Cultured vascular endothelial cells secrete a protein factor(s) that promotes the contraction of collagen lattices made with fibroblasts

Conditioned media collected from arterial endothelial cells contain protein factor(s) that promotes the contraction of collagen lattices made with skin fibroblasts. Based on the lattice contraction-promoting activity, a protein with an apparent molecular weight of 22 kDa was identified. This 22-kDa protein stimulated lattice contraction in both serum-containing and serum-free media. When assayed at a 30% equivalent of the conditioned medium, the contraction-promoting activity of the purified factor was about 50 to 60% of that elicited by the unfractionated conditioned medium. Some contraction-promoting activity was also present in certain subfractions of the conditioned medium generated during the separation of the 22-kDa protein. Taken together, the results indicate that the lattice contraction-promoting activity in the endothelial cell-conditioned medium is probably aided by multiple active principles. The biochemical and biological characteristics indicated that the 22-kDa protein is not a transforming growth factor-beta-related factor nor a fibroblast growth promoter.     

Endothelial cells secrete a factor that promotes fibroblast contraction of hydrated collagen gels

Bovine aortic endothelial cells (BAEC), grown in vitro, are shown to synthesize and secrete factor(s) that stimulate fibroblasts to contract collagen matrices. The amount of contraction-promoting activity in the conditioned media is dependent on conditioning time and the number of cells in the culture. Production of the contraction-promoting activity continues at a high stable level for at least 5 d in serum-free medium but is abolished when the cells are exposed to an inhibitor of protein synthesis. The mechanism of action of the contraction factor(s) derived from endothelial cells was compared with that of unidentified serum factors. The endothelial cell-secreted factor(s) depends on active protein synthesis by the target cell but does not need to be present during the contraction process. The serum factors on the other hand promote collagen contraction in the absence of de novo protein synthesis but need to be continuously present. Preliminary biochemical characterization of the contraction-promoting factors produced by endothelial cells revealed properties similar to those of previously identified growth factors. However, the BAEC-secreted factor was found to be distinct from a previously identified contraction-promoting transforming growth factor beta.     

Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.     

Purification and properties of a single-chain urokinase-type plasminogen activator form produced by subcultured human umbilical vein endothelial cells

Single-chain Mr 54,000 u-PA (scu-PA) was isolated, in the presence of aprotinin, from 3-liter batches of 60-h serum-free conditioned media obtained from subcultured (4-6th passage) human umbilical vein endothelial cells (HUVECs, approximately 1.8 x 10(9) cells). In the presence of heparin and endothelial cell growth factor, subcultured human umbilical vein endothelial cells produced u-PA proteins consisting of about 85-90% Mr 54,000 scu-PA and 10-15% two-chain Mr 54,000. The major scu-PA form was purified to homogeneity by ion-exchange chromatography on CM-Sephadex C-50, immunoadsorption on purified anti-u-PA IgG-Sepharose and affinity chromatography on p-amino-benzamidine-Agarose. Typically, about 8-10 micrograms of purified scu-PA protein (antigen/protein ratio = 1) was isolated from 3-liter batches of heparin-containing serum-free conditioned media with a yield of about 41% of the total starting u-PA antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified u-PA protein showed a single Ag-stained band (nonreduced and reduced), with an estimated molecular weight of about 54,000, which exhibited very low fibrinolytic activity. Purified HUVEC-derived scu-PA did not incorporate 3H-labeled diisopropyl fluorophosphate. This protein did, however, exhibit very low amidolytic activity (approximately 5,000 IU/mg) on the u-PA-specific synthetic substrate pyroglu-Gly-Arg-p-nitroanilide, very low plasminogen-dependent fibrinolytic activity on 125I-labeled fibrin coated plates, and directly activated 125I-labeled plasminogen following Michaelis-Menten kinetics with high affinity, Km = 0.72 microM and low turnover number, kcat = 0.0005 s-1. Treatment with plasmin rapidly converted the HUVEC-derived scu-PA to the active two-chain Mr 54,000 u-PA form (approximately 90,000 IU/mg). Binding to fibrin clots, using antigen quantitation, indicated about 20, 10, and 90% binding for equimolar amounts of HUVEC-derived scu-PA, two-chain u-PA, and tissue plasminogen activator standards, respectively. These results indicate that subcultured HUVECs synthesize and secrete their u-PA protein as a single-chain molecule with low intrinsic amidolytic and fibrinolytic activity, high affinity for plasminogen and no specific affinity for fibrin. The role of scu-PA in endothelial cell-mediated vascular function has yet to be clearly defined.     

