Decrease in transforming growth factor beta 1 binding during differentiation of rat adipocyte precursors in primary culture

Transforming growth factor beta 1 (TGF-beta 1) binding and action were investigated during differentiation of adipocyte precursors freshly isolated from rat inguinal fat-pad cultivated in defined medium. The data presented in this paper indicate that TGF-beta 1 inhibits differentiation of adipocyte precursors with a 50% effective dose of 9 pM. Time course experiments demonstrate that TGF-beta 1 is active only when it is added to the cells while they are still undifferentiated. If added after the cells have started to differentiate, TGF-beta 1 is less active or becomes inactive. 125I-TGF-beta 1 binding studies on adipocyte precursors before and after differentiation indicate a 10-fold decrease in the number of TGF-beta 1 binding sites after the cells have differentiated. Blocking of the differentiation process by treating the cells with fetal bovine serum or with prostaglandin F2 alpha prevented the decrease in the number of TGF-beta 1 receptors, thereby demonstrating that this change in binding was specifically linked to the differentiation process. Experiments cross-linking 125I-TGF-beta 1 to adipocyte precursors showed that 125I-TGF-beta 1 is specifically cross-linked to two bands with molecular weights of 92,000 and 70,000. After differentiation, a decrease in the intensity of the cross-linked bands was observed. These results demonstrate that loss of cell surface TGF-beta 1 binding sites follows differentiation of adipocyte precursors.     

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.     

Antidiabetic AD4743 enhances adipocyte differentiation of 3T3 T mesenchymal stem cells

AD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet-poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0-200 micrograms/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose-dependent manner in medium containing 10% FBS once they reached confluence. The data suggest that the ability of AD4743 to inhibit growth may also be coupled with its ability to enhance differentiation. In addition, AD4743 (1-10 micrograms/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (less than 5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80-95% differentiation could be achieved in just 4 days, compared to 8-12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to a greater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation-specific genes. AD4743 at 1-10 micrograms/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin-dependent pathway.     

The seminiferous growth factor induces proliferation of TM4 cells in serum-free medium

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.     

Growth requirements of ferret tracheal epithelial cells in primary culture

In mass cell culture conditions, protease dissociated ferret tracheal epithelial cells (FTE) proliferated in growth factor-supplemented F12 medium to high cell densities (0.5 X 10(5) cells/cm2) with an average population doubling time of 24 hr. The growth factor constituents of the F12 medium included epidermal growth factor (25 ng/ml), insulin (1 microgram/ml), transferrin (10 micrograms/ml), hydrocortisone (18 ng/ml), hypothalamus extract (30-100 micrograms/ml), and conditioned medium from mouse 3T3 fibroblasts. Growth of these cells under clonal conditions was achieved by the partial replacement of F12 medium with M199 medium which was attributed, in part, to the presence of vitamin A in M199 medium. Serum did not stimulate the growth of FTE cells. The epithelial cell nature of these cells in culture was confirmed by ultrastructural features and by immunofluorescent staining for fibronectin.     

Growth of human foreskin fibroblasts in a serum-free,defined medium without platelet-derived growth factor

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Wheresas thrombin and hydrocortisone were major growth stimulants, platelet-derived growth factor was not. Human foreskin fibroblasts grew in a serum-free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), trasferrin (10 μg/ml), thrombin (1 μg/ml), ascorbic acid (10 μg/ml), and hydrocortisone (5 × 10^?5M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet-derived growth factor, fibroblst growth factor, nor somatomedin activity increased proliferation. This serum-free medium designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.     

Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium

Rapid clonal growth of primary cultures of human costal chondrocytes in a defined medium has been achieved. The basal nutrient medium used for such growth is MCDB 104. It is prepared without linoleic acid and supplemented with 1 microgram/ml insulin, 100 ng/ml fibroblast growth factor, 1.0 x 10(-7) M dexamethasone, and 5 micrograms/ml mixed lipids, presented to the cells in the form of liposomes. The lipid supplement contains soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. No expression of cartilage-like differentiation occurs in the defined medium. However, colonies grown for several days in the defined medium and then grown for an additional period of time in medium F12 supplemented with fetal bovine serum and chicken embryo extract synthesize large amounts of refractile matrix that is stained intensely by acidified alcian green, thus demonstrating that the cells growing in the defined medium are capable of expressing cartilage matrix in a permissive environment. Good clonal growth and expression of differentiation can also be obtained by inoculating primary cultures of human chondrocytes directly into the F12-serum-embryo extract medium.     

