Modulation of endothelial cells growth induced by heparin

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1% serum concentration and was abolished at 10%. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth.     

Culture and properties of cells derived from Kaposi sarcoma

We describe the establishment of four continuous cell cultures isolated from pleural or peritoneal fluid of patients with Kaposi sarcoma (KS) and show evidence that these cells are derived from vascular endothelium. Although provision of an extracellular matrix (fibronectin, laminin, or matrigel) was essential, the cell cultures were not dependent on exogenously added growth factors (platelet-derived growth factor, epidermal growth factor with or without heparin) for continuous culture. Specific staining for endothelial cell (EC) markers (factor VIII, Ulex europaeus type 1 lectin) and the secretion of endothelin, a vascular EC product, were demonstrated. The KS cells secreted large amounts of cytokines (granulocyte-macrophage-CSF, TNF-alpha, IL-1 beta, and especially IL-6). Conditioned media from the KS cells caused normal capillary EC to proliferate. The KS cells synthesized fibroblast growth activity in amounts sufficient to induce the proliferation of normal EC and fibroblasts. These data support the existence of a paracrine pathway of EC proliferation in KS and suggest that KS cells could sustain their own growth via an autocrine mechanism.     

The effect of heparin on fibronectin and thrombospondin synthesis and mRNA levels in cultured human endothelial cells.

Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 micrograms/ml) and endothelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No change in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of heparin on EC FN synthesis was independent of whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cells. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-translationally regulated.     

Structural features in heparin that interact with VEGF165 and modulate its biological activity.

The 165 amino acid form of vascular endothelial growth factor (VEGF165) is a heparin-binding growth factor with mitogenic activity for vascular endothelial cells. We examined activities of various heparin derivatives toward their interactions with VEGF165 using an enzyme-linked immunosorbent assay and elucidated the structural features in heparin for the interactions. Native heparin interacted with VEGF165, whereas N-desulfated, N-acetylated (N-DS, N-Ac-) heparin, and 6-O-desulfated (6-O-DS-) heparin did not. The 2-O-desulfated (2-O-DS-) heparin retained the ability for the interaction with VEGF165. In contrast, the 2-O-DS-heparin exhibited no ability for the interaction with FGF-2 and HGF. Thus, structural requirements in heparin for the specific interaction with VEGF165 are distinct from those with FGF-2 and HGF which require a high content of 2-O-sulfate groups. In a cell proliferation assay, native heparin and 2-O-DS-heparin exhibited inhibitory abilities for VEGF165-induced proliferation of human umbilical vein endothelial cells (HUVECs) with their high concentrations (more than 64 microg/ml), while only native heparin could enhance the proliferation of the chlorate-treated cells. These results suggested that a high content of 2-O-sulfate groups is not required for the specific interaction with VEGF165alone, although it is essential for the mitogenic activity of the growth factor.     

Transcription-Independent Activation of Ornithine Decarboxylase Activity by Heparin in Cloned Cerebral Endothelial Cells

Abstract: Heparin, a highly sulfated glycosaminoglycan, is known to be obligatory for long-term endothelial cell cultures; it potentiates the mitogenic activities of endothelial cell growth factors and prolongs the replicative life span of the cells. Here we have shown that besides its growth factor-supportive role, heparin exerts a specific action on cerebral capillary endothelial cells (cECs), unrelated to serum or growth factors, by increasing activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in these cells. For our experiments we have used two different types of cloned cECs: type I cECs, grown in the presence of endothelial cell growth factor and heparin, and type II cECs, usually cultivated without growth factors. Heparin action on ODC activity was shown to be dose dependent within the range of 1–100 μg/ml. Increasing concentrations of or depletion of endothelial cell growth factor from type I cultures had no effect on ODC activity. The increase in enzyme activity was highest after 30 min to 1 h of heparin treatment. As evidenced by northern analysis, the heparin-mediated enhancement of ODC activity was not accompanied by changes of ODC mRNA levels. Studies of DNA replication revealed that in the absence of heparin-binding growth factors, heparin did not affect the proliferative activity of cloned cECs.     

Effect of inflammatory cytokines on human endothelial cell proliferation.

Neovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN-gamma) (10-100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)-stimulated EC proliferation. Recombinant interleukin 1 (rIL-1) and recombinant tumor necrosis factor-alpha (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN-gamma and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by 51Cr release. In contrast, the combination of rIFN-gamma and rIL-1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were 1) the stimulation of proliferation by IFN-gamma at a low concentration and 2) the inhibition by both rIL-1 and rTNF of ECGF-stimulated proliferation.     

