Inhibition of endothelial cell proliferation by gamma-interferon.

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose-dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor-ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.

Serial propagation of human endothelial cells in vitro.

Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.

Organizational behavior of human umbilical vein endothelial cells.

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.

Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the Src pathway correlates with migration.

FGF regulates both cell migration and proliferation by receptor-dependent induction of immediate-early gene expression and tyrosine phosphorylation of intracellular polypeptides. Because little is known about the disparate nature of intracellular signaling pathways, which are able to discriminate between cell migration and proliferation, we used a washout strategy to examine the relationship between immediate-early gene expression and tyrosine phosphorylation with respect to the potential of cells either to migrate or to initiate DNA synthesis in response to FGF-1. We demonstrate that transient exposure to FGF-1 results in a significant decrease in Fos transcript expression and a decrease in tyrosine phosphorylation of the FGFR-1, p42(mapk), and p44(mapk). Consistent with these biochemical effects, we demonstrate that attenuation in the level of DNA synthesis such that a 1.5-h withdrawal is sufficient to return the population to a state similar to quiescence. In contrast, the level of Myc mRNA, the activity of Src, the tyrosine phosphorylation of cortactin, and the FGF-1-induced redistribution of cortactin and F-actin were unaffected by transient FGF-1 stimulation. These biochemical responses are consistent with an implied uncompromised migratory potential of the cells in response to growth factor withdrawal. These results suggest a correlation between Fos expression and the mitogen-activated protein kinase pathway with initiation of DNA synthesis and a correlation between high levels of Myc mRNA and Src kinase activity with the regulation of cell migration.

Human endothelial cells are chemotactic to endothelial cell growth factor and heparin.

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.

Bovine fibroblast growth factor: comparison of brain and pituitary preparations.

Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl-3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints.

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II.

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.

Hormonal requirements for growth of arterial smooth muscle cells in vitro: and endocrine approach to atherosclerosis.

In this study the hormonal requirements for the growth of arterial smooth muscle cells in vitro were determined. A serum-free, biochemically defined medium, supplemented with the relevant hormones, permitted proliferation and propagation of normal diploid mammalian arterial smooth muscle cells. Serum-free, hormone-supplemented cultures spontaneously formed atherosclerotic plaque-like nodules. Thus atherosclerosis may be mediated by a complex endocrine system.

Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain.

Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

An endocrine approach to the control of epidermal growth: serum-free cultivation of human keratinocytes.

Human keratinocytes, derived from the skin of newborns and of adults, were grown in the complete absence of serum, in a hormone-supplemented medium on fibronectin-coated cell culture dishes at low seed density. The cell culture medium consisted of Medium 199 containing epidermal growth factor, triiodothyronine, hydrocortisone, Cohn fraction IV, insulin, transferrin, bovine brain extract, and trace elements. Removal of the brain extract from the hormone supplement had a greater negative impact on proliferation of the keratinocyte cultures than did the removal of epidermal growth factor, hydrocortisone, and triiodothyronine or Cohn fraction IV. The growth of keratinocytes in this hormone-supplemented medium suggests that control of keratinocyte growth depends in large part on endocrine stimulation by other body organs, including the brain.

Extension of the life-span of human endothelial cells by an interleukin-1 alpha antisense oligomer.

The proliferative potential of human diploid endothelial cells is finite, and cellular senescence in vitro is accompanied by the failure of the endothelial cell to respond to exogenous growth factors. Senescent human endothelial cells were shown to contain high amounts of the transcript for the cytokine interleukin-1 alpha (IL-1 alpha), a potent inhibitor of endothelial cell proliferation in vitro. In contrast, transformed human endothelial cells did not contain detectable IL-1 alpha messenger RNA. Treatment of human endothelial cell populations with an antisense oligodeoxynucleotide to the human IL-1 alpha transcript prevented cell senescence and extended the proliferative life-span of the cells in vitro. Removal of the IL-1 alpha antisense oligomer resulted in the generation of the senescent phenotype and loss of proliferative potential. These data suggest that human endothelial cell senescence in vitro is a dynamic process regulated by the potential intracellular activity of IL-1 alpha.

Modulation of the sis gene transcript during endothelial cell differentiation in vitro.

Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.

Site-directed neovessel formation in vivo.

Angiogenesis is an important component of organogenesis and wound repair and occurs during the pathology of oncogenesis, atherogenesis, and other disease processes. Thus, it is important to understand the physiological mechanisms that control neovascularization, especially with methods that permit the molecular dissection of the phenomenon in vivo. Heparin-binding growth factor-1 was shown to bind to collagen type I and type IV. When complexed with gelatin, heparin-binding growth factor-1 can induce neovascularization at polypeptide concentrations that are consistent with the biological activity of the mitogen in vitro. The adsorption strategy induces rapid blood vessel formation at and between organ- and tissue-specific sites and permits recovery of the site-specific implant for examination and manipulation by molecular methods.

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization.

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.

Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence.

