal proteins. In ductus arteriosus closure, several SMC phenotypes have been observed in the underlying media of the ‘intimal cushions’. It has been suggested that, in response to pathological stimuli, some medial SMCs maintain their differentiation, whereas others undergo dedifferentiation [5, 7, 10]. These observations are supported by our ultrastructural analysis. Dedifferentiated SMCs, characterized by a dramatic increase in rough reticulum endoplasmic, were observed in the subendothelial part of slightly thickened intima. A more differentiated state was noted in the SMCs of the juxtamedial part of intima with a persistence of small sarcomers and poorly developed endoplasmic reticulum. These SMCs were not morphologically different from those in the inner region of media, and even appeared to be continuous with them. These observations suggest that the emergence of SMCs in the intima could be due to migration from the tunica media with fragmentation of internal elastic lamina more than a rapid proliferation. The presence of dedifferentiated SMCs in the media could suggest that the dedifferentiation process should be necessary before SMC migration. Moreover, contrasting with the dedifferentiated cells in the other parts of intimal thickening, the presence of SMCs with an intermediate phenotype in the subendothelial part of the narrowest part of coarted aorta suggest that redifferentiation of SMCs could participate in the pathogenesis of narrowing in aortic coartation. Such a mechanism has been evoked for ductus arteriosus though sensibility to vasoconstrictive agents.

As the patterns of SMC differentiation in human coarctation seem similar to those described for ductus arteriosus, two questions can be defined, the initiation of coarctation during fetal life and participation of ductal cells in the coarctation narrowing process. Concerning the first point, our study cannot eliminate the hypothesis of initiation of coarctation during fetal life. On the contrary, the absence of medial atrophia and the absence of proliferating SMC suggest that SMC migration cannot totally explain the severity of intimal thickening and that a prior SMC proliferation phase could participate in the pathogenesis during fetal life. Concerning the origin of SMC participating to intimal thickening of aortic coarctation, the main result is the apparent heterogeneity of the differentiation. This heterogeneity could be due to the presence of SMC from the same origin but at a different step in the differentiation process (differentiation, dedifferentiation, redifferentiation) [32]. The origin of the embryonic vascular SMC has been described from neural crest and locally differentiating mesenchyme [33]. DeRuiter has evidenced that embryonic endothelial cells may transdifferentiate into vascular SMC. The different origin of SMC could also explain their heterogeneity in coarctation. However as the site of aortic coarctation is near the end of the ductus arteriosus, we cannot exclude the hypothesis that some SMC are in fact persistent ductal cells.

The understanding of cellular mechanisms in aortic coarctation could have great implications in clinical practice. Balloon dilatation of native aortic coarctation will be followed by persistence of intimal thickening that could lead to recurrence. Incomplete surgical excision of areas with intimal thickening in coarcted aorta could explain the rate of restenosis reported in subclavian flap aortoplasty [15].

Although the mechanisms underlying intimal thickening in aortic coarctation are still unclear, our observations provide further evidence for a similarity between this process and the formation of ‘intimal cushions’ during closure of the ductus arteriosus.

Time for primary review 20 days.


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

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