E. Betz

Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty.

Smooth muscle cell (SMC) proliferation is known to be an important factor for the development of restenosis after percutaneous transluminal coronary angioplasty. To determine the time course of intimal and medial SMC proliferation and morphological changes after experimental angioplasty, an intimal atheroma was produced with repeated weak electrical stimulations in the right carotid artery of 45 male New Zealand White rabbits. Angioplasty was subsequently performed in 35 rabbits, and the proliferative responses were analyzed with histomorphological and immunohistological criteria at 3, 7, 14, 21, 28, and 42 days after intervention. A hemodynamic relevant stenosis after angioplasty was found in eight (23%) of 35 dilated arteries. In five rabbits the stenosis was due to a mural thrombus, and in three animals restenosis was caused by intimal SMC proliferation. In all dilated arteries the intimal wall thickness increased from 13 +/- 5 intimal cell layers (after electrical stimulation) to 33 +/- 14 cell layers during 28 days after angioplasty (p less than 0.05). Later than 4 weeks after angioplasty, no additional increase of intimal thickening occurred. Application of bromodeoxyuridine 18 and 12 hours before excision of the vessels allowed determination of the percent of cells undergoing DNA synthesis in the intima and media using monoclonal antibody against bromodeoxyuridine. SMCs were identified by alpha-actin staining. Immunohistological quantification of intimal SMC proliferation showed a maximum of cells undergoing DNA synthesis within the first 7 days after angioplasty (p less than 0.01). In contrast, medial proliferation of SMCs was delayed and showed a small but significant increase 21 days after dilatation (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Growth characteristics and cytoskeletal organization of cultured smooth muscle cells from human primary stenosing and restenosing lesions.

Growth characteristics of human plaque cells selectively extracted from advanced primary stenosing and fresh restenosing lesions by percutaneous transluminal atherectomy were studied in vitro. Cells were isolated either by explant technique or by enzymatic disaggregation, and they were identified as smooth muscle cells (SMC) by positive reaction with antibodies against alpha-smooth muscle actin. Endothelial cells were not found in the atherectomized tissue. The cells of primary stenosing tissue (ps-SMC) exhibited a significantly low growth rate (0.16 +/- 0.04 population doublings per day) in comparison to the cells of restenosing lesions (re-SMC, 0.64 +/- 0.15 population doublings per day). Furthermore, ps-SMC became senescent and remained quiescent after two passages, whereas re-SMC retained a high proliferative activity and became quiescent by passage 8 to 10. Both types of cells responded to increasing serum concentrations in a dose-dependent manner. Ps-SMC failed to respond to purified platelet-derived growth factor (PDGF) and a mitogen mixture isolated from bovine brain (ECGF), but their proliferative activity was increased by the addition of re-SMC-conditioned culture medium. Despite their high basic growth rate, the proliferative activity of re-SMC was significantly stimulated by PDGF and ECGF in a dose-dependent manner. PS-SMC and re-SMC populations consisted of two distinct subpopulations, which could be discriminated by cell size measurements and cell adhesion: 1) relatively small (cell diameter, 18.6 +/- 5 microns), low-adhesive, predominant cells, and 2) enlarged (cell diameter, 27.1 +/- 3 microns), high-adhesive, fibroblast-like cells with abundant microfilaments. Neither ps-SMC or re-SMC stained with antibodies against desmin, but did express vimentin. The organization patterns of vimentin and tubulin were unaltered in comparison to each other and to smooth muscle cells cultured from the media of nonatherosclerotic human arteries.

