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.

Orientation response of arterial smooth muscle cells to mechanical stimulation.

Arterial smooth muscle cells from rabbit aortic media in primary culture and subculture were grown on hydrophilized and collagen-coated silicone membranes which were then subjected to cyclic and directional stretches and relaxations at a frequency of 60 times/min. The membranes were stretched with various amplitudes ranging from 2% to 20%. Smooth muscle cells on unstretched membranes in the same incubation chamber served as controls. In long-term experiments the stretching and relaxing of the membranes was continued for several days. While the smooth muscle cells grown on unstretched membranes remained in random orientation in all experiments, the cells which underwent mechanical stimulation showed a high degree of orientation. The angle of cell orientation varied in direct relation to the stretching amplitude and became steeper in correlation to the intensity of the mechanical stimulus. The angle of cell orientation was reversible, as preoriented cells changed their orientation when another stretching amplitude was applied. To study the role of cytoskeleton in the process of cell orientation, we examined the behaviour of the intracellular actin filament system. In short-term experiments the smooth muscle cells were exposed for 3 to 12 h to cyclic and directional stretches and relaxations with an amplitude of 10%. We observed a rearrangement of the intracellular actin filament system prior to the orientation of the whole cell bodies. The present study provides evidence that stretching the artery wall by blood pulsation may result in an orientation response of the intracellular actin cytoskeleton and in the orientation of the smooth muscle cells within the media of artery walls.

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.

Effect of PDGF-AB heterodimer on a corneal epithelial cell line.

Exposure of cells of a rabbit corneal epithelial cell line (SIRC) to platelet-derived growth factor-AB heterodimer (PDGF-AB) resulted in a rapid and transient elevation of cytosolic free calcium concentration with a maximum at 2 to 3 min after stimulation. The kinetics of the calcium response were dose-dependent, e.g., higher concentrations of PDGF-AB caused a faster rise in cytosolic free calcium concentration. Maximum response was achieved with 10 ng/ml PDGF; higher concentrations up to 100 ng/ml did not further enhance cytosolic free calcium concentration. The ED50 was calculated to be 5 ng/ml PDGF-AB. After complexing extracellular calcium, PDGF-AB still caused a significant rise in cytosolic free calcium concentration indicating a mobilization of calcium from intracellular stores. This rise, however, was less pronounced than in the presence of extracellular calcium. The elevation in cytosolic free calcium concentration was not accompanied by an increased mitotic or proliferative activity of the cells as checked by [3H]thymidine incorporation and counting of cell numbers after 3 days of continuous incubation with various concentrations of PDGF-AB or by alterations in cell size and cell volume. In contrast, alterations in cell shape with a remarkable amount of rounded and partially detached SIRC cells after addition of PDGF-AB were observed within 24 h. Moreover, PDGF-AB caused a reversible distortion of cytoskeletal components such as actin-containing microfilament bundles, microtubules, and vimentin filaments. The results suggest that PDGF-AB may act only as a competence factor for the stimulation of SIRC cells via modification of the intracellular calcium homeostasis.

Cytoskeletal reorganization in NIH 3T3 fibroblasts expressing the ras oncogene.