Heparin-binding growth factor-1 (HBGF-1) is an angiogenic polypeptide mitogen for mesoderm- and neuroectoderm-derived cells in vitro and remains biologically active after truncation of the amino-terminal domain (HBGF-1 alpha) of the HBGF-1 beta precursor. Polymerase chain reaction mutagenesis and prokaryotic expression systems were used to prepare a mutant of HBGF-1 alpha lacking a putative nuclear translocation sequence (amino acid residues 21 to 27; HBGF-1U). Although HBGF-1U retains its ability to bind to heparin, HBGF-1U fails to induce DNA synthesis and cell proliferation at concentrations sufficient to induce intracellular receptor-mediated tyrosine phosphorylation and c-fos expression. Attachment of the nuclear translocation sequence from yeast histone 2B at the amino terminus of HBGF-1U yields a chimeric polypeptide (HBGF-1U2) with mitogenic activity in vitro and indicates that nuclear translocation is important for this biological response.

Effects of endothelial cell growth factor on bone remodelling in vitro.

Endothelial cell growth factor (ECGF) alpha was studied for its effects on bone formation in cultured fetal rat calvariae and on bone resorption in cultured fetal rat long bones. ECGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA, an effect enhanced by heparin. Treatment with ECGF for 24 h decreased the incorporation of [3H]proline into collagen but treatment for 48-96 h increased collagen and noncollagen protein synthesis, an effect that was concomitant with an increase in DNA content. ECGF did not alter collagen degradation in calvariae or 45Ca release from long bones, which indicated it had no effect on bone resorption. Although ECGF increased prostaglandin E2 concentrations, its effect on DNA synthesis was not prostaglandin-mediated. In conclusion, ECGF stimulates calvarial DNA synthesis, which is an effect that results in a generalized increase in protein synthesis, but ECGF has no effect on matrix degradation or bone resorption.

In vivo cyclooxygenase expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and streptococcal cell wall arthritis.

Cyclooxygenase (COX), or prostaglandin (PG) H synthase, plays a role in inflammatory diseases, but very limited data exist on the regulation of COX in vivo. We, therefore, studied the in vivo expression of COX in synovia from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), as well as joints of rats with streptococcal cell wall (SCW) and adjuvant arthritis. Extensive and intense intracellular COX immunostaining, which correlated with the extent and intensity of mononuclear cell infiltration, was observed in cells throughout RA synovia. Significantly less or equivocal staining was noted in OA and normal human synovia. Similarly, COX immunostaining was equivocal in the joints of normal and arthritis-resistant F344/N rats. In contrast, high level expression developed rapidly in euthymic female Lewis (LEW/N) rats throughout the hindlimb joints and overlying tissues including skin, preceding or paralleling clinically apparent experimental arthritis. COX was expressed in the joints of athymic LEW.rnu/rnu rats 2-4 d after injection of SCW or adjuvant but was not sustained. Physiological doses of antiinflammatory glucocorticoids, but not progesterone, suppressed both arthritis and COX expression in LEW/N rats. These observations suggest that, in vivo, (a) COX expression is upregulated in inflammatory joint diseases, (b) the level of expression is genetically controlled and is a biochemical correlate of disease severity, (c) sustained high level up-regulation is T cell dependent, and (d) expression is down-regulated by antiinflammatory glucocorticoids.

Coexpression of phosphotyrosine-containing proteins, platelet-derived growth factor-B, and fibroblast growth factor-1 in situ in synovial tissues of patients with rheumatoid arthritis and Lewis rats with adjuvant or streptococcal cell wall arthritis.

Fibroblast growth factor (FGF)-1 and PDGF-B-like factors have been implicated in the pathobiology of RA and animal models of this disease. Since the receptors for FGF-1 and PDGF are tyrosine kinases, we examined the expression of tyrosine phosphorylated proteins (phosphotyrosine, P-Tyr) in synovial tissues from patients with RA and osteoarthritis (OA), and rats with streptococcal cell wall (SCW) and adjuvant arthritis (AA). Synovia from patients with RA and LEW/N rats with SCW and AA arthritis, in contrast to controls, stained intensely with anti-P-Tyr antibody. The staining colocalized with PDGF-B and FGF-1 staining. Comparative immunoblot analysis showed markedly enhanced expression of a 45-kD P-Tyr protein in the inflamed synovia. Treatment with physiological concentrations of dexamethasone suppressed both arthritis and P-Tyr expression in AA. P-Tyr was only transiently expressed in athymic nude Lewis rats and was not detected in relatively arthritis-resistant F344/N rats. These data suggest that (a) FGF-1 and PDGF-B-like factors are upregulated and may induce tyrosine phosphorylation of proteins in vivo in inflammatory joint diseases, (b) persistent high level P-Tyr expression is T lymphocyte dependent, correlates with disease severity, and is strain dependent in rats, (c) corticosteroids, in physiological concentrations, downregulate P-Tyr expression in these lesions.