Expression of matrix Gla protein and osteonectin mRNA by human aortic smooth muscle cells.

BACKGROUND: Recent data indicate that matrix proteins such as matrix Gla protein (MGP) and osteonectin (ON) influence not only mineralization of vasculature but smooth muscle cell (SMC) differentiation. METHODS: We examined whether MGP and ON are expressed by human aortic SMCs in vivo using Northern blotting, in situ hybridization and immunohistochemistry. RESULTS: MGP and ON mRNAs were strongly expressed in the aorta without atherosclerosis from newborn and four young subjects up to 10 years old. In the aorta from 15 adult cases, MGP and ON mRNAs were decreased as atherosclerosis developed. We determined cell type and distribution of the MGP- and ON mRNA-expressing cells by in situ hybridization and immunohistochemistry. In the aorta obtained from newborn and young subjects, SMCs in the media and thin intima expressed MGP mRNA and, to a lesser extent, ON mRNA. In the adult aorta with fibrous thickening, MGP mRNA was expressed by intimal SMCs and subpopulation of medial SMCs. Osteonectin mRNA was expressed mainly by intimal SMCs and few medial SMCs. Double immunohistochemical staining revealed that both MGP- and ON protein-expressing cells were positive for anti-alpha-smooth muscle actin antibody, aortic SMCs. CONCLUSIONS: These results suggested that MGP and ON expression by aortic SMCs might be regulated by the degree of atherosclerosis and SMC differentiation in human aorta.     

Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis

Endothelin-1 is a potent vasoconstrictor and exhibits a mitogenic activity on vascular smooth muscle cells (SMCs). Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin-1 processing. We studied the immunolocalization of ECE in human coronary atherosclerotic lesions with different disease stages. Frozen sections of normal coronary arteries with diffuse intimal thickening (n=13) and those of coronary arteries with early (n=10) or advanced atherosclerotic plaques (n=13) were studied. Monoclonal antibodies used were directed against SMCs, macrophages, endothelial cells, and ECE. For the identification of cell types that express ECE, double immunostaining analysis was also used. In normal coronary arteries, ECE immunoreactivity was observed in luminal endothelial cells and medial SMCs. Early atherosclerotic plaques, which consisted predominantly of SMCs, showed enhanced ECE expression in luminal endothelial cells and intimal SMCs. In advanced atherosclerotic plaques, distinct ECE expression was found in accumulated macrophages and in endothelial cells of intraplaque microvessels, while luminal endothelial cells showed relatively weak immunoreactivity for ECE. In conclusion, the present study demonstrates that the major cell types expressing ECE within the plaques are different between early and advanced stages of human coronary atherosclerosis. Enhanced ECE expression and possible endothelin-1 generation may contribute to SMC proliferation and vasoconstriction in early atherosclerotic stages, and may promote plaque destabilization in advanced atherosclerotic stages.     

Subnormal shear stress-induced intimal thickening requires medial smooth muscle cell proliferation and migration

Arterial intimal thickening is consisted of predominately smooth muscle cells (SMC). The source of these SMCs and mechanisms response for their changes have not been well cleared. Using a model of rabbit common carotid artery (CCA) shear induced intimal thickening, we sought to identify and describe the source of SMCs in intima. The enlarged CCA 28 days after arteriovenous fistula (AVF) creation was subjected to subnormal wall shear stress (WSS) for 1, 3, and 7 days by closure of the AVF. To determine SMC proliferation, BrdU pulse labeling of SMCs was performed. BrdU-labeled SMCs were tracked over time to further confirm SMC migration. In response to subnormal WSS intimal thickening developed progressively. BrdU-labeled SMCs localized in the subendothelial area. When the BrdU-labeled medial SMCs were tracked 1 day after AVF closure, progenies of these BrdU-incorporated SMCs increased by 4.8-fold with 75% of them in the intima. They were 12-fold increased with 83% in the intima 7 days after. En face examination showed an accumulation of SMCs in internal elastic lamina gap after AVF closure, which later migrated into subendothelial area. In situ hybridization revealed increased TGF-beta1 mRNA expression in intimal SMCs. This study demonstrates that the medial SMCs are the predominant cells in subnormal WSS-induced intimal thickening. Early expression of TGF-beta1 may play an important role in the process of intimal thickening.     

Clonal architecture of normal and atherosclerotic aorta: implications for atherogenesis and vascular development.

