Retention of aggregated LDL by cultured human coronary artery endothelial cells

Aggregated LDL (AgLDL) accumulates within the subendothelial space of developing atherosclerotic lesions. We were interested to learn whether endothelial cells can interact with AgLDL. Incubation of endothelial cells with AgLDL resulted in apparent cholesterol retention. Microscopic examination revealed that cholesterol retention resulted mainly from endothelial cell surface attachment of AgLDL. Little AgLDL entered endothelial cells consistent with the small amount of endothelial cell degradation of AgLDL. Although endothelial cell retention of AgLDL was inhibited by LDL, AgLDL retention was not blocked by lactoferrin, C7 anti-LDL receptor monoclonal antibody, or receptor-associated protein, suggesting that LDL receptor family members did not mediate this retention. Surface retention of AgLDL depended on microtubule function and could be regulated by the protein kinase C activator, PMA. Treatment of endothelial cells with PMA either before or during, but not after incubation with AgLDL, inhibited retention of AgLDL. Our findings show that endothelial cells can retain AgLDL but internalize and metabolize little of this AgLDL. Thus, it is unlikely that endothelial cells can transport AgLDL out of atherosclerotic lesions, but it is likely that retention of AgLDL affects endothelial function.     

Characterization of beta adrenoceptors on cultured endothelial cells by radioligand binding

The properties of beta-adrenoceptors present on the cultured bovine pulmonary artery endothelial cells were studied by radioligand binding. These cell contain a high density of beta-adrenoceptors (approximately 16,000 receptors/cell) having high affinity (Kd 18 pM) for 125I-iodocyanopindolol (ICYP). Competition binding experiments suggested the presence of more than two subtype of beta-adrenoceptors. 25% of the total population of receptors was found to be of beta 1-type. The remaining 75% represented a mixed population containing what is suggested to be a mixture of beta 2- and atypical beta-adrenoceptors.     

Characterization of cultured rat aortic endothelial cells.

We describe a new method to obtain rat aortic endothelial cells without contamination by vascular smooth muscle cells. The endothelial cells were characterized up to the 20th passage by low density lipoprotein incorporation, the absence of alpha-smooth muscle actin, the production of endothelium derived relaxing factor, and an elevation in intracellular free calcium concentration in response to bradykinin and ATP but not to AMP and angiotensin II.     

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.     

Purine metabolism in cultured aortic and coronary endothelial cells

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.     

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.     

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.     

Platelet thrombi produced on cultured endothelial cells by the dye/light method.

Platelet adhesion and aggregation were induced on cultured endothelial cells using the fluorescent dye/light method. A cone-and-plate apparatus was newly developed to observe interactions between platelets and cultured endothelial cells under a shear flow condition. The platelet deposition grew on the light-irradiated area of the cells. Degree of endothelial cell injury induced by the dye/light reaction seemed to depend on the dye concentration. Application of either aspirin or indomethacin significantly inhibited the growth of platelet aggregation, but was not effective for the platelet adhesion to endothelial cells. The platelet thrombi were formed on endothelial cells without their denudation. It was found by transmission electron microscopy that platelets directly adhered to endothelial cells which were not seriously damaged. This thrombus model is expected to be applicable to some physiological and pharmacological studies investigating platelet-endothelial cell interaction and mechanism of platelet thrombus formation in blood vessels.     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

Characterization of beta-adrenergic receptors in cultured human and bovine endothelial cells

We used radioligand binding methods to characterize beta-adrenergic receptors on endothelial cells cultured from adult human iliac vein (HIVE) and bovine fetal aorta (BFAE). For comparison, we also studied the well-characterized C6 glioma cell line (C6). Both human and bovine endothelial cells showed specific saturable binding of [125I]iodopindolol. There was no difference in the binding affinity (KD) of iodopindolol to membranes from the three cell types. However, the beta-receptor density (Bmax) was greater on HIVE cells and BFAE cells than on C6 cells. Displacement of ligand from HIVE and BFAE cells by zinterol or from BFAE cells by ICI 89,406 was consistent with binding to the beta 2-subtype. In contrast, displacement of ligand from C6 cells by zinterol or ICI 89,406 was consistent with binding to both beta 1- and beta 2-subtypes. Exposing BFAE cells in culture to 10 microM isoproterenol for 6 h resulted in a 55% decrease in Bmax without a change in KD. We conclude that 1) human and bovine endothelial cells in culture contain a substantial population of beta-adrenergic receptors, which are predominantly of the beta 2-subtype, and 2) endothelial beta-receptors exhibit downregulation by beta-agonists in culture.     

Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Specific binding sites for synthetic porcine endothelin (pET), a novel potent vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells, and its effects on cytosolic free Ca2+ concentrations ([Ca2+]i) and phosphatidylinositol (PI) response were studied in cultured rat aortic vascular smooth muscle cells (VSMC). Binding of 125I-labeled-pET to rat VSMC was time- and temperature-dependent and the cell-bound 125I-labeled-pET was resistant to dissociate. Scatchard analysis of binding studies indicated the presence of a single class of high-affinity binding sites: the apparent Kd was 2-4 X 10(-10) M and the maximal binding capacity was 11,000-13,000 sites/cell. The binding was highly specific for pET because neither well-recognized vasoconstrictors, peptide neurotoxins, nor Ca2+-channel blockers affected the binding. pET dose-dependently (10(-9)-10(-7) M) induced a transient and sustained increase in [Ca2+]i in fura-2-loaded cells of which effect was largely dependent on extracellular Ca2+, whereas it had no significant effect on PI response in 3H-myoinositol-prelabeled cells. The present data clearly demonstrates the presence of specific receptors for pET distinct from those of the well-recognized vasoconstrictors and voltage-dependent Ca2+-channels in cultured rat VSMC, and suggest that pET-induced increase in [Ca2+]i is involved in the mechanism of its vasoconstriction.     

Urokinase produced by cultured human kidney embryo cells

Urokinase was obtained from cultured cells of human fetal kidneys. Ultrafiltration on an Amicon cell and purification by gel filtration chromatography (Ultrogel ACA54) yielded two molecules capable of activating plasminogen into plasmin. Their molecular weights were respectively 47,500 and 31,500 daltons. The first one showed more active than the latter. In this experiment, only small amounts of Urokinase were harvested. The yield could be enhanced using activators (pronase, glycine) or adapting fetal cells to large scale cultures.     

Analysis of extracellular matrix proteins produced by cultured cells

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.     

Extracellular matrix produced by cultured corneal and aortic endothelial cells contains active tissue-type and urokinase-type plasminogen activators

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of ¹²⁵I-fibrin in a time-and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-and anti-t-antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-β-D-), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties. © 1993 Wiley-Liss, Inc.     

Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.     

Basal lamina formation by cultured microvascular endothelial cells

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.     

Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate-specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans.     

Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.