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts leads to a set point shift of cell volume regulation and causes an increase in cell volume by activation of Na+/H+ exchange and Na+, K+, 2Cl- cotransport. Since both ion transport systems are thought to be governed by the cytoskeleton, the aim of this study was to examine the alterations in growth characteristics and cytoskeletal organization due to the expression of the oncogene. The experiments were performed on NIH 3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct and expressing the oncogene after treatment with low serum medium and 1 mumol/l dexamethasone (+ras cells). Transfected cells not expressing the oncogene (-ras cells) and treated with low serum medium, but without the addition of dexamethasone, served as controls. The growth characteristics were examined and the cytoskeletal architecture was visualized by indirect immunofluorescence microscopy using specific antibodies and fluorescent dyes. Expression of the ras oncogene was accompanied by a significant and serum-independent increase in proliferative activity irrespective from the coating of the dishes with attachment factors (poly-L-lysine, collagen type I). Both, -ras and +ras cells, proliferated slower on substrates coated with poly-L-lysine than on tissue culture plastic or collagen type I. Expression of the ras oncogene also resulted in a significant increase in cell volume which was independent from the substrate. +ras Cells became more elongated, exhibited long cytoplasmic protrusions and tended to detach when compared with -ras cells. Examination of the cytoskeletal architecture in +ras and -ras cells revealed marked differences such as a depolymerization of the stress fiber network to strongly fluorescent "focals" as well as the absence of vinculin-containing attachment plaques (focal contacts), a disorganization of non-muscle myosin and of cell surface fibronectin in +ras cells. In addition, a retraction of microtubules and vimentin filaments to the perinuclear region was also observed in +ras cells. For comparison, NIH 3T3 fibroblasts which were not transfected with the ras oncogene (0ras cells) and which were also subjected to the experimental conditions described above (low serum medium +/- dexamethasone), did not exhibit the cytoskeletal alterations as observed for +ras cells. The results demonstrate that the expression of ras oncogene causes not only profound alterations in the proliferative activity, cell volume and cell morphology, but also a marked reorganization of cytoskeletal architecture, which may participate in the altered regulation of volume-regulatory ion transporters in the cell membrane.

Activation of Na+/H(+)-exchanger by transforming Ha-ras requires stimulated cellular calcium influx and is associated with rearrangement of the actin cytoskeleton.

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts (+ras cells) results in growth factor-independent proliferation and marked alteration of cytoskeletal architecture including breakdown of actin stress fiber network. Compared to identical cells not expressing the oncogene (-ras cells), +ras cells exhibit a more alkaline intracellular pH (pHi) and a larger cell volume (CV), both of which are important mitogenic elements. They are due to a set point shift for activation of the Na+/H(+)-exchanger. Moreover +ras cells respond to stimuli like 0.5% fetal calf serum or bradykinin with sustained oscillation of the cell membrane potential (PD) due to stimulated Ca2+ entry which triggers pulsatile release of calcium from internal stores and subsequent activation of calcium-sensitive K+ channels. 10 mumol/l bepridil inhibit oscillations of PD and protect +ras cells against actin stress fiber depolymerization. It is shown that bepridil blocks both cellular calcium entry as measured by Mn2+ quenching of fura-2 fluorescence and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene. Inhibition of the Na+/H(+)-exchange with 10 mumol/l HOE 694, on the other hand, does not significantly alter Ha-ras stimulated calcium entry or cytoskeletal rearrangement. In -ras cells ionomycin (0.1 mumol/l) leads to a transient increase in Cai. This effect is paralleled by a transient depolymerization of actin stress fiber network which cannot be inhibited by HOE 694. Disruption of the actin cytoskeleton in -ras cells by cytochalasin D does not alter steady state cell volume or Na+/ H(+)-exchange activity. However, stimulation of cytochalasin-treated -ras cells with bradykinin leads to cell swelling which can be blunted by HOE 694. The results show that both cytoskeletal rearrangement and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene require stimulated calcium influx and Cai oscillations. The depolymerization of the actin cytoskeleton is permissive for the Na+/ H(+)-exchanger to cause cell swelling upon stimulation with bradykinin.

Effects of calcium channel blockers on NIH 3T3 fibroblasts expressing the Ha-ras oncogene.