X chromosome inactivation studies indicate that smooth muscle cells in human atherosclerosis are monoclonal. Monoclonality could arise either by 1) proliferation of a single cell in the adult intima, eg, by selection or mutation, or 2) proliferation of many cells within a large, pre-existing clonal patch that formed during development. To determine whether clonal expansion occurs concomitantly with plaque growth or as part of normal development, X chromosome inactivation patterns were mapped in microdissected samples of aortic smooth muscle, using the human androgen receptor locus as a marker. As expected, 43% of plaque samples were skewed toward one X chromosome, indicating a monoclonal population. Surprisingly, 25% of normal medial samples and 31% of diffuse intimal thickening samples also were skewed toward one X chromosome, indicating a relatively large patch size. Furthermore, 30% of diffuse intimal thickening and 22% of medial samples showed contiguous regions of 4 mm skewed to the same allele, showing that patch length often exceeded 4 mm. Intima and overlying media typically were skewed to the same allele (73% concordance), suggesting common cells of origin. Because patch size is large in normal arteries, X-inactivation analysis cannot discriminate between a monoclonal and a polyclonal origin of plaque smooth muscle cells. We propose that human arteries grow by expanding coherent smooth muscle clones, with little mixing of adjacent clones. Determining whether plaques arise by clonal expansion will require other approaches, such as analysis of somatic mutations; the finding of large X-inactivation patches raises the possibility that plaques arise from a pre-existing (developmental) clone.     

Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody.

To elucidate the deposition of advanced glycation end products (AGEs) in aortic atherosclerosis, aortic walls were obtained from 25 autopsy cases and examined immunohistochemically and immunoelectron microscopically with a monoclonal antibody specific for AGEs, 6D12. Among the autopsy cases, atherosclerotic lesions were found in the aortas of 22 cases and were composed of diffuse intimal thickening, fatty streaks, atherosclerotic plaques, and/or complicated lesions. In these cases, intracellular AGE accumulation was demonstrated in the intimal lesions of aortic atherosclerosis in 12 cases. Compared with the diffuse intimal thickening, intracellular AGE accumulation was marked in the fatty streaks and atherosclerotic plaques. Immunohistochemical double staining with 6D12 and monoclonal antibodies for macrophages or muscle actin or a polyclonal antibody for scavenger receptors demonstrated that the AGE accumulation in macrophages or their related foam cells was marked in the diffuse intimal thickening and fatty streak lesions and that almost all macrophages and macrophage-derived foam cells possessed scavenger receptors. Immunoelectron microscopic observation revealed the localization of 6D12-positive reaction in lysosomal lipid vacuoles or electron-dense granules of the foam cells. These results indicate that AGE accumulation occurs in macrophages, smooth muscle cells, and their related foam cells.     

Phenotypic features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening. Biochemical and morphologic studies

Balloon catheter denudation of rat carotid artery that results in significant medial damage is followed by marked intimal smooth muscle cell (SMC) proliferation associated with limited endothelial regrowth. In this report we demonstrate that: (a) SMC of the carotid media, preceding their intimal proliferation, develop a cytoskeletal profile and morphology consistent with a de-differentiated SMC phenotype; and (b) both medial and intimal SMC subsequently revert to a cytoskeletal profile and morphology reflecting incomplete but significant re-differentiation toward normal SMC phenotype. Specifically, early after balloon injury, SMC of the media and those that have migrated into the intima contain decreased amounts of actin, desmin, and tropomyosin and increased amounts of vimentin; moreover, beta-actin becomes the dominant actin isoform, whereas alpha-actin decreases as compared with that found in normal medial SMC. Late after balloon injury, actin is still less abundant, however, desmin, tropomyosin, and vimentin return toward normal values and both medial and intimal SMC again show a predominance of alpha-actin, although the endothelium does not regenerate over the central surface of intimal thickening in this model. The SMC surface to volume ratio significantly decreases early after balloon injury, whereas it is not significantly different late after balloon injury as compared with that of SMC of the normal carotid media. We demonstrate, furthermore that: (c) adjacent luminal SMC are interconnected by gap junctions and develop focal tight junctions, a feature not reported previously to occur in smooth muscle; these cells however do not form any well defined membrane specialization with the leading edge of endothelium, supporting the view that presence of modified SMC on the luminal surface of chronically denuded vessels is not responsible for the cessation of endothelial regrowth.     

