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.

Cytological and ultrastructural study of the lymphoid-like and X cell populations in mouse bone marrow during the neonatal period.

During the recovery period following a sublethal irradiation, lymphoid-like cells accumulate in the bone marrow of the mouse. Among these cells, a peculiar element called 'X cell' has been previously described. Its morphological aspects are different from the precursors of the various hemopoietic lines. The 'X cell' could be an ancestral element normally repressed in the adult marrow. The aim of the present work is to determine whether this peculiar element can be detected in the bone marrow of young mice during the first month of life. The myelograms showed that on the day of birth, the marrow was almost exclusively composed of erythrocytic cells. During the postnatal period up to day 20, there was a progressive increase of granulocytic cells. The percentage of lymphoid-like cells increased from 5 per cent at birth to 28 per cent on day 20. The absolute number of these elements, calculated from the percentages obtained on the myelograms and from the number of nucleated cells per two femurs, increased from 20,000 at birth to 7,000,000 at day 30. The ultrastructural analysis demonstrated that the lymphoid-like population was composed of small lymphocytes, lymphoblasts and X cells, the proportions of which varied in relation to the age of the animals. X cells and lymphoblasts were found to be especially numerous around the 4th or 5th day, whereas the small lymphocytes were scarce.

Human vascular smooth muscle cells in culture: growth characteristics and protein pattern by use of serum-free media supplements.

This study demonstrates that cultivation of vascular smooth muscle cells from human artery wall is possible under completely serum-free conditions. The effects of attachment factors on cell spreading and cell proliferation are described in detail as well as routine cultivation methods under serum-free conditions (clone cultures, cell migration, subcultivation by use of an exogenous trypsin inhibitor, cryopreservation and readaptation of cells). After a careful adaptation period, only two (BMS and Ultroser G) of the four commercially available serum-free media supplements tested were used successfully for a routine cultivation of the smooth muscle cells over several passages. With both supplements cell proliferation rates were comparable with those obtained in medium containing 10% fetal calf serum. The addition of platelet-derived growth factor or transferrin to serum-free cultures had no growth-stimulating effect. The addition of endothelial cell growth factor isolated from bovine brain caused a significant increase in proliferative activity of cells cultivated with BMS, but not with Ultroser G. Moreover, we report that under the serum-free culture conditions described here, the gamma-actin content of the cells is largely reduced (51% +/- 13% (means +/- SD) for cells cultivated in Ultroser G, and 12% +/- 4% (means +/- SD) for cells cultivated in BMS) when compared with cells cultivated under serum-containing conditions (gamma-actin content = 100%). The alpha-actin content was observed to be unaltered. Even after a careful readaptation of serum-free cultured cells to serum conditions, the gamma-actin content remained reduced.

Intra- and interindividual variation in measurements of beta-carotene, retinol, and tocopherols in diet and plasma.

Twenty-four men were studied for 4 wk to estimate intra- and interindividual variation of retinol, beta-carotene, and tocopherols in diet and plasma. Ratios of intra- to interindividual variances ranged from 0.62 for beta-carotene in plasma to 6.02 for vitamin E in diet. Results indicate that 3-9 independent measurements may be required to distinguish reliably even large differences (approximately 1 SD) in these micronutrients; 28-86 independent measurements may be needed for small differences (approximately 0.25 SD). If uncontrolled, large ratios of intra- to interindividual variance can constitute an important source of error in studies of micronutrients.

Densitometric measurement of increased endothelial permeability in arteriosclerotic plaques and inhibition of permeability under the influence of two calcium antagonists.

A densitometric technique was established to investigate quantitative changes in endothelial permeability for horseradish peroxidase (HRP), mol. wt. 40,000 daltons, in rabbit carotid artery. Repeated weak electrical stimulations of rabbit carotid arterial walls with implanted electrodes lead to fibromuscular plaques mainly beneath the anode. It could be demonstrated that there exists a typical growth curve of the plaques dependent on the number of days of electrostimulation, with a fast proliferation rate of smooth muscle cells in the first 2 weeks of electrostimulation, and an increasing retardation of proliferation during the next 4 weeks. Endothelial permeability for HRP increases in close relation to the plaque development. Intravenous applications of single doses of the calcium entry blockers flunarizine or nimodipine are able to inhibit the increased permeability of the endothelial lining covering arteriosclerotic plaques. The intensity of the inhibitory action of these calcium antagonists correlates with the size of the arteriosclerotic plaques in inverse proportion, but nevertheless in large plaques an inhibitory effect is seen.