Growth factor responses of human arterial endothelial cells in vitro

Summary Human arterial endothelial cells were cultured in vitro for up to 40 cumulative population doublings. Culture conditions similar to those required for long-term propagation of human umbilical vein endothelial cells were employed. These included fibronectin-coated culture vessels, 5 to 20% fetal bovine serum, endothelial cell growth factor, and heparin. Thoracic aorta endothelial cells were larger than iliac artery endothelial cells. Both cell types stained positively for Factor VIII antigen by immunofluorescence. A decrease in confluent density as a function of population doubling level was correlated with the appearance of large, senescent cells in the cultures. Serum growth factors to which the arterial endothelial cells responded included insulin, transferrin, epidermal growth factor, thrombin, and somatomedins. The effect of thrombin did not require the availabilty of the active site of the protease. The effect of the somatomedins was only seen in the presence of heparin. Neither platelet-derived growth factor nor hydrocortisone induced arteiral endothelial cell proliferation. These growth factor responses were also observed on the part of human umbilical vein endothelial cells. This work was supported in part by Public Health Service grants HL01030, HL01734, and AG00599.     

Mechanism of estrogen action on cellular proliferation: evidence for indirect and negative control on cloned breast tumor cells

Human MCF7 breast tumor cells grew as estrogen-dependent tumors in nude mice. In contrast, they were not estrogen-dependent for proliferation in serumless culture media. Charcoal-dextran stripped female human serum supplemented media (5% to 40%) inhibited their proliferation in a dose dependent pattern. Estrogens reversed this inhibition. Concentrations of 2% of this serum allowed for maximal yield regardless of the presence of estrogens. Charcoal-dextran stripped fetal bovine serum was also inhibitory but less potent than the human serum. Non-estrogenic steroids, insulin, epidermal growth factor and transferrin failed to overcome the inhibitory effect of human serum. These results suggest that 1) human and bovine sera contain an inhibitor of the proliferation of estrogen-sensitive cells, and 2) estrogens promote cell proliferation by neutralizing this serum-borne inhibitor.     

Long-term culture of human endothelial cells

Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.     

Modulation of endothelial cell proliferation,adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin

Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence -Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependet and reversible. Thus, the antiproliferative activities of the thrombospondin peptides and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

Effects of an extract of human brain containing growth factor activity on the proliferation of human vascular endothelial cells in primary culture

Lesions of vascular human EC play an important role in the development of thrombi and atherosclerosis. The factors which control the repair of vascular lesions are not well known. In addition, they are difficult to study because vascular EC from large vessels are fastidious cells to grow in tissue culture. We have investigated some of the factors that may be important in human umbilical vein EC growth in primary culture. Because of reported species differences in EC culture, we have decided to culture human EC only in the presence of biological culture reagents of human origin. Human umbilical vein EC, at low seed density, can be grown to confluency on a human FN matrix or on human ECM providing the medium is supplemented with a high concentration (30%) of human serum. The optimal proliferation of EC (even when seeded at clonal density) is obtained if HBE is added. HBE cannot completely replace serum, but EC proliferate to a similar extent whether they are grown on FN or on ECM in the presence of 30% human serum of 10% human serum plus HBE. Thus, HBE contains a growth factor activity for human EC which stimulates cell growth and DNA replication. Further work is needed to purify HBE and to compare it to other endothelial cell growth factors isolated from bovine brain and bovine eye.     

Human arterial smooth muscle cells in culture. Effects of platelet-derived growth factor and heparin on growth in vitro

Human arterial smooth muscle cells (hASMC) were cultured from explants of the inner media of uterine arteries obtained at hysterectomy. The presence of alpha-actin and smooth muscle-specific actin isoforms and the microscopic appearance of the cells in secondary culture established their smooth muscle origin. The hASMC were diploid and had no signs of transformation. Plasma-derived serum failed to stimulate their proliferation in vitro. Their rate of proliferation was, however, proportional to the concentration of whole blood serum in the medium. Anti-PDGF IgG at high concentrations inhibited the stimulatory effect of whole blood serum on cell proliferation. This suggests that hASMC depend on exogenous PDGF for their growth. In PDS or bovine serum albumin cell numbers remained constant for 7 days in culture and the thymidine index was below 1% per 24 h. When reexposed to whole blood serum these cells started to proliferate within 2 days. This indicates that hASMC when deprived of PDGF enter a quiescent state that is fully reversible upon rexposure to the mitogen. Heparin is a powerful growth inhibitor for SMC. In our system, heparin caused a dose-dependent inhibition of cell proliferation despite optimal concentrations of whole blood serum. This inhibition was reversible upon withdrawal of heparin. At heparin concentrations which caused a half-maximal inhibition it was also competed for by increasing concentrations of whole blood serum. Quiescent hASMC expressed the PDGF receptor on their surface as judged from immunofluorescence with a monoclonal antibody. This was true irrespective of whether growth arrest was achieved by serum depletion or by the addition of heparin to serum-containing medium. Cells growing in the presence of whole blood serum did not, however, express the receptor antigen. These observations suggest that heparin may interfere with PDGF or with its binding and further processing at the level of the cell-surface receptor.     