NIH 3T3 fibroblasts expressing the ras oncogene (+ras cells) respond to bradykinin, bombesin or serum with sustained oscillations of cell membrane potential reflecting oscillations of intracellular calcium activity and subsequent activation of calcium-sensitive K+ channels. In contrast, identical cells not expressing the oncogene (-ras cells) respond to bradykinin with a single, transient hyperpolarization of the cell membrane. Furthermore, +ras cells are characterized by a serum-independent proliferation, an increase in cell volume and a marked reorganization of the cytoskeleton. It has been shown previously that the calcium channel blocker nifedipine, but not verapamil and diltiazem, inhibits oscillations of cell membrane potential as well as proliferation. In this study, we have examined the effect of several calcium channel blockers (bepridil, nifedipine, verapamil, diltiazem) on the proliferation, volume and cytoskeletal reorganization of +ras cells. Bepridil (10 mumol/l), which is also shown here to inhibit oscillations of cell membrane potential, and nifedipine (10 mumol/l) caused a decrease in cell number, whereas verapamil and diltiazem (10 mumol/l each) resulted in growth rates which did not differ from untreated +ras cells. The increase in cell volume as observed in untreated +ras cells was also observed for cells treated with verapamil and diltiazem, whereas cell volumes of +ras cells treated with bepridil and nifedipine were markedly reduced and similar to the values obtained for -ras cells. In addition, bepridil and nifedipine markedly inhibited cytoskeletal rearrangement, i.e depolymerization of actin-containing stress fibers. This inhibitory effect was not observed for verapamil and diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Aluminium phthalocyanines-induced photolysis of human vascular wall cells in culture and the effect of fluoride on photodynamic action.

BACKGROUND: Phthalocyanines, second-generation photosensitizers with several attractive properties for use in photodynamic therapy, have been shown to accumulate in malignant lesions and atherosclerotic plaques. After exposure of phthalocyanines-loaded tissues to visible light, the targeted cells become injured and eventually die. In vitro, when fluoride is added before exposure to the light, it can protect some cell types against photodynamic action sensitized by chloroaluminium phthalocyanine (AIPc) and its derivatives. METHODS: The effect of 50 mumol/l chloroaluminium phthalocyanine tetrasulfonate (AIPcS4) and 5 mumol/l AIPc, with and without the addition of 5 mumol/l NaF, on the viability of cultured human endothelial cells (HuEC), human smooth muscle cells (HuSMC), and skin fibroblasts (HuSF) was examined. A 150 W quartz halogen light bulb equipped with a cut-off filter (lambda > 605 nm) was used to activate the phthalocyanines. Cell viability was examined 24 h after irradiation by staining of the cells with fluorescein diacetate/ethidiumbromide and by counting the number of attached cells with a cell counter. RESULTS: In the case of AIPc, the viability of all cell types tested was reduced, in a dose-dependent manner, to about 50% of that of the corresponding controls for a maximum irradiation time of 600 s. When we used AIPcS4, both HuEC and HuSF were considerably less sensitive than HuSMC. The addition of fluoride to AIPc-loaded cells before exposure to light protected HuEC and HuSF, but not HuSMC. In the case of AIPcS4, with and without fluoride, only HuSMC were sensitive. CONCLUSIONS: The addition of fluoride to AIPc or the use of AIPcS4 without fluoride could be a valuable approach, selectively destroying HuSMC without affecting HuEC and HuSF, for the reduction of restenosis rates after recanalization of stenosed or occluded arteries. Moreover, because neither type of phthalocyanines causes cutaneous phototoxicity, these second-generation photosensitizers seem to be best suited to the photodynamic treatment of atherosclerosis.

Photodynamic therapy of vascular stenosis: results from cell culture studies on human endothelial cells.

BACKGROUND: Experimental results from human cell culture studies have shown that smooth muscle cells derived from human atherosclerotic plaques react more sensitively to photoactivated dihematoporphyrin-ester and -ether (DHE) than smooth muscle cells from human nonatherosclerotic arteries. A therapeutic concept of photodynamic therapy of vascular stenosis appears, therefore, to be promising. The prerequisite for an intravascular application is a relatively harmless application of the photosensitizing agent to the luminal lining of endothelial cells. METHODS: The effect of DHE with and without photoactivation was examined on endothelial cell cultures from human saphenous veins. The cellular uptake of DHE in relation to the serum content of the culture medium was evaluated, as well as its effect on proliferative activity, cell size, cell volume, and cellular viability. RESULTS: Intracellular uptake of DHE decreased significantly when higher serum concentrations were present in the culture medium. Incubation of cells with the photosensitizer for 9 days in the dark without light activation resulted in a significant decrease of endothelial cell proliferation only at concentrations higher than 2.5 micrograms/mL (= 2.5 mg/kg body weight for systemic application in vivo). Additional photoactivation caused no reduction of cell viability at DHE concentrations of 1 microgram/mL, but at 2.5 micrograms/mL and 5 micrograms/mL, a reduction of viable cells within 24 hours was observed in relation to the energy densities used for irradiation. CONCLUSIONS: Because smooth muscle cells from atherosclerotic plaques are, however, much more sensitive to photodynamic treatment, the concept of a photodynamic therapy of vascular stenosis may provide a good tool in the reduction of restenosis rates after recanalization of severely stenosed or even occluded arteries.

