Localization and smooth muscle cell composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits.

Morphological techniques (histology and electron microscopy), as well as immunofluorescence assays, were applied to the study of the localization and smooth muscle cell (SMC) composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits during a 4.5-month period. Vascular segments from different arteries (carotid, coronary, and iliac arteries) or from the same vessel at different levels (aorta) of animals at days 7, 15, 30, 40, 60, 90, and 135 showed that the atherosclerotic lesion first became visible at the level of the aortic arch in 60-day-old WHHL animals. Histological examination of serial cryosections from this vascular region indicated that the vascular lesion arose from a cavity in the media layer, located anatomically at the level of the juncture of the ligamentum arteriosum with the aortic arch. This aortic arch cavity is formed during the postnatal closure of the ductus arteriosus and is characterized by the presence of a thickened intima, which was absent in the other vascular regions examined. Immunofluorescence comparison of normal and atherosclerotic tissues from the aortic arch cavity wall with the use of monoclonal antibodies specific for smooth muscle and nonmuscle myosin isoforms revealed the existence of distinct SMC populations. SMCs in the thickened intima showed a myosin isoform pattern peculiar to cells with a degree of maturation intermediate between the fully differentiated and the developing (fetal) aortic SMCs. By contrast, SMCs present in atherosclerotic lesions displayed a predominant fetal-type pattern of myosin isoform expression. The achievement of this myosin isoform content seems to be correlated with the accumulation of lipids in the intima. In the media subjacent to the intimal thickening or atherosclerotic lesion, SMCs primarily displayed an intermediate degree of maturation. In older WHHL animals and at this aortic level, the SMC composition of the atherosclerotic lesion did not change, whereas in the subjacent media, the cells of intermediate type almost disappeared. In the vascular regions in which the atherosclerotic lesion appeared at later stages, such as near the aortic bifurcation, the distribution of fetal and intermediate cell types in the atherosclerotic wall was similar to that taken at the aortic arch level. These results indicate that there is 1) a preferential anatomic site from which atherogenesis initiates in WHHL rabbits; 2) a time correlation between the accumulation of lipids in the wall and the phenotypic change of SMCs toward a poorly differentiated cell type; and 3) the tendency for SMCs to follow the same differentiation pattern in early atherosclerotic lesions, irrespective of the site and time at which they develop.

Aortic intimal thickening and myosin isoform expression in hyperthyroid rabbits.

The potential effect of thyroid hormones on the expression of cytoskeletal and cytocontractile proteins of vascular smooth muscle cells (SMCs) was examined by a panel of monoclonal antibodies and immunocytochemical procedures. L-Thyroxine was administered to adult New Zealand White rabbits for as long as 26 days, and the aortic SMC composition was studied at days 1, 2, 7, 15, and 26 from the beginning of hormonal treatment. A diffuse intimal thickening of the aorta became visible after 7 days of thyroxine administration. Histological and histochemical examination of intimal tissues from hyperthyroid rabbits revealed the presence of a homogeneous Sudan black-negative cell population. In immunofluorescence tests the intimal cells were found to be negative for antibodies specific for monocyte/macrophage or desmin and homogenously reactive (positive) for antibodies to vimentin and smooth muscle (SM) alpha-actin, thus indicating that cells present in the thickened intima were of the SM type. In addition, intimal SMCs from aortas of hyperthyroid rabbits showed a myosin isoform content similar to that found in normal developing aortic SM and in a specific medial SMC subpopulation of aortas from adult euthyroid animals. In the media underlying the intimal thickening, almost all the SMCs switched their myosin isoform expression toward the "immature" phenotype after 2 days of thyroxine treatment. When the level of thyroid hormones was reduced by propylthiouracil treatment, the medial SMC subpopulation with the immature myosin isoform content present in euthyroid rabbits completely disappeared. The study of DNA synthesis-related bromodeoxyuridine incorporation in aortas from hyperthyroid rabbits showed the presence of labeled nuclei in medial SMCs before the appearance of the intimal thickening as well as in the thickened intima and in the underlying media at days 7 and 15. These results are consistent with a specific role for thyroid hormones in inducing proliferation/migration of medial SMCs into the intima. Moreover, the switch in the expression of myosin isoforms induced by thyroid hormones appears to precede the accumulation of medial SMCs in the intima.

