Vascular smooth muscle cells of male and female rats in culture, migration, proliferation and chromosome number

The authors evaluated the migratory and proliferative properties and the chromosome number of cultivated male and female smooth muscle cells (SMC) obtained by the explanation method from the thoracic aorta of rats of a conventional and a specific pathogen-free (SPF) breed. It was found that male SMC, in most cases, began to migrate from the explants sooner than female SMC and that they migrated from a higher total number of explants. The time needed for the number of cells in the culture to double (doubling time) was practically the same for male and female SMC, but male SMC attained a higher maximum population density. Male SMC cultures (2nd passage) contained cells with a hyperploid chromosome number, whereas female SMC were diploid. It was also found that SMC from conventional rats, in which the presence of pathogens could be presumed, displayed higher migratory and proliferative capacity than the SMC of SPF rats. The capacity of the SMC of male rats for migration and proliferation could have been potentiated by the effect of a different composition of the intercellular matrix and a different chromosome number, and in conventional rats by the presence of pathogens.     

Rat cerebral microvascular smooth muscle cells in culture

This report describes the development and establishment of long-term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 microns nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase-elastase-treated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2-3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered "hill and valley" pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle alpha-actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early (less than P 15)- and late (greater than P 15)-passage RV- and RA-SMC populations revealed markedly different cell growth responses. Representative growth curves of early- and late-passage RA-SMC showed a significantly higher growth rate (two- to fourfold) than RV-SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 micrograms/ml inhibited the growth of RV-SMC during the first 3 days after addition in both exponential and growth-arrested culture states, whereas RA-SMC cultures showed no inhibitory response. These studies indicate that long-term RV-SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.     

Gender-related differences in adhesion, growth and differentiation of vascular smooth muscle cells are enhanced in serum-deprived cultures

In 1-day cultures with 10% serum, the number of rat aortic smooth muscle cells (VSMC) adhering to the growth support was similar in cells from both sexes, whereas in 1% serum, the number of VSMC from male donors was lower. In 10% serum medium, the doubling time was significantly shorter and the number of [3H]thymidine-labelled nuclei was higher in cells of high passage from male rats. In serum-free medium, these differences increased and were also seen in cells of low passage number. Morphologically, the cells in male-derived cultures at higher passage number were mainly spindle-shaped, formed well-developed 'hills and valleys' and possessed longitudinally oriented bundles of alpha-actin-containing microfilaments. Most cells from female rats were flat, polygonal, the multilayered 'hills' were less prominent, with alpha-actin microfilaments forming a mesh-like network.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

Differential proliferation of rat aortic and mesenteric smooth muscle cells in culture.

Smooth muscle cells (SMC) from various arterial origins have been successfully maintained in culture. The present study evaluates the proliferative activity of aortic and mesenteric SMC in culture. Aortic and mesenteric SMC were obtained from male Wistar rats by explant and enzyme digestion techniques, respectively. Vascular SMC obtained by either method exhibited a characteristic hill-and-valley growth pattern in culture after confluence and were positively labelled with either anti-smooth muscle actin or myosin by an indirect immunofluorescent method. The rate of incorporation of thymidine into DNA and cell number counting were used as indices of proliferation in vitro. Vascular SMC from passages 4-33 were first synchronized with either Dullbecco's Modified Eagle's Medium (DME) or Ham's F-12 medium, supplemented with insulin-transferring-selenium (ITS), for 72 hours. SMC were then stimulated with 10% bovine serum for either 24 or 72 hours with the former processed for scintillation counting, the latter for cell number determination. The incorporation of tritiated thymidine into DNA following a 2 hour incubation was determined by scintillation counting after perchloric acid extraction. In terms of cell numbers, proliferative responses to bovine serum were determined by Coulter counting. Autoradiography was also carried out in some cultures to determine both thymidine and mitotic labelling indices. The rate of thymidine incorporation in aortic cells was 2-3 fold higher than in mesenteric cells. Aortic and mesenteric SMC lines exhibited similar cell cycle intervals in terms of total duration and individuals cycle parameters. However, the total thymidine index was higher in the aortic than mesenteric SMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

A sex-related difference in the hypertrophic versus hyperplastic response of vascular smooth muscle cells to repeated passaging in culture