Repression of enhancer activity by the adenovirus E1A tumor protein(s) is mediated through a novel HeLa cell nuclear factor.

We have examined the mechanism for the host cell-dependent repression of enhancer activity by the adenovirus early region 1A (E1A) proteins. The enhancer used in this study, from the human BK virus P2, functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2. In addition, enhancer activity can be stimulated by the E1A gene products in these cells. However, cis-enhancer activity is repressed in the HeLa cell line, and we demonstrate here that further repression can be induced by the E1A proteins. We show that the binding site for the negative regulatory factor involved in cis-repression, designated BK virus enhancer factor 1 (BEF-1), is also required for E1A-induced repression. Using gel mobility retardation assays, we demonstrated a 4-fold increase in active BEF-1 in nuclear extracts containing the E1A proteins. However, the E1A proteins did not change the binding pattern or the strength of binding of BEF-1 to its target sequence. BEF-1 was identified as a 98-kDa nuclear factor, and phosphorylation was shown to be important for DNA binding. Three potential nuclear factor 1 (NF-1) sites are present in the BEF-1-binding site. Using a known NF-1 site as competitor DNA in a gel mobility retardation assay, we provide evidence that BEF-1 may be a newly identified NF-1 family member. In addition, the sequence TGA present in the repressor-binding site was shown to be essential for high affinity binding of BEF-1. Overall, our data demonstrate that an enhancer can be repressed by the trans-activation of a negative regulatory factor.     

Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line

24 established melanoma cell cultures were screened for their secretion of plasminogen activators and plasminogen activator inhibitors into the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by conventional and reverse fibrin autography. Among the cell lines investigated, 22 cell lines predominantly secreting tissue type plasminogen activator (t-PA) and four cell lines additionally secreting urokinase were found. The conditioned media of two cell lines (KRFM and MJZJ) were found to contain plasminogen activator inhibitor (PAI) activity at a Mr position of approximately 50,000. The PAI of one of the two melanoma cell (MJZJ)-conditioned media found to contain PAI activity was purified to apparent homogeneity employing concanavalin A-Sepharose chromatography, gel filtration on Sephadex G-150, chromatography on Affi-Gel blue, and affinity chromatography on a Sepharose 4B immobilized monoclonal anti-t-PA IgG column. The purified melanoma PAI was found to be a single chain protein, acid stable, immunologically related to the endothelial derived PAI. In contrast to endothelial PAI, melanoma PAI presented itself in the conditioned media of the melanoma cells and in the purified preparation to an appreciable extent in its active form.     

Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the 16-h assay period. Several hormones and growth factors were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.     

Density-dependent endothelial cell production of an inhibitor of smooth muscle cell growth

Embryonic data and ultrastructural analyses suggest that the primitive endothelium signals undifferentiated mesenchymal cells to migrate to the forming blood vessel and subsequently regulates mural cell growth and behavior. Upon maturation of the blood vessel, chemotactic and mitogenic signals are apparently diminished and differentiated smooth muscle cells normally remain quiescent. This homeostasis is seemingly upset in conditions which lead to pathologies characterized by smooth muscle cell hyperplasia such as atherosclerosis. By culturing endothelial cells at different densities, we attempted to re-create the various stages of vascular development. Whereas media conditioned by sparse endothelial cells stimulate smooth muscle cells, media conditioned by dense endothelial cell cultures are inhibitory. Culture of sparse smooth muscle cells in media conditioned for 3 days by postconfluent endothelial cell cultures leads to dose-dependent and reversible smooth muscle cell inhibition. Furthermore, in the presence of the endothelial cell-derived inhibitor, smooth muscle cells are rendered refractory to mitogens such as fibroblast growth factor and platelet-derived growth factor. The inhibitory activity is not attributable to the well-characterized inhibitors of smooth muscle cell growth, transforming growth factor type-β, prostaglandin I₂, or heparan sulfate proteoglycan. Partial characterization of the inhibitory conditioned media suggests that the active molecule is smaller than 1,000 da, and stable to boiling as well as proteinase K and heparinase digestion. These findings support the concept that there is intercellular communication between endothelial cells and smooth muscle cells and provide evidence for a novel endothelial cell-derived smooth muscle cell growth inhibitor.     