Adipocyte conversion of CHEF cells in serum-free medium

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serum-free medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, greater than 90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.     

Transforming growth factor type beta is a specific inhibitor of 3T3 T mesenchymal stem cell differentiation

In the current studies we examined the effects of transforming growth factor type beta (TGF-beta) on the control of differentiation of BALB/c 3T3 T stem cells. We report that TGF-beta is a potent, reversible inhibitor of adipocyte differentiation (50% inhibition at approximately 0.06-0.08 ng/ml), while other biologically active polypeptides, such as epidermal growth factor (EGF), human growth hormone (hGH), and somatomedin C, have no specific effect on differentiation at even higher concentrations (200 ng/ml). We also report that TGF-beta inhibits differentiation in a cell cycle-dependent manner by its effect on a specific phase in the differentiation process. We therefore suggest that if TGF-beta is an important regulatory factor, one of its critical mechanisms of action may be its ability to inhibit the process of cell differentiation.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Neural differentiation of ectodermal explants of the early gastrula from Rana temporaria under the action of various mitogens and kainic acid

The following mitogens: concanavalin A (con A), phytohemagglutinin (PHA), hydra growth factor (HGF) as well as neurotoxic agent kainic acid, caused neural differentiation (N) effects differed in value and also in character of dependence on concentration of the agent. The lowest effective concentration of con A was 75 micrograms/ml (15% neural differentiation, treatment during 3 h), and the effect reached maximum of 50-60% at 100-200 micrograms/ml. Con A concentration 50 micrograms/ml showed no effect but after 1% rabbit gamma-globulin was added, 17% neural differentiation was detected. N-effects observed after treatment of explants with con A (200 micrograms/ml, 3h) at 2 degrees and 21 degrees were similar (58 and 42% respectively). Minimum PHA concentration used (6 micrograms/ml, 18h) led to neural differentiation in 5% of explants. N-effect of PHA increased along with the concentration of the lectin and was most pronounced at 25 micrograms/ml. However, further increase in concentration (up to 200 micrograms/ml) resulted in decrease of its N-effect to 13%. At 12 micrograms/ml PHA exerted not only neural differentiation, but also lens-inducing (32%) action on the ectoderm. N-effect of HGF (2.5, 25 and 250 micrograms/ml) was lower as compared with the maximum effects of con A and PHA (30-35%). No correlation of HGF inducing action with its concentration was observed. Kainic acid showed weak N-effect (20-30%) at 1 and 10 micrograms/ml. Higher concentration (100 micrograms/ml) had no N-effect, but in 27% of explants "free" lentoids were found. Oubain (10(-3) and 10(-4) M) and HEPES (20 mM) did not affect the differentiation of explants.     

Cultivation of rat granulosa cells in a serum-free chemically defined medium--a useful model to study lipoprotein metabolism