Activation of growth of vascular smooth muscle cells (VSMC) in adults participates in pathogenesis of dysplastic diseases of the vascular system. In this study, we examined the impact of gender of rat donors on the degree of hyperplastic and hypertrophic responses of VSMC in cultures subjected to repeated passaging. The cells were derived from the outgrowth zone of explants of the thoracic aorta and were studied up to passage 45. Under these conditions, the cells undergo repeated growth stimulation by the serum growth factors mimicking some pathological situations in vivo. At lower passages (5-7), the cells from both sex donors did not differ significantly in their doubling time, maximum population density, protein content and ploidy. At higher passages (40-45), we found that the hyperplastic response, monitored by doubling time and BrdU-revealed DNA synthesis, was more intense in VSMC of male origin. In contrast, female-derived cells reacted by more prominent hypertrophic changes. The latter included a relatively higher increase in the volume and protein content of cells. As indicated by the DNA content histograms and chromosome numbers, these cells also showed a higher degree of passage-dependent polyploidization. In addition, the female-derived VSMC were found to be more effective in adhesion to the growth support evidenced by wider spreading and higher resistance of these cells to trypsin-mediated detachment as well as higher expression of some integrin and cytoskeletal molecules. These features could partly account for the slower proliferation and polyploidization of these cells. The results suggest that rat VSMC populations of male and female origin contain cells which are intrinsically different with respect to their capability of reacting to growth stimuli. The lower responsiveness of female-derived cells to growth stimuli may contribute to less frequent formation of hyperplastic vascular lesions in female organisms.     

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.     

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

Characteristics of human arterial smooth muscle cell cultures infected with cytomegalovirus

Summary Distinct, sequential events occurring during the destruction and simultaneous regrowth of human arterial smooth muscle cell (SMC) cultures infected with cytomegalovirus (CMV, AD169 strain) were characterized. The events were influenced by the typical phenotypic diversity reflecting relative states of differentiation of the SMC cultures. Progenitors of regeneration were a surviving population of small, undifferentiated or relatively undifferentiated SMCs. As these cells reached confluence focally, the number of cells reactive with antismooth muscle serum, i.e. differentiating, increased, and in some postconfluent foci the organization of SMCs resembled the topography of uninfected cultures. Thus, infected SMC cultures had a limited capacity to repopulate, to organize typically, and to differentiate. However, continuing cytopathic effects gradually destroyed much of the regrowth, and a relatively large, nondividing SMC with prominent cytoplasmic filaments, similar to SMCs in terminal, uninfected cultures, predominated. Infected cultures consisting overwhelmingly of the large terminal phenotype were far less productive of infectious CMV than cultures populated by SMCs with continuing capacity to divide. Gradually, cultures consisting of the terminal phenotype deteriorated as a result of sporadic cytopathic effects of CMV and an effect resembling “senescent” degeneration in uninfected, nondividing cultures in late passage. The infected, terminal phenotype could be a latent or persistent source of CMV antigen or nucleic acid-positive cells detected by different investigators in normal and in atheromatous, human tissue, assuming that it exists and survives for an extended period in vivo after infection of vascular SMC. The derivation of smooth muscle cell lines used in this investigation was supported through National Research and Demonstration Center grant HL-17269-07 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.     

Heparin-like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells

The effect of heparin on the binding of epidermal growth factor (EGF) to vascular smooth muscle cells (SMC) was examined. Heparin pretreatment of SMC obtained from bovine aortic explant tissue resulted in significant reductions in the amount of EGF bound. Decreases in mitogen binding were observed with both growth arrested as well as exponentially growing cultures. The heparin concentrations (10-100 micrograms/ml) and pretreatment times (48-72 h) necessary for suppression of EGF binding correlated with the concentrations and temporal requirements necessary for growth inhibition. Chondroitin sulfate, which has negligible antiproliferative activity, had no effect on EGF binding. However, a highly inhibitory heparan sulfate species obtained from postconfluent SMC suppressed EGF binding by 45%. Platelet-derived growth factor and insulin-like growth factor-1 binding were unaffected by heparin. Scatchard analysis revealed that heparin induced 50 to 60% reductions in the numbers of high and low affinity EGF receptors without detectable changes in the binding affinity or ratio of high to low receptors. Experiments were also performed with enzymatically dispersed SMC. These cultures were inhibited by heparin in a time dependent manner which was partially reversible in the presence of EGF. Subsequent studies revealed that heparin suppressed EGF binding in these cultures by 20 to 40%. In summary, heparin reduces the number of EGF receptors on both explant and enzyme dispersed SMC by a mechanism which closely parallels the antiproliferative effects of this glycosaminoglycan.     

Ontogeny of pituitary gonadotrophin secretion in the fetal and post-natal pig in response to LHRH in vitro

Monolayer cultures of anterior pituitary cells from male or female pigs of 60, 80, 105 days of fetal life or of 60, 160 and 250 days of post-natal life were prepared and treated with LHRH (1 pM to 10 nM). Dose-related increases of LH were first seen at 80 days of gestation in both sexes, while only female fetuses responded to maximal LHRH at 60 days. Basal and stimulated LH release doubled in cultures from 105-day-old fetuses when compared with those at 80 days. Compared to late fetal stages LH release was 20- to 30-fold higher in cell cultures from 60-day-old (post-natal) donors without further change during the post-natal period. In all pre- and post-natal age groups basal and maximal LH release of pituitary cells from males was lower than that of females. FSH stimulation started in male and female cells at 80 days of gestation only at LHRH concentrations exceeding or equal to 0.1 nM. By 105 days FSH secretion was dose-related and pituitary cells of females responded with higher FSH values than did those of males. In general, post-natal cells released much higher amounts of FSH than did prenatal cells. Basal and maximal release of FSH decreased during post-natal development in both sexes. Basal as well as maximal FSH release of cultures from female donors was higher than that found in cultures from male donors. Determination of total LH and FSH content in fetal pituitary cell cultures indicated that the developmental increase in gonadotrophin release potential is a function of the total gonadotrophin content in vitro. We conclude that (1) the in-vitro release of gonadotrophins to LHRH is dose-, age- and sex-dependent; (2) in the female fetal pig LH responsiveness develops earlier than FSH responsiveness; (3) apparently, these maturational changes mainly reflect alterations in pituitary gonadotrophin content; and (4) there is no simple relationship between in-vitro release and circulating gonadotrophins.     