Induction of angiogenesis by smooth muscle cell-derived factor: Possible role in neovascularization in atherosclerotic plaque

The development of atherosclerotic plaque is associated with neovascularization in the thickened intima and media of vascular walls. Neovascularization may have a role in the progression of atherosclerotic plaque as well as in the development of intraplaque hemorrhage. However, the mechanism and stimulus for neovascularization in atherosclerotic plaque are unknown. We postulated that smooth muscle cells (SMCs), a major cellular component in the vascular wall, might contribute to the induction of neovascularization in atherosclerotic plaque through the secretion of an angiogenic factor. We observed that endothelial cells (ECs) cultured on collagen gel with SMC-conditioned medium became spindle shaped, invaded the underlying collagen gel, and organized a capillary-like branching cord structure in the collagen gel. The conditioned medium also stimulated EC proliferation and increased the EC-associated plasminogen activator activity. The angiogenic factor in SMC-conditioned medium was retained in a heparin-Sepharose column and eluted with 0.9 M NaCl. Neutralizing anti-vascullar endothelial growth factor (VEGF) antibody attenuated the angiogenic activity in the conditioned medium, including the induction of morphologic changes in ECs, mitogenic activity, and increased plasminogen activator activity associated with ECs. Immunoblotting analysis confirmed the secretion of VEGF from SMCs. These observations indicate that SMC may be responsible for the neovascularization in atherosclerotic plaque through the secretion of VEGF. © 1995 Wiley-Liss, Inc.     

Adenoviral E4 Gene Stimulates Secretion of Pigmental Epithelium Derived Factor (PEDF) that Maintains Long-term Survival of Human Glomerulus-derived Endothelial Cells

Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region also supported survival of human glomerulus-derived endothelial cells in serum-free media. Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT-PCR, Western blot and ELISA analysis. PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells.Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease (1). Despite their unique features and their importance, it was a challenge to study these cells comparing to other endothelial cells because of difficulty in maintaining them in culture (2, 3). Primary cells have limited capacity to divide in culture and quickly reach a nonproliferative state known as senescence. Senescence or mortality checkpoint (M1)¹ is characterized by the absence of cell division because of the inhibition of the cell cycle (4). Cells can overcome M1 when they lose the cell cycle inhibitory signals, such as functional Rb or p53 proteins. These cells extend their replicative life span but eventually reach the second mortality checkpoint (M2) also known as crisis (4) which is associated with shortening telomere sequences (5). Culturing primary endothelial cells requires supplementation with serum and pro-angiogenic factors, such as VEGF-A, FGF-2, EGF and IGF. Deprivation of growth factors or serum results in a rapid cell death (6). Thus the limited life span and dependence on serum and growth factors put a considerable limitation to studies that can be conducted with primary endothelial cells. Human primary glomerulus-derived endothelial cells (PrGECs) either transformed with a simian virus 40 (SV40) (7) or conditionally immortalized with temperature-sensitive SV40 large T antigen and telomerase using retroviral vectors (8) retain the ability to grow up to 60 passages and maintain endothelial cell markers. However, culturing these transformed or immortalized cell lines still requires supplementation with serum and growth factors and their withdrawal leads to quick cell death (9). Previously, expression of adenoviral early region 4 (Ad E4) or E4ORF1 gene product was shown to support long-term survival of different types of primary endothelial cells including human umbilical vein endothelial cells and human testicular endothelial cells, while preserving these cell''s potential for tubulogenesis and sprouting (6, 10, 11). Introduction of AdE4ORF1 into primary endothelial cells increases their survival in serum and growth factor -free media without stimulation of cell proliferation. The mechanism by which Ad E4ORF1 induces cell survival is not yet known.In the present study, we showed that media conditioned with human epithelial embryonic kidney cells stably transfected with AdE4 (293-E4 cells) supported survival of primary glomerulus-derived endothelial cells (PrGECs) in serum-free and growth factor-free media without the need to transform the primary cells with AdE4 gene expression vector. Supplementation with the media conditioned with 293-E4 cells (293-E4 media) prolonged the life-span of the PrGECs and also human primary glomerulus-derived endothelial cells transformed with SV40 large T antigen (HGECs). The mass-spectrometry analysis of the 293-E4 media identified pigmental epithelium derived factor (PEDF) that was absent in the control media conditioned with the parental 293 cells (293 media). PEDF expression in 293-E4 cells was validated by RT- PCR, Western blot, and ELISA analysis. PEDF was also observed in mouse glomeruli by immunostaining. Supplementation with recombinant PEDF increased PrGECs and HGECs survival in serum-free media, protecting the cells from necrosis and apoptosis while preserving their ability for FGF-2 stimulated growth and tubulogenesis.     

IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice

Mononuclear phagocytes can interact with mesenchymal cells and extracellular matrix components that are crucial for connective tissue rearrangement. We asked whether blood monocytes can alter matrix remodeling mediated by human lung fibroblasts cultured in a three-dimensional collagen gel. Blood monocytes from healthy donors (>95% pure) were cast into type I collagen gels that contained lung fibroblasts. Monocytes in coculture inhibited the fibroblast-mediated gel contractility in a time- and concentration-dependent manner. The concentration of PGE(2), a well-known inhibitor of gel contraction, was higher (P < 0.01) in media from coculture; this media attenuated fibroblast gel contraction, whereas conditioned media from either cell type cultured alone did not. Three-dimensional cultured monocytes responded to conditioned media from cocultures by producing interleukin-1beta and tumor necrosis factor-alpha, whereas fibroblasts increased synthesis of PGE(2). Antibodies to interleukin-1beta and tumor necrosis factor-alpha blocked the monocyte inhibitory effect and reduced the amount of PGE(2) produced. The ability of monocytes to block the fibroblast contraction of matrix may be an important mechanism in regulating tissue remodeling.     

Production of interleukin 1 by SK hepatoma tumor cells

SK hepatoma cells and SK hepatoma conditioned media contain an 18,000-dalton factor which is pyrogenic, stimulates collagenase and prostaglandin production in skin and synovial fibroblasts, induces bone resorption, and stimulates the proliferation of murine thymocytes. These results are consistent with the finding that this tumor cell line produces interleukin 1 [Doyle, M. V., Brindley, L., Kawasaki, E., & Larrick, J. (1985) Biochem. Biophys. Res. Commun. 130, 768-773] since all these activities have been associated with this cytokine. Greater than 80% of the cellular activity has a molecular weight of 30,000, while in contrast, greater than 80% of the activity in the tumor-conditioned media has a molecular weight of 18,000. When active material from the cells is incubated with trypsin, this high molecular weight material is completely converted into an active 18,000 molecular weight species. The isoelectric point of all active material is always between pI 4.0 and 5.1, regardless of molecular weight. All of these results are consistent with the hypothesis that active, high molecular weight interleukin 1 alpha is first synthesized and stored by the tumor cell. This cytokine is then cleaved by a trypsin-like protease to an active, lower molecular weight species which can be secreted into the media.     

Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF(165/121) antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.     

Age dependent production of a competence factor by human fibroblasts

Several cell types such as Balb/c 3T3 have been shown to require platelet-derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin-C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12-wk embryo, newborn, and 3-yr-old were exposed to serum-free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3-yr-old cells with the addition of plasma alone, whereas conditioned medium from the 3-yr-old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3-yr-old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3-yr-old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age-dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and supplemented with plasma alone.