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1.
We have studied the ability of bovine adrenal capillary cells cultured in vitro to uptake and metabolize heparin. We have previously demonstrated that endogenous heparin can be extracted from human plasma (Vannucchi, S. et al., (1985) Biochem. J. 227, 57-65), and here we show that also endothelial cells contain heparin. However, experiments with (35S)sodium sulfate labeling indicate that these cells do not synthesize de novo heparin, but they uptake it from culture serum. Bovine adrenal capillary endothelial cells are able to bind and uptake (3H)heparin added to culture medium and they also release its low molecular weight degradation products, thus indicating a metabolism of heparin. We discuss about the role of endothelial cell-mediated uptake and metabolism of endogenous heparin in relationship with circulating heparin. We also discuss about these events as related to some of the antithrombogenic properties of the endothelium.  相似文献   

2.
Inhibition of human endothelial cell proliferation by heparin and steroids   总被引:1,自引:0,他引:1  
Previous works have reported the controversial effects of heparin and steroids on angiogenesis. In this study, we investigated the effect of these compounds on human endothelial cell (EC) growth in vitro. An antiproliferative heparin activity was found in low human serum concentrations (2%). When EC were exposed to heparin (10(-6) M), their proliferation index was reduced in the presence of endothelial cell growth factor added 6 hours or more later. These results suggest that there is an intracellular effect of heparin which reduces 3H-methylthymidine uptake. Hydrocortisone acetate and tetrahydroS induced inhibition of EC growth in a dose-dependent manner. Steroids inhibited proliferation of EC in culture medium in the presence or the absence of growth factor and in different human serum concentrations. These results suggest a possible synergistic antiangiogenic action of heparin plus steroids.  相似文献   

3.
Summary Derivatized dextrans exert a stimulatory effect on the in vitro growth of human umbilical vein endothelial cells (HUVEC). Measurements of growth were monitored by [3H]thymidine uptake and cell numbers. Our results show that some derivatized dextrans at 4 μg/ml (88 nM) increase the [3H]thymidine incorporation, whereas starting dextran (40 000 Da), dextran sulfate, and carboxymethyl dextran have no effect. In addition, heparin under similar experimental conditions shows a slight inhibitory effect on the HUVEC growth. The stimulatory effect of derivatized dextrans was also found when HUVEC grew during 7 days in medium containing 2% fetal bovine serum. We also observed that derivatized dextrans had no effect on the mitogenic activity of acidic fibroblast growth factor, a mitogenic factor for several cell types including HUVEC. By assessment of [3H]thymidine uptake at 48 h without serum, we concluded that the exogenous growth factors were not involved in the proliferative activity of these components. The stimulatory effects are related to the chemical nature and the proportion of substituents on the synthetic polysaccharides. The data indicate that benzylamide sulfonated groups play a key role in the stimulation of HUVEC growth. Neither carboxyl nor sulfate groups alone exhibit this effect. Thus, the stimulatory capacity of dextran derivatives depends strongly on the respective ratios of the functional groups.  相似文献   

4.
We investigated the effect of 1 alpha,25-dihydroxyvitamin D3 (1,25 (OH)2 vit D3) on the 3H-thymidine uptake by Balb/c 3T3 cells and by human skin fibroblasts stimulated by normal human serum or by purified PDGF. We found an inhibitory effect of 1,25 (OH)2 vit D3 on the DNA synthesis of Balb/c 3T3 cells grown in the presence of human serum as well as in the presence of PDGF. At 5% human serum this effect is minimal at 10(-12) M 1,25 (OH)2 vit D3 and is maximal at 10(-9) M. On the DNA synthesis of human fibroblasts stimulated by human serum or by PDGF a modulatory effect of 1,25 (OH)2 vit D3 was shown. On these cells the vitamin had a stimulatory effect between 10(-11) and 10(-9) M and an inhibitory effect at very high concentrations (10(-7) M). Our results suggested that the effect of 1,25 (OH)2 vit D3 on fibroblast DNA synthesis could be mediated by interactions with its specific intracellular receptor. 1,25 (OH)2 vit D3 had no any action on the growth of human fibroblasts stimulated by fibroblast growth factor.  相似文献   

5.
Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.  相似文献   

6.
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.  相似文献   

7.
8.
Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.  相似文献   

9.
Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.  相似文献   

10.
Summary Eleven human endothelial cell lines have been produced by introducing sequences from the DNA tumor virus SV40 into human umbilical vein endothelial cells either by strontium phosphate coprecipitation or electroporation. The resultant lines were confirmed as being endothelial in origin by their production of endothelial-specific von Willebrand factor. The growth characteristics of the different lines in normal and reduced levels of serum was determined, as was their cellular response to endothelial cell growth supplement in combination with heparin, basic fibroblast growth factor, transforming growth factor-alpha, and epidermal growth factor.  相似文献   

