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Correspondence to: Johan Thyberg, Dept. of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden.
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Summary |
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Earlier in vitro studies suggest opposing roles of laminin and fibronectin in regulation of differentiated properties of vascular smooth muscle cells. To find out if this may also be the case in vivo, we used immunoelectron microscopy to study the distribution of these proteins during formation of intimal thickening after arterial injury. In parallel, cell structure and content of smooth muscle -actin was analyzed. The results indicate that the cells in the normal media are in a contractile phenotype with abundant
-actin filaments and an incomplete basement membrane. Within 1 week after endothelial denudation, most cells in the innermost layer of the media convert into a synthetic phenotype, as judged by loss of actin filaments, construction of a large secretory apparatus, and destruction of the basement membrane. Some of these cells migrate through fenestrae in the internal elastic lamina and invade a fibronectin-rich network deposited on its luminal surface. Within another few weeks a thick neointima forms, newly produced matrix components replace the strands of fibronectin, and a basement membrane reappears. Simultaneously, the cells resume a contractile phenotype, recognized by disappearance of secretory organelles and restoration of
-actin filaments. These findings support the notion that laminin and other basement membrane components promote the expression of a differentiated smooth muscle phenotype, whereas fibronectin stimulates the cells to adopt a proliferative and secretory phenotype. (J Histochem Cytochem 45:837-846, 1997)
Key Words: arterial injury, neointima, smooth muscle cells, phenotype, actin, laminin, fibronectin, immunoelectron microscopy
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Introduction |
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Smooth muscle cells (SMCs) build up the arterial media and, together with macrophages and T-lymphocytes, are the main cell types that take part in the formation of atherosclerotic and restenotic lesions (
Earlier studies in our laboratory have indicated that a substrate of fibronectin promotes transition of arterial SMCs from a contractile to a synthetic phenotype during the first few days in primary culture, whereas substrates of the basement membrane proteins laminin and collagen Type IV hold the cells back in a contractile phenotype (
In a recent study, we used electron microscopic and stereological techniques to describe the formation of neointimal thickenings in the rat carotid artery after balloon injury. Within 1 week after removal of the endothelium, the cells in the innermost part of the media took on a synthetic phenotype, as revealed by loss of myofilaments and outgrowth of an extensive endoplasmic reticulum and a large Golgi complex. Later, some of these cells migrated into the intima via fine openings in the internal elastic lamina. Here, they began to divide and secrete ECM components, and within another week a thick neointima had formed. Subsequently, the majority of the cells eventually resumed a contractile phenotype with a cytoplasm largely occupied by myofilaments (
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Materials and Methods |
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Arterial Injury
Four- to 5-month-old male Sprague-Dawley rats were used (400-450 g). The left carotid artery was denuded of endo-thelium with a Fogarty 2F embolectomy catheter as previously described (
Electron Microscopy
After repeated rinsing in PBS, the tissue was dehydrated in graded ethanol (70, 95, and 100%) and embedded in LR White (London Resin Company; Reading, UK). For the latter purpose, the specimens were first incubated in a mixture of equal parts ethanol and LR White (v/v) for 30 min and then left in pure resin for 12-15 hr at 4C. After two additional incubations in LR White (30 min each), the carotid rings were put into gelatin capsules completely filled with resin and tightly capped. The capsules were placed in a UV polymerization unit (Agar Scientific; Stansted, UK) for 12-15 hr. Thin sections were cut with diamond knives on an LKB Ultrotome IV, picked up on nickel grids coated with a carbon-stabilized formvar film, stained as described below, and finally examined in a JEOL EM 100CX operated at 60 kV.
Antibodies
As primary antibodies, mouse anti-smooth muscle -actin (N-terminal synthetic decapeptide; no. 2547; Sigma Chemical, St Louis, MO), rabbit anti-laminin (Engelbreth-Holm-Swarm mouse sarcoma; Sigma no. 9393), and rabbit anti- fibronectin (human plasma; Sigma no. 3648) were used. These products react with human as well as animal tissues and show a high degree of antigen specificity. As secondary antibodies, goat anti-mouse IgG (Sigma no. 7652) and goat anti-rabbit IgG (Sigma no. 7402) conjugated to 10-nm colloidal gold particles were used. Control stainings without primary antibodies and with nonimmune IgG fractions were negative.
Immunostaining
The grids were rinsed in water, placed on a droplet of PBS with 0.25% bovine serum albumin (PBS/BSA) for 5 min to block unspecific binding sites, transferred to primary antibodies diluted in PBS/BSA, and incubated for 2-3 hr in a humid atmosphere. The grids were then rinsed repeatedly with PBS, placed on a droplet of PBS/BSA for 5 min, transferred to secondary antibodies diluted in PBS, and incubated for 1-2 hr. After renewed rinsing with PBS, the sections were postfixed with 2% glutaraldehyde in PBS and then rinsed again, first with PBS and then with water. Finally, contrast staining was made with aqueous uranyl acetate for 30 min followed by alkaline lead citrate for 30 sec.