Ultrastructural evidence for collagen degradation in the walls of varicose veins

In the myointimal thickening of human varicose veins morphologically distinct collagen fibrils within the cytoplasm of intimal smooth muscle cells (SMC) were observed, which could be interpreted as a possible image of collagen phagocytosis. Human varicose veins were obtained surgically from five patients and studied both histologically and electronmicroscopically. A myointimal thickening of a different degree was established in all cases examined. In three cases a large number of intimal SMC displayed intracellular membrane-bound collagen-like fibrils. The fibrils were single or packed together in small groups, showed the typical periodicity of collagen, and were surrounded by an ill-defined homogeneous “matrix” material. Within other SMC cytoplasmic vacuoles were found, including collagen fibrils in a more advanced stage of lysosomal decomposition. Characteristically the collagen-containing intimal SMC were localized predominantly in close proximity to newly formed capillaries of the thickened intima, where a relatively great quantity of bloodborne monocytes was also observed. It is suggested that vascular SMC are capable of phagocytosis and decomposition of collagen. Thus they may be part of the cellular basis for collagen breakdown in the remodeling of the blood vessel wall.     

Apoptosis participates in cellularity regulation during rat aortic intimal thickening.

Intimal thickening induced after endothelial denudation of rat aorta is though to be due to migration and proliferation of smooth muscle cells (SMC). When the reendothelialization is achieved, intimal thickening shows an important decrease in cellularity. Using in situ end labeling of fragmented DNA and electron microscopy, we show that this remodeling is accompanied by apoptosis of SMC. The number of apoptotic SMC becomes important 15 days after endothelial injury and reaches a maximum at 20 days; at 45 days the intimal thickening is reendothelialized and no more apoptotic SMC are detected. Apoptotic SMC show nuclear and cytoplasmic condensation as well as cytoplasmic vacuolization. Our results indicate that apoptosis is an important mechanism in the regulation of intimal thickening evolution.     

Structural Changes Within the Media of Coronary Arteries Related to Intimal Thickening

From observations on 454 coronary arteries from subjects ranging in age from prematurely newborn to 90 years, six structural patterns of medial smooth muscle are interpreted as representing, or related to, the proliferation and/or migration of medial smooth muscle into intima. By measuring the extent of inner medial circumference occupied by four of the six patterns, it was possible to calculate a numerical value designated the medial proliferatice and / or migratory activity (MP-MA) of each artery. During the first three decades, nonatherosclerotic diffuse intimal thickening was the characteristic intimal process, and during this phase of the arterial maturation span, the MP-MA of the arteries was significantly related to the degree of intimal thickening. Following a peak MP-MA level by the end of the third decade, there was a progressive decrease in the MP-MA level as the incidence and severity of atherosclerotic intimal thickening increased. At advanced stages of atherosclerotic intimal thickening, which were associated with thinning of adjacent media, intimal-medial structural patterns indicating a relationship between medial smooth muscle proliferative activity and the expanding atherosclerotic plaque were also apparent. The observations support the concept that the movement of medial smooth muscle into intima is a critical step preceding and during the evolution of the atherosclerotic plaque.     

Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions.

The authors have performed immunocytochemical investigations of the distribution of various cell types in human atherosclerotic plaques using monoclonal antibodies specific to smooth muscle cells (CGA7 [Gown et al, J Cell Biol 1985, 100:807-813] and HHF35 [Tsukada et al, Am J Pathol (In press)] ); lymphocytes (T200 antigen); endothelial cells (Factor VIII and the Ulex europeus agglutinin); and macrophages, the latter with a new macrophage-specific antibody HAM56. All studies were performed on methanol-Carnoy's-fixed, paraffin-embedded tissues. In areas of grossly normal aorta, significant numbers of macrophages were noted within areas of diffuse intimal thickening. The cellular composition of the following three types of raised lesions were analyzed: fibro-fatty lesions, which, despite their gross appearance, consistent with fibrous plaques, were composed almost exclusively of macrophages and lymphocytes and almost devoid of smooth muscle cells; fibrous plaques, which were predominantly composed of smooth muscle cells displaying considerable morphologic heterogeneity and an admixture of blood-borne cells; advanced plaques, which were characterized by complex layers of smooth muscle cells and macrophages with considerable variation from region to region. Also noted were foci of medial and even intimal vascularization subjacent to the more advanced plaques. These studies demonstrate the application of monoclonal antibody technology to the study of the cellular composition of human atherosclerotic lesions.     