Recruitment and dynamics of leukocytes in the formation of arterial intimal thickening--a comparative study with normo- and hypercholesterolemic rabbits.

Leukocyte involvement in intimal thickening was investigated as a function of time and diet. Fibromuscular or foam cell-rich thickings were induced by electrical stimulation (ES) of carotid arteries in rabbits either on a normal or a high (1%) cholesterol diet. Under both dietary conditions granulocytes (predominantly neutrophils), monocytes and lymphocytes migrated through and accumulated beneath a continuous, yet structurally altered endothelium already after 1 day of ES. This preceded the occurrence of smooth muscle cells (SMCs) in the intima. Under normocholesterolemia, leukocyte attachment to the endothelium decreased with continued ES, which coincided with the re-establishment of a normal endothelial cell pattern. Neutrophils ceased to invade the stimulated intima and disappeared from the lesion after 14 days. The proportion of mononuclear leukocytes was also reduced in the thickened intima, finally amounting to 5.5 +/- 5.9% in the 4-week-old fibromuscular lesion where SMCs prevailed. Hypercholesterolemia did not affect neutrophil involvement in response to ES. However, it provoked lipid deposition first in macrophages, then in SMCs and resulted in elevated amounts of mononuclear leukocytes both within the foam cell-rich thickening and in association with the overlying endothelium. These data indicate adaptive behavior of leukocytic infiltration in the development of fibromuscular thickening, and a shift to a chronic inflammatory response under additional hypercholesterolemia.

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.

Target cells and thymus microenvironment in the pathogenesis of thymic lymphomas in C57BL/Ka mice.

In C57BL/Ka mice, the induction of thymic lymphomas either by inoculation of radiation leukemia virus (RadLV) or by a split dose irradiation requires complex cellular events: Target cells are found among the population of thymic subcapsular blast cells, or, alternatively, of marrow or spleen prothymocytes; Progression of target cells to lymphoma growth requires a multi-step process, which occurs only within thymic microenvironment; Target cells are rapidly induced as "preleukemic" cells; After inoculation of RadLV, the initial events occur when target cells are in close association with cells of a specialized component of thymic epithelium, i.e., the so-called "nurse cells"; The leukemogenic agents induce damages to the thymic microenvironment itself; Lymphoma prevention by marrow grafting after irradiation results from mechanisms still unknown which inhibit the progression of "preleukemic" cells to neoplastic growth.

Covered stents for prevention of restenosis. Experimental and clinical results with different stent designs.

RATIONALE AND OBJECTIVES: Metallic stents in small vessels go along with a significant risk of restenosis and reocclusion. Different models of stents and covering materials have been purported to prevent intraluminal neointimal proliferation by cover-based closure of the spaces in the wire mesh. METHODS: Tantalum stents covered with polyethylacrylate/polymethylmethacrylate (PEM) were implanted in the infrarenal aorta of six New Zealand white rabbits by aortotomy and compared with eight rabbits treated with uncovered tantalum stents. For deployment, covered and uncovered stents necessitated a 7-French (F) and 5-F sheath, respectively. In addition, nine human patients with arteriosclerotic lesions of the superficial femoral arteries (stenosis > 5 cm or total occlusion) were treated percutaneously with a Dacron-covered nitinol vascular stent via a 9-F sheath. Patients were followed for a mean of 13.5 months, and control angiography was performed after 6 months. RESULTS: Experimental placement of the tantalum Wiktor stent was feasible technically in all cases. Five of six stents covered with PEM were occluded 3 days after placement despite the intravenous use of heparin and aspirin. In the group with uncovered stents, no area of stenosis greater than 10% was observed. There was a neointimal layer of 89 +/- 68 microns around the stent wires. Stent placement was successful in all patients. In four patients, a hyperergic reaction occurred, resulting in noninfectious periarteriitis. This complication was treated successfully with nonsteroidal antiinflammatory drugs. The primary patency was 50%, and the secondary patency (after application of a second covered stent in two patients) was 63%. CONCLUSIONS: The uncovered stent induces little neointimal proliferation around the stent wires. The insertion of stents covered with PEM into the rabbit aorta was accompanied by a strong thrombotic reaction, despite sufficient anticoagulation. Dacron-covered nitinol stents showed a surprisingly high restenosis rate after 9 months of follow-up. Further research concerning the in vivo properties of new covering materials is mandatory before routine vascular clinical application.