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Results |
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Smooth Muscle -Actin as Phenotypic Marker During Neointima Formation
The fine structural characteristics of the vessels and the changes occurring after balloon injury were the same as described previously (-actin (Figure 1a). The majority of the SMCs in the media of the injured arteries likewise retained a differentiated phenotype as judged by these criteria (Figure 1b). However, 1 and 2 weeks after the operation, most of the SMCs in the inner layer of the media were modified and showed a reduced content of filaments positive for smooth muscle
-actin (Table 1; cf. Figure 5a). Such cells were also observed migrating from the media to the intima via small holes in the internal elastic lamina. In the 1- and 2-week-old neointima, a further modification of the SMCs was evident, with a prominent endoplasmic reticulum and Golgi complex, few filaments, and only a weak reactivity for smooth muscle
-actin (Figure 2). Five weeks after the operation, the secretory organelles had again become smaller and most of the cytoplasm was instead filled with filaments stained for smooth muscle
-actin (Figure 3; Table 1).
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Changes in Distribution of Laminin During Neointima Formation
In the media of both the control and the injured carotid arteries, an incomplete basement membrane with a positive staining for laminin surrounded the SMCs (Figure 4a and Figure 4b). On the other hand, no or only weak staining for laminin occurred around the phenotypically modified SMCs in the inner layer of the media and in the neointima 1 and 2 weeks after the operation (Figure 5a and Figure 5b). In connection with the redifferentiation of the SMCs in the neointima, laminin was again found both underneath the regenerating endothelial cells (recognized by their thin shape and the presence of Weibel-Palade bodies) and around the SMCs themselves (Figure 6a and Figure 6b). At this time, faint laminin staining was also seen in the endoplasmic reticulum and Golgi complex of the endothelial cells and SMCs.
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Changes in Distribution of Fibronectin During Neointima Formation
All layers of the control vessels (intima, media, and adventitia) showed only weak staining for fibronectin. In the injured vessels, a distinct reaction was obtained in the neointima and the most superficial parts of the media, close to the fenestrae in the internal elastic lamina. The other parts of the media and the adventitia remained weakly stained. One week after the operation, a thin layer of degranulated platelets typically covered the luminal surface of the vessels. Under this layer, a fine meshwork with conspicuous staining for fibronectin was found (Figure 7a). It was of variable overall dimensions and was laid down before SMCs had started to migrate into the intima. Accordingly, the first SMCs that appeared here were covered by fibronectin, whereas only little stainable material occurred between them and the internal elastic lamina. As the neointima grew in thickness, the platelets disappeared from the inner vessel surface and were replaced by SMCs that penetrated the fibronectin meshwork. The cells in the young neointima were thus surrounded by a loosely arranged matrix dominated by fibrillar strands with a positive reaction for fibronectin (Figure 7b). Two and 5 weeks after the operation, the neointima had grown further in size and was made up of multiple layers of SMCs encircled by a dense matrix of collagen fibrils and elastic fibers. At the same time, a successive reduction in the amount of fibronectin in the pericellular matrix was noted (Figure 8a and Figure 8b).
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Discussion |
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Immunoelectron microscopy was used to study the formation of a neointima in rat carotid arteries after endothelial denudation with a balloon catheter. Special interest was paid to the change in differentiated properties of the SMCs and the composition of their pericellular matrix. For this purpose, stainings were made with antibodies against the cytoskeletal marker protein smooth muscle -actin, the basement membrane protein laminin, and the plasma/ECM protein fibronectin. The results indicate that the SMCs in the normal arterial media are in a contractile phenotype with a cytoplasm filled with
-actin filaments. After 1 week, most of the SMCs in the inner medial layer had converted into a synthetic phenotype and showed a markedly reduced content of
-actin filaments as well as an enlarged endoplasmic reticulum and Golgi complex. Some of these cells were seen to migrate into the intima via holes in the internal elastic lamina, whereas cells in a contractile phenotype were never observed to do so. On the basis of these findings, we conclude that the cells that give rise to the neointima are derived from phenotypically modified SMCs in the inner part of the media. The observation that smooth muscle
-actin is lost before the SMCs migrate into the intima is in accordance with the proposal that an essential function of this actin isoform is to prevent cell motility (
After the passage through the internal elastic lamina, the modification of the SMCs was completed by an additional loss of -actin and a further growth in size of the secretory organelles. Hence, the proliferating and matrix-producing cells in the neointima were in a synthetic phenotype. In a similar manner, cultured SMCs typically show an inverse relation between the expression of
-actin and the rate of cell growth (
-actin filaments. Biochemical analyses have likewise revealed a reversible change in cytoskeletal profile during the formation of neointimal thickenings in the rat carotid artery (
Staining for laminin showed that the SMCs in the control arteries and most of the cells in the media of the injured arteries were enclosed by an incomplete basement membrane. In contrast, no distinct reaction was found around the phenotypically modified SMCs in the inner medial layer and the developing neointima of the latter vessels. However, the ability to produce laminin was retained and a basement membrane subsequently re-formed around the redifferentiating SMCs. These observations corroborate the notion that laminin and other basement membrane components promote the differentiation of vascular SMCs (
Only weak staining for fibronectin was detected in the media. However, earlier light microscopic studies have demonstrated that this protein is present in the rat arterial media and that an alternative form containing extra Domain A appears in the neointima after balloon injury (
The concentration gradient created in this way could be an important factor behind the observed change in SMC phenotype as well as the subsequent migration of cells into the intima. In support of this idea, it has been shown that freshly isolated SMCs adhere to a substrate of fibronectin and in a few days of serum-free culture convert from a contractile to a synthetic phenotype (
The SMCs of the growing neointima secrete ECM components and, with increasing time, an increasing fractional volume of the tissue is occupied by a collagen- and elastin-rich matrix (
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Acknowledgments |
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Supported by the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, the Loo and Hans Osterman Fund, and the Karolinska Institute.
Received for publication October 7, 1996; accepted January 29, 1997.
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