The in-vitro effect of antineoplastic agents on proliferative activity and cytoskeletal components of plaque-derived smooth-muscle cells from human coronary arteries.

BACKGROUND: Restenosis after successful percutaneous transluminal coronary angioplasty remains the major clinical problem limiting the long-term efficacy of the treatment. Recent advances in the understanding of the biology of restenosis indicate that its cause is predominantly a multifactorial stimulation of smooth-muscle cell proliferation. The aim of this study was to investigate the in-vitro effect of antineoplastic agents on smooth-muscle cells isolated from human coronary plaque material. METHODS: Atherosclerotic tissue from coronary arteries was extracted from 15 patients of both sexes by thrombendarterectomy. Cells were isolated using enzymatic disaggregation and identified to be smooth-muscle cells with fluorescent antibodies for smooth-muscle-specific alpha-actin. The antineoplastic agents cytarabine (500-0.005 micrograms/ml), doxorubicin (50-0.0005 micrograms/ml), and vincristine (10-0.0001 micrograms/ml) were added to the cultures. Six days after seeding, the cells were trypsinized and then counted. RESULTS: All three antineoplastic agents had a strong dose-dependent antiproliferative effect on cultured smooth-muscle cells. After the application of cytostatic agents, cells either became rounded or underwent complete lysis. Cytoskeletal elements, such as actin, microtubules, and vimentin, were largely altered. CONCLUSION: This investigation examined the potential role of antineoplastic therapy in the prevention of restenosis after coronary angioplasty. The development of new intravascular delivery systems, such as coated stents, may open the way for local antiproliferative strategies in interventional cardiology.

Corticosteroid agents inhibit proliferation of smooth muscle cells from human atherosclerotic arteries in vitro.

We studied the in vitro effect of steroid agents on smooth muscle cells from human atherosclerotic arteries. Recent advances in the understanding of the biology of restenosis indicate that restenosis is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. The steroid agents prednisolone (0.0075-750 micrograms/ml), hydrocortisone (0.0125-1250 micrograms/ml), and dexamethasone (0.0004-40 micrograms/ml) were added to the cultures. Six days after seeding the cells were trypsinized and the cell number was measured by a cell counter. All three steroid agents exhibited a significant antiproliferative effect on smooth muscle cell proliferation. At high concentrations of hydrocortisone, cytoskeletal elements of smooth muscle cells such as actin, microtubules, and vimentin, were largely altered. Our data indicate that the proliferation of smooth muscle cells from human atherosclerotic arteries in vitro can be inhibited by steroid agents and thus may open the way for local post-angioplasty treatment strategies.

A prescreening system for potential antiproliferative agents: implications for local treatment strategies of postangioplasty restenosis.

BACKGROUND: Recent advances in the understanding of the biology of restenosis indicate that it is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. The aim of this study was to investigate the in vitro effect of five potential antiproliferative agents on smooth muscle cells from human atherosclerotic femoral arteries. METHODS AND RESULTS: Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. Dalteparin sodium (0.001-100 anti-Xa units/ml), cyclosporine A (0.005-500 micrograms/ml), colchicine (0.00004-4 pg/ml), etoposide (0.002-200 micrograms/ml), and doxorubicin (0.0005-50 micrograms/ml) were added to the cultures. Six days after seeding, cells were trypsinized and cell number was measured by a cell counter. All five agents tested exhibited a significant inhibition of smooth muscle cell proliferation (P < 0.001). After an incubation time of 48 h, the cytoskeletal components, alpha-actin, vimentin, and microtubules were investigated. At peak concentrations, all five tested agents except dalteparin sodium caused severe damage to the cytoskeleton. CONCLUSIONS: All five potential antiproliferative agents exhibited a significant inhibition of smooth muscle cell proliferation. The development of new intravascular delivery systems may open the way for local antiproliferative treatment strategies in interventional cardiology.