The effect of pentoxifylline on endothelial permeability of rabbit carotid artery wall.

Weak electrical stimulation of the rabbit carotid artery wall with direct current impulses over a period of 45 min increased the endothelial permeability for horseradish peroxidase (HRP), molecular weight 40,000 Da, beneath the anode region of the electrodes. The stimuli caused a massive accumulation of the reaction products of peroxidase in the subendothelial space. With a microdensitometric technique, it was possible to quantify the amount of the peroxidase reaction products in the subendothelium, and to compare the amounts in the non-stimulated regions with those in the stimulated regions. Intravenous administration of pentoxifylline before stimulation inhibited the transendothelial transport of peroxidase. Inhibition was pronounced in the stimulated regions, whereas the uptake of HRP in the non-stimulated regions was only slightly lower than that in non-treated animals. It was demonstrated that the extent of inhibition was dose-dependent (3, 10 and 30 mg pentoxifylline/kg). Electron microscopic examination showed that the main route of permeation of HRP was through the interendothelial clefts. However, electrical stimulation also caused an increase in the vesicular uptake of HRP into endothelial cells. Both pathways seem to be influenced by pentoxifylline.

In vitro model of the inner parts of a vessel wall with cultured human vascular cells.

BACKGROUND: Transfilter culture systems with enzymatically isolated human vascular cells were established to imitate the morphologic situation of the inner parts of a vessel wall. METHODS: In transfilter cultures, only smooth muscle cells were seeded on one side of the filter, whereas in transfilter cocultures, smooth muscle cells were cultivated in the presence of confluent or nonconfluent (injured) endothelial cells on the other side of the filter. The filter mimics the porous internal elastic lamina. Fourteen days after seeding, cultures were fixed, embedded in Araldite (Serva, Heidelberg, Germany), and prepared for histologic examination (light microscopy, indirect immunofluorescence staining of von Willebrand factor- and alpha-smooth muscle actin-antigen). RESULTS: In transfilter cultures, smooth muscle cells migrated through the pores to the opposite side of the filter, replicated there, and formed fibromuscular proliferates (two to four layers of smooth muscle cells). The proliferation rates of the smooth muscle cells were similar on both sides of the filter and showed an optimum rate on day 4 in culture as determined by 5-bromo-2'-deoxyuridine labeling. By cocultivating a confluent endothelial cell layer on one side of the filter, migratory activity of smooth muscle cells was inhibited. However, when smooth muscle cells were cultivated together with proliferating endothelial cells (injured state), proliferation of smooth muscle cells was massively stimulated (up to 12 layers of smooth muscle cells). CONCLUSIONS: The results indicate that the confluency of the endothelial cells and their proliferation rate influence the migratory and proliferative behavior of smooth muscle cells. The transfilter system may be a suitable model for prescreening of potential antiproliferative and antiarteriosclerotic drugs.

Staff manuals for teaching patients.

In addition to outlining what to teach patients, the Staff Manual for Teaching Patients with Diabetes and the Staff Manual for Teaching Patients with Hypertension outline how patient and family learning can be promoted. A chapter specifically on the teaching-learning process is included in each manual with concrete examples relevant to the disease process being discussed. Each manual also includes a brief chapter on how to organize and set up patient education in a hospital setting using a team approach.

The CARDIA dietary history: development, implementation, and evaluation.

To meet the objectives for dietary assessment in the Coronary Artery Risk Development in Young Adults (CARDIA) prospective study, we developed a dietary history to provide accurate and reliable quantitative data on habitual individual nutrient intakes at baseline. The CARDIA dietary history was an interviewer-administered method that included a short questionnaire regarding general dietary practices followed by a comprehensive food frequency questionnaire about typical intake of foods using the previous month as a reference for recall. For each broad category of foods, participants were questioned in detail about specific foods only if they indicated that they consumed foods from that category. Follow-up questions for selected foods concerned serving size, frequency of consumption, and common additions to these foods. Provision was made for reporting foods not found in the food frequency list. The interview took approximately 45 minutes. Cue cards prompted responses and plastic food models assisted in estimating usual amounts consumed. A precoded format standardized coding for reported items and established the detail needed for recall during the interview. Baseline nutrient analyses from the CARDIA dietary history provided estimates that agreed reasonably well with expected caloric intake for body mass index according to the age- and sex-specific Recommended Dietary Allowances, but were higher than those reported from 24-hour recalls for comparable age, sex, and race groups in the second National Health and Nutrition Examination Survey. The CARDIA dietary history is a comprehensive assessment tool that can provide a dietitian with detailed information regarding habitual eating patterns and nutrient intakes among adults.

