Biotin and choline replace the growth requirement of Madin-Darby canine kidney cells for high-density lipoproteins

MDCK cells maintained on extracellular matrix (ECM)-coated dishes and exposed to Dulbecco's modified Eagle's medium (DME) supplemented with transferrin and either high-density lipoproteins (HDLs) or phosphatidyl choline (PC) liposomes have a growth rate and final cell density similar to those of cultures exposed to serum-supplemented DME. When MDCK cells are exposed to a medium consisting of a mixture (1:1) of DME and F12 medium (D/F), the addition of transferrin (10 μg/ml) alone supports cell growth and the presence of HDLs or PC liposomes is no longer required. MDCK cells exposed to D/F medium supplemented with transferrin can be passaged for more than 50 generations in total absence of serum. The F12 components that support growth in the absence of HDLs or PC liposomes are biotin (which is absent in DME) and choline (which is present in insufficient concentration in DME). Supplementation of DME with transferrin, biotin (3.6 ng/ml), and choline (10 μg/ml) allows optimal growth of MDCK cells and permits serial propagation through more than 50 generations. The growth requirement of MDCK cells for HDLs or PC liposomes can therefore be replaced by adequate concentrations of biotin and choline. The widely observed fact that a combination of DME/F12 medium is more effective than DME alone in supporting cell growth may be due in part to the lack of biotin and suboptimal choline concentration in DME.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.     

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells

Summary Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovinn vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any grwoth-promoting activity. This work was supported by Grants HL 20197 and HL 23678 from the National Institutes of Health, Bethesda, MD.     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.     

Rapid purification and activity of apolipoprotein CI on the proliferation of bovine vascular endothelial cells in vitro

The growth-promoting activity of human high-density lipoproteins (HDL) and of their apolipoprotein components on bovine vascular endothelial cells in vitro has been compared. When maintained on plastic culture dishes and exposed to medium containing lipoprotein-deficient serum and fibroblast growth factor, these cells do not proliferate. Addition of either HDL or the total HDL apolipoproteins induces significant cell proliferation. Apolipoprotein C_I, purified by chromatography on the ion-exchanger resin Polybuffer exchanger 94, has an effect on the cell growth similar to that of the total apolipoproteins of HDL.     

Primary cultures of rat aortic endothelial and smooth muscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity

Summary Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 μM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morpholgic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Growth requirements of low-density rabbit costal chondrocyte cultures maintained in serum-free medium

The factors required for the active proliferation of low-density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low-density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high-density lipoprotein (300 micrograms/ml), transferrin (60 micrograms/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10(-6) M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low-density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum-free chondrocyte cultures seeded at low density and maintained on ECM-coated dishes gave rise to a homogeneous cartilage-like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.     

Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells

The hypothesis that, in the case of clonal or low-density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low-density vascular endothelial cell cultures maintained on either plastic or ECM-coated dishes and exposed to various combinations of media and sera. Proliferation of low-density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI-1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM-coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to become confluent. In the case of cultures exposed to RPMI-1640 or medium 199 plus thymidine, it was even toxic. When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low-density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum or FGF. These restrictions cannot be relieved by maintaining cells on ECM-coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM. These results suggest that, when low-density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Secretion of ribonucleases by normal and immortalized cells grown in serum-free culture conditions

Summary The requirement of serum in cell culture is a major limitation for studies on secreted ribonucleases (RNases) because serum contains a high amount of ribonucleolytic activity. Defined culture condition is thus of interest to improve our knowledge of the RNase biology. We report here that cells from three different types and origins, Chinese hamster lung fibroblasts, bovine smooth muscle cells, and human endothelium-derived EA.hy926 cells, proliferate consistently in the presence of a basal medium supplemented with bovine serum albumin, high-density lipoproteins, basic fibroblast growth factor, insulin, and transferrin. Using a new quantitative radio-RNase inhibitor assay, two distinct ribonucleolytic assays, and a radioimmunoassay against angiogenin, it is shown that RNases became apparent in media conditioned by cell monolayers. Both the hamster lung fibroblast and the EA.hy926 cell lines secreted larger amounts of RNase inhibitor-interacting factors and RNase activity than normal smooth muscle cells. The serum-free medium represents an alternative way to grow these cells and allows investigation of biosynthesis and functions of RNases in culture. It should be useful to identify and quantitate unambiguously specific members of the RNase family secreted by normal versus tumor cells in culture.     

Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries

Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.     

Lipoproteins increase growth of mitogen-stimulated arterial smooth muscle cells

The relationship between lipoproteins and growth of aortic smooth muscle cells has been a matter of controversy. We therefore reexamined this issue using serum-free defined media methodology. By themselves, LDL or HDL (50-500 micrograms/ml) from normolipemic human or bovine plasma produced little or no growth of homologous aortic smooth muscle cells incubated in serum-free medium that was supplemented with insulin and transferrin to maintain cell viability. In fact, LDL prepared in the absence of an antioxidant (BHT) was toxic to these cells. However, in the presence of maximally effective concentrations of platelet-derived growth factor (PDGF), LDL or HDL consistently increased the growth of homologous smooth muscle cells (up to twofold increased in DNA accumulation in 48 hr). Lipoproteins also augmented the growth response of arterial smooth muscle cells to fibroblast growth factor or epidermal growth factor. The mechanism of this effect was investigated further with HDL, because, in contrast to LDL, HDL apoproteins are water-soluble. Neither HDL delipidated by solvent extraction (apoHDL), purified bovine apoA-I, nor cholesterol added in the form of phospholipid vesicles appreciably increased PDGF-induced growth of bovine smooth muscle cells. However, HDL-like particles reconstituted by sonication of apoHDL with cholesterol and phospholipids did increase the growth of cultures of bovine smooth muscle cells treated with PDGF. Uptake of tritiated thymidine by cultures incubated with partially purified PDGF alone (10 micrograms/ml) was 5,693 +/- 235 dpm/24 hr compared to 10,381 +/- 645 dpm/24 hr (p less than 0.01) in the presence of both PDGF and reconstituted HDL-like particles (250 micrograms protein/ml). Thus both the lipid and protein components of HDL may be necessary for optimal potentiation of growth of mitogen-stimulated cells. These results indicate that lipoproteins from normolipemic sera are not bona fide growth factors but can potentiate the growth of mitogen-stimulated cells, perhaps by supplying exogenous cholesterol required for membrane biogenesis. This finding might be important in arterial injury when the release of PDGF and exposure to plasma lipoproteins could act in concert to stimulate the proliferation of smooth muscle cells.     

The production and localization of laminin in cultured vascular and corneal endothelial cells

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20-fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10- and 30-fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells

Summary Endothelial and smooth muscle cells were isolated from human adult large blood vessels to compare their proliferative response to hormones and growth factors. Neural extracts and the medium from differentiated hepatoma cells were used as concentrated sources of required hormones and growth factors that supported both cell types. Active hormones and growth factors were identified from the neural extracts and hepatoma medium by substitution or direct isolation and biochemical characterization. Epidermal growth factor, lipoproteins, and heparin-binding growth factors elicited growth-stimulatory effects on both endothelial and smooth muscle cells. Both types of human vascular cells displayed 7600 to 8600 specific heparin-binding growth factor receptors per cell with a similar apparent dissociation constant (Kd) of 200 to 250 pM. Heparin modified the response of both endothelial and smooth muscle cells to heparin-binding growth factors dependent on the type of heparin-binding growth factor and amount of heparinlike material present. In addition, heparin exerted a growth factor-independent inhibition of smooth muscle cell proliferation. Platelet-derived growth factor, insulinlike growth factors, and glucocorticoid specifically supported proliferation of smooth muscle cells with no apparent effect on endothelial cell proliferation. Growth-factorlike proteinase inhibitors had an impact specifically on endothelial cell proliferation. Transforming growth factor beta was a specific inhibitor of endothelial cells, but had a positive effect on smooth muscle cell proliferation. The results provide a framework for differential control of the two vascular cell types at normal and atherosclerotic blood vessel sites by the balance among positive and negative effectors of endocrine, paracrine and autocrine origin. This research was supported by NIH grants CA37589, HL33847, and AM35310 from the National Institutes of Health, Bethesda, MD; grant 1718 from the Council for Tobacco Research; and a grant from RJR/Nabisco, Inc.