Orientation of Cultured Arterial Smooth Muscle Cells Growing on Cyclically Stretched Substrates

Arterial smooth muscle cells from rabbit aortic media were grown in first subcultures on hydrophilized and collagen-coated silicone membranes which were then subjected to directional cyclic stretches and relaxations at a frequency of 50 times/min. The membranes were stretched 2, 5 and 10% beyond their resting length. Cells on unstretched and stationary membranes in the same chamber served as controls. The cells which were stretched with an amplitude of 2% remained in random orientation after 14 days of continuously performed cyclic stretching. The cells which were stretched 5% for 12 days orientated at an angle of 61 +/- 9 degrees to the direction of stretching, while the cells which were stretched with an amplitude of 10% for 6 days orientated at an angle of 76 +/- 5 degrees. The cells on the stationary and unstretched membranes remained in random orientation. We were able to confirm that the angle of orientation is reversible, i.e. preorientated cells changed their orientation during application of another stretching amplitude. The results suggest that stretching of the artery wall by blood pulsation may be a factor influencing the orientation of smooth muscle cells within the media of the artery wall and of those smooth muscle cells which proliferate into the subendothelial space after mechanical injury of the endothelium or electrical stimulation of the artery wall. An apparatus is presented which produces cyclic and directional mechanical stimuli similar to those which may occur in the artery wall.

Inhibition of cellular proliferation after experimental balloon angioplasty by low-molecular-weight heparin.

Background The proliferative response induced by balloon angioplasty is known to be an important factor in the development of restenosis after successful coronary angioplasty. Methods and Results To study the effects of low-molecular-weight heparin (LMWH) on cellular proliferation after experimental balloon angioplasty, LMWH (3.9 kd, 400 anti-Xa units/kg/day) was given to 20 male New Zealand White rabbits. After an intimal fibromuscular plaque was induced by electrical stimulation in the right carotid artery, LMWH was applied during the 7 days after balloon dilatation. As the control group, 20 other rabbits underwent balloon angioplasty without application of LMWH. The vessels were excised 3, 7, 14, and 28 days after balloon treatment. During the final 18 hours before the rabbits were killed, bromodeoxyuridine was applied. Intimal wall thickness increased from 13±5 cell layers (preangioplasty control group) to 20±6 cell layers in the LMWH-treated group at 28 days (p < 0.05). In contrast, histological examination of control animals 28 days after angioplasty revealed a significant increase to 35±15 cell layers (p < 0.01). Immunohistological quantification showed a significant increase (p < 0.001) of cells undergoing DNA synthesis at 3 (10.2±4.2%) and 7 (7.7±4.8%) days after balloon dilatation in control animals. In contrast, at 3 and 7 days after balloon treatment, the percentage of cells undergoing DNA synthesis in LMWH-treated rabbits was lower (3 days, 2.7±1.8%; 7 days, 1.9±0.3%) than the corresponding untreated controls but showed a significant increase (p < 0.01) compared with the preangioplasty controls. The differences between the two groups were statistically significant, however (3 days, p < 0.01; 7 days, p < 0.05). As early as 14 days after angioplasty, the extent of cellular proliferation was normalized and was comparable to the preintervention levels in both groups. Conclusions Our data indicate that the proliferative response after balloon angioplasty can be reduced in vivo by early treatment with LMWH and thus encourage further clinical investigations.

Cell constitution and characteristics of human atherosclerotic plaques selectively removed by percutaneous atherectomy

The Simpson atherectomy device used for the recanalization of severely stenosed peripheral arteries is able to collect plaque material which can be further characterized. This study reports histological, immunohistochemical and transmission electron microscopic findings on advanced human primary atherosclerotic plaques of peripheral arteries percutaneously removed by a Simpson atherectomy catheter. Material from stenosing plaques consisted of dense connective tissue with abundant amounts of concentrically arranged elastic fibers and lamellae. This meshwork contained numerous cells, often arranged in clusters and oriented with their longer axis parallel to the direction of blood flow. The vast majority of these cells could be easily identified as vimentin-positive and desmin-negative smooth muscle cells containing lipid deposits in the perinuclear region and numerous glycogen particles. Monocytes/macrophages were observed only very infrequently. Plaque tissue contained a range of smooth muscle cell phenotypes. Most of the cells were of an intermediate phenotype, i.e. sparsely filled with myofilament bundles at the cell periphery and a high amount of organelles such as mitochondria, rough endoplasmic reticulum and Golgi cisterns. An intact lining of pieces of intimal tissue with endothelial cells was not observed. Two-dimensional gel electrophoresis of plaque tissue showed the presence of alpha-, beta- and gamma-actin isoforms with a clear predominance of the beta-isoform.