Current status of the Strecker stent.

Knitted flexible tantalum stents proved to be a valuable adjunct to percutaneous transluminal angiplasty (PTA) in the case of insufficient PTA results, and their use was established in the distal aorta, the iliac, the femoro-popliteal, the renal, and the coronary arteries. Recently, long arterial occlusions were defined as new indications for primary stenting; stent indications were further extended to the subclavian, the carotid, and the splanchnic arteries. Due to higher incidence of acute and late complications after stent treatment of small diameter arteries, patients have to be selected thoroughly. Newly designed drug-releasing stents tested in animal experiments promised to be suitable to diminish the incidence of late restenosis due to intinal hyperplasia, thus providing better long-term patency.

Spontaneous immunoglomerulonephritis in Wistar rats.

A strain of rats bred at the Nuclear Study Center of Mol (C.E.N.) develops a nephropathy characterized by granular deposition of immunoglobulin and complement in the renal glomeruli and tubules. We have attempted to specify the complement activation pathways (classical or alternate) in this disease by looking for their initial components (C1q, C3 and C4) in tissue lesions. The immunohistologic and histologic course of the disease has been followed in male and female rats. Immunohistologic results show that glomerular and tubular injury in CEN Wistar rats may be mediated by the classical pathway of complement activation. Histologic data demonstrate that the tubular, glomerular, and interstitial lesions appear in both sexes, but are more extensive and develop earlier in male rats. The lipopigments found in tubular cells suggest a lysosomal dysfunction which might contribute to the progress of the disease.

Early arteriosclerotic changes of the carotid artery wall induced by electrostimulation. A study by scanning and transmission electron microscopy.

Using the method described by Betz and Schlote, 1979, it is possible to produce local smooth muscle cell proliferation in the intima of arterial walls by electrostimulation. If local electrostimulation is combined with a high cholesterol diet, atheromatous plaques arise at the anodal region of the stimulation electrodes. This is achieved by implanting a cuff around the common carotid artery of rabbits. By means of the electrodes which are situated in the cuff, DC-impulses are applied. The advantages of the described method are the following: the site of alteration can be determined, the stimulation intensity can be exactly adjusted and an initially induced coarse endothelial injury can be avoided. In this study the very first morphological changes have been observed by scanning and transmission electron microscopy. After an initial enlargement of the subendothelial space a migration of myocytes into this space is seen followed by a transformation of smooth muscle cells into modified cells. They develop a more active metabolism, undergo mitosis and produce proliferations after 4 days of stimulation. The development of the plaques shows a striking agreement with the results of other authors, although the used methods are completely different. Therefore, it can be assumed that the walls of arteries respond to various types of alterations with an unspecific and uniform reaction. Concerning our results, at least, an endothelial desquamation is not a prerequisite for a proliferative reaction of arterial smooth muscle cells. It can be concluded that, at the beginning of the arteriosclerotic development, a functional event predominates, which depends in our experiments on the electrical charge applied to the artery wall at the anode. Because of its functional character, this triggering mechanism cannot be investigated by morphological methods alone.

Structural alterations in vascular endothelial tight junctions in the course of their gradual degradation in vitro.

Rabbit aorta explants in organ culture maintained their endothelium as a confluent cell layer for 1-6 days. Depending on culture time, interendothelial tight junctions underwent gradual morphological changes in their substructure, as seen in freeze-fracture replicas. The formation of a P-face associated groove and concurrent confluence of tight junction particles on E-faces after 24 hr in vitro was followed by a rarefaction of particles and shortening of tight junctional strands. By day 6 in vitro, almost all tight junctions have disappeared. We interpret these findings as signs of a degradation of tight junctions in vitro, involving three different substructural components: a component facing the protoplasm, tight junction particles and a component facing the extracellular space. The degradation was inhibited by culturing under increased ambient pressure (910 mmHg).