Neurite outgrowth induced by an endothelial cell mitogen isolated from retina

Retina-derived growth factor (RDGF) is a polypeptide growth factor purified from salt extracts of bovine retinas on the basis of its mitogenic activity for capillary endothelial cells (EC) and BALB/c 3T3 cells. RDGF is angiogenic in vivo. We show here that RDGF induces neurite extension by PC12 cells and that this neurite outgrowth is dramatically potentiated by heparin. Neurite formation elicited by RDGF in the presence of heparin cannot be distinguished from that elicited by nerve growth factor (NGF) either by the time course of neurite formation or by the morphology of the neurites at the level of the light microscope. Neurite outgrowth induced by either purified RDGF or by a crude retinal extract is not blocked by antibodies to NGF. Furthermore, neurite outgrowth induced by NGF is not potentiated by heparin and NGF is not mitogenic for capillary EC. Thus, RDGF has profound regulatory effects on cell types of very different embryonic origins. These results indicate that the physiological role for this growth factor may be far more complex than previously suspected and suggest that the formation of neural connections and the process of vascularization may unexpectedly share common regulatory elements.     

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.     

Mitogen-induced disorganization of capillary-like structures formed by human large vessel endothelial cells in vitro

Endothelial cells (EC) from human aorta, umbilical vein and pulmonary artery were grown in Medium 199 supplemented with 20% human serum (HS), endothelial cell growth factor (ECGF) from bovine and human brain (200 micrograms/ml) and heparin (100 micrograms/ml) in gelatin-coated flasks. Under these conditions cells rapidly proliferated and survived 15-25 passages (40-60 cumulative population doublings). When cells were cultured on plastic substrate and without growth factors a capillary-like network appeared after 3-4 weeks of growth. According to TEM, this network consisted of tubes with the lumen encircled by one or several cells. The reduction of serum concentration in the medium or the replacement of plasma-derived serum (PDS) for HS reduced the time of network formation to 3-5 days. S-180 conditioned medium mitogenic for EC induced a rapid spreading of the cells and a partial reversion to a two-dimensional monolayer structure. Trypsin inhibitor did not abolish the effect of tumour conditioned medium. Other EC mitogens, e.g. ECGF and fibroblast growth factor (FGF), also disorganized the capillary-like network. In a day or two the network was completely restored. In contrast, culturing EC on gelatin-coated substrate is a sufficient condition for monolayer formation from tubes and long-term maintenance. We suggest that mitogens can influence the EC morphology but that it is the nature of the substrate that determines the stage of large vessel EC differentiation.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Inhibition of cell proliferation and protease activity by cartilage factors and heparin

Proliferating rat smooth muscle cells and fibroblasts have membrane-associated protease activity. High concentrations of heparin inhibited membrane-associated protease activity and cell proliferation, while low concentration of heparin promoted smooth muscle cell proliferation. The inhibition of protease activity and proliferation was abolished when heparin was treated with protamine sulfate or when acid treated fetal calf serum was used. Heparin required the presence of an acid labile factor(s) in serum for the inhibition of protease activity and proliferation. Heparin and antithrombin III in the presence of acid-treated fetal calf serum did not inhibit cell proliferation or protease activity. Cartilage factors isolated from bovine nasal cartilage containing trypsin inhibitory activity, but not papain inhibitory activity, inhibited rat smooth muscle and fibroblast proliferation and surface associated protease activity. The cartilage factors did not require acid-labile components in the fetal calf serum for the inhibitory activity. The inhibitory activity due to heparin and cartilage factors was not permanent under our experimental condition. Protein synthesis was not inhibited by heparin or the cartilage factors. In rat smooth muscle cells and fibroblasts, the expression of surface-associated protease activity was related to the proliferative state of the cells. Surface protease activity was only present on proliferating cells. When surface protease activity was inhibited by high concentrations of heparin in the presence of an acid-labile serum component(s) or cartilage factors, cell proliferation was also inhibited.