11.
Summary The development of pulmonary hypertension in a wide variety of human disease states and experimental animal models characterized by chronic alveolar hypoxia is mediated by two pathologic vascular processes, a) vasoconstriction and b) vasoconstruction (structural remodeling). The anatomic changes seen within the pulmonary circulation include a) increased deposition of collagen and elastin in the adventitial layer and b) aberrant pulmonary vascular smooth muscle cell proliferation and maturation in the medial segments. Despite the demonstrated ability of pharmacologic manipulation in the experimental animal to ameliorate both the structural and hemodynamic changes, the actual etiologic mechanisms are only beginning to be explored. Using the cell culture technique of co-cultivation, we have investigated the potential role of bovine pulmonary arterial endothelial cell-derived factors in mediating abnormal bovine smooth muscle cell growth under conditions of reduced oxygen tension. We have demonstrated that these cultured endothelial cells exposed in vitro to reduced levels of atmospheric oxygen concentrations of 5.0% and 2.5% O2 for durations of 24 to 72 h produce and secrete soluble growth factor(s) which stimulate smooth muscle cell proliferation when compared to cells maintained under standard tissue culture oxygen conditions of 95% room air. This growth-stimulatory effect required the concomitant presence of serum factors (0.5% fetal bovine serum), was inhibited by heparin, was distinct from platelet-derived growth factor, and seemed to have a molecular weight greater than 14 000 Da. We conclude that reduced levels of oxygen tension in vitro can selectively induce pulmonary arterial endothelial cells to release mitogen(s) which can stimulate vascular smooth muscle replication. Furthermore, we speculate that this in vitro finding may be of importance as an etiologic mechanism to explain the accelerated smooth muscle cell growth characteristic of hypoxic pulmonary arteriopathy.  相似文献   

12.
R Barra 《Cytobios》1987,52(209):99-107
Glycyl-histidyl-lysine (GHL) has been shown to have growth stimulatory effects on a number of different cell types including hepatocytes and hepatoma cells. In this study, the effects of GHL on Morris hepatoma 7777 cells were investigated. The greatest stimulatory effects on 3H-thymidine and 3H-leucine incorporation were observed at a GHL concentration of 2 ng/ml. In randomly proliferating cells, the incorporation of 3H-thymidine into DNA increased by 50% and that of 3H-leucine into protein by 29%. In addition, synergistic effects were observed when insulin and glucagon were included with GHL in the incubation mixture. Experiments with cells rendered quiescent by serum starvation indicated that cells in the G1 phase of the cell cycle are more sensitive to GHL stimulation. In these experiments, 3H-thymidine incorporation increased earlier and peaked at a higher value than in the control cells. This finding suggests that GHL may play a role in stimulating quiescent cells to re-enter the cell cycle.  相似文献   

13.
Growth factor responses of human arterial endothelial cells in vitro   总被引:2,自引:0,他引:2  
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.  相似文献   

14.
15.
Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: 1) maximal response to PM was 2-5 times greater; 2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; 3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.  相似文献   

16.
Present studies deal with the role of inhibin in proliferation and growth. The effect of inhibin on incorporation of 3H-thymidine in prostatic DNA in vivo as well as by NRK-49F and Balb/c3T3 cell lines in vitro, was investigated. Also studied the immunocytochemical localization of inhibin in normally proliferating and differentiated tissues of human prostate and endometrium. The in vivo studies revealed a suppression of 3H-thymidine uptake both in ventral (33%) and dorsolateral (26%) lobes of rat prostate. Interestingly, the histology of inhibin treated rat prostate manifested amidst the epithelial lining, an appearance of apoptotic bodies which are considered to be indicative of cell death. Further, the immunocytochemical studies for localization of inhibin showed intense staining in the differentiated human prostate and endometrium as compared to the respective proliferative tissues. Is inhibin kept suppressed in these proliferating tissues, because it is antiproliferative? The present in vitro experiments demonstrated that, at low inhibin concentrations, the incorporation of 3H-thymidine is stimulated while at higher doses it is suppressed. Thus, it is clear that prostatic inhibin seems to have a concentration-dependent dual role in the regulation of DNA synthesis.  相似文献   

17.
In investigating the role of cell-extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell-collagen interactions were examined. Exponentially growing Balb/c-3T3 fibroblasts were radiolabelled with 3H-thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70-90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10-50% when heparin was present from 0.1-100 micrograms/ml. Cell-collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell-collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (greater than 40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr less than or equal to 6KD) with type I collagen was analyzed by affinity co-electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell-collagen and heparin-collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative activity in vivo and in vitro.  相似文献   

18.
Heparin has a potent angiogenic effect in experimental animals and patients with ischemic diseases; however, the precise mechanism behind this angiogenesis remains to be clarified. The aim of this study was to determine whether the administration of heparin affects the levels of heparin-binding angiogenic factors in human plasma, and to identify the molecule responsible for heparin-induced angiogenesis. Plasma levels of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were measured before and after administration of 100 U, 3,000 U or 10,000 U of heparin in patients with coronary artery disease. Administration of 3,000 U or 10,000 U of heparin caused significant increases in plasma HGF (40- and 54-fold, respectively), in absence of obvious increases in bFGF and VEGF levels. Furthermore, compared with the serum collected before heparin administration, the serum collected after heparin administration had more prominent growth-promoting and vascular tube-inducing properties on endothelial cells, and these increased activities were completely inhibited by neutralization of HGF, whereas neutralization of bFGF and VEGF had no effect. These findings suggest that HGF plays a significant role in heparin-induced angiogenesis.  相似文献   

19.
1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.  相似文献   

20.
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.  相似文献   

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