Macrophage differentiation in atherosclerosis. An in situ immunohistochemical analysis in humans.

The differentiation of macrophages present in diffuse intimal thickening, fatty streaks, and atheromatous plaques, was analyzed with immunohistochemical methods, using segments of aorta, coronary, and carotid arteries obtained at autopsy. Various differentiation antigens were studied with the monoclonal antibodies anti-HLA-DR, EBM-11, Leu M3, OKM1, and OKM5. Adjacent sections were stained for lipids (oil red O) and lysosomal activity (acid phosphatase). Almost all macrophages identified with the pan-macrophage antibody EBM-11, also stained with the anti-HLA-DR antibody. Diffuse intimal thickening showed a predominance of Leu M3+ cells; fatty streaks also showed OKM1+ and OKM5+ macrophages. Classical atheromatous plaques showed a gradual shift in phenotypic expression towards the center of the lesion. Cells in the superficial layers were positive only with Leu M3, deeper localized cells showed double expression of Leu M3 and OKM1 or double expression of OKM1 and OKM5. Cells that were localized adjacent to the atheromatous debris stained only with OKM5. The phenotypic changes occurred in parallel with an increase in both fat uptake and lysosomal activity of the macrophages. This shift in phenotypic expression suggests a process of differentiation and maturation of the macrophages involved. The results indicate that macrophages within the arterial intima are activated and mature towards cells that express receptors for adhesion proteins and complement during the development of atherosclerotic plaques. This may imply that the macrophages involved in lipid metabolism also have a potential to act as effector cells in a chronic inflammatory process, and thus, may contribute to the progression of an atherosclerotic plaque. Functional studies of macrophage subpopulations are needed to verify this hypothesis.     

A 90-kd surface antigen from a subpopulation of smooth muscle cells from human atherosclerotic lesions.

A monoclonal antibody, designated 10F3, that reacts with an antigen with a molecular weight of 90,000 daltons has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) originally isolated from fetal human aorta (fSMC). In adults the antigen is present on venous, arterial and capillary endothelial cells of heart, kidney, liver, spleen, intestine, skin, uterus, placenta, and arteries only, as shown by immunohistochemical investigation using the PAP technique. The antigen 10F3 is also present on the mesenchymal cells of human fetal tissues (7 and 18-week-old fetuses) and on SMC of 7-week-old fetal aorta, and a subpopulation of cells reacting with 10F3 antibody also has been found in atherosclerotic intima. Double staining using 10F3 antibody and muscle actin-specific monoclonal antibody HHF-35 showed that the antigen-positive cells are smooth muscle cells. In primary culture of adult SMC, antigen-positive cells were detected 2 days after seeding (about 90% positive in medial and intimal cultures). It is suggested that 10F3 is a mesenchymal antigen that, lost during differentiation by cells other than endothelium, but expressed again by the SMC involved in atherogenesis.     

Identification of macrophages and smooth muscle cells in human atherosclerosis using monoclonal antibodies

Sections of human atherosclerotic plaques were stained by the indirect immunoperoxidase technique using three rat anti-human monoclonal antibodies: YAML 501.4 (anti-'leukocyte-common' (T200) antigen; YTH 8.18 (antimacrophage cytoplasm); and YPC 1/3 . 12 (anti-smooth muscle cell). The cells of diffuse intimal thickening were almost all smooth muscle cells but there were rare subendothelial macrophages. Focal lesions, in contrast, contained numerous macrophages as well as smooth muscle cells. Macrophage 'foam cells' were particularly numerous in fatty streaks and in advanced fibro-fatty plaques, but were less conspicuous in focal fibro-elastic lesions. The results confirm that macrophages are an important component of atherosclerotic plaques and suggest that they may have a significant role in atherogenesis in man.     