Enhanced endothelial permeability and invasion of leukocytes into the artery wall as initial events in experimental arteriosclerosis.

The temporal sequence of the very early events in arteriosclerosis, as induced by electrical stimulation in carotid arteries of rabbits, was examined by combined light microscopy and transmission electron microscopy. After one session (30 min) of DC impulses, the endothelial permeability to horse-radish peroxidase was increased mainly beneath the anode, judging from the massive accumulation of reaction products of peroxidase in the subendothelium. After a further stimulation period, some of the endothelial cells displayed alterations in pattern and size and heavy cytoplasmic deposition of silver salt. However, the endothelium was maintained as a continuous lining. During this initial phase, a considerable number of granulocytes and monocytes was found adhering to the endothelium of the stimulated region and also within the subendothelial space. The invasion of the leukocytes preceded the migration of smooth muscle cells from the media into the intima, a process which began after two days of the electrical stimulation schedule. These initial phases of plaque development may represent a special form of an inflammatory response.

Effect of etofibrate on the development and the regression of atheromas in a rabbit model of atherosclerosis.

The study presents the effect of 2-[2-(p-Chlorophenoxy)-2-methylproprionoxy]ethyl nicotinate (Etofibrate, Lipo-Merz) on progression and regression of experimentally induced atheromas. The atheromas (intimal proliferates) were produced in carotid arteries of rabbits fed for 4 weeks a diet containing 1% cholesterol. One carotid artery was stimulated transmurally with weak electrical pulses via chronically implanted electrodes during the 4 weeks in which the animals received the cholesterol-enriched food. The standardized stimulus induced an atheroma in the stimulated region of the artery in all animals. Quantitation was carried out by counting cell layers in serial sections of the stimulated artery region. Lipid accumulation was measured in oil red stained histological sections with a semiquantitative technique. Additionally serum cholesterol, serum triglycerides, and HDL-cholesterol were determined. Addition of 200 mg Etofibrate/kg body weight per day to the cholesterol-enriched food caused a significant inhibition of the proliferation of the smooth muscle cells in the intimal proliferate which consequently resulted in a less pronounced plaque. Stainable lipids were found in all controls and all Etofibrate-treated animals. HDL-cholesterol levels were higher in the treated animals than in the controls while the other lipid parameters were not influenced by Etofibrate. In another set of experiments two groups of rabbits were treated as described above. After the production of an atheroma (4 weeks) they were fed normal food. One of the groups received daily 200 mg Etofibrate/kg body weight, the other not. After the regression period of one year the arteries were excised and sectioned for histology and lipid levels were measured as above.(ABSTRACT TRUNCATED AT 250 WORDS)

Human endothelial cells are stimulated and vascular smooth muscle cells are inhibited in their proliferation and migration by heparins.

The actions of two natural heparins and a semi synthetic low molecular weight heparin with low anticoagulant activity have been studied on the migration and proliferation of human vascular endothelial and vascular smooth muscle cells in vitro. In a migration assay non irradiated confluent cultures of endothelial cells and smooth muscle cells were "wounded" with a sharp razor blade in such a way, that the migration of individual cells from the wound edge into a region void of cells could be measured. The maximum distance migrated (perpendicularly to the wound edge) three days after wounding, was taken as an index for migratory activity. All tested heparins reduced migration of vascular smooth muscle cells and enhanced migration of vascular endothelial cells in a concentration dependent manner. Cell proliferation was studied in clone culture. All heparins tested were found to inhibit smooth muscle cell growth. The number of clones, as well as the size of single clones, were smaller with increasing concentrations of natural and low molecular weight heparin. The endothelial cells, however, exhibited contrary responses; with increasing concentrations of heparin, the cloning efficiency and the cell number of individual endothelial cell clones increased. This opposite effects of natural heparins and low molecular weight heparin could be of importance in preventing secondary stenosing intimal proliferations after angioplasty, bypass operation and embolectomy or even atherogenesis. Since the low molecular weight heparin has only a low anticoagulatory activity, it may be more appropriate than natural heparins for long term therapy to prevent artery stenoses caused by intimal SMC proliferation.