Characterization of marginal and Claudius' cells growing from cochlear explants in vitro.

Tissue specimens of stria vascularis together with spiral ligament were transferred from the guinea pig cochlea to tissue culture dishes. To characterize and identify cells growing out from the explants, indirect immunofluorescence microscopy was used. The expression of the intermediate-sized filaments vimentin and cytokeratin 18 in cells on the surface of tissue specimens and in cells growing out from the explants after different cultivation periods were compared. Basically, three types of cells grew from the explants during several days: marginal cells, Claudius' cells and fibroblast-like cells. In primary cultures of explants, growth of marginal cells was observed in 25% of the dishes. Their proliferative activity, estimated by the use of the BrdUrd-DNA antibody, started in the stria vascularis and continued across the attachment of Reissner's membrane down to the bottom of the cell culture dish. The newly-formed marginal cells expressed cytokeratin 18 in the same way that original marginal cells on the tissue specimen do. If the newly-formed marginal cells were in contact with fibroblast-like cells or were forming groups (domes) on the bottom, they expressed vimentin. In 3% of the dishes growth of Claudius' cells was observed. Proliferative activity of these cells was found at the point where the basilar membrane was attached to the spiral ligament. New Claudius' cells spread at the opposite side of an explant when compared with the location of new marginal cells. Original as well as newly-formed Claudius' cells contained cytokeratin 18. Fibroblast-like cells were commonly present in cultures and contained only vimentin.

Nephrotoxic effects of di-(2-ethylhexyl)-phthalate (DEHP) hydrolysis products on cultured kidney epithelial cells.

1. Di-(2-ethylhexyl)-phthalate (DEHP) possesses a great industrial value as a plasticizing agent and has become an ubiquitous environmental contaminant. In most species it is rapidly metabolized to mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (2-EHA). Evaluation of toxicity of DEHP and its primary metabolites has been focussed on reproductive toxicity and hepatocarcinogenic properties. The aim of this study was to determine the nephrotoxic potential of both DEHP metabolites by use of cultured kidney epithelial cells (Opossum kidney cells; OK cells). 2. For this purpose, OK cells were exposed for 3 days to MEHP and 2-EHA at concentrations ranging from 0.1 -500 micromol/L and the toxicity as well as the effects on migratory activity and intracellular cytoskeleton were studied by cell biological, morphological and morphometric methods. 3. When compared with corresponding controls, treatment of OK cells with MEHP and 2-EHA, respectively, showed marked differences in cell viability between both DEHP metabolites. MEHP caused a dose-dependent decrease in cell viability (ED50 = 25 micromol/L) accompanied by a moderate swelling of the cells at concentrations up to 25 micromol/L. MEHP concentrations higher than 25 micromol/L caused a dose-dependent shrinkage of the cells and the occurrence of a high amount of cell debris as a result of cell lysis. 2-EHA did not cause a reduced viability or an altered cell volume. The migratory activity of OK cells was not significantly influenced by both metabolites. Moreover, MEHP toxicity resulted in a largely reduced and altered organization of F-actin (stress fibers), but not of myosin, microtubules and vimentin. 4. The study indicates that cultured epithelial cells can be used as a prescreening system to assess the nephrotoxicity of hazardous substances such as DEHP. As demonstrated in this study, only MEHP, but not 2-EHA, has a marked nephrotoxic effect in vitro.

Sodium monochloroacetate causes cytotoxic effects, an increased lactate and pyruvate level and induces ultra structural and cytoskeletal alterations in cultured kidney and liver epithelial cells.