A nifedipine-sensitive smooth muscle cell population is present in the atherosclerotic rabbit aorta.

We evaluated the ability of the Ca2+ channel blocker nifedipine to influence the severity of atherosclerotic lesions and the pattern of aortic smooth muscle cell (SMC) differentiation in cholesterol-fed New Zealand White rabbits. The animals were fed a 1% cholesterol-enriched diet for 12 weeks. After 4 weeks of the diet, some rabbits were given nifedipine (20 mg b.i.d.) for another 8 weeks without discontinuation of the cholesterol-enriched diet (experiment 1). Another group of rabbits was treated with nifedipine from the beginning of the cholesterol-enriched diet for the entire 12 weeks (experiment 2). The severity of ahterosclerotic lesions was determined by computerized planimetry, and qualitative effects of nifedipine on SMCs were studied by monoclonal antibodies specific for smooth muscle and nonmuscle myosins. In the aortic media of normal rabbits, these antibodies can identify an SMC population with an "immature" type of myosin pattern; a marked increase in the number of these cells is observed during atherogenesis. In experiment 1, we observed a marked decrease of medial SMCs with the immature type of myosin pattern, without any significant reduction in atherosclerosis severity. In experiment 2, disappearance of the previously mentioned medial SMC population was accompanied by a dramatic slowing of intimal lesion development. These results indicate that nifedipine treatment is effective in reducing atherosclerotic lesions only when given from the beginning of a cholesterol-enriched diet. Delay of nifedipine administration until the fourth week of the cholesterol-enriched diet fails to halt progression of the disease. The observed antiatherosclerotic activity can be attributable to a direct effect of the drug on the medial SMC population, which increases during the course of experimental atherogenesis.

Troponin T- and troponin I-like proteins in bovine vascular smooth muscle.

We have tested the hypothesis whether proteins with biochemical and immunochemical properties similar to those of troponin T (TnT) and troponin I (TnI) are expressed in bovine vascular smooth muscle (SM). Three monoclonal anti-TnT antibodies (TT-1, TT-2, and RV-C2) specific for the two isoforms of TnT present in the bovine cardiac muscle and two monoclonal antibodies (TI-1 and TI-5) reacting with cardiac TnI were used in this study. Anti-TnT antibodies were found to be unreactive with 1) skeletal and nonmuscle isoforms of glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme that shares some structural homologies with skeletal TnT, and 2) calponin, a TnT-like calmodulin/tropomyosin binding protein with some antigenic properties in common with TnT. When tested on SM extracts from aorta or coronary arteries by Western blotting, the anti-TnT antibodies were able to react exclusively with one or two polypeptides whose electrophoretic mobility corresponds to the cardiac TnT subunits. Similarly, anti-TnI antibodies specifically recognized a component in the aortic or coronary SM extracts with electrophoretic properties identical to the cardiac TnI. Immunofluorescence analysis performed on the vascular SM cells of bovine aorta, coronary arteries, and intramural branches of coronary vessels confirmed the existence of cardiac troponin immunoreactivity in these tissues. In addition, differences in the distribution of cardiac TnT- and TnI-like proteins were evidenced in nonvascular and vascular SM cells. This study shows for the first time that polypeptides with some structural properties in common with cardiac TnT and TnI can be found in the vascular SM system.

Myosin isoform expression and smooth muscle cell heterogeneity in normal and atherosclerotic rabbit aorta.