Identification of macrophages and smooth muscle cells with monoclonal antibodies in the human atherosclerotic plaque

Summary Sections of human atherosclerotic plaques, obtained from 21 autopsy cases with various degrees of atherosclerosis, were stained with the indirect immunoperoxidase technique using specific monoclonal antibodies against macrophages and smooth muscle cells. Distinctive results were found in differing stages: Single blood monocytes were observed in diffuse intimal thickening and the foam cells seen in fatty streaks were mostly identified as mature tissue macrophages, while only very few blood monocytes were present. The spindle cells observed in fibroelastic plaques showed positive reactions to antibodies against desmin, which points to their derivation from smooth muscle cells, whereas only a few macrophage-derived foam cells were seen in these lesions. In the complicated lesions the majority of foam cells were macrophage-derived, but there was also a small number of foam cells positive to antibodies against desmin, suggesting a smooth muscle cell derivation. - Our results confirm that in human atherosclerotic plaques the majority of the foam cells are obviously macrophage-derived, which emphasizes the important role of macrophages in the morphogenesis of these lesions.Supported by Landesamt für Forschung Nordrhein-Westfalen     

Phenotypic variants of the smooth-muscle cells in an atheromatous plaque of the human aorta

The authors investigated the expression of cytoskeletal proteins and the ultrastructure of cells in normal intima and atheromatous plaque of human aorta. It has been established, using double immunofluorescent method and a set of antibodies that intimal smooth muscle cells /SMC/ of normal aorta express myosin, vimentin, alpha-actin and actin but not desmin. In seven out of 28 atherosclerotic plaques the cells contained desmin and all other SMC cytoskeletal proteins were found. These cells had the ultrastructural features of SMC, i.e. well-developed endoplasmic reticulum and Golgi apparatus. Besides, some cells in 13 atherosclerotic plaques proved to be myosin-, alpha-actin- and desmin-negative. The cells were stained with monoclonal antibodies specific to SMC but not with macrophage-specific antibody. Ultrastructurally, the cytoplasm of the cells was filled with rough endoplasmic reticulum and a developed Golgi complex, but a certain portion of the cells retained basal lamina and myofilament bundles. The peculiarities of cytoskeletal protein in expression and ultrastructure of cells in human aortic atherosclerotic plaques may be explained by a phenotypic modulation of vascular SMC.     

Atherosclerotic lesions in humans. In situ immunophenotypic analysis suggesting an immune mediated response

The immunophenotypical features of the cellular infiltrates in different types of human atherosclerotic lesions, including diffuse intimal thickening as a potential but controversial precursor lesion, have been examined using monoclonal antibodies. Special emphasis is put on monocytes/macrophages, lymphocytes, and their possible interactions. Immuno-double staining techniques have been employed to study these aspects. T lymphocytes and macrophages were detected in diffuse intimal thickening, fatty streaks, and atheromatous plaques. In some lesions a predominance of suppressor/cytotoxic lymphocytes was found, whereas in other lesions mixtures of T suppressor/cytotoxic cells and T helper/inducer cells were found in ratios varying from 1:1 to 4:1. A substantial number of T cells and macrophages was considered to be immunoactivated because of the expression of HLA-DR and, to a lesser extent, of I12 receptor molecules. The activation was particularly evident at sites of close cell-to-cell contact between monocytes/macrophages and lymphocytes. These observations suggest that a specific in situ immune mediated hypersensitivity reaction is associated with the development of atherosclerosis.     

Increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ in human atherosclerosis

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-α correlated with that of PPAR-γ in FS and AP. The PPAR-γ protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-γ protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-γ may be involved in the early stages of human atherosclerosis.     

Effect of renal and genetic hypertension and endotoxin injections on cultivated smooth muscle cells derived from different regions of the aorta

The proliferation capacity of cultured smooth muscle cells (SMC) from standardized explants of different aortic layers or regions was investigated. SMC outgrowth from explants was compared in renal and genetic hypertensive rats and in renal hypertensive and staphylolysine injected minipigs. SMC proliferation in the minipigs was always greater in the upper thoracic aorta than in the lower thoracic aorta. In the latter, the intima was more reactive than either of the medial layers. A significant increase in proliferation was observed in hypertensive minipigs in the intimal and inner medial layer of the upper and in the outer medial layer of the lower thoracic aorta. Staphylolysine injections led to a significant increase in proliferation of the intimal and outer medial layer of the upper thoracic aorta. Renal and genetic hypertensive rats showed significantly higher levels of SMC proliferation in the aortic arch and the upper thoracic aorta. In the lower thoracic aorta, only explants from genetic hypertensive rats showed significantly greater outgrowth. These data show that pathological SMC proliferation is dependent on the kind of stimulus as well as on the anatomical position of the SMC in the aortic wall.