Inhibition of smooth muscle cell proliferation and endothelial permeability with flunarizine in vitro and in experimental atheromas

SummaryRepeated weak electrical stimulations of rabbit carotid artery walls with implanted electrodes cause intimal proliferations of smooth muscle cells (SMC) and lead to fribromuscular plaques beneath the anode. If the animals receive a cholesterol-enriched diet the plaques become typical atheromas. The endothelial lining is maintained. The procedure for the production of an atheroma with 11 ± 4 layers of SMC lasts 4 weeks. Addition of the calcium antagonist Flunarizine to the food during the stimulation periods inhibits the growth of the plaque. The inhibition is dose-dependent.Whether the drug inhibits atherogenesis by direct action on SMC or via an effect on permeation of macromolecules through the endothelium has been studied by measurement of (1) peroxidase (MW 40,000 dalton) permeability through the stimulated endothelium of the artery and (2) the inhibitory effects of Flunarizine on cultures of arterial SMC.Endothelial permeability increases for some hours after stimulation mainly beneath the anode. Flunarizine inhibits the permeation of peroxidase through the endothelial lining for the most part by its action on intercellular transport. The drug also inhibits the growth of SMC in mass cultures and clone cultures. The inhibition of proliferation is not specific for SMC. Skin fibroblasts obtained from the same animals as the artery smooth muscle cells are also inhibited in mass cultures and clone cultures.From the results it can be concluded that Flunarizine exerts its inhibitory action not only by its effect on the permeation through the endothelial lining but by a combined action on permeability and proliferation of cells in the artery wall.

Interaction of H+ and Ca++ in the regulation of local pial vascular resistance.

SummaryMock CSF of various compositions was injected into the perivascular space of different small pial arteries by means of micropipettes. Acid CSF (pH 6.5) caused local vascular dilatation, as did normal CSF with the addition of EDTA or EGTA. Systemic acidosis caused by CO2 respiration also caused pial vasodilatation. Tiny silver-chloride electrodes were placed directly on the surface of small pial arterioles. Current-constant stimulation with series of DC-impulses (2–5 mA strength, duration 1 ms/imp.; stimulation lasted 10 sec) caused local vasoconstriction of the stimulated vessel. The constricted region of the vessel was of a different shape. Most frequently spindle-shaped constrictions with an extention not exceeding the length of the indifferent electrode were seen. In some cases the constriction was unilaterally placed near the different electrode (100 μm ∅) and in a few cases the constriction was propagated along the whole vessel.Stimulation after application of acid CSF or during CO2-respiration caused a constrictory action of about the same degree as in normal CSF. The acidotic dilatation, however, remained visible because the resulting vascular diameter after stimulation remained greater than in the controls with normal CSF. In Ca++-free CSF the stimulation elicited a small response and during EGTA no constriction appeared after stimulation.In CSF with EDTA the constrictory response was distinctly diminished. From the experiments it cannot be excluded that the constriction of pial vessels caused by electrical stimulation is based on another mechanism than that caused by extravascular alkalosis. Ca++ is necessary for both mechanisms.

Responses of cultured smooth muscle cells from human nonatherosclerotic arteries and primary stenosing lesions after photoradiation: Implications for photodynamic therapy of vascular stenoses

Cultured smooth muscle cells from human nonatherosclerotic arteries and from primary stenosing lesions were labeled with dihematoporphyrinester and ether, a photosensitizing probe used mainly for the detection and photodynamic therapy of tumors. After labeling for 24 h, cells were irradiated with ultraviolet light (wavelength 365 nm, energy densities ranging from 30 to 1,200 mJ/cm2). Twenty-four hours after photoradiation, 80% of smooth muscle cells from nonatherosclerotic arteries and only 20% of smooth muscle cells from atherosclerotic plaques were viable and still adherent. Moreover, dynamic cell and cytoskeletal alterations in response to irradiation are described. The differential sensitivity of smooth muscle cells from nonatherosclerotic arteries and from atherosclerotic plaques provides evidence that a photodynamic treatment might be a valuable therapeutic approach to vascular stenosis.