Two monoclonal antimyosin antibodies, Western blotting experiments, and immunofluorescence procedures were used to investigate myosin isoform expression in normal and atherosclerotic aortas of adult rabbits. The SM-E7 antibody reacted with the two myosin heavy chain (MHC) isoforms of smooth muscle (SM) type (SM-MHC-1 and SM-MHC-2) expressed in the adult rabbit aorta. The NM-G2 antibody recognized an epitope shared by the nonmuscle (NM) myosin heavy chains (NM-MHC) present in fibroblasts, macrophages, lymphocytes, and platelets. Two smooth muscle cell (SMC) populations were identified in the medial layer of normal adult aorta, namely cells that contained SM myosin exclusively and cells that showed the coexistence of SM and NM myosin isoforms. The size of the cell population with double myosin isoform content increased markedly during experimental atherogenesis and represented by far the predominant SMC phenotype in the atherosclerotic plaque. Western blotting analysis performed on crude extracts from the atherosclerotic plaque showed the presence of SM-MHC-1 and NM-MHC isoforms in this tissue. Co-expression of SM and NM myosin at the molecular and the cellular level were found in aortic tissue during the early stages of development. These results indicate that in experimental atherosclerosis, the accumulation in the plaque of SMC with an "immature" pattern of myosin isoform expression is accompanied by similar modifications in the differentiation pattern of SMC of the underlying media.

Myosin isoforms and cell heterogeneity in vascular smooth muscle. I. Developing and adult bovine aorta.

Monoclonal anti-smooth muscle (SM-E7, SM-F11, and BF-48) and anti-nonmuscle (NM-A9 and NM-G2) myosin antibodies, Western blotting, and immunocytochemical procedures were used to study myosin isoform composition and distribution in the smooth muscle (SM) cells of bovine aorta differentiating in vivo and in vitro. Two myosin heavy chain (MHC) isoforms were identified by SM-E7 in adult aorta: SM-MHC-1 (Mr = 205 kDa) and SM-MHC-2 (Mr = 200 kDa), respectively. When tested with the SM-F11 antibody, SM-MHC-2 isoform showed distinct antigenic properties compared to SM-MHC-1. Two bands of 205 and 200 kDa were also present in the aortic SM tissue from 3-month-old fetus and were equally recognized by the BF-48 antibody. The 200-kDa SM myosin isoform was labeled by SM-F11 but not by SM-E7, thus indicating the existence of a fetal-specific SM-MHC-2 isoform. At the cellular level, both developing and adult bovine aortic tissues showed the existence of distinct patterns of myosin isoform expression. Three or even more aortic cell populations are differently distributed in areas which appear as (1) a network of interconnecting sheet-like or compact tissue (early fetus) and (2) enriched of collagenous-elastic or muscular tissue (adult animal). In addition, the SM-MHC-2 isoform of the fetal type appears to be uniquely distributed in cultured SM cells grown in vitro from adult bovine aortic explants. Our data indicate that in bovine aorta (1) MHC isoform expression is developmentally regulated and (2) the distribution of myosin isoforms is heterogenous both among and within aortic cells. These findings may be related to the distinct physiological properties displayed by SM during vascular myogenesis.

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells.

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.

Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle.

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.

Myosin heavy-chain isoforms in human smooth muscle.

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody. The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.

Neonatal myosin heavy chains are not expressed in Ni-induced rat rhabdomyosarcoma.

Myosin heavy chain (MHC) composition of chemically-induced rhabdomyosarcoma (RMS) was analyzed by gel electrophoresis and Western blotting using a panel of monoclonal antimyosin antibodies specific for embryonic-, neonatal-, slow- and adult fast-type MHC isoforms. Myosin extracted from tumours and electrophoresed on 6%-sodium dodecyl sulfate (SDS)glycerol gels was found to migrate as three distinct MHC components. These polypeptides were present in different relative amounts in the five RMS studied. Western blotting experiments revealed that variable proportions of embryonic-, slow- and adult fast-, but not neonatal-type, MHC isoforms are consistently expressed in RMS. Indirect and double immunofluorescence procedures applied to cryosections of tumoral tissue showed that: (a) RMS cells were unreactive with antineonatal-type-MHC antibody, (b) the majority of neoplastic, desmin-positive, cells contained embryonic- as well as adult fast-type MHCs and (c) a minority of cells were labelled by anti-slow MHC antibody. The results of this study indicate that there is no obligatory sequence of MHC isoform expression in the molecular transition (emb----neo----adult) which occurs during rat skeletal myogenesis.