Heparin regulates the collagen phenotype of vascular smooth muscle cells: induced synthesis of an Mr 60,000 collagen

The effect of heparin on the biosynthetic phenotype of rat vascular smooth muscle cells (SMC) was investigated in vitro. Addition of heparin to the culture medium of early passage rat SMC resulted in a marked (3-15-fold) increase of a cell layer-associated Mr 60,000 protein that was sensitive to digestion by purified bacterial collagenase and contained significant amounts of hydroxyproline. Pulse-chase analysis of heparin-treated SMC revealed that the Mr 60,000 collagen was a primary and abundant product of these cells and was not processed extracellularly to a smaller form. The inductive effect of heparin could be mimicked by iota carrageenan or dextran sulfates but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfates. The induction was concentration dependent with a maximal effect observed at a heparin concentration of 10 micrograms/ml. Synthesis of the Mr 60,000 collagen increased 18-24 h after addition of heparin to the cultures. Following induction and subsequent removal of heparin, synthesis of the protein remained maximal for at least 12 h and required 72 h to return to a basal level. These data demonstrate that the biosynthetic phenotype of vascular SMC in vitro can be controlled, at least in part, by heparin and related polyanions and suggest a role for similar molecules endogenous to the vessel wall in the regulation of SMC function.     

Prostaglandins E1 and E2 stimulate the proliferation of pulmonary artery smooth muscle cells.

Prostaglandins E1 and E2 are thought to be inhibitors of the growth of systemic vascular smooth muscle cells (SMC). However, their effect on the proliferation of SMC from the pulmonary artery (PA) has not been described and was the subject of this investigation. Cultures of bovine PA SMC were exposed to PGE1 and PGE2 under various conditions and their growth was assessed. PGE1 and PGE2 did not inhibit the growth of PA SMC in 10% fetal calf serum (FCS), but instead caused a dose dependent (10 ng - 1 microgram/ml) increase in [3H]-thymidine incorporation when added to cultures containing 0.5% FCS; the highest doses resulted in 95% and 75% increases in [3H]-thymidine uptake at 24 hours with PGE1 and PGE2 respectively. This was accompanied by a modest increase in actual cell numbers (e.g., 20% with 1 microgram/ml PGE1). Furthermore, PGE1 could mimic insulin-like growth factor (IGF-1) by potentiating the stimulation of SMC growth by fibroblast growth factor, suggesting that PGE1 may act as a progression factor in the growth cycle of these cells. There was, however, no effect of PGE1 on the proliferation of bovine aortic SMC. We conclude that, contrary to most reported effects on systemic SMC, PGE1 and PGE2 do not inhibit the proliferation of PA SMC but rather stimulate it.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Glycosaminoglycan production in cultures of early and late passage human endothelial cells: the influence of an anionic endothelial cell growth factor and the extracellular matrix

An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL less than or equal to 20) and late (CPDL greater than 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on a matrix from early passage endothelial cells than when grown on matrix from late passage cells. We conclude that EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; early passage cells contain more GAG but less heparan sulfate than late passage cells, extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered growth properties of normal human cells induced by phorbol 12,13-didecanoate

The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cells with 10(-7) or 10(-8) M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4 alpha-phorbol 12,13-didecanoate failed to elicit similar cellular responses.     

Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall

Summary Smooth muscle cells (SMC) were cultured from atherosclerotic plaques and uninvolved arteries to determine if differences exist between growth characteristics or ultrastructure of the cultured cells. Eighteen aortic punch biopsies provided the uninvolved tissue, and 58 carotid plaques provided the atherosclerotic tissue. Eighty percent of the sample yielded viable cultured cells, which reached a maximum population doubling time during log phase growth of 72 h (seeding density=1.0×10⁴ cells/cm², 2nd passage). Growth characteristics of both normal and plaque-derived cells were the same in vitro. Growth rate declined with time in culture, and cell division ceased by the 5th or 6th passage. In culture, spindle shaped cells formed the “hill and valley” configuration typical of SMC. Plaquederived SMC were ultrastructurally similar to SMC from uninvolved vessel wall. Proliferative potential did not vary with age of sex, with method of culture, or with whether the cells were plaque derived or not. This study was supported in part by National Institutes of Health Grant HL-17269