1. Monochloroacetic acid (MCAA) and its sodium salt, sodium monochloroacetate (SMCA) are widely used in chemical industries as intermediates in the synthesis of carboxymethylcellulose, phenoxyacetic acid, thioglycolic acid, glycine, indigoid dyes and others. Moreover, MCAA has been found as a by-product of the chlorination disinfection of drinking water and as an environmental contaminant of the atmosphere from the photodechlorination reactions of chlorinated hydrocarbons. Little is known about the mode of action of both compounds on the cellular level. From cases of accidental poisoning of man it is known that MCAA accumulates in liver and kidney. 2. In this study, the cytotoxicity of SMCA on cultured liver (Chang liver cells) and kidney epithelial cells of the proximal tubule (Opossum kidney cells) was investigated and its effect on metabolism, ultrastructure and organization of cytoskeleton was examined. 3. Independent from the growth state of the cells (proliferating or quiescent), the results clearly show that SMCA causes a dose-dependent decrease in cell viability after an exposure period of 24 h. In all experiments, proliferating cells were more sensitive than quiescent and confluent cells. Liver cells were less sensitive against SMCA treatment than kidney epithelial cells. In contrast to liver cells, kidney cells exhibited a dose-dependent decrease in cell volume. The decrease in cell viability was accompanied by an increase of lactate and pyruvate concentrations released into the culture medium. In the case of Opossum kidney cells, lactate and pyruvate levels increased 5 - 6-fold, whereas in the case of Chang liver cells the increase was approximately twofold. While the ultrastructure of liver cells remained unaltered after drug treatment, kidney cells exhibited cytoplasmic vacuolization, membraneous disruption and especially mitochondrial alterations. In accordance with the changes in the ultrastructure of Opossum cells, was the reorganization of cytoskeletal elements with an increased stress fiber network at the basolateral surface as well as a partial depolymerization of microtubules and vimentin filaments. A cytoskeletal reorganization was not observed for Chang liver cells after SMCA treatment. 4. The results demonstrate that SMCA causes a dose-dependent cytotoxicity which is accompanied by metabolic, mitochondrial and cytoskeletal alterations in the cells.

[Growth characteristics of bovine retinal pericytes in culture]. / Wachstumscharakteristika von bovinen retinalen Perizyten in Kultur.

The loss of retinal pericytes is one of the earliest changes in diabetic retinopathy. In order to study this phenomenon in vitro, an optimal isolation and cultivation system has to be established. Therefore, pericytes from bovine retinae were isolated enzymatically with 0.4% collagenase in phosphate-buffered saline and identical immunologically by positive staining with antibodies against smooth muscle alpha-actin. Routine cultivation of pericytes was performed by using DMEM supplemented with 10% fetal calf serum. Dependent on the in vitro age of cells, the effect of the following reagents on proliferative activity was determined: fetal calf serum, heparin, ECGF, ECGF+heparin, and glucose. Increasing serum concentrations stimulated the proliferation of pericytes, although the degree of stimulation was reduced with increasing in vitro age. Heparin inhibited the growth in a dose-dependent manner; the achieving 50% inhibition was extrapolated to be 25 micrograms/ml. ECGF increased pericyte proliferation significantly, with a maximum at 10 microliters/ml. In addition, ECGF reversed the inhibitory effect of heparin. Furthermore, all tested glucose concentrations (5.5-27.75 mmol/l) did not show any influence on growth rates of pericytes. The results demonstrate that routine cultivation of retinal pericytes is possible. Moreover, they indicate that enhanced blood glucose concentrations, as observed in diabetic patients, are not the only important factor in the loss of retinal pericytes.

[Ascorbic acid. Cytotoxic effect on cultivated bovine lens epithelium cells]. / Die Askorbinsäure. Zytotoxischer Effekt auf kultivierte bovine Linsenepithelzellen.