Induction of Cell-Rich and Lipid-Rich Plaques in a Transfilter Coculture System with Human Vascular Cells

Cell-to-cell interactions are mainly involved in the control of the proliferation, migration, differentiation and function of different cell types in a wide range of tissues. In the arterial vessel wall, human arterial endothelial cells (haEC) and smooth muscle cells (haSMC) coexist in close contact with each other. In atherogenesis, haSMC can migrate from the media to the subintimal space to form fibromuscular and atheromatous plaques. In the present study, a transfilter coculture system is described, in which the interface between haSMC and confluent or proliferative haEC can be studied in detail. Cells were cocultured on the opposite sides of a porous filter which separates both cell types like the internal elastic lamina in vivo. In cocultures containing proliferative haEC, haSMC growth was significantly stimulated (33.4 +/- 5.7 cells/section, p < 0.05) compared to haSMC monocultures (22.9 +/- 2.5 cells/section) and cocultures containing confluent haEC (15.6 +/- 2.9 cells/section). If confluent haEC were injured mechanically, haSMC growth increased highly significantly (71.3 +/- 16.8 cells/section, p < 0.001). Thus, cell-rich proliferates containing 5-7 layers of haSMC embedded in extracellular matrix were formed after 14 days. On the other hand, after haSMC migration to the endothelial side had occurred, the addition of LDL and monocytes to cocultures with arterial media explants and haEC resulted in the formation of lipid-rich, low-cellular structures. After 28 days, characteristic in vitro plaque growth was induced; the plaque contained a lipid core with predominantly necrotic cells, extracellular lipid accumulations, atypically shaped lipid-loaded haSMC and macrophages, similar to in vivo foam cells, as well as an increased amount of extracellular matrix (collagen I, III and IV). These areas were surrounded by typical fibromuscular caps consisting of smooth muscle alpha-actin-positive haSMC. Finally, the formation of capillaries by haEC could also be observed within these structures.

Morphological changes and smooth muscle cell proliferation after experimental excimer laser treatment.

BackgroundLittle is known about the mechanism(s) in the development of restenosis after excimer laser angioplasty. Thus, the rationale of this study was to determine the time course of intimal and medial smooth muscle cell (SMC) proliferation and histomorphological changes after experimental excimer laser treatment. Methods and ResultsLaser ablation was performed in the right carotid artery of 34 New Zealand White rabbits after development of a fibromuscular plaque by repeated weak electrical stimulations. The vessels were excised 3, 7, 14, 21, 28, and 42 days after excimer laser treatment. Staining of a-actin was used to identify SMCs. In five rabbits (15%), a stenosis of more than 50% of luminal area was due to intimal proliferation of SMCs, and in four other rabbits, a total occlusion was due to organized thrombi. After the initial ablation of the preformed plaque (13±6 intimal SMC layers) a continuous increase of intimal wall thickness was found from 7 + 6 SMC layers at 7 days to 28±5 intimal SMC layers at 28 days after excimer laser ablation (p < 0.01). After 42 days, no additional increase of intimal thickening occurred. After bromodeoxyuridine labeling, the extent of cell proliferation (percent of cells undergoing DNA synthesis) in the intima and media was determined using a monoclonal antibody against bromodeoxyuridine. Immunohistological quantification of SMC proliferation in the intima revealed a significant increase of cells undergoing DNA synthesis at 3 (p <0.05) and 14 (p <0.01) days after laser treatment. Medial proliferation of SMCs was delayed and had a significant increase 7 days (p < 0.05) after intervention. Twenty-one days after laser treatment, SMC proliferation in the intima as well as in the media was normalized. ConclusionsThe proliferative response of SMCs after experimental excimer laser treatment will occur as a dynamic process with a maximum of SMCs undergoing DNA synthesis during 14 days after laser ablation, resulting in an increase of intimal thickening within 4 weeks after laser treatment. The extent of intimal hyperplasia due to SMC proliferation after excimer laser treatment is comparable with the effect of transluminal balloon angioplasty in this experimental model.