We have developed a chemically defined, serum-free medium for the culture of rat granulosa cells. This medium contains Dulbecco's modified Eagle's medium/Ham's nutrient F12 (DME:F12) (1:1) plus insulin (2 micrograms/ml), hydrocortisone (100 ng/ml), transferrin (5 micrograms/ml) and fibronectin (2 micrograms/cm2). Granulosa cells grown in this medium have an absolute requirement for added cholesterol-rich lipoproteins for steroidogenesis. When cells are cultured in basal medium, progestin production is low; when cells are cultured in the presence of follicle-stimulating hormone (FSH) or dibutyryl cAMP [Bu)2 cAMP), progestin secretion is increased 10-100-fold. Both heterologous and homologous lipoproteins synergistically increased the effects of (Bu)2 cAMP or FSH: e.g., addition to the medium of human (h)-HDL3 produced a significant increase in both basal (approx. 15-fold) and (Bu)2 cAMP-stimulated (approx. 1000-2000-fold) progestin production. LDL were less effective than HDL at equivalent concentrations of lipoprotein cholesterol. FSH invoked changes similar to that of (Bu)2 cAMP, although the magnitude of the FSH-induced change was less dramatic than that seen with (Bu)2 cAMP. The effect of h-HDL3 and h-LDL on both basal and hormone-stimulated progestin production was concentration- and time-dependent. The maximum effect of h-HDL3 was achieved at a protein concentration of 500 micrograms/ml, with an ED50 of approx. 90 micrograms/ml. In contrast, h-LDL was most effective at a concentration of 30-40 micrograms protein/ml. Likewise, rat (r-)HDL and r-LDL supported steroidogenesis in a concentration-dependent manner. Maximal responses to all additions were observed after 72 h of treatment. Granulosa cells secreted 20 alpha-hydroxypregn-4-ene-3-one as the predominant steroid in response to (Bu)2 cAMP. However, with the addition of h-HDL3, the major secreted product was progesterone. In conclusion, rat granulosa cells maintained in the described serum-free medium are exquisitely sensitive to supplied cholesterol-rich lipoproteins. When cultured in the presence of both lipoproteins and stimulatory agents, they produce from 1000-2000-times the progestins made by comparable cells maintained in medium alone. This responsiveness of the cells to both lipoprotein and hormone stimulation makes them uniquely suitable for studies involving the uptake and metabolism of lipoproteins during steroidogenesis.     

Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells.

We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.     

Keratinocyte differentiation inversely regulates the expression of involucrin and transforming growth factor beta1

Extensive skin loss from a variety of conditions such as severe thermal injury is associated with significant functional morbidity and mortality. In recent years, the healing quality has been improved for patients who suffer burns due in part to the usage of skin replacement mainly prepared from multi-layered sheets of cultured keratinocytes. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-beta1 (TGF-beta1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor and has any functional influence on dermal fibroblasts. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms express different levels of TGF-beta1. To address this hypothesis, keratinocytes were grown in serum free medium (KSFM) supplemented with bovine pituitary extract (50 microg/ml) and EGF (5 microg/ml). When cells reached confluency, conditioned medium was removed and replaced with 50% KSFM with no additives and 50% DMEM without serum and cells were allowed to form multi-layers and differentiate. The conditioned medium was then collected every 48 h up to 24 days and the level of TGF-beta1 and the efficacy of a keratinocyte released fibroblast mitogenic factor were evaluated by ELISA and (3)H-thymidine incorporation, respectively. Northern analysis was also employed to evaluate the expression of TGF-beta1, involucrin, TIMP-1, and 18 S ribosomal RNA in keratinocytes at different times of the onset of differentiation. The microscopic morphology of keratinocytes at different times of induction of cell differentiation showed detachments (nodules) of many regions of keratinocyte sheet from culture substratum within 1-2 weeks. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. The results of TGF-beta1 evaluation revealed that mono-layers of cultured keratinocytes which were round, attached, and proliferating in KSFM + BPE and EGF containing medium released a significantly higher level of TGF-beta1 (196 +/- 58 pg /ml) relative to those grown in multi-layer forms (28 +/- 7.8 pg/ml). A longitudinal experiment was then conducted and the results showed that cells on the onset of differentiation released even greater level of TGF-beta1 (388 +/- 53 pg/ml) relative to those grown in KSFM + BPE and EGF. This finding was consistent with the expression of TGF-beta1 mRNA evaluated in keratinocytes grown in test medium for various duration. In general, the level of TGF-beta1 protein and mRNA gradually reduced to its lowest level within 12 days of growing cells in our test medium. When aliquots of the collected keratinocyte conditioned medium were added to dermal fibroblasts, the level of (3)H-thymidine incorporation increased only in those cells receiving aliquots of conditioned medium containing high levels of TGF-beta1. When involucrin was used as a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. In conclusion, high involucrin expressing differentiated keratinocytes seem to be quiescent in releasing both TGF-beta1 and a fibroblast mitogenic factor.     

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.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.