Ascorbic acid is one of the main components (1.16 mM/l) of the aqueous humor. The molarity of this molecule is 25 times higher than in the plasma of the cow, man or horse. Now the question arises as to which function ascorbic acid has in this extremely high concentration referring to the proliferation of the lens epithelial cells. Thus, the effect of ascorbic acid was investigated upon bovine lens epithelial cells (BLEC) in the range of 0-3 mM/l. These cells were cultivated under various culture conditions (serum-free, serum-containing, aqueous-humor-containing medium) and also incubated with such mitogens as retinal extract (RE), crude endothelial cell growth factor (cECGF), basic fibroblast growth factor (bFGF) or with calcium. In each culture condition 1 mM/l ascorbic acid caused remarkable inhibition of the proliferation of BLEC. Higher concentrations (> 1.5 mM/l) revealed cytotoxic effects. These effects were independent of small variations in the pH value caused by ascorbic acid. In addition, the effect of 2 mM/l ascorbic acid in combination with catalase in a concentration of 500 Um/ml and 1000 Um/ml, respectively, was investigated. It could be shown that catalase is capable of preventing the cytotoxic effect of ascorbic acid. These results show the inhibitory effect of ascorbic acid in its physiological concentration in the proliferation of BLEC.

[Effect of heparin on proliferation of cultivated bovine lens epithelial cells]. / Wirkung von Heparin auf die Proliferation kultivierter boviner Linsenepithelzellen.

The treatments proposed to date for the prevention of secondary cataract have shown limited efficacy or have not been satisfactory due to ocular toxicity. Since it has been demonstrated that heparin can inhibit the proliferative activity of smooth muscle cells and fibroblasts in vitro and in vivo, we examined the effect of heparin at concentrations ranging from 20 to 200 micrograms/ml on the proliferation of cultured bovine lens epithelial cells (BLEC) under various culture conditions: (1) serum-free medium (SFM); (2) SFM + aqueous humor 1:1; (3) SFM +1 and 10% fetal calf serum; (4) SFM +1% retinal extract; (5) SFM +50 micrograms/ml endothelial cell growth factor; (6) SFM +10 ng/ml epidermal growth factor; (7) SFM +10 ng/ml basic fibroblast growth factor. Heparin caused no cytotoxic effects in any of the experiments. With medium 2 and 3, heparin caused dose-dependent inhibition of cell proliferation at concentrations ranging from 10 to 50 micrograms/ml. Cells cultivated in medium 4-7 with the addition of 50 micrograms/ml heparin revealed increased proliferative activity when compared with the corresponding controls. The antiproliferative activity on BLEC in medium containing aqueous humor suggests that heparin is a valuable tool for the prevention of secondary cataract in vivo.

Response of cultured endothelial cells to mechanical stimulation.

Endothelial cells covering the luminal surface of vessels are exposed to at least two different mechanical forces: 1) fluid shear stress produced by the circulation of blood, and 2) periodic stretching and relaxing as a result of the diameter oscillations caused by blood pulsation. In this study we present an apparatus which was constructed to imitate the volume pulse with its typical incisura of the abdominal aorta. Using this apparatus, we exposed cultured endothelial cells to continuously produced cyclic and directional stretching and relaxation for three days. In all experiments cells remained attached and viable when subjected to mechanical stimulation. The vast majority of endothelial cells which underwent mechanical stimulation became elongated and oriented with their longer axis perpendicular to the direction of stretching (angle of cell orientation: alpha = 88.7 degrees +/- 12 degrees; means +/- SD), whereas cells on unstretched membranes had a cobblestone-like appearance and remained in random orientation. In the stretched cells, the factor of elongation was f = 6.8 +/- 1.3; means +/- SD; unstretched cells which exhibited a polygonal shape had a factor of elongation of f = 1.8 +/- 0.8; means +/- SD. In addition, the behavior of cytoskeletal components such as microfilaments and microtubules was examined in the process of cell orientation as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented in parallel alignment perpendicular to the stretch direction (angle of actin filament orientation: beta = 90.4 degrees +/- 9 degrees; means +/- SD). A distinct orientation of microtubules was not observed, although a noticeable number of microtubules was observed to be in parallel alignment. Furthermore, microtubules of cells which underwent mechanical stimulation exhibited a pronounced asymmetric intracellular distribution with strongly fluorescent cytoplasmic areas in which microtubules seemed to be accumulated. The results indicate that endothelial cell elongation and orientation in vitro can be induced by periodic stretching and relaxation comparable to the periodic oscillations of the vessel wall due to